Microbiology and Biochemistry

Microbiology And Biochemistry

r

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MICROBIOLOGY

ANn BIOCHEMISTRY

Dr. Madan Lal Bagdi

MANGLAM PUBLICATIONS DELHI-II0053 (INDIA)

Published by: MANGLAM PUBLICATIONS L-2111, Street No. 5, Shivaji Marg, Near Kali Mandir, J.P. Nagar, Kartar Nagar, West Ghonda, Delhi -110053 Phone: 9968367559,9868572512 Email: [email protected] manglam. [email protected]

Microbiology And Biochemistry

©Reserved First Edition: 2009 ISBN 978-81-906785-0-6

All rights reserved no part ofthis work may be reproduced, stored in a retrieval system, or transmitted in any form or by any means, electronic, mechanical, phtocopying, recording or otherwise, without the prior permission in writting from publisher of this book.

PRINTED IN INDIA Published by D. P. Yadav for Manglam Publications, Delhi-110053, Printed at Sachin Printers, Moujpur Delhi-53

Preface The present title Microbiology and Biochemistry is an authoritative text book compilated for under-graduate and post-graduate students of various Indian Universities offering this subject. It would· be equally useful as a text in courses in molecular biolo,gy, pharmacology and certain other desciplines of biology. All kinds ofmicroorganisms have been touched to create an impression about the divl!rsity. The scope and practices of using different micro-orgabisms have been shown which may attract future generation. The enormous prospect of application of microbiology and biochemistry have been indicated The author expresses his thanks to all those friends, colleagues, and research scholars whose continuous inspirations have initiated him to bring this title. The author wishes to thank the Manglam Publications, printer and staff mambers for bringing out this book. Constructive criticisms and suggestions for iniprovement of the book will be thankfully acknowledged. Author

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Contents 1.

Introduction ......................................................... 1-35 1.1 Host-Parasite Relations ........................................ 2 1.2 1.3 1.4

1.5

1.6

Diagnosis of Parasitic Infections ............................. 4 Laboratory Procedures ......................................... 5 Procedures for Intestinal Parasites ........................... 6 1.4.1 Collection and Handling of Fecal Specimens ... 6 1.4.2 Gross Examination of Feces ...................... 8 Procedures for Microscopic Examination ..................................... 8 1.5.1 Calibration and Use of an Ocular Micrometer. 9 1.5.3 Direct Wet Mount.. ............................... lG 1.5.4 Concentration Procedures ........................ 12 1.5.5 Permanent Stains .................................. 17 (ii) Unpreserved specimens with PV A fixative .................................. 18 (iii) PV A fixative-preserved specimens ......... 18 Egg Counts ......................................... 21 1.5.6 Duodenal Material ................................ 21 1.5.7 Sigmoidoscopic Material ......................... 12 1.5.8 Abscess Material ................................ , . 22 1.5.9 Cellophane Tape ................................... 23 1.5.10 Examination of Cellophane Tape ................ 23 1.5.11 Culture for Amoebae ............................ ,. 23 1.5.12 1.5.13 Larval Maturation ................................. 24 Adult Worms ....................................... 25 1.5.14 Blood and Tissue Parasites ................................... 25 (i)

CONTENTS

(ii)

1.7

1.8

1.9 1.10 1.11 1.12 1.13 1.14

Collection and Handling of Blood Specimens .............. 7fj 1.7.1 Tissue ............................................... Z7 1.7.2 Aspirates of Bone Marrow or Spleen ........... 28 1.7.3 Fluids ............................................... 28 1.7.4 Skin Snips .......................................... 28 l. 7.5 Concentration Procedures for Blood ............. 28 1.7.6 Membrane Filter Concentration for Filariae .. 19 1.7.7 Saponin Lysis Concentration for Filariae ...... 19 Staining Procedures ........................................... 30 l.8.1 Giemsa Stain Procedure .......................... 30 1.8.2 Gram-Weigert Stain Procedure .................. 30 1.8.3 Culture Procedures for Blood and Tissue Parasites ................................................. 31 Urine ............................................................ 32 Sputum .......................................................... 32 VAginal Material ............................................. 32 Referral of Materials ....................... ~ ................. 32 Safety ........................................................... 33 Quality Assurance ............................................. 35

2.

Origin of Microbiology .......................................... 36--51 2.1 Beginnings of Microscopy .................................... 37 2.2 The First Microscopes ........................................ 38 2.3 Microorganisms and the Origin of Life .............................................. 42 2.4 "Diseases" of Wines .......................................... 46 2.5 Pasteurization .................................................. 47 2.6 Pasteur on Specificity of Disease ........................... 48 2.7 Pasteur of Spontaneous Generation .......................... 48 2.8 Modem Style ................................................... 49 2.9 Chemical Evolution ........................................... 49

3.

Microbiology of Fungi ....••.......•.....••.......•....•........• 52--82 3.1 Characterization ............................................... 52 3.2 Collection and Storage of Specimens ....................... 53 3.3 Direct Exainination ........................................... 53 3.4 Culture and Isolation .......................................... 56 3.5. Identification ................................................... 63

(iii)

CONTENTS 3.5.1 3.5.2 3.5.3 3.5.4 3.5.5 3.5.6 3.5.7 3.5.8 3.5.9 3.5.10 3.5.11 3.5.12

4.

Aureobasidium Spp ................................ 70 Cladosporium Spp ................................. 70 Curvularia Spp ..................................... 71 Drechslera Spp ................... _................ 72 Exophiala Spp ...................................... 74 Fonsecaea spp ...................................... 76 Phaeococcomyces Spp ............................. 77 Phialophora Spp .................................... 78 Rhinocladiella Spp ................................ 79 Scedosporium Spp. . ............................... 79 Scytalidium Spp .................................... ~ Sporothrix Spp. . ................................... ~

Microbiology of Bacteria ...................•.................. 83-105 Shape of Bacteria ............................... " ...... '" .... S4 4.1.1 Size of Bacteria ................................... 85 4.2 The Bacterial Cell ............................................ 87 4.2.1 Cytoplasmic Membrane .......................... 87 4.2.2 Cell Wall ........................................... 88 4.2.3 Capsules ............................................ 89 4.2.4 Polysaccharide Structures ........................ 92 4.2.5 Nucleus ............................................. 92 4.2.6 Metachromatic Granules ......................... C)l 4.2.7 Fat Globules ....................................... 95 4.2.8 Motility ............................................. 95 4.2.9 Motion of Colonies ................................ 99

4.1

5.

Microbiology of Viruses .............................•........ 106-167 The Nature of the Virus Particle. ... ..... .... ....... ...... 108 The Classification of Viruses .............................. 115 The Virus Host .............................................. 116 Quantification of Viruses ................................... 117 General Features of Virus Reproduction ................. 120 Early Events of Virus Multiplication ..................... 123 Viral Genetics ............................................... 128 General Overview of Bacterial Viruses .................. 130 RNA Bacteriophages ........................................ 131

5.1 5.2 5.3 5.4 5.5 5.6 5.7 5.8 5.9

(iv)

CONTENTS 5.10 5.11 5.12 5.13 5.14 5.15 5.16

Single-Stranded Icosahedral DNA Bacteriophages ....................................... Single-Stranded Filamentous DNA Bacteriophages ....................................... Double-Stranded DNA Bacteriophages................... Large Double-Stranded DNA Bacteriophages ....................................... Temperate Bacterial Viruses: Lysogeny .................. A Transposable Phage: Bacteriophage Mu ........................................... General Overview of Animal Viruses. . . . . . . . . . . . . . . . .. . ..

134 138 139 143 147 156 160

6.

General Metabolism .......................•.•..•.............. 168-178 6.1 Characterization of Metabolism.. . . . . . . . . . . . . . . . . . . . .. .. .. 168 6.2 Energy Cycles in Animate Nature ........................ 170 6.3 Energetics of Biochemical Reactions......... ............ 173 6.4 Hight-Enegry and Low-Energy Phosphates: General Considerations ............................................... 175 6.5 Energy Transfer in Biochemical Processes ............... 177

7.

Metabolism of Saccharides ................................... 179-193 7.1 Carbohydrage Catabolism in Tissues ...................... 179 7.1.1 Pentose Phosphate Cycle ....................... 180" 7.1. 2 Interrelation of the Pentose Phosphate Cycle and Glycolysis............... 183 7.1.3 The Biological Function of the Pentose Phosphate Cycle. . . . . . . . . . . . . . . . ... 184 7.2 Biosynthesis of Carbohydrates in Tissues. . . . . . . . . . . .. .. .. 186 7.2.1 Gluconeogenesis ................................. 186 7.2.2 Biosynthesis of Glycogen (Glycogenogenesis) 189 7.2.3 Biosynthesis of Other Oligosaccharides and Polysaccharides ........ 190 7.3 Carbohydrate Metabolism Control in the Organism .... 191

8.

Metabolism of Fats and Glycerides .......................... 194-214 8.1 Degradation of Lipids in Tissues .......................... 194 8.1.1 Intracellular Hydrolysis of Lipids ............. 194 8.1.2 Oxidation of Glycerol ........................... 195 8.1.3 Oxidation of Fatty Acids ....................... 195

CONTENTS

8.2

8.3 8.4 8.5 9.

(v)

Biosynthesis of Lipids in Tissues .......................... 8.2.1 Biosynthesis of Fatty Acids .................... 8.2.2 Biosynthesis of Triglycerides .................. 8.2.3 Phospholipid Biosynthesis ....................... 8.2.4 Biosynthesis of Ketone Bodies ................. 8.2.5 Biosynthesis of Cholesterol ..................... Regulation of Lipid Metabolism in the Organism ............................... Pathology of Lipid Metabolism ............................ Applications of Lipids and Their Components in Pharmacotherapy ..........................

200 200 203 204 206 200 2fJ)

211 213

Metabolism of Nucleic Acid ••..•...••••.••.••••...••........ 215-257 9.1 Functional Roles of DNA .................................. 215 9.1.1 DNA as the Genetic Material ................. 215 9.1.2 Cellular Location of DNA ..................... 217 9.1.3 Clinical Comment. . . . . . . . . .. .. . .. . ............. 220 Other Conformations of DNA ................. 221 9.1.4 The "Central Dogma" .......................... 222 9.1.5 Strand Separation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 223 9.1.6 DNA Polymerases ............................... 224 9.1.7 Bacterial DNA Polymerases ................... 224 9.1.8 Stages of DNA Synthesis ....................... 226 9.1.9 9.1.10 Bidirectional Synthesis ......................... 727 9.2 DNA Synthesis in Animal Cells ........................... 230 9.2.1 DNA Polymerase a ............................. 230 9.2.2 DNA Polymerase b ............................. 231 9.2.3 DNA Polymerase g ............................. 231 9.2.4 Reverse Transcriptase .......................... 231 9.2.5 Nucleosome Formation ......................... 234 9.2.6 Transposable Genetic Elements ............... 235 9.3 Molecular Basis of Mutation ............................... 236 9.3.1 Mutagens ......................................... 237 9.3.2 Physical Agents .................................. 239 9.3.3 Excision Repair .................................. 239 9.3.4 Postreplication Repair .......................... 240 9.4 Chemical Carcinogenesis ................................... 241

CONTENTS

(vi)

9.5 9.6

9.4.1 Initiating Agents ................................. 9.4.2 Promoting Agents ........................... , ... , 9.4.3 Oncogenes ........................................ DNA Sequence Analysis ................................... Recombinant DNA Technology in Medicine ............. 9.6.1 Restriction Endonucleases ...................... 9.6.2 Restriction Maps ................................ 9.6.3 Cloning of Recombinant DNA ................ , 9.6.4 Clones ............................................. 9.6.5 Libraries of Genomic DNA .................... Detection of Recombinant DNA .............. 9.6.6 Clinical Comment .......................... , ... , 9.6.7 Reverse Genetics ................................ 9.6.8 Polymerase Chain Reaction .................... 9.6.9 9.6.10 Cloning and Sequencing the Human Genome .............................

242 242 243 246 248 248 249 249 250 253 254 255 256 256 257

1

Introduction Parasitic diseases continue to cause significant morbidity and mortality in the world, particularly in lessdeveloped tropical and subtropical countries. In the United States, indigenous malaria was eradicated long ago, and indigenous nematode infections such as ascariasis, trichuriasis,· and hookworm infection have markedly decreased in both incidence and severity. Some other infections are increasing. Giardiasis is a frequent public health problem, with outbreaks related to water supplies and day care centers for children. Giardia, ameba, and Cryptosporidium infections are increasing in male homosexuals. Pneumocystis carinii, Cryptosporidium species, Strongyloides stercoralis, and Toxoplasma gondii are increasingly important causes of serious infections in immunoco-mpromised hosts, especially those with AIDS (acquired immune deficiency syndrome). In addition to infections which are indigenous to the United States, a wide variety of infections may be seen in U.S. citizens who have traveled or worked in foreign countries or in foreign nationals who are visiting or now residing in the United States. Many of these people, such as persons infected with malaria, may be asymptomatic for months or years before disease develops or relapses occur. Some people are recognized as having malaria only when a recipient of their blood develops transfusion-induced malaria or when a baby develops congenital malaria. Other diseases such as echinococcosis may require years before becoming clinically evident. Efforts to eradicate parasite infections have had variable success. Sanitary fecal disposal, improved water supplies, and improved hygiene in food production and preparation have aided in the control of intestinal parasites. However, much of the earlier enthusiasm for

2

MICROBIOLOGY AND BIOCHEMISTRY

the eradication of malaria has been tempered by the realization that malaria eradication is going to be difficult because parasites are becoming resistant to chemotherapeutic agents, mosquito vectors are becoming resistant to common insecticides, and the use of some insecticides may harm the environment. Human modifications of the environment, such as the building of dams and irrigation systems, have provided an appropriate environment for vectors such as snails and thus allowed diseases such as schistosomiasis to flourish in areas where these diseases had been uncommon. In addition, immunization programs for parasite infections have developed more slowly than was anticipated.

1.1 HOST-PARASITE RELATIONS A knowledge of parasite life cycles is crucial in the understanding of the ways infection is acquired and spread, the pathogenesis of disease, and the ways in which disease might be controlled. Some parasites which infect only humans, such as Enterobius vermicularis (pinworm), have a narrow host specificity, whereas others such as Trichinella spiralis infect numerous species. When oth~ animals harbor the same parasite stage as humans, these animal species may serve as reservoir hosts. Humans infected with a parasite stage usually seen in other animal species are referred to as accidental hosts. In the simplest life cycle, the parasite stage from humans is immediately infective for other humans, as in pinworm infection or giardiasis. In other infections such as ascariasis or trichuriasis, a maturation period outside the body is required before the parasite is infective. However, for many parasite infections, a second or even a third host is required for completion of the life cycle. Hosts are defmed as intermediate hosts if they do not contain the sexual stage and as definitive hosts if they do contain the sexual stage. Some protozoa, such as the amebae, flagellates and hemotlagellates, do not have a recognized sexual stage. In the intermediate host, there may be a massive proliferation of organisms, as occurs in -humans harboring malaria parasites or snails harboring schistosome intermediate stages, or there may be no proliferation, as in mosquitoes which harbor microfilaria undergoing maturation. There may be proliferation in definitive hosts, as in mosquitoes harboring the sexual stage of malaria in which thousands of sporozoites are produced, or there may be no proliferation, as in helminth infections in which one adult is developed from each infective larva. However, in the latter, the adult helminths do produce numerous eggs or larvae.

INTRODUCTION

3

TABLE 1.1 : INCIDENCE OF INTESTINAL PARASITES IN 322,735 FECAL SPECIMENS EXAMINED BY STATE HEALTH DEPARTMENT LABORATORIES No. of % of positive Parasite examinations specimens Protozoa 12,947 4.0 Giardia lamblia 2,409 Entamoeba histolytica 0.8 1,880 0.6 Dientamoeba fragilis 7 Balantidium coli 1 Isospora spp. 21,120 6.5 Nonpathogenic Nematodes 5,481 1.7 Trichuris trichiura 4,630 Ascaris lumbricoides 1.4 4,344 Enterobius vermicularis 1.4 2,035 Hookworm 0.6 Strongyloides stercoralis 602 0.2 Trichostrongylus spp. 14 Trematodes Clonorchis-Opisthorchis 205 0.06 Schistosoma mansoni 48 Fasciola hepatica Paragonimus westermani Cestodes Hymenolepis nana 1,068 0.3 Taenia spp. 251 0.08 Diphyllobothrium latum 20 Hymenolepis diminuta 12 Dipylidium caninum 7 In some helminth infections, a migration through various body tissues is essential for maturation, as in ascarasis or schistosomiasis, whereas in other infections, the larva leaves the egg and simply matures in the intestinal tract, as in trichuriasis and enterobiasis. Host tissues involved vary depending upon the parasite. In severely immunocompromised patients, sites may be involved that are not involved in normal hosts.

4

MICROBIOLOGY AND BIOCHEMISTRY

Parasites of humans proliferate tremendously at certain stages, with thousands or even millions of forms being produced for every one that survives to perpetuate the parasite. Parasites may be quite hardy. For example, certain stages, particularly eggs and cysts, may survive for weeks or months in the environment. Parasites have often developed unique ways of protection from the defense mechanisms of the host. The survey does not include laboratories in Guam, Puerto Rico, or Virgin Islands. One or more parasites were found in 14.7% of specimens. Percentages are not calculated for parasites identified less than 100 times. These mechanisms include the ability to change antigenic characteristics so that although the host forms antibody, the antibody does not react with the modified parasite, or the parasite may be coated with host immunoglobulins, as in schistosomiasis, so that the host does not recognize the parasite as foreign. Macrophages and both cell-mediated and humoral immunities appear to be important in the host response to infection. Eosinophils are particularly important in the defense against tissue-invading helminths.

1.2 DIAGNOSIS OF PARASITIC INFECTIONS The diagnosis of most parasitic infections is dependent upon the laboratory. For intestinal and blood parasites, morphologic demonstration of diagnostic stage(s) is the principal means of diagnosis, whereas for tissue infections, immunodiagnostic teChniques are generally more important. During the early stages before diagnostic forms are produced (prepatent period), patients may be symptomatic. For example, patients may have pulmonary symptoms and eosinophilia due to ascaris larva migration at a time when eggs are not produced. In such patients the physician may suspect parasite infection, but the actual diagnosis must be based on a clinical impression or immunodiagnostic tests, or diagnosis must await the production of diagnostic stages. In establishing a diagnosis, the clinician places a great deal of trust in the laboratory. This trust can be misplaced if laboratory personnel are not competent to identify or exclude parasites. The literature clearly documents instances in which outbreaks have been overlooked due to incompetent laboratory diagnosis or in which inflammatory cells or other objects have been identified as parasites and outbreaks have been diagnosed when none existed. The results of proficiency-testing programs also suggest that laboratories have

5

INTRODUCTION

difficulty with the identification of some parasites, especially intestinal protozoa. Identification may be by gross examination for adult helminths or, more commonly, by microscopic examination for protozoa, helminth eggs, and larvae. The diagnostic forms of some parasites, such as the eggs of Ascaris spp., are present on a regular basis. Other forms, such as malaria parasites, Taenia eggs, or Giardia cysts, vary from day to day. TABLE 1.2 : PARTICIPANT PERFORMANCE No. of Parasite

Avg correct specimens

identification

(%) Formalin-fixed fecal specimens Ascaris lumbricoides eggs Hookworm Strongyloides stercoralis larvae Trichuris trichiura eggs Diphyllobothrium latum eggs Hymenolepis diminuta eggs Taenia sp. eggs Paragonimus westermani eggs Giardia lamblia cysts Entamoeba coli cysts No parasite seen PVA-fixed specimens Entamoeba histolytica Entamoeba coli Endolimax nana Negative for parasites

6 6 4

6 6 5 6 5 8 9 6 5 4 4

3

90 92 88 93 81 91 87 83 65 88 92 73 52 51 77

Most immunodiagnostic tests used today for parasitic infections detect antibody. In recent years, the sensitivity and specificity of many such tests have improved. A number of antigen detection tests have recently been described and show promise, but none of these tests are currently available commercially.

1.3 LABORATORY PROCEDURES Many methods for diagnostic parasitology have been described.

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MICROBIOLOGY AND BIOCHEMISTRY

'There are advantages and disadvantages to each method. Some are particularly valuable for epidemiologic studies or for evaluations of new therapeutic agents, whereas other methods are used primarily for laboratory diagnosis. In this chapter we emphasize the diagnostic procedures. From the numerous methods, we have selected those which are widely used in this country and which are sensitive and relatively easy to perform. These methods should prove adequate for most laboratories. For additional procedures, laboratory manuals or parasitology books should be consulted. When alternative methods or methods for specific parasites are indicated, references will be given, but the methods will not be described.

1.4 PROCEDURES FOR INTESTINAL PARASITES Intestinal and biliary parasites are generally diagnosed by finding diagnostic stages in feces or other intestinal material such as duodenal or sigmoidoscopic aspirates. Studies have shown that the eggs of most parasites are uniformly distributed in the fecal mass due to the mixing action of the colon, although some, such as schistosome eggs, which originate in the distal colon, may be more numerous on the surface of formed fecal specimens. The distribution of protozoan forms is more variable. There may be fewer protozoan trophozoites in the first part of an evacuation than in the last because they have deteriorated while in the lower colon. 1.4.1 Collection and Handling of Fecal Specimens The numbers and times of collection for fecal specimens depend somewhat on the diagnosis suspected. As a routine, because some organisms are shed in a variable pattern, it is advisable to examine multiple specimens before excluding parasites. The general recommendation is to collect a specimen every second or third day, for a total of three specimens. From a hospitalized patient, one specimen each day for three days may be more cost effective. A number of substances may interfere with stool examination. Particulate materials such as barium, antacids, kaolin, and bismuth compounds interfere with morphologic examination, and oily materials such as mineral oil create small, refractile droplets that make examination difficult. Antimicrobial agents, particularly broadspectrum antimicrobial agents, may suppress amebae. If any of these substances have been used, specimens should not be submitted until the substances have been cleared (generally 5 to 10 days). A fecal specimen may appear satisfactory by gross examination when there is still barium, etc., which can interfere with microscopic examination.

INTRODUCTION

7

Fecal specimens are best collected into widemouthed, water-tight containers with tight-fitting lids such as waxed, pint-sized ice cream cartons or plastic containers. Usually patients can defecate directly into such containers. Urine should not be allowed to contaminate specimens, as it is harmful to some parasites. If specimens are to be collected in a bed pan, the patient should micturate into a separate container before the specimen is collected. Toilet paper should not be included with the specimen. Stool should not be retrieved from toilet bowl water, as various freeliving protozoa in water might be confused with the parasites. In addition, water is harmful to some parasites such as schistosome eggs and amebic trophozoites. If the patient is producing formed specimens, stool may be collected by having the patient squat over waxed paper to defecate. Purgation with sodium sulfate or buffered phosphosoda may be helpful in the diagnosis of amebiasis in some patients. Purgation is usually done after a series of fecal specimens have been negative, and it requires the order of a physician. Prior arrangements must be made with the laboratory, and specimens must be collected during regular laboratory hours. The patient is given the appropriate salt solution orally. In approximately 1 to 1.5 h, the patient will begin to pass stool specimens, and each specimen should be promptly transported to the laboratory for examination. Clinical information such as the suspected diagnosis, travel history of the patient, and clinical findings should be included on the requisition. In addition, the time the specimen was passed and the time it was placed in fixative should be noted. If the specimen is in fixative, the consistency of the original specimen should be stated, or a portion of unfixed specimen should be included with the fixed specimen. A laboratory may have specimens placed in fixatives in the home or patient care area immediately after passage, may place portions of specimen in fixatives at the time they are received in the laboratory, or may examine the specimen unfixed. Many laboratories use a combination of these methods depending on the location of the patient, consistency of the specimen, time of day, and laboratory work load. Prompt examination or fixation is particularly important for soft, loose, or watery specimens, which are most likely to contain protozoan trophozoites. Formed specimens, which are likely to contain protozoan cysts or helminth eggs or larvae, can remain satisfactory for a number of

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MICROBIOLOGY AND BIOCHEMISTRY

hours at room temperature or overnight in a refrigerator. Soft and liquid specimens should be examined or placed in fixatives promptly (within 1 h). Specimens which cannot be examined or fixed promptly should be either refrigerated or left at room temperature. They should not be incubated, as incubation speeds the deterioration of the organisms. Feces for parasite examination must not be frozen and thawed. The fixative system generally used is a two-vial technique with one vial containing 5 to 10% buffered Formalin and the other vial containing polyvinyl alcohol (PV A) fixative. A portion of the specimen is added to the fixative in a ratio of approximately 3 parts fixative to 1 part specimen and thoroughly mixed to ensure adequate fixation. An alternative to Formalin is Merthiolate-iodine-formaldehyde (MIF), which fixes and stains at the same time. If unfixed specimens are processed in the laboratory, fecal films may be prepared and immediately fixed in Schaudinn fixative.

1.4.2 Gross Examination of Feces Specimens should be examined grossly to determine the consistency (hard, formed,loose, or watery), color, and presence ofgross abnormalities such as worms, mucus, pus, or blood. It may be profitable to examine flecks of mucus, pus, or blood for parasites. If adult worms or portions of tapeworms are sought, the feces may be carefully washed through a screen. (Small worms may be difficult to see if gauze is used.) The identification characteristics of adult worms are not discussed in this chapter, so parasitology books should be consulted.

1.5 PROCEDURES FOR . MICROSCOPIC EXAMINATION The three principal microscopic examinations performed on stool specimens are direct wet mount, wet mount after concentration, and permanent stain. Although each examination can contribute to diagnosis, the yield of some methods is small with certain kinds of specimens. As a minimum, formed specimens should be examined by a concentration procedure. Soft specimens should be examined by concentration and permanent stain, and, if submitted fresh, by direct wet mount. Loose and watery specimens should be examined by wet mount and permanent stain. If specimens are received in fixative and the consistency is not known, concentration and permanent stain should be performed. Other examinations may be helpfuL Special procedures which may assist in the diagnosis of specific parasites are noted below in discussions of the parasites.

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INTRODUCTION

1.5.1 'Calibration and Use of an Ocular Micrometer Size is important in the differentiation of parasites and is most accurately determined with a calibrated ocular micrometer, thus, each laboratory performing diagnostic parasitology must have such a micrometer. An ocular micrometer is a disk on which is etched a scale in units from 0 to 50 or 100. To determine the micrometer value of each unit in a particular eyepiece and at a specific magnification, the unit must be calibrated with a stage micrometer. A stage micrometer has a scale 2 mm long ruled in fine intervals of 0.01 mm (10 p.m). 1.5.2 Calibration of the Ocular Micrometer 1. Insert the micrometer in the eyepiece so that the micrometer rests on the diaphragm, with the etched scale facing the eye. In many new microscopes, the micrometer can be dropped in and secured with a ring retainer. (It is helpful to have an extra ocular in which the micrometer may be left.) 2. Place the stage micrometer on the microscope stage. TABLE 1.3: LABORATORY EXAMINATIONS FOR VARIOUS TYPES OF FECAL SPECIMENS Type of specimen

Direct wet mount

Unpreserved Formed + ++ Soft + + Loose and watery ++ Preserved Formalin Formed or soft + Loose or liquid PVA fixative Formed Soft, loose, or liquid Essential for basic examination;

Permanent stain

++ ++

±

±

++

+ +

++

+ +,

Method Concentration

+

± ++ +,

recommended for basic examination;

±, optional for basic examination. 3. Focus on the etched scale. Since the micrometer must be calibrated for each objective, begin with the lowest magnification

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MICROBIOLOGY AND BIOCHEMISTRY

(e.g., x 10). 4. Align the two scales so that the zero points are superimposed. 5. Find a point far down the scales at which a line of the stage micrometer coincides with a line of the ocular micrometer. Count the number of ocular units and the number of stage units from zero to these coinciding lines. 6. Multiply the number of stage micrometer units by 1,000 to convert millimeters to micrometers. 7. Divide the. product of step 6 by the number of ocular units to determine the 'value of an ocular unit. Repeat the calibration for each objective. Keep a record of the unit value for each objective for each microscope used. Calibration must be done separately for each microscope and must be repeated if an ocular or objective is changed. Use of the micrometer. Insert the eyepiece containing the calibrated ocular micrometer in the microscope. Count the number of ocular units which equal the structure to be measured. Multiply the number by the micrometer value of the ocular unit for the objective being used. If an ocular micrometer is properly used, parasites which are similar in appearance but different in size can be readily differentiated. 1.5.3 Direct Wet Mount The direct wet mount made from unconcentrated fresh feces is most useful for the detection of the motile trophozoites of intestinal protozoa and the motile larvae of Strongyloides spp. It is also useful for the detection of protozoan cysts and helminth eggs. For fixed feces, the direct wet mount may allow the detection of parasites which do not concentrate well. This method is also useful for the examination of specific portions of feces, such as flecks of blood or mucus. Direct wet mounts are prepared by placing a small drop of 0.85% saline toward one end of a glass slide (2 by 3 in. [ca. 5 by 7.5 cm]) and a small drop of appropriate iodine solution (see below) toward the other end. With an applicator stick, a small portion of specimen (1 to 2 mg) is thoroughly mixed in each diluent, and a no. I cover slip (22 mm) is added. The density of fecal material should be such that newspaper print can be read with difficulty through the smear. The material should not overflow the edges of the cover slip. Grit or debris may prevent the cover slip from seating and may be

INTRODUCTION

11

removed with a comer of the cover slip or an applicator stick. Mounts may be sealed with Vaspar (50% petroleum jelly, 50% paraffin) which is melted on a hot plate (not over an open flame). A cotton applicator or small brush is used to apply small drops of Vaspar to opposite comers to attach the cover slip and then to seal it with even strokes. The amount of Vaspar on top of the cover slzip should be minimal. Sealing slows drying and allows oil immersion magnification to be used. Alternatively, drying can be slowed by placing wet gauze or paper toweling in a petri dish, laying portions of applicator sticks or glass rods on the moist material, laying the slide on the .sticks or rOds, and replacing the lid of the dish. The iodine solution should be that of Dobell and O'Connor (1 %) or a 1:5 dilution of Lugol iodine. Iodine solution, if too weak, will not stain organisms properly, and if too strong, it will cause clumping of fecal material. Stock iodine solution should be stored in a tightly stoppered brown bottle away from sunlight. Keep the iodine and saline solutions in small dropper bottles, and replace (don't replenish) the solutions weekly. Iodine solution keeps longer if it is refrigerated. Iodine stain solution can be quality controlled by the observation of appropriate staining in positive clinical specimens or Formalin-fixed specimens kept for that purpose. For the examination of wet mounts, the light of the microscope must be properly adjusted. To achieve optimal resolution, the condenser should be centered and focused for Kohler illumination (racked up). To achieve contrast of the objects in the field, light intensity is diminished with the iris diaphragm of the condenser rather than by lowering the condenser. The entire saline wet mount cover slip should be systematically scanned with x 100 to X 200 magnification. Suspicious objects are confirmed at higher magnification. In addition, the preparation should be scanned at higher power (X 400 to x 500) for a couple of passes across the cover slip to look for protozoan cysts which might be missed with lower power. Screening a slide should take an experienced microscopist about 10 min. If debris is covering a suspicious object, the debris may be removed by pressing or tapping on the cover slip with an applicator stick. This pressure may also help in reorienting an egg, as when one is looking for an operculum. The saline wet mount is best for the detection of helminth eggs and larvae, and it is especially good for protozoan cysts, which appear refractile. The principal usefulness of the iodine mount is to study

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the morphology of protozoan cysts, as this stain shows nuclear detail and glycogen masses (but does not stain chromatoid material). If suspicious objects are seen, they can be examined under oil immersion ( xl, (00). If definite or possible protozoan cysts or trophozoites are detected which cannot be identified in wet mounts, permanent stains are required. A solution of buffered methylene blue (pH 3.6) may be used as a vital stain for the examination of fresh specimens for protozoa. The wet mount is prepared as described above, with buffered methylene blue substituted as diluent and 5 to 10 min allowed for the dye to become incorporated in the organisms before examination. Organisms become overstained in 20 to 30 min.

1.5.4 Concentration Procedures Concentration procedures are used to separate parasites from fecal detritus. These procedures are based on differences in the specific gravity of parasite forms and fecal material. In sedimen-tation, the parasite forms are heavier than the solution and are found in the sediment, whereas in flotation, solutions of high specific gravity are used, and parasite forms float to the surface. An initial washing step removes some of the soluble or fmely particulate material, and straining removes larger portions of debris. A wide variety of sedimentation and flotation methods have been described. The Formalin-ethyl acetate modification of the Formalin-ether sedimentation technique and a zinc sulfate flotation technique are widely used and are the only methods described in this chapter. Both methods require that centrifugation be performed in centrifuges with free-swinging carriers. Squeeze bottles for Formalin, saline, or water simplify the processing of large numbers of specimens. 1.5.4.1 Formalin-ethyl acetate centrifugal sedimentation The original procedure from which the Formalinethyl acetate centrifugal sedimentation technique was adapted was the Formalinether concentration method of Ritchie. The Formalin-ethyl acetate procedure avoids problems with the flammability and storage of ether. This procedure can be performed on specimens which have been fixed in Formalin for a time or on specimens with Formalin added during the processing. The procedure can also be performed on material fixed in MIF. The procedure with Formalin-preserved specimens is as follows. 1. Thoroughly mix the formalinized specimens.

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2. Depending on the density of the specimen, strain a sufficient quantity through gauze into a 15-ml conical centrifuge tube to give the desired amount of sediment. (Wet gauze in a 4-oz [ca. 120-ml] conical paper cup with the tip cut off can be used for straining.) 3. Add tap water or saline, mix the solution thoroughly, and centrifuge it at 650 x g for 1 min. The amount of the resulting sediment should be about 1 ml. The amount of sediment may be adjusted by the addition of more feces and centrifugation again or by the addition of water, suspension again, the removal of an appropriate amount of material, and then recentrifugation. 4. Decant the supernatant, and wash it again with tap water, if desired. 5. To the sediment, add 10% Formalin to the 9-ml mark, and mix the solution thoroughly. 6. Add 4 ml of ethyl acetate, stopper the tube, and shake the tube vigorously in an inverted position for 30 s. Remove the stopper with care. 7. Centrifuge the solution at 450 to 500 x g for 1 min. Four layers should result: ethyl acetate, plug of debris, Formalin, and sediment. 8. Free the plug of debris from the sides of the tube by ringing the tube with an applicator stick, and carefully pour the top three layers into a discard container. With the tube still tipped, use a swab to remove debris from the sides of the tube. This step is very important, for lipid droplets which reach the sediment make examination difficult. 9. Mix the remaining sediment with the small amount of fluid that drains back down from the sides of the tube (or add a drop of saline or Formalin). If mounts are to be prepared later, a small amount of Formalin may be added to the sediment and the tube may be stoppered. 10. Prepare wet mounts as described above, and examine them. The procedure for Formalin-ethyl acetate centrifugal sedimentation with fresh specimens is as follows. 1. Comminute a portion of stool about 1.5 cm in diameter in 10 ml of saline or water. 2. Strain about 10 ml of the fecal suspension into a 15-ml conical centrifuge tube. 3. Centrifuge the suspension at 650 x g for 2 min. This step

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should provide about 1 ml of sediment. If not, adjust the amount of sediment as described above. 4. Wash the sediment again if desired. 5. To the sediment, add 10% buffered Formalin lo the 9-ml mark, mix thoroughly, and allow the mixture to stand for 5 min or longer. 6. Proceed as for step 6 of the procedure for fixed specimens. (Note that either saline or water can be used. Tap water will lyse Blastocystis hominis. If schistosomiasis is suspected, the specimen should be preserved in Formalin before concentration to prevent hatching.)

1.5.4.2 Zinc sUlfate centrifugal flotation The zinc sulfate concentration method originally described by Faust et al. may be performed on unfixed or Formalinfixed specimens, although the specific gravity of the zinc sulfate solution required differs. The disadvantages of the zinc sulfate concentration are: (i) dense schistosome eggs do not concentrate well; (ii) opercula often pop, and thus operculate eggs may be missed; and (iii) larvae and cysts may collapse. The modified procedure with Formalin-fixed feces slows the collapse of larvae and cysts and largely prevents the popping or opercula. The advantages are that it leaves a rather clean background, has less grit than the sedimentation procedure, and is better for the concentration of some parasites, such as Giardia cysts. The procedure for Formalin-preserved specimens is as follows. The specific gravity of the zinc suflate must be 1.20. Centrifugation must be performed in round-bottomed tubes such as 16- by l00-mm disposable tubes. 1. The Formalin-preserved fecal material is mixed, strained through one layer of cheesecloth into a conical paper cup, poured into the tube to a level about 1 cm from the top, and then centrifuged. 2. The tubes are centrifuged for 3 min at about 650 x g. There should be 1 to 1.5 cm of sediment. 3. Decant the supernatant from each tube, and drain the last drop against a clean section of paper towel. 4. To the packed sediment of each tube, add zinc sulfate to within 1 cm of the rim. 5. Insert two applicator sticks, and thoroughly mix the packed sediment. 6. Immediately centrifuge the suspension at 500 rpm for 1.5 min.

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7. Very carefully transfer the tubes to a rack of the proper size, so that the tubes remain vertical. Do not disturb the surface ftlms, which now contain the parasites. Allow the tubes to stand for 1 min to compensate for any movement. The countertop must be vibration free. 8. With a loop which is bent at a right angle, transfer to a slide (2 by 3 in.) two loops of surface material beside I drop of saline and two loops beside I drop of iodine. With the heel of the loop, mix first the saline and then the iodine with the surface material. Cover each mixture with a 22-mm no. I cover slip. The slide should be made within 20 min. 9. To retard drying, place each prepared slide on a bent glass rod or portions of applicator sticks in a petri dish containing a damp paper towel. Petri dishes may be placed in the refrigerator if examination will be delayed. Alternatively, cover slips may be sealed with Vaspar. The procedure with fresh specimens is as follows. / 1. Comminute a fecal specimen about 1 cm in diameter in a tube (16 by 100 mm) half filled with tap water. Add additional water to within 1 to 2 cm of the top. 2. Centrifuge the tube at 650 x g for 1 min. 3 .. Discard the supernatant, and add a zinc sulfate solution of specific gravity 1.18 to within 1 cm of the rim. 4. Proceed as from step 5 above. 1.5.4.3 Sheather sugar ./lotation She ather sugar flotation is recommended for the concentration of Cryptosporidium cysts. Although these oocysts will concentrate when the Formalin-ethyl acetate or zinc sulfate technique is used, they are more readily detected with the Sheather sugar flotation, for they stand out sharply from the background in this solution of high specific gravity. This procedure may be performed on unfixed or Formalin-fixed feces. The procedure for Sheather sugar flotation is outlined below. l. (a) Fonned stool. Place approximately 0.5 g of stool in a tube (16 by 100 mm) about half full of Sheather sugar solution. Mix the solution thoroughly, and then add more sugar solution to within 1 cm of the rim. (b) Watery stool. Centrifuge the fecal specimen and mix 0.5 to 1 ml of sediment with Sheather solution as described above.

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2. Centrifuge the solution at 400 x g for 5 to 10 min. 3. Remove the top portion of the sample with a wire loop bent at a right angle. Place severalloopfuls on a glass slide (2 by 3 in.). Cover the specimen with a 22-mm cover slip, and examine the slide with a x 40 objective. Oocysts are found just beneath the cover slip and are refractile. Saline or iodine is not used in the pr~paration of these mounts.

1.5.4.4 Baermann concentration The Baermann concentration technique has greater sensitivity for the detection of strongyloides larvae than do the standard concentration techniques described above. This technique is useful clinically for the diagnosis and monitoring of therapy of strongyloides infections, and it is useful epidemiologically for the examination of soil for the larvae of nematode parasites. A funnel with a clamped rubber tube on the stem is placed in a ring stand. A circular mesh screen is placed across the funnel approximately one-third from the top, a portion of coarse fabric such as muslin is placed on the screen, and feces is added. Tap water at 37°C is added so that the water just touches the feces. Let the specimen stand I h, remove 2 ml of fluid from the stem, and centrifuge the sample at 300 x g for 3 min. Prepare a wet mount of sediment, and examine it for larvae. 1.5.4.5 Hatching technique for viable schistosome eggs Place a large amount of feces (5 to 10 g) in a large flask (1 to 2 liters), and add water while mixing to break up the feces to a fine suspension. Bring the water level to 2 to 5 em from the top of the flask. Cover the sides of the tlask with foil or other material to shield all but the top of the liquid from light. Allow the tlask to stand at room temperature for several hours. With a hand lens, examine the material at the top of the tlask neck for swimming miracidia. Remove the miracidia with a Pasteur pipette for examination with a x 10 objective. It is not possible to determine the species of schistosome from the miracidia. 1.5.4.6 Other concentration procedures Concentration procedures have been described for feces preserved in MIF, sodium acetate-Formalin, or PYA fixative. MIF- or sodium acetate-Formalin-fixed feces may be used in place of Formalin-fixed feces in the Formalin-ethyl acetate concentration procedure. Some workers feel that organisms do not concentrate as well from material

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fixed in PV A fixative or from material which has been in MIF for extended periods. If large amounts of specimen are to be concentrated, as when specimens of C)ggs are prepared for teaching, gravity sedimentation is usualiy used. The feces is thoroughly mixed in liquid (water, saline, or 10% Formalin) and allowed to settle in a sedimentation jar or funnel for several hours ot overnight. Supernatant fluid is discarded, and the sediment is again suspended and allowed to settle. This procedure can be continued if desired until the supern-atant is clear.

1.5.5 Permanent Stains Permanent stains of fecal smears are most needed for the detection and identification of protozoan trophozoites, but they are also used for the identification of cysts. Wet mounts of fresh feces, even with stains such as methylene blue, are not as sensitive for trophozoites and therefore do not substitute for permanent stains. It is sometimes difficult to identify cysts which are detected in wet mounts; thus, for each specimen, regardless of consistency, it may be worthwhile to fix a portion in PV A fixative or to prepare two fecal films fixed in Schaudinn fixative so that permanent stains can be performed if needed. Permanent stains also provide a permanent record and are easily referred to consultants if there are questions on identification. A number of staining procedures have been described. Some stains, such as chlorazol black, require fresh specimens and are not widely used. A variety of stains for fecal smears preserved by Schaudinn or PV A fixative have been described, including various hematoxylin stains. The stain most widely used in the United States is the Wheatley trichrome stain, which is the only permanent stain described in this chapter. The trichrome staining procedure uses reagents with a relatively long shelf life and is easy to perform. Note that there are differences in staining times depending on whether the specimen is fixed in Schaudinn or PV A fixative, as penetration is slower in the latter. Preparation of smears. (i) Unpreserved specimens with Schaudinn fixative. 1. To prepare thin, uniform smears, place a drop of saline on a glass slide (l by 3 in. [ca. 2.5 by 7.5 cm]). With an applicator stick, transfer a small, representative portion of the specimen to the drop of saline, and mix the two. Spread the solution into a film by rolling the applicator stick along the surface. Remove any lumps. Before watery specimens are smeared, apply an adhesive such

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as serum or albumin to the slide. Liquid specimens may be centrifuged, and the sediment may be used for smear preparation. 2. Place fresh smears immediately into Schaudinn ftxative. Do not allow the smears to dry at any time before they are stained. Smears should ftx for at least I h at room temperature or for 5 min at 50°C; however, they can be left in ftxative for several days. After ftxation, slides may be kept in 70% alcohol indeftnitely before they are stained. (ii) Unpreserved specimens with PV A fixative. 1. On a slide (l by 3 in.), thoroughly mix I drop of unftxed

specimen ~ith I drop of PYA ftxative. 2. Spread the specimen as described below. 3. Allow the smear to dry, preferably overnight, before it is stained. (iii) PVA fixative-preserved specimens.

1. Preserve I part specimen in 3 parts PV A fixative. Mix thoroughly. Fix for at least I h. Specimens keep indefinitely. 2. Add I drop of PV A-fixed specimen to a slide. (a) If there is little sediment, remove a portion of the sediment with a Pasteur pipette. (b) If there is abundant sediment, mix the specimen thoroughly, and add I drop of specimen to a slide with applicator sticks or a Pasteur pipette. 3. Spread the material over the center third of the slide by rolling the specimen with an applicator stick. Remove any lumps. The film should extend to both the top and bottom edges of the slide, as this helps prevent peeling. 4. Allow the slide to dry overnight at room temperature or 35°C. In an urgent situation, the slide can be dried for 4 h at 35°C and then stained.

1.5.5.1 Trichrome staining procedure Table elsewhere outlines the steps in the trichrome staining procedure. Permanently stained slides may be mounted with a cover slip or may be air dried and examined after oil is added. Slides should be examined at a magniftcation of x 400 to X 500 or greater after they are scanned under lower power to fmd optimal areas. A x 50 oil immersion objective is particularly helpful, as it allows the easy use

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of aX 100 oil immersion objective for the detailed examination of organisms while allowing more rapid screening with a x50 objective. Oculars of x 5 or x 6 can provide the same result. A x20 dry objective may also assist in screening. Permanently stained slides should be kept for 2 years.

1.5.5.2 Stain reactions In an ideal stain, the cytoplasm of cysts and trophozoites is bluegreen tinged with purple. Entamoeba coli cyst cytoplasm is often more purple than that of other species. Nuclear chromatin, chromatoid bodies, erythrocytes, and bacteria stain red or purplish red. Other ingested particles such as yeasts often stain green. Parasite eggs and larvae usually stain red. Inflammatory cells and tissue cells stain in a fashion similar to that of protozoa. Color reactions may vary from the above. Incompletely fixed cysts may stain predominantly red, and organisms which have degenerated before fixation often stain pale green. Poor fixation due to an inadequate mixing of the specimen in fixative may result in both of these" appearances. In some specimens, degeneration has occurred before the specimen is placed in fixative, either in the patient before the specimen was evacuated or because of delay in fixing the specimen. 1.5.5.3 Troubleshooting the trichrome stain Except for problems with delayed or inadequate fixation as noted above, problems with the trichrome stain are usually related to reagents other than the stain. If crystalline material is apparent after the specimen is stained, the crystals are probably mercuric chloride in the fixative which was not adequately removed because the iodine in the alcohol-iodine solution was too weak or because the slide was in this solution too short a time. If crystals are present after treatment with proper-strength iodine-alcohol, they are present in the specimen, which is thus unsatisfactory, and another specimen should be requested. If the stain appears washed out, it is likely that the slide was destained too much. This washed-out appearance can be either because the specimen was left too long in the acid-alcohol destain or because the alcohol wash after the acid-alcohol destain had become acidic as a result of transfer by previous slides. The trichrome may become diluted by carry-over alcohol if more than 10 slides per day are stained in one Coplin jar. To restore the

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stain, the lid may be left off for several hours to allow alcohol to evaporate, and then the volume is replaced with new stain. Control slides should be used to monitor the staining. Specimens containing protozoa are best for controls; however, feces containing inflammatory cells or added buffy-coat leukocytes also are satisfactory.

1.5.5.4 Restaining Should the stain be unsatisfactory, the slide can be destained by placing it in xylene to remove the cover slip or immersion oil and then placing it in 50 % alcohol for 10 min to hydrate the slide. Destain the slide in 10% acetic acid in water for several hours, and then wash it thoroughly first in water and then in 50 and 70% alcohols. Place the slide in stain for 8 min, and then complete the stain procedures. It is helpful to e.Jiminate or shorten the destain step.

1.5.5.5 Acid-fast stain for Cryptosporidium sp. Acid-fast staining for Crypiosporidium. sp. has recently become important because this parasite is now recognized as a cause of severe diarrhea in immunodeficient patients such as those with AIDS, and it can cause transient diarrhea in immunocompetent individuals. The modified acid-fast stain recommended is similar to that used to stain Nocardia spp. in that it uses milder acid decolorization. A variety of acid-fast and fluorochrome staining procedures have been described for Cryptosporidium spp., and all the procedures appear to work. The following procedure is useful for staining Nocardia species as well as Cryptosporidium species. This procedure may be used on fresh or Formalinfixed material or on material from concentration procedures. If the specimen is liquid, centrifuge it, and use the sediment to prepare a smear. 1. Pick a portion of material with an applicator stick, mix the material in a drop of saline, spread it on a glass slide (1 by 3 in.), and allow it to dry. 2. Fix the dried film in absolute methanol for 1 min, and air dry the slide. 3. Flood the slide with Kinyon carbol-fuchsin, and stain the smear for 5 min. 4. Wash the slide with 50% ethyl alcohol in water, and immediately rinse it with water. 5. Destain the smear with 1 % sulfuric acid for 2 min or until no color runs from the slide. 6. Wash the slide with water.

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7; Counterstain the smear with Loeffler methylene blue for 1 min. 8. Rinse the slide with water, dry it, and examine the smear with oil inunersion. The results are that Cryptosporidium oocysts stain bright red, and background materials stain blue or pale red.

1.5.6 Egg Counts Egg-counting methods are used in clinical studies to assess the intensity of infections (especially infections by intestinal nematodes) and the efficacy of therapeutic agents, and these methods are commonly used for epidemiologic studies. Methods used for scientific studies, such as Kato thick smear or Stoll egg counting, require greater accuracy than methods used for patient care. The simplest, most practical method is to use a standard fecal suspension which contains approximately 2 mg of feces mixed in a drop of saline and covered with a cover slip. The entire cover slip is examined at a magnification of 100 x, field by field, and the number of eggs is counted. For research work, the density of the smear can be standardized with a light meter, but this standardization is not essential for patient care. The number of eggs per cover slip provides a rough index of the severity of the infection. 1.5.7 Duodenal Material The examination of duodenal fluid or duodenal biopsy material may be useful for the diagnosis of giardiasis, strongyloidiasis, or other upper intestinal parasite infections in patients in whom parasites cannot be detected in the feces. In addition, duodenal fluid occasionally can be useful in showing whether helminth eggs are originating in the biliary or intestinal tract. Duodenal material may be obtained by passing a tube through the nose and stomach into the upper small intestine and then aspirating enteric fluid. As an alternative, a string test may be used. A weighted gelatin capsule attached to a string is swallowed, and the proximal end of the string is taped to the face of the patient. Over a period of several hours, helped with small sips of water, the string reaches the upper small intestine. After 4 to 5 h, the string is retrieved, and the material on the bilestained portion is stripped from the string and examined for parasites with direct wet mounts or with permanent stains when wetmount fmdings are questionable. Aspirated duodenal fluid is examined in a similar fashion. The material for permanent stains can be fixed in Schaudinn or PV A fixative, although the latter may adhere better

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to the slide. If question able organisms are seen in the direct wet mount, the coverslip can be removed, the material can be mixed with a drop of PV A fixative, and a film can be made for later permanent staining. A duodenal biopsy can be used to demonstrate Giardia organisms. A biopsy is usually obtained by a swallowed biopsy capsule. In searching for Giardia spp., it is generally preferable to make both impression smears and sections of biopsy tissue. Giardia spp. are usually present in mucus or attached to epithelium rather than in tissue. Biopsies occasionally can confirm a diagnosis of strongyloidiasis or cryptosporidiosis. 1.5.8 Sigmoidoscopic Material Materials obtained by sigmoidoscopy may be helpful in the diagnosis or monitoring of amebiases, schistosomiasis, or cryptosporidiosis. Patients suspected of having amebiasis may have ulcerations of the l:olon which can be visualized by sigmoidoscopy or colonoscopy. Scrapings or aspirates of material from ulcers can be examined by direct wet mounts and permanent stain as described above. The rmding of typical, erythrophagocytic, motile trophozoites in direct wet mounts or in permanently stained preparations allows a diagnosis of amebiasis. Material is best aspirated with a pipette or scraped with an instrument. Swabs should not be used, as the parasites may be killed or trapped by swab material. Biopsy material for amebiasis should be processed for surgical pathology and then examined for ulcers containing amebae. The periodic acid-Schiff stain counterstained with hematoxylin is particularly helpful because amebae stain more positively with periodic acid-Schiff stain than do inflammatory cells, and amebae show typical amebic nuclei. Of course, there are no amebic cysts in tissue. Biopsy material for schistosomiasis is better examined in teased preparations than in sections, as the entire thickness can be examined at once, and the viability of eggs can be determined by observation of the movement of the larvae within the eggs. In cryptosporidiosis, biopsy material shows organisms at the luminal surface of the epithelial cells, but the organisms are small, and the study of structural detail requires electron microscopy. 1.5.9 Abscess Material Abscesses suspected of being caused by Entamoeba histolytica may be aspirated, and the material may be submitted to the laboratory.

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The last material aspirated is most likely to contain amebae. Material may be examined microscopically in wet mounts and permanent stains, and in addition, it can be cultured for amebae if bacteria are also added to the culture as described below. Abscess material is often thick and difficult to examine. It may be treated with streptokinase and streptodonase enzymes to liquefy the specimen. 1. Reconstitute streptokinase and streptodonase per the instructions of the manufacturer. 2. Add 1 part enzyme solution to 5 parts aspirated material. 3. Incubate the mixture at 35 to 37°C for I h. Shake the mixture at intervals. 4. Centrifuge the mixture at 300 to 400 x g for 5 min. 5. The sediment may be used for microscopic examinations for amebae (wet mounts and permanent stains) and for the culture of amebae.

1.5.10 Cellophane Tape Cellophane tape is used for finding the eggs of Enterobius vermicularis or Taenia species from the perianal area. The tape used must be clear cellophane and not slightly cloudy or opaque. Alternatively, a Vaspar swab may be used. Specimens from more than 1 day may be required to diagnose light infections.

1.5.11 Examination of Cellophane Tape 1. If the specimen is difficult to examine, raise the tape from the front of the glass slide, add a drop of toluene to the slide, and replace the tape smoothly with an applicator stick. (Remember. Enterobius spp. and Taenia solium eggs are infective!) 2. Examine the entire tape, including the edges, with x 100 magnification (x 10 objective). 3. Confirm suspicious objects with high dry objectives (x40 to x50).

1.5.12 Culture for Amoebae Cultures for amoebae have improved detection in some studies, but they are not widely used. Although Giardia spp. have been cultured in research laboratories, cultures are not useful for diagnosis. A variety of culture media for amebae have heen described, and some may be purchased from commercial meUlum manufacturen. The method described here uses the modified charcoal agar slant diphasic medium described by McQuay.

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1. Place 3 ml of sterile 0.5% phosphate-buffered saline on a charcoal agar slant. 2. Add approximately 30 mg of sterile rice starch to the tube. 3. Warm the medium to 35°C before it is inoculated. 4. (a) Inoculate the medium with fecal specimen (approximately 0.5 ml of liquid specimen or a 0.5-cm sphere of formed specimen) which is mixed with the saline overlay. (b) If abscess material is cultured, bacteria must be inoculated into the culture in addition to the inoculation with 0.5 ml of specimen. A heavy inoculum with Clostridium perjringens or Escherichia coli is satisfactory. 5. Incubate the culture at 35°C. 6. At 24 and 48 h, remove I drop of liquid from the lowest point of the overlay, and prepare a wet mount. 7. Examine the wet mount for amebae. 8. Permanent stains can be prepared by the fixation of sediment in PV A fixative, with the subsequent preparation of smears and staining. 1.5.13 Larval Maturation Larval maturation studies, sometimes referred to as cultures, can be performed on fecal specimens applied to wet filter paper. Nematode larvae such as Strongyloides spp. or hookworm mature to the filariform stages in the culture container and migrate from feces into water, where they are detected microscopically. The procedure can be performed in a petri dish with a square of filter paper or in a large test tube with a strip of filter paper. 1. Smear approximately 0.5 g of feces on the filter paper. 2. (a) For the tube ·method, insert the filter paper strip into the tube so that the bottom of the strip is in 3 ml of water. The fecessmeared portion of the strip need not be immersed in the water. (b) For the petri dish method, place feces on one half of a piece of filter paper. Lay the feces-bearing end of the filter paper on a glass rod or a portion of an applicator stick in the petri dish. Add approximately 3 ml or sufficient water so that the feces-free end of the filter paper is in the liquid. 3. Leave the tube or dish at room temperature in the dark. Add water as needed to ensure that the filter paper is in contact with the water. 4. Examine the liquid for larvae either by direct microscopic

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examination with an inverted microscope or by examination of a wet mount of sediment from the liquid. With the petri dish method, the surface of the feces also may be examined with a dissecting microscope. 5. Examine the specimen on days 3, 5, and 7. Strongyloides filariform larvae are found on days 2 and 3, and hookworm larvae are found on days 5 through 7. Larvae are identified by their morphological characteristics. 1.5.14 Adult Worms Adult worms, or objects suspected to be adult worms, may be submitted to the laboratory. The laboratory must determine if these are helminths and, if so, if they are parasites. Identification characteristics are described in standard references. Tapeworm proglottids, particularly those of the Taenia species, are difficult to differentiate grossly unless they are cleared so that the internal structure can be seen and the number of lateral uterine branches can be counted. One procedure for clearing the proglottids is outlined below.

1.5.14.1 Clearing Taenia proglottids and other helminths Proglottids are first relaxed by placing them in warm saline (56°C) for I h and then clearing them in carbolxylene while they are kept flat. They may be kept flat in a number of ways. One way is to press the proglottid between two glass slides held together with membrane clips or string. Clearing takes from several hours to overnight. The proglottid is examined under a dissecting microscope or with a hand lens, and the uterine branching is observed. Glycerine and beechwood creosote can also be used with good results. Cleared proglottids may be mounted or stained if desired. Small nematodes may also be cleared in carbolxylene or beechwood creosote and mounted in permount or balsam. This method is particularly good for hookworm adults.

1.6 BLOOD AND TISSUE PARASITES Blood and tissue parasites whose diagnostic forms circulate in the peripheral blood are generally diagnosed by the demonstration of parasites in Giemsastained thick or thin films of blood. Special concentration techniques may be helpful for the diagnosis of some diseases such as filarial or trypanosornal infection. Other tissue parasites which do not circulate in the blood may be diagnosed by the detection of parasites in skin snips, lesion scrapings, body fluids,

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MICROBIOLOGY AND BIOCHEMISTRY

or biopsy material or by the detection of antibody or antigen in serum or other body fluids.

1. 7 COLLECTION AND HANDLING OF BLOOD SPECIMENS The timing of the collection of blood specimens depends on the parasite disease suspected. For example, for certain filarial infections, specimens are best obtained between 10:00 p.m. and midnight, whereas for other infections, specimens are best obtained during the day. In malaria, the numbers and stages of parasites in the peripheral blood vary with different parts of the cycle. Blood films are best made from blood which is not anticoagulated, such as that obtained from finger stick or ear lobe puncture. Anticoagulants may interfere with parasite morphology and staining. Care should be taken that the alcohol disinfectant is allowed to dry before the area is punctured, or there may be fixation of erythrocytes, which will interfere with the preparation and staining of thick films. Both thick and thin films can be prepared from blood obtained by venipuncture, although it is best if the blood remaining in the needle of the venipuncture device is used, because it is anticoagulant free. Thick and thin films can be prepared from blood that is anticoagulated, but the staining characteristics are not as good. EDTAanticoagulated blood is better for staining than citrate- or heparinanticoagulated blood. Both thick and thin blood films are useful. Thick films are more sensitive because the same amount of blood can be examined in a thick film in 5 min as can be examined in a thin film in 30 min. However, thin films allow the study of the effects of parasites on erythrocytes and provide better parasite morphology. Thick and thin films may be prepared on separate slides or on the same slide, with the thick film at one end and the thin film at the other end. The thick film is prepared by spreading 1 drop or puddling several small drops of blood into an area approximately 1.5 cm in diameter. A properly prepared thick film should be thin enough so that newspaper print can barely be read through it. If the film is too thick, it will fragment and peel, and if the film is too thin, the increased sensitivity will be lost. Thick films should be allowed to dry overnight and should be stained within 3 days. They must not be heated, and they should be protected from dust. If the erythrocytes are fixed, they will not dehemoglobinize. If prompt examination is

INTRODUCTION

27

required, prepare a slightly thinner thick film, dry it for I h, and stain it. The thin film is prepared in the same manner as a film for a differential leukocyte count. A small drop of blood is placed on one end of a microscope slide. A second slide held at an acute angle of 30 to 45 0 is backed into the drop of blood, which spreads along the junction of the slides. The spreader slide is then pushed along the slide, and it pulls the drop of blood along behind the angled edge of glass. A properly prepared thin film should have a significant area near the end which is only one erythrocyte thick and in which the erythrocytes show good morphology. The angle and speed of the spreader slide and the size of the drop of blood will influence the thickness and size of the film. Slides with only a thin film can be fixed by being immersed in absolute methyl or ethyl alcohol for I min and allowed to air dry. If the thick film is on' one end of the slide and the thin film is on the other end, the thin film is fixed by a brief flooding or by immersion in alcohol and allowed to air dry, while the thick film is protected from alcohol or alcohol fumes. In a well-ventilated area, the slide may be dried vertically with the thick film up or horizontally after the thick film is covered with a dry paper towel. Thick and thin films are best stained with Giemsa stain, as it provides the most detailed and intense staining of parasites. Wright stain can be used for thin films but not for thick films, as it contains alcohol, which will fix the erythrocytes. Wright stain does not stain parasites as well as Giemsa stain. The staining procedure is outlined below. 1. 7 .1 Tissue Biopsy or necropsy tissue may be examined by histology sections or impression smears. To prepare impression smears, tissue should be blotted to remove as much blood or other fluid as possible and then pressed against glass slides (1 by 3 in.) to make a series of impressions. Tissue should stick to the slide slightly and leave an irregular film on the slide. Similar impressions may be made on multiple slices from the same portion of tissue. Portions of biopsy tissue with different gross appearances can be used with the impressions from each portion placed in a longitudinal row. Impressions must be close together, preferably with slight overlapping to make slide scanning easier. Impressions from small fragments may be placed in a small area (1

28

MICROBIOLOGY AND BIOCHEMISTRY

cm in diameter). After being dried, the area with impressions is circled with a diamond marker to facilitate the location and scanning of the material. Fixatives and stains appropriate for the parasites suspected are used. If amebiasis is suspected, impression smears must be fixed promptly in Schaudinn fixative and not allowed to air dry. For most other parasites, the slides are allowed to dry before fixation in methyl alcohol. Giemsa is the usual stain, but other stains such as Gram-Weigert or hematoxylin may be used depending on the parasite suspected. 1.7.2 Aspirates of Bone Marrow or Spleen Aspirates of bone marrow or spleen may be useful in the diagnosis of infections such as leishmaniasis, trypanosomiasis, and occasionally malaria. In such instances, Giemsa stains of alcoholfixed bone marrow films are most useful. Splenic aspiration is r~ely performed in the United States because it is dangerous. 1.7.3 Fluids Fluids such as tissue aspirates, cyst fluid, bronchial washings, cerebrospinal fluid, pleural fluid, and peritoneal fluid can be examined directly, or they can be centrifuged and the sediment examined by wet mounts or stains (or both), depending on the parasite suspected, as described above for abscesses or tissue. 1.7.4 Skin Snips Skin snips may be useful in the diagnosis of microfilarial infections such as onchocerciasis in which the parasites circulate in the skin and not the blood. A small (2-mm) skin snip is taken with a needle and a knife. The needle point is stuck into the skin, and the skin is raised. With a sharp knife or razor blade, the skin is excised just below the needle. Alternatively, a scleral punch may be used. The skin snip is then placed in a small volume (0.2 ml) of saline in a tube or a microtiter well, teased, and allowed to stand for 30 min or more. The microfilariae migrate from the tissue into the saline, which is then examined microscopically to demonstrate the wiggling microfilariae. 1. 7.5 Concentration Procedures for Blood A number of procedures have been described for the concentration of blood specimens. Most of these procedures have been developed to diagnose filarial infections. The three most widely used methods are membrane filter, saponin lysis, and Knotts concentration. Procedures for the first two methods

INTRODUCTION

29

will be given here, as these methods are the most sensitive. Membrane filter techniques use 5- or 3-lLm filters. Both filters give satisfactory results, but the procedures with the Nuclepore filters do not require the lysis of erythrocytes. Parasites on filters are often not as suitable for morphologic study as are those in thick films.

1.7.6 Membrane Filter Concentration for Filariae 1. Collect approximately 7 ml of blood in EDTA. 2. With a syringe and firm pressure, pass 5 to 7 ml of blood through a 5-lLm Nuclepore filter held in a Swinney adapter. 3. Wash the membrane several times with a small amount of distilled water or physiologic saline. 4. The moist filter may be e}t'cUtlined directly or fixed and stained in the usual fashion for a thin blood film.

1. 7.7 Saponin Lysis Concentration for Filariae The saponin lysis method can be performed on either EDT'\- or citrate-anticoagulated blood. Saponin solution to lyse erythrocytes is available in most laboratories for use with automated hematology instruments. 1. Centrifuge up to 10 ml of blood at 150 x g for 10 min. 2. Remove and discard the plasma. 3. Mix the packed erythrocytes with 50 ml of 0.5% saponin solution in 0.85% saline. 4. Mix the solution at intervals for 15 min. 5. Centrifuge the solution at 650 x g for 10 min. 6. Decant and discard the supernatant (there should be about 1 ml of sediment). 7. Spread several drops of sediment on a glass slide (1 by 3 in.), and examine two such uncovered wet mounts for motile microfilariae. Allow the wet mounts to dry before they are fixed and stained. 8. Prepare four or five similar wet mounts and examine them as described above. To. each slide, immediately add 2 drops of 1 % acetic acid solution and mix it well (microfilariae will be killed and straightened). Allow the slide to air dry. 9. Dip the dried slides in buffered methylene bluephosphate solution. 10. Rinse the slides in distilled water, and let them air dry. 11. Stain the mounts for 10 min in a 1:20 dilution of Giemsa

30

MICROBIOLOGY AND BIOCHEMISTRY

stain in buffered water. 12. Examine the slides microscopically.

1.8 STAINING PROCEDURES 1.8.1 Giemsa Stain Procedure The procedures for staining thick. and thin fIlms differ. Staining is usually done in a Coplin jar. The ,&tain must be made fresh each day. Stain slides with only a thin fIlm as follows. 1. Fix and dry the blood film as described above. 2. Prepare a 1:40 dilution of stock Giemsa stain in neutral buffered water, pH 7.0 to 7.2 (generally, 2 ml of Giemsa stock plus 38 ml of buffered water with 0.01 % Triton X-l00). 3. Stain the film for approximately 60 min (the time, which will vary slightly with different lots of stock Giemsa stain, can be determined by the staining of leukocytes and erythrocytes). 4. Wash the slide brietly by dipping it in buffered water. 5. Air dry the slide in a vertical position. Note that, alternatively, a 1:20 dilution for 20 to 30 min may be used. Stain slides with only a thick fIlm as follows. .,1. Do not fix the slide . 2. Prepare a 1:50 dilution of stock Giemsa stain in neutral buffered water (pH 7.0 to 7.2). 3. Stain the film for approximately 50 min (the optimal time may vary with different lots of stain). 4. Wash the slide by placing it in buffered water for 3 to 4 min. 5. Air dry the slide in a vertical position. For combination thick and thin fIlms, the procedure is as follows. 1. Fix the thin film but not the thick film as described above. 2. Stain the film in a 1:50 dilution of Giemsa stain in neutral buffered water (pH 7.0 to 7.2) for approximately 50 min. 3. Rinse the thin film brietly by dipping it in buffered water. Wash the thick film by immersing it in buffered water for 3 to 5 min. 4. Dry the slide in a vertical position with the thick film down. 1.8.2 Gram-Weigert Stain Procedure The Gram-Weigert stain is used to stain the cyst walls of P. carinii cysts. It also stains fungi and many bacteria. Impression

INTRODUCTION

31

smears, sediment smears, or sections are fixed in methanol and air dried. For sections, when reagents are added, flood the slide gently from the end opposite the section, and rinse the slide carefully so that the tissue is not washed from the slide. The stain procedure is as follows. 1. Stain the slide with eosin Y for 5 min. 2. Wash the slide with water. 3. Stain the slide with crystal violet for 5 min. 4. Rinse the crystal violet from the slide with Gram iodine solution. 5. Leave the iodine solution on the slide for 5 min. 6. Rinse the slide with water. 7. Blot the smears carefully (do not blot the sections). 8. Wipe the reverse of the slide. 9. Air dry the slide completely. 10. Decolorize the smear in aniline-xylene, agitating the slide gently until no purple runs from it (the use of a second Coplin jar of aniline-xylene after the majority of blue stain has been removed aids the visual assessment of decolorization). 11. Rinse the slide in xylene. 12. Air dry the slide, add immersion oil to it, and examine it. P. carinii cysts and fungi stain dark blue and somewhat irregularly. Cell nuclei may stain blue if they are inadequately decolorized, but they are not as dark as P. carinii cysts. 1.8.3 Culture Procedures for Blood and Tissue Parasites Culture procedures have been developed for a number of blood and tissue parasites, but these procedures are used primarily in research. The culturing of Leishmania spp. and Trypanosoma cruz; may be helpful for diagnosis, and the procedures are easy to use. Biopsy or blood specimens may be cultured for Leishmania spp. or T. cruzi with Novy-MacNeal-Nicolie (NNN) medium. Biopsy specimens are ground in a small amount of saline. Biopsies from skin lesions or other tissues which may contain bacteria may have penicillin (0.1 ml of 1,000 U/ml) added to the medium with the inoculum. The inoculum is 1 drop of ground tissue or blood. Incubate the culture at room temperature (22°C), and at days 3 and 7, examine a direct mount of liquid from the bottom of the slant at x400 magnification. These cultured organisms are potentially infective for humans.

32

MICROBIOLOGY AND BIOCHEMISTRY

1.9 URINE Urine specimens usually are examined for the eggs of Schistosoma haemotobium or the trophozoites of Trichomonas vaginalis, although occasionally the larvae of Strongyloides stercoralis may be found in patients with hyperinfection syndrome. Urine is the usual specimen for the diagnosis of Trichomonas infection in males. See below (Vaginal Material) for culture method. Urine is centrifuged, and the sediment is examined microscopically.

1.10 SPUTUM Sputum may be examined to diagnose Paragonimus infection or hyperinfection due to Strongyloides stercoralis. Occasionally an amebic abscess or hydatid cyst may rupture, and amebic trophozoites or hydatid sand, respectively, may be found in sputum. Entamoeba histolytica must be differentiated from Entamoeba gingivalis, which may be found in the oral cavity of over 30% of people. Occasionally, the migrating larvae of ascarids, strongyloides, or hookworm can be found. Sputum may be examined directly by wet mount or treated with a mucolytic agent such as Nacetyl-cysteine and then concentrated by simple centrifugation, with subsequent examination of the sediment.

1.11 VAGINAL MATERIAL T. vagina lis frequently infects the vagina, and Enterobius vermicularis adults or eggs occasionally may be found. Direct wet mounts of vaginal material for typical, tumbling T. vagina lis organisms are widely used and generally allow the diagnosis of symptomatic infection, but wet mounts are not as sensitive as culture methods. Vaginal material is best submitted as liquid in a tube, although swabs submitted in a small amount of saline may be used. A drop of the material is covered with a cover slip and examined with reduced light. To culture, 1 or 2 drops of urine sediment or vaginal exudate are inoculated into tubes of warmed, modified Diamond medium. If vaginal swabs are submitted, the swab is immersed in the medium and pressed against the side of the tube to express material. Tubes are incubated at 35°C, and drops of culture are examined by wet mount at 48 and 72 h for motile trophozoites.

1.12 REFERRAL OF MATERIALS Few laboratories perform complete parasitological examination, whereas many perform limited studies, and some perform none. Referral laboratories may provide services not available in the individual

33

INTRODUCTION

laboratory and can provide consultation on specimens with questionable laboratory findings. Referral laboratories with a special interest and competence in parasitology may be found in major cities, university medical centers, and state public health laboratories. The major national resource is the Centers for Disease Control in Atlanta, Ga. Specimens for the Centers must be sent via the state health laboratory, and appropriate clinical information must be provided. Of course, the recommendations of the specific referral laboratory should supersede' these guidelines.

1.13 SAFETY The parasitology laboratory has infection hazards for personJil.el. Blood, feces, and other body materials as well as parasite cultures may be infective. Eggs of Ascaris spp. can survive and embryonate even in Formalin, and Cryptosporidium oocysts are hardy. In fresh fecal specimens, the cysts of Entamoeba histolytica and Giardia spp .. the oocysts of Cryptosporidium spp., the eggs of Enterobius vennicularis. Taenia solium, and Hymenolepis nana, and the lanae of Strongyloides stercoralis may be infective. In addition, feces may contain other infectious agents such as hepatitis A, rotavir~s, Salmonella spp .. Shigella spp., and Campylobacter spp. Blood and tissue specimens can be infectious for trypanosomes, Leishmania sp~., malaria, and Babesia spp., as well as for non-A, non-B hepatids, hepatitis B, and possibly AIDS.

TABLE 1.4 : HANDLING OF SPECIMENS FOR Specimen Feces, for Helminths Protozoa

Cryptosporidium spp.

Material from suspected amebic abscess

Duodenal aspirate

REFERRA~

Handling Fix in 10% buffered Formalin. Fix a portion in 10% buffered Formalin and either fix a portion in PVA fixative or prepare three Schaudinn-fixed fecal films. Fix a portion in 10% buffered Formalin. Place the last material aspirated in a sterile tube and send it on ice for culture (do not freeze). Prepare Schaudinn-fixed fecal films, or fix a portion in PVA fixative. Obtain serum for serology. Centrifuge, and remove the supernatant.

34

MICROBIOLOGY AND BIOCHEMISTRY Prepare two films from sediment. Fix in Schaudinn or PYA fixative. Preserve the remainder of sediment in 10% Formalin.

Urine, for Trichomoniasis

Schistosomiasis

Sputum, for Nematode larvae or Paragonimus eggs

Amebae

Blood Malaria and babesiasis

Filariasis

Trypanosomiasis

Centrifuge. Cover the sediment with sterile saline and send it on ice (not frozen) for direct mounts and culture. Centrifuge entire midday urine. Add an equal volume of 10% buffered Formalin to the sediment.

Break up mechanically or digest I part sputum plus 5 parts 3% NaOH for 1 h. Centrifuge, and preserve the sediment in an equal volume of 10% buffered Formalin. Prepare films fixed in Schaudinn fixative, or fix a portion in PYA fixative.

Send unstained and, if available, Giemsastained thick and thin films. Fix thin film (but not thick) in alcohol before it is sent. Send 5 ml of citrate- or EDTA-anticoagulated blood on ice (not frozen). Unfixed thick films may be sent in addition. Send serum for serologic tests. Send 5 ml of anticoagulated blood as for filariasis (above).

Send fixed thin films. Cerebrospinal fluid Trypanosomes, toxoplasma, leishmania, trichinella Free-living amebae Sigmoidoscopic material Tissue

Send on ice (not frozen). Send in a sterile container without refrigeration. Fix films in Schaudinn fixative or mix material with PYA fixative. For impression smears when E. histolytica

INTRODUCTION

35 is suspected, fix in Schaudinn or PVA fixative. When toxoplasma, leishmania, Pneumcoystis spp., or Trypanosoma cruzi is suspected, prepare multiple impression smears and fix in methyl alcohol. For surgical pathology, fix the tissue in buffered Formalin.

Whole worms or proglottids

Wash debris from the specimen and send it in saline. If there are mUltiple worms or proglottids, some may be fixed in Formalin.

~eagents such as mercury-containing fixatives may be toxic, and solvents such as ether may be flammable. These materials must be handled and discarded properly.

1.14 QUALITY ASSURANCE The parasitology laboratory must have an up-todate procedure manual and appropriate reference materials which might include color atlases or 35-mm slide collections permanently stained glass slides, wet fecal material containing parasites, and one or more standard reference books on laboratory methods or general medical parasitology. The persons performing parasitic examinations must be competent in the identification of parasites which might be found in patients from whom they receive specimens. Methods should allow the ready use of outside consultants, if there is a question of diagnosis. Personnel may maintain proficiency through participation in formal courses or workshops, review of self-study sets, and periodic review of known positive materials. Participation in external survey programs is particularly valuable, as the performance of the laboratory in the identification of unknown specimens can be compared with the performance of other laboratories. If a laboratory is unable to do accurate parasitology because of either the types of procedures offered or the quality of personnel available, it should arrange to have specimens appropriately prepared and submitted to a reference laboratory.

tI

2

Origin of Microbiology Before the dawn of civilization in the Mesopotamian regions and farther east some 7000 to 8000 years ago there was little exact knowledge of either the causes or nature of natural phenomena. However, scholarly thinkers and their works were not wholly lacking. By the time writing and written history had been "invented" 5000 to 6000 years ago 'in Sumeria, Egypt, Syria and adjacent regions, many keen and ambitious minds in the ancient priesthoods, secular upper classes and royal families had learned of the medicinal and poisonous properties of certain plants and of the venoms of certain snakes and insects. They knew how to exploit nature for political and other purposes. For thousands of years after the beginnings of civilization magic, incantation, abracadabra and witchcraft passed for science and usually also for religion. Even as recently as the Middle Ages (c. 500-1400 AD.) and later in the European Renaissance (c. 1400-1700 A.D.) astrology (aided by imaginative charlatans, with weird grimaces and impressive passes) passed for astronomy; alchemy (strongly flavoured with wizardry) masqueraded as chemistry; the most outrageous quackery was accepted, even by royalty, as medicine. As always, however, honest, imaginative and inquisitive men here and there were still capable of analytical and creative thought and the proposing of working hypotheses to be tested experimentally. They were sometimes reviled, persecuted and tortured for their supposed dealings with "The Evil One." Century after century these pioneer scientists (seekers after experimentally demonstrable truth) began to establish a system of knowledge based on accurate, purposeful 36

ORIGIN OF MICROBIOLOGY

37

observation; logical inference; imaginative hypothesis; and ingenious experiments designed to establish indisputable fact or destroy fallacy. Because of great difficulties in travel and communications, ancient scholars shared little of one another's learning. As the centuries passed, exploration began and travel became more common, populations increased, and vast interminglings of peoples occurred because of wars and trade. Scientific information thus began to spread from country to country and, more recently, from continent to continent. Instead of a few great scholars who were thought (even by themselves!) to know everything, men began to realize that there were boundless deserts and plains and illimitable dark forests of ignorance only awaiting the axe and plow of the devoted researcher to yield rich crops of wonderful, golden knowledge. Men also realized the awesome truth that knowledge is power-to create or to destroy utterly. Eventually scientific thought, experimentation and communications became permissible and even respectable. They also became incalculably profitable, and frightening. Scientists interested in the mysteries of life collected, over the centuries, a considerable mass of reasonably accurate information about such living things as could be seen with the naked eye, and even with "magnifying glasses" (magnifications of about 10 diameters). By 350 B.C. Aristotle and his students had drawn up a systematic, though limited and (as we now know) often erroneous classification of hundreds of plants and animals. Accumulating knowledge of living organisms slowly became arranged into a more or less orderly system and the study of life was eventually dignified with a given name: biology (Gr. bios = life; logos = study or description). Most of biology was at first largely descriptive of outward form and macroscopic (Gr. makros = large; skopion = to see) anatomy. These descriptions became the basis of classifications and taxonomy-major preoccup-ations of most early botanists and zoologists. Until the seven-teenth and eighteenth centuries chemistry and physics were almost completely separate fields of study and little used in biology. Life and living substance were commonly thought of as mysterious and beyond physical and chemical analysis.

2.1 BEGINNINGS OF MICROSCOPY Until about 1660 A.D. all knowledge of the form, structure and life processes of plants and animals was narrowly restricted to what could be seen with the naked (or very feebly assisted) human eye. Microorganisms were merely "fabulous monsters." Visual limitations

38

MICRnBIOLOGY AND BIOCHEMISTRY

of the pitiably restricted eye of man had always stood, like an impenetrable curtain, between man and the fantastic and glittering cosmos of the microscopic world. Unaided human vision fails to see objects less than about 100 p, (0.004 or 11254 inch) in diameter or to perceive as separate objects

TABLE 2.1 SOME LINEAR MEASURES COMMONLY USED IN MICROBIOLOGY 1 inch = 2.54 cm. 10 mm. 1 mm.:::: 1000 p. 1 cm. 1 p. = 0.001 mm. = 0.00003937 or 1125,400 inch = 1000 mp. 0.001 p. = 10.0 Angstrom (A) I mp' 1 A = .0001 p. = 0.0000001 mm. ;:; 1/254,000,000 inch (i.e., resolve) particles separated by distances less than about 100 p.. Microscopic linear units are shown in Table 1. 1. The development of practical, relatively high-power microscopy about the middle of the seventeenth century was like turning on a SOD-watt lamp in a pitch-dark curiosity shop. It gave men the power to see a universe of objects, living and inanimate, so minute that their very existence had never before even been suspected.

2.2 THE FIRST MICROSCOPES By the end of the thirteenth century simple lenses (magnifying glasses) had already been used in various ways for many years. Such lenses, however, did not magnify very highly. About 1590, a Dutch spectacle maker, Zacharias J anssen, used a second lens to magnify the image produced by a primary lens. This is the basic principle of the compound microscope used by every microbiologist today. Galileo invented an improved compound microscope in 1610. Robert Hooke (1635-1703) made and used a compound microscope in the 1660's and described his fascinating explorlttions of the newly discovered universe of the microscopic in his classic "Micrographia" (1665), published at request of the Royal Society in London. Although Hooke's highest magnifications were possibly enough to reveal bacteria, he apparently made no observations of them. probably because he studied mainly opaque objects in the dry state by reflected light, conditions that, as will be explained, are not optimal for observation of microorganisms. However, his pictures of "white mould" (probably a Mucor species) are very informative and accurate.

ORlGIN OF MICROBIOLOGY

39

f'iiure 2.1 : Hooke's compound microscope: drawn by himself.

A contemporary of Hooke, and the man mainly responsible for revealing the hitherto unknown and unseen world of microorganisms, did not use a compound microscope. He was the Dutch investigator, Antonj van Leeuwenhoek (1632-1723), a linen merchant by trade and a man of public and commercial affairs in the city of Delft. He was nO! a trained scientist but was self-educated, and amused himself by means of his skill and craftsmanship in glass blowing and fine metal work. He lived in relatively easy circumstances with leisure time for his avocation of making minute, simple but powerful lenses. With these he delighted in examining a great variety of objects: saliva, pepper decoctions, cork, the leaves of plants, circulating blood in the tail of a salamander , ~minal nuid, urine, cow dung. scrapings from the teeth and &0, on. ln' many of these be saw living creatures , some of which we now know were protowa and bacteria but all of which he called "animalcules." In spite of the fact that his microscopes were not compound he obtained remarkable results with them. he showed rare ingenuity and expert craftsmanship in the grinding and mounting of his simple lenses, a skill which he zealously kept to himself; and in spite of the requests of his learned friends, he refused to disclose the secret of his success. I..eeuwennoek's instruments are not true microscopes at all in

40

MICROBIOLOGY AND BIOCHEMISTRY

Figure 2.2 : Drawing of "white mould".

the sense in which we ' think of microscopes, but rather simple magnifying glasses generally consisting of a small, single, biconvex lens. The object, and not the lens, was moved into focus by means of screws.

Figure 2.3 : Antonj VllJI Leeuwenhoek .. A fanciful delineation based on a famous portrait. The picture shows accurately the size and shape of the first microscopes. the manner in which they were used. and the simple laboratory apparatus of the "Father of Bacteriology. "

To adjust the lens to the object was so long and tedious a task that it is not surprising that Leeuwenhoek used an individual lens for each object.... The magnification varied and at best did not exceed

ORIGIN OF MICROBIOLOGY

41

two hundred to three hundred diameters .... The size of objects which Leeuwenhock examined was determined by comparison. For this purpose he used at various times a grain of sand, the seed of millet or mustard, the eye of a louse, a vinegar eel, and still later hair or blood corpuscles. In this way he secured fairly accurate measurements of a great variety of objects . . . . he was forced to admit that the sand grain was more than one million times the size of one of the animalcules. Leeuwenhoek was so interested in the things he observed that, like Hooke, he wrote minutely detailed reports about them to the Royal Society in London, beginning in 1674. He was later elected a fellow of the Royal Society. Some of his observations are at once quaint and epochmaking. For example, after examining material which he scraped from between his teeth, he said: Though my teeth are kept usually very clean, nevertheless when I view them in a Magnifying Glass, I fmd growing between them a little white matter as thick as wetted flour; in this substam.:e, though I could not perceive any motion, I judged there might probably be living Creatures.

Figure 2.4 : One of Leeuwenhoek's microscopes: front, back and side views. The tiny spherical or hemispherical lens is held in the slightly raised structure in the upper part of the metal plate. The object to be examined was mounted at the tip of the sharp-pointed mounting pin. Focusing was accomplished by means of the three· thumbscrews to which the mounting pin is attached. These are approximately actual size.

I therefore took some of this flour and mix it either with pure rain water wherein were no animals; or else with some of my spittle (having no Air bubbles to cause a motion in it) and then to my great surprise perceived that the aforesaid matter contained very small living animals, which moved themselves very extravagantly. The

MICROBIOLOGY ANO:BIOCHEMISTRY

42

biggest-sort had the .shape of A. Theirnidtron was stron.g -arid nimble; and they darted themselves through the' water or spittle~ 'as a: JaCK ot Pike· does' through the water. These' were 'generally not many; 'In number.· The .second sort had the' shape of B. These sjnm 'abOut like a top, or took 'a' course sometimes on one side; as is 'shown' at C arid D: They were more in number than' the first. In the third s~rt I could n6t well distinguish the Figure, for sometimes It seem'd to' be an oval, and other times a circle. These were so small they'seem'a no bigger than E and therewithal so swift, 'that I can compare them to nothing better than a swarm of Flies or Gnats, flying and tUrning among one another in a small space. -

cPdA<=>

'." .',;

,

Figure 2.5 : Leeuwenh;~;~ drawings bacteria. Heii; may be seen cocci, bacilli and (probably) a spirochet~. The motion of one of the bactli is clearly indicated. Today such observations are conbnonplace. But l,eeuwenhoek itas seeing them for the first time in the history of the human race! iwas as momtfuous a discovery as that of Columbus -a new world! " . ,.!. ... '

,. : Note, that, unlike Hooke, Leeuwenhoek made many of his observations' by light transmitted through the' object' and that the ri'lic:too'rganisms were Slispended in vaiiolls f\uids,:nOl ipiInobiliied oi-'l:)therwise altered by drying. ' ,

2.'3:':, MICROORGANISMS AND':~·' ,:,'<:THE' ORIGIN OF LIFE" ", -

.,

',-c., ..

,",.' (

>,:i

"

1'lie"aricients Iffiew nothing of ~ctQOrgani~rits, of-~vQ1Qt[~n,: of!:ijie 'fut:t 'that only 'living things could beget liyini( tiirog~':' r'tiey' befieveci, that creatures like frogs, mice, bees 'and 'other sprang'

:6r

ariiiDais -

'ORIGIN OF MICROBIOLOGY

43

fully formed from fertile mud, decaying carcasses, warm rain or fog . and the like. Van Helmont (1577-1644) devised a method for manufacturing mice. He recommended putting some wheat grains with soiled linen and cheese into an appropriate receptacle and leaving it undisturbed for a time in an attic or stable. Mice would then appear. This observation may still be experimentally confirmed but the conclusions drawn from it differ today. Belief in spontaneous generation lived on for years, as it !lad for centuries. For example, an elderly lady of the writer's early acquaintance complained bitterly that she had been cheated by a merchant who sold her a woolen coat which was of such a quality that it turned entirely into moths when left undisturbed in a closed for SOme months! . In the earlier years, in the absence of exact knowledge of microorganisms· or chemistry, there had arisen much skepticism and bitter feeling over the question of the origin of life. One "scientist" who still held to the ancient ideas says of the views of another who doubted, So may we doubt whether, in cheese and timber, worms are generated, or if beetles and wasps in cow dung, or if butterflies, locusts, shell-fish, snails, eels, and such life be procreated of putrefied matter which is to receive the forms of that creature to which it is by formative power disposed. To question this is to question reason, sense and experience. If he doubts this let him go to Egypt and there he will fmd the fields swarming with mice begot of the mud of Nylus, to the great calamity of the inhabitants. There Was a great deal of such acrid discussion by wordy savants of the-times, who tried to settle everything by argument. Experimentation was regarded as rather undignified and even smacking of relations with the devil. Francesco Redi (1626-1679). The experimental method was, however, being invoked here and there .. For example, it had always been· supposed that the maggots in decaying meat were derived spontaneously from transformations of the putrid meat itself. Francesco Redi, a physician of Arezzo, questioned this hypothesis. He placed meat and fish in jars covered with very fine gauze and· saw flies approach the jars and crawl on the gauze. He saw the eggs of the flies caught on the gauze and observed that the meat then putrefied without maggot formation. Maggots developed only when the flies' eggs were deposited on the meat itself. Obviously the meat

44

MICROBIOLOGY AND BIOCHEMISTRY

itself did not turn into maggots. Redi's work was not widely noted, however, and it was not until much later that another series of experiments was made. Louis Joblot (1645-1723). After Leeuwenhoek's disc-overy of microor~anisms it was thought by many who belie-ved in the Aristotelian doctrine concerning spontaneous generation of life that animal or vegetable matter contained a "vital or vegetative force," capable. of converting such matter into new and different forms of life. A popular notion was that geese and lambs could grow from certain kinds of trees. Leeuwenhoek's animalcules were hailed by many as proof of this. In 1710, Louis loblot observed that hay, when infused in water and allowed to stand for some days, gave rise to countless animalcules, or infusoria (bacteria and protozoa). The hay was thought by some to change into animalcules; anyone today can observe the development of these for himself. loblot, however, boiled hay infusion and divided it into two portions, placing one in a carefully baked (sterilized) and closed vessel, which he heated thoroughly and kept closed. The other portion was not heated and was kept in an open vessel. The infusion in the open vessel teemed with microorganisms in a few days. In the closed vessel no life appeared as long as it remained closed, thus showing that the infusion alone, once freed of life by heat, was incapable of generating new life. spontaneously. John Needham (1713-1781). Similar experiments carried out by an English scientist, John Needham, gave conflicting results. Life developed in Needham's heated closed vessels as well as in the open unheated ones. He therefore believed in spontaneous generation. We shall see later that this result was due to insufficient heating which failed to kill heat-resistant forms of bacteria called spores. But nothing was known about spores at that time. Lazzaro Spallanzani (1729-1799). Spallanzani, an Italian naturalist, published the results of a whole series of the same type of experiments which disagreed with those of Needham. He showed that if heating was prolonged sufficiently and the vessels kept closed to exclude dust and air, no animalcules developed in hay infusions or in any other kind of organic matter, such as urine and beef broth. Needham, in reply, said that the prolonged heating destroyed the "vegetative force" of the organic matter which, he said, was necessary for the spontaneous generation of life. Spallanzani answered Needham's objections by showing that the heated infusions in the

ORIGIN OF MICROBIOLOGY

45

closed flasks could still develop animalcules when exposed to air (i.e., when microorganisms were introduced with dust). In 1775 Lavoisier discovered oxygen and the relation between air and life: This renewed the controversy about spontaneous generation, the objection to Spallanzani's results being that it was the exclusion of air (oxygen) from the flasks which prevented the development of ,life. Schulze and Schwann. New experiments were perfo-rmed in which unheated air was admitted freely to the previously heated infusions of meat or hay, but only after passing through sulfuric acid or potassium hydroxide solutions (Schulze, 1836) or through very hot glass tubes (Schwann, 1836), the idea being that the air itself introduced the germs of life into the infusions. When the infusions exposed to air so treated failed to develop any life, it was claimed by others that this was not due to a destruction of any germs of life in the air by the sulfuric acid or hot glass, but that the "life-giving" power of the air had been destroyed by these methods, thus preventing spontaneous generation. , Schroder and von Dusch. This objection was oyercome by Schroder and von Dusch (1854-1861) who performed similar experiments in which the air Was not heated or passed through acid or alkali but merely filtered through cotton wool. This method prevented the appearance of animalcules in the heated broth or infusions until the vessels were opened. It was therefore apparent that the method of treatment of the air had nothing to do with the development of animalcules and that these did not develop spontaneously, but that there were particles of living matter floating on dust in the air which not only were killed by heat, acids and alkalis, but which could be caught and withheld by the cotton wool alone. The presence of the microorganisms in the cotton wool was later proved by Pasteur. The experiments of Schroder and von Dusch were the origin of our present-day use' of cotton plugs for bacteriological culture tubes and flasks. In spite of these demonstrations, long and bitter controv-ersies still raged. Schroder and von Dusch were not convinced by their own ' experiments and admitted the possibility that spontaneous generation might occur under natural condi-tions. Louis Pasteur (1822-1895). Pasteur, one of the most famous French scientists, was born in Dole, December 27, 1822. Son of a moderately prosperous tanner who had fought for and been decorated

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'ORIGiN OF'MicROBIOLOdv 47 would de~~i~p: -~h~n'icer{ai~ dr'cum~t~nc~~ 'of' temp:r~~~re, -~f atmospheric variations, of exposure to air, would favour )heir evolution or their introduction into wines? ... I have indeed re~ched this result that the alterations ("diseases"1 of wines ate co-eXistent witIi 'the presence arid multipliCation of microscopic' vegetatlons. ' Pasteur had; found that acid wines, "ropy" wines, bitter wines, sour beer and so on were caused by the growth in them bf undesirable contaminating organisms which produced, these so-called diseases. , The solution of the problem, as later proved 'by Pasteur, lay in preventing the' growth of foreign orgartisms, '''wild'' yeasts and bacteria, which caused the 'uridesirable conditions. After' considera15le experimentation along these liIles he discovered that the wine; did not spoil in transit if it were held for some '~nutes at a temperatUre between 50 0 and 6(j°C.' He said, l have '. ascertained that wine was 'neverilltered by th'at preliminary operation (healing), and as nothing 'prevents it afterwards from undergoing . '. . improvement with. ageit is evident that this process [heating] offers every advantage. Ris ~l>eriDiems\ were ~'Stlccessftll'Utat !-a 'Practical :test; oftlie efficacy of his methods was :made, He wrote. to a friend: .. , . experiments on the heating of wines will be made by the M~ter of the Navy. Great quantities of heated and of non-heated wine are to be sent to Gabpn so as to test the process; at, present our ,cplonial crews h;lVe to ,drink mere vipegar. ~asteur . laid, down· three great principles: 1. Every alteration. either of beer or of wine, depends -on the devel9pm~llt in. it of 'Jl}icrool1ganisms which are ferments, or ."diseases" of the beer or wine;' ,.' .. 2",These "germs or ferments" are btoughtby the'air, by the ingredients or by the apparatuS used in breweries . . . 3. Whenever beer 'or, wiriecontains nO living microorg-'anistris 'it remains unchanged.

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, : IIqbe same "Y~y~, wille$ could be pn:;l!ervedbybeat~g irom varipus c~ses of alteration, bQtt}.ed beer c~>ulq es«ape the develppment ofdiseas~ ferlnen~ by being brought to a te~rature of 50· to 55 0 t;,: The 'app1lcation of this pr,ocess soon gave tise to,the neW word. ')Jasteuri?-eiJ" beer, whi~ti"be~e cUI;fent in technicallangQage_, Today" Pilsteuiization of ,millc(hea~ing at 63°C., for 3Q minutes) .isroutine·. The heating kilis pathogemc (disease-producrng)' microorganisms.

48

MICROBIOLOGY AND BIOCHEMISTRY

2.6 PASTEUR ON SPECIFICITY OF DISEASE Pasteur foresaw the consequences of his studies, and wrote in his book on beer: When we see beer and wine subjected to deep alterations because they have given refuge to micro-organisms invisibly introduced and now swarming within them, it is impossible not to be pursued by the thought that similar facts may, must, take place in animals and in man. It was obvious from Pasteur's studies that each special kind of fermentation or disease of beer or wine was the result of the growth and activity in it of a special, distinct form of yeast or other microorganism, depending on the type of fermentation or disease under investigation. This furthered an idea, already old, of the specificity of biological action, and supported the view that animal and human diseases also, like different sorts of putrefaction and fermentation, were each caused by a single, specific type of microorganism.

2.7 PASTEUR OF SPONTANEOUS GENERATION After Pasteur's views of the nature of fermentation had been made public he became involved in the bitter quarrel over the apparently mysterious appearance of "germs" in fermentable or putrescible liquids like wine, beer, urine and broth. Pasteur carried out many ingenious experiments to answer the various objections and fallacies of previous workers and to show that the animalcules in spoiled beer and wine were merely descendants of microorganisms that had gained access to the fluids from dust in the air and that, by their growth and metabolism, caused fermentation and putrefaction. First, he redemonstrated that living creatures float in the air attached to particles of dust. Then he showed, as Schulze and Schwann had done, that when they could be excluded from vaTious substances such as sterilized broth and urine, these substances did not ferment or putrefy. By using flasks with long open necks having several vertical bends in them, he showed that although unheated, untreated and unfiltered air communicated freely with the interior, the dust was caught by gravity in the bends of the neck, and no life appeared in the infusions. Not until the flask was tilted so that the fluid came into contact with this dust and was allowed to run back into the flask, or until the neck of the flask was broken off close to the body, did growth occur in the fluids. Some of Pasteur's flasks which were

ORIGIN OF MICROBIOLOGY

49

sterile in 1864 have been preserved and are still sterile (if they have not been destroyed by wars) after over a century!

2.8 MODERN STYLE In 1864 Pasteur received a prize from the French Academy of Science for his studies that conclusively disproved the Aristotelian doctrine of spontaneous generation. Since that time, studies of organic chemistry have produced a mass of evidence to revive the doctrine of spontaneous generation in a modem form called chemical evolution. There is now good reason to believe that life evolved during many millions of years from combinations of a few elements (chiefly C, H, 0, N, S and P) by a series of reactions that were compatible with natural laws (second law of thermodynamics or law of entropy) and were actually inevitable under what are called "primitive earth" or prebiotic conditions. These are conditions that are thought to have existed during the later stages of the formation of the earth over 4,000,000,000 years ago, eons before the Cambrian period that began as recently as 600,000,000 years ago-practically yesterday, geologically speaking.

2.9 CHEMICAL EVOLUTION Prior to 1828 the formation of organic compounds was believed to be absolutely restricted to living organisms (hence organic). In 1828 Friedrich Wohler, a German chemist, produced urea, 0=C=(NH2)2' an organic substance common in the wastes of many animals, by the simple process of evaporating an aqueous solution of ammonium cyanate, 0=C=N(NH4), an inorganic substance. The old-time distin-ction between organic and inorganic evaporated with the water from Wohler's solution; Wohler had demonstrated that "spontaneous generation" of organic substances from inorg-anic ones could occur. Everyone was naturally excited with the implications for the spontaneous generation of life. If urea could be made to appear "spontaneously," what about other organic substances? Could living substance, or anything like it, be made to appear? Without recounting the research studies dealing with or formaldehyde under the influence of ultraviolet and gamma rays. These sugars are, as will be detailed later, parts of the most important and absolutely essential materials, ribonucleic and deoxyribonucleic acids (RNA and DNA, respectively), of all living cells and also of viruses. Certain purines and pyrimidines, other essential parts of DNA and RNA, are also reported to have appeared under experimental

MICROBIOL~)(,Y

50

EVOLUlJONARY 'SUCCESSION Of liFE' IQRM$ , (It. f.w r.pr...ntati•• types)

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Figure 2.7 : Geologic time scale (left) showing ancient origin of bacteria and other microorganisms in pre·Cambrian times (entirely s~culative) at foot of evolutionary scale. Pictures I, 2, protozoa; 3, 4, bacteria; 5, spi;ochete (protozoa.like bacterium); 6, marine worms; 7, mold·like bacteria; 8, aquatic fungus (Saprolegnia); 9, trilobite (fossil marine arthropod); 10, 13, bacteria·like algae (Cyanophyceae, desmids); 11, higher algae; 12, fossil fish; 14, cotylosaur (fossil reptile); 15, Psilopsida, first vascular land plants (Silurian); 16, Triceratops, 'a dinosaur; 17, cycad tree (Jurassic); 18, fossil man·like ape; 19, hybrid (1968) daiSies; 20, man in space. *Oldest rocks dated by modern measurements of radioactivity :

prebiotic conditions: adenine in solutions of amn10nium cyanide, uracil in mixtures of CH4 , H2 and HP, and soon. Thus we see actually demonstrated the "spontaneous" formation

ORIGIN OF MICROBIOLOGY

51

of the organic molecules like amino acids, simple sugars, purines and pyrimidines and a number of others that are the "building stones" of the enormously complex macromolecules (fats, proteins, polysaccharides, nucleic acids, corijugates such as apoenzymes, lipoproteins, respiratory metalloproteins and so on) that make up living cells.

Figure 2.8 : Reconstruction of a Middle Cambrian sea floor about 600,000,000 years ago. The fauna includes siliceous sponges (the upright cones), jellyfish, and two genera of trilobites (Paradoxides, the large form, and Ellipsocephalus, the small form). BacteTla had probably already been in existence for millions of years.

Unfortunately (or fortunately!) human attempts, even by the most advanced methods, to combine these "building stones" into complex macromolecules as they occur in living cells have failed. Some sugars, some enzyme-like complexes and some genelike structures have been made synthetically in minute quantities at great cost. The last steps, the formation of the type of integrated colloidal complexes that are found in living cells, are still far in the future. (It would be so profitable if we could cheaply synthesize cane sugar or beef!)

3 Microbiology of Fungi 3.1 CHARACTERIZATION Dematiaceous fungi are characterized by the development of a brown-to-olive-to-black color in the cell walls of their vegetative cells, conidia, or both. This cell coloring results in colonies that are olive to black. These ubiquitous and cosmopolitan opportunistic pathogens are normally associated with soil and plants, but occasionally they may cause ,infections in humans and animals. In medical mycology, dematiaceous fungi often are thought of as being exclusively hyphomycetes. This idea is in error because some ascomycetes, basidiomycetes, coelomycetes, and zygomycetes may be dematiaceous. Mycotic infections caused by dematiaceous fungi include chromoblastomycosis, mycetoma, phaeohyphomycosis, and sporotrichosis. In this chapter, only chromoblastomycosis, phaeohyphomycosis, and sporotrichosis will be considered. Sporotrichosis is treated here because the etiologic agent is dematiaceous in culture, even though the yeast form in tissue is hyaline. Deciding whether a particular dematiaceous fungus is involved in the disease process can at times be difficult, since these fungi occasionally are recovered from clinical specimens as contarni-nants. Documentation of a dematiaceous fungus as the etiologic agent of a mycotic infection necessitates sound evidence that the infection is compatible with a mycosis, that the suspected etiologic agent is seen in clinical specimens, that the morphology of the fungus in the clinical specimens is compatible with the suspected etiologic agent, and that the recovered fungus is properly identified. The repeated recovery of a suspected etiologic agent, especially from more than one type of clinical specimen, is highly significant. The recovery of a fungus 52

MICROBIOLOGY OF FUNGI

53

from body sites that normally are sterile is important.

3.2 COLLECTION AND STORAGE OF SPECIMENS Clinical specimens must be collected aseptically and then promptly transported to the clinical laboratory in a properly labeled sterile container. Specimens collected on swabs or transported to the clinical laboratory in a transport medium are unacceptable for mycological study. An adequate quantity of clinical material is necessary if the information obtained in the laboratory is to be meaningful. The most frequently submitted specimens for the recovery of dematiaceous fungi include aspirates, biopsy material, scrapings, and tissue specimens. Specimens other than skin scrapings must be protected from dehydration at all times. This protection can be accomplished by ensuring that a few drops of sterile saline or distilled water is added to the specimens at the time of their collection. Biopsy and tissue specimens can be kept moist by placing them between two pieces of sterile gauze moistened with sterile saline or distilled w~ter. Clinical specimens should never be placed on cotton pads, since cotton fibers can be confused with hyphae in the direct microscopic examination. In addition, it is often impossible to recover all of the. clinical specimen from among the cotton fibers. Direct microscopic examination of the specimens and subsequent plating must be done promptly.

3.3 DIRECT EXAMINATION Clinical specimens obtained for the recovery of dematiaceous fungi usually do not require extensive processing. If aspirated' specimens contain a substantial amount of purulent material, this can be dissolved with N-acetyl-L-cysteine without sodium hydroxide. Tissue specimens and biopsy material should be homogenized in a tissue homogenizer after highly suspicious areas consisting of necrotic, purulent, or caseous material are selectively, examined microscopically and inoculated onto isolation media. Specimens are typically examined microscopically in 10% KOH. The clearing process can be accelerated by gently heating the KOH preparation. The dematiaceous nature of fungal elements in clinical specimens should be determined only by bright-field microscopy. Phase-contrast microscopy is an excellent method for examining specimens, but it does not always permit the demonstration of the dematiaceous nature of these fungi. In some instances, the dark color

54

MICROBIOLOGY AND BIOCHEMISTRY

of these fungi can be seen in tissue sections stained with hematoxylin and eosin. However, if a dematiaceous fungus is suspected, an unstained tissue section should be examined microscopically by brightfield microscopy. A drop of immersion oil can be placed directly onto a paraffin section mounted on a microscope slide, and then the section is examined microscopically. The etiologic agents of chromoblastomycosis may be filamentous at the surface of the skin. In the deeper subcutaneous tissues, they occur as muriform cells (sclerotic bodies). Muriform cells are typically chestnut brown and variable in size, with thick cross walls arranged in a muriform manner. The cells result from vegetative growth without the elongation seen in hyphae. The presence of muriform cells in clinical specimens is diagnostic of chromoblastomycosis. However, the various etiologic agents of this mycosis cannot be identified solely on the basis of their morphology in tissue. Phaeohyphomycosis is characterized by the presence in tissue of dematiaceous yeastlike cells, hyphae, or both. The hyphae may be regular and uniform in diameter or irregular in shape with many swollen. cells, and they can be either short or very long. The name phaeohyphomycosis is not meant to be restricted to hyphomycetes, but it encompasses all dark hyphae causing disease in tissue, regardless of the taxonomic classification of the etiologic agent. As with chromoblastomycosis, the etiologic agents of phaeohyph-omycosis c.umot be identified in clinical specimens. These fungi must be grown on laboratory culture medium before they can be identified.

Figure 3.1 : Alternaria alternata. Taken by phase-contrast microscopy after 2 weeks on potato dextrose agar. Bar equals 10 /Lm.

MICROBIOLOGY OF FUNGI

55

Most mycologists consider it fruitless to directly examine clinical specimens for the yeast form of Sporothrix schenck;;. The number of yeast cells in the specimen is typically very limited, and their small size and shape are not distinctive. The yeast form of S. schenck;; is not easily seen in sections of tissue stained with hematoxylin and eosin.

Figure 3.2 : Aureobasidium pullulans. Taken by phase-contrast microscopy after 2 weeks on potato dextrose agar. Bar equals IO /Lm.

The fungus usually can be seen in tissue sections when the sections first are treated with diastase and then stained by the Gomori or periodic acid-Schiff technique. Fluorescent-antibody-specific conjugates are ideal, although they are available only at certain reference laboratories at the present time. Even though the yeast form may be difficult to see in specimens, there is generally no difficulty in recovering this fungus by the cultivation of clinical materials on media that are routinely used for the isolation of fungi.

Figure 3.3 : Cladosporium carrionii. Taken by phas~:-dlltJ:ast microscopy after 2 weeks on potato dextrose agar. Bar equals 10 /Lm. • .

56

MICROBIOLOGY AND BIOCHEMISTRY

3.4 CULTURE AND ISOLATION Dematiaceous fungi are easily isolated on most routine media. Some of them are sensitive to cycloheximide, and for that reason, Sabouraud dextrose agar (2 % glucose) should be used in conjunction with a medium containing cycloheximide. Most dematiaceous fungi grow well at 30°C; some species grow poorly or not at all at 37°C. The majority of the pathogenic dematiaceous fungi usually are visible on isolation media within a week. However, cultures should not be discarded as negative until 6 weeks. Once a dematiaceous fungus is isolated, it must be determined whether or not the isolate is a pure culture. If the culture is not pure, then the fungus must be purified by the isolation of hyphal tips or individual germinating conidia. This

Figure 3.4 : Cladosporium bantianum. Taken by phase-contrast microscopy after 2 weeks on potato dextrose agar. Bar equals 10 "m.

purification is extremely important because many of the opportunistic dematiaceous pathogens are polymorphic; that is, they can produce several different kinds of conidia in the 'same culture. For an accurate identification, it must be known whether the various types of conidia present in a culture were formed by one fungus or by several fungi. The colony characteristics and microscopic morphology used for the identification of dematiaceous fungi are based upon cultures that are approximately 2 weeks old and have been grown at 25 to 30°C on a medium such as potato dextrose agar or cornmeal agar. These media

MICROBIOLOGY OF FUNGI

57

usually stimulate the formation of conidia. If a suspected pathogen cultivated as described above does not produce conidia, exposure to a naked daylight-type bulb for several days in a 12-hlight, 12-h-dark cycle while the pathogen is growing on a medium such as 2 % water agar, sterile wooden sticks, potato dextrose agar, cornmeal agar, or filter paper may stimulate it to form conidia. The culture also can be lyophilized and then regrown, a procedure which often stimulates the development of conidia. Exposure to UV light often stimulates the development of conidia and other kinds of structures.

Figure 3.5 : Curvularia lunata. Taken by phase-contrast microscopy after 2 weeks on potato dextrose agar. Bar equals 10 !LID.

It has been suggested that pathogenic and nonpathogenic Cladosporium isolates can be distinguished from each other by their inability or ability, respectively, to hydrolyze casein, gelatin, or Loeffler serum medium. Although this test may be of some value for designating a Cladosporium species as a saprophyte, it cannot be used in place of morphological studies. When isolates are believed to be S. schenckii, they should be subcultured on an enriched medium such as blood agar to determine whether they are dimorphic; that is, whether they grow vegetatively as hyphae at 25°C and as yeast cells at 35°C. The conversion from the mold form to the yeast form is enhanced by incubation of the inoculated medium in a candle jar. When temperature studies are conducted, it is important to concurrently incubate an additional tube of medium inoculated with the fungus at 25°C to ensure viability of the inoculum. For an isolate to be considered dimorphic, only a few cells of its typical tissue form need

TABLE 3.1: DIAGNOSTIC FEATURES FOR SOME MEDICALLY IMPORTANT DEMATIACEOUS FUNGI Genus

Diagnostic characteristics

Comments

Selected references

Alternaria

Conidiophores dark, septate. simple or branched. Conidia muriform, obclavate, with a beak, darkly pigmented. in simple or branched acropetal chains.

Recognized by the distinctive muriform, obclavate conidia with a beak.

3,4,6, ll, 12,19

Aureobasidium

Conidiophores hyaline to chestnut brown, undifferentiated from hyphae. Conidia borne laterally, hyaline, one celled, oflen producing secondary blastoconidia. Large, dark, one- or two-celled, thickwalled arthroconidia commonly present.

Differentiated from Hormonema spp. by the production of conidia in a synchronous manner. Differentiated from Phaeococcomyces spp. by the lack of dematiaceous yeast cells.

10-12,19

Conidiophores dark, erect, often septate. Conidia one to several celled in some species, with dark hila, occurring in fragile, branched acropetal chains. Conidia at base of chains usually shield shaped.

C. bantianum grows at 42 to 43°C and

Cladosporium

Curvularia

Conidiophores dark, erect, geniculate due to sympodial development. Conidia

VI 00

~

?)

~

-5 t:x:I

5, 19,21

forms long, sparsely branching chains of conidia from hyphalike conidiophores, with conidia ca. 6.4 I!m long. C. carrionii grows up to 36°C and forms short, branching chains of conidia from distinct conidiophores, with conidia ca. 5 I!m long. Differentiated from Drechslera spp. by possessing conidia which have an

0

Cl

-< > Z 0

-g t:x:I

:I:

11,12, 19

Table 3. J Contd.

trI

3: ~

~

~

Table 3.1 Contd.

Diagnostic characteristic~

Comments

multiseptate, usually curved, with central cell larger and darker than end cell wall approximately the same.

enlarged and darker central cell and narrow septa. cells and thickness of septa and outer

Conidiophores dark, erect geniculate due to sympodial development. Conidia multiseptate, cylindrical to oblong, dark, with septal walls thickened.

Differentiated from Curvularia spp. by possessing conidia that are oblong to cylindrical with thickened septal walls,

11, 12, 19

Exophiala

Conidiophores hyaline to subhyaline, hyphalike or distinct. Conidiogenous cells annellides that are cylindrical to lageniform. Conidia one to several celled (one species), hyaline to pale brown, accumulating in balls at the apices of the annellides. Phaeococcomyces synanamorph often present.

E. werneckii has annellides reduced to yeast cells, one to two celled, the latter predominant, tapering towards the end bearing annellations. E. jeanselmei has cylindrical-to-lageniform annellides produced from conidiophores, with some annellides intercalary. Growth up to ca. 3TC. E.jeanselmei IS differentiated from W dermatitidls by lack of the ability to grow at 40°C and by the development of annellides instead ofphialides.

10,17-19,23

Fonsecaea

Conidiophores pale brown, usually erect swollen apically due to sympodial development. Conidia one celled, pale

F pedrosoi is differentiated from F compacta by the formation of conidia that are more elongate and in loose

19

Genus

Drechslera

Selected references

~

o

Table 3.1 Contd.

5 Q o'T1

2lz

-

o

VI

IQ

~

Table 3.1 Contd.

Genus

Diagnostic characteristics

Comments

brown; primary conidia function as

conidial heads. sympodial conidiogenous cells to produce secondary conidia. Tertiary conidia may be formed in the same manner. Rhinocladiella, Cladosporium, or Phialophora synanamorphs often are present.

Phaeococcomyces Conidiophores and hyphae absent. Yeast cells one celled, pale brown to black; pseudo hyphae may be formed. May occur as a synanamorph associated with species of Exophiala, Phialophora, Wangiella, and other genera. Phialophora Conidiophores absent or present, pale brown. Conidiogenous cells phialides with distinct collarettes. Conidia one celled, hyaline to pale brown, accumulating as balls at the apices of the phialides.

Often will produce synanamorphs when grown on either cornmeal agar or potato dextrose agar.

Selected references

8,10, 19

~

n ~

t::!1

o

P. verrucosa produces flask-shaped phialides with cup-shaped, dark, often deep collarettes. P. parasitica produces phialides of variable length, some isolates forming extremely long phialides; phial ides often swollen near base, with prominent encrustations on the cell wall. Conidia elliptical to cylindrical, often curved. P. repens produces intercalary

11-13, 19

5 Cl >-<:

> ~ t::!1

o n ::t: tI1

-;3 ~

Table 3.1 Contd.

CIl

>-<:

....("')a;::

Table 3.1 Contd. Genus

Diagnostic characteristics

Rhinocladiella

Conidiophores pale brown, erect, usually with distinct scars, and sympodial in development. Conidia one celled, fusiform to obovate, pale brown, with a dark basal scar.

Scedosporium

Conidiophores hyaline, short or long. Predominant conidiogenous cells annellides with slight swelling just below the apex. Conidia one celled, obovate. truncate, subhyaline to light black. single or in balls. Hyp!lae produce one- to two-ce\led arthroconidia, pale brown to brown, subglobose to ellipsoidal. H. toruloidea

Scytalidium

Comments phiCllides without basal septa or phialides cylindrical to slightly lageniform with a delicate collarette. P. richardsiae produces phial ides of variable size and shape. some phial ides long with flaring, flattened collarettes. Conidia of two shapes: globose conidia from phial ides with flattened collarettes and cylindrical conidia that are often curved from other phialides. Conidia occur along a rachis.

Selected references

~

0

....0t:tI

5

Cl

-<

0

'Tl 'Tl

c::: Z

....

Cl

11,12.19,26

Several species of ascomycetes besides Pseudallescheria boydii may produce a S. apiospermum anamorph.

19.22

May occur as synanamorphs with Aureobasidium spp.

19,27

Table 3.1 Contd

0'1 .-

Table 3.1 Contd. Genus

Comments

Selected references

Conidiophores erect, hyaline, and sympodial in development. They may be apically swollen or geniculate and gently tapering. Conidia of two types: one celled, hyaline, arising on denticles from sympodial conidiophores, and one celled, thick walled, black, arising laterally from the hyphae in some isolates.

S. schenckii is a dimorphic fungus capable

9,19

Conidiophores hyphalike, subhyaline to pale brown. Conidiogenous cells phialides without distinct collarettes, intercalary or laCeral from hyphae. Phial ides cylindrical with rounded apices. Conidia one celled, subglobose, pale brown, occurring as balls that slip down the conidiogenous cells. Annellidic and apical sympodial development also may occur. Phaeococcomyces synanamorph often present.

W dermatitidis is differentiated from E. jeanselmei and similar fungi by the production of phial ides and the ability to grow at 40'C.

Diagnostic characteristics synanamorph may be produced by some isolates.

Sporothrix

Wangiella

of growing as a yeast form at 37°C and as a maid at 25°C. Black aleurioconidia are often no longer produced after repeated subculture.

16, 19,24

MICROBIOLOGY OF FUNGI

63

to be present; the entire colony does not have to be converted to its corresponding tissue form.

Figure 3.6 : Drechslera spicifera. Taken by phase-contrast microscopy after 2 weeks on potato dextrose agar. Bar equals 10 /Lm.

Figure 3.7 : Helm inthosporiurn solani. Taken by phase-contrast microscopy after 2 weeks on potato dextrose agar. Bar equals 10 /Lm.

3.5 IDENTIFICATION The identification of dematiaceous fungi ultimately rests upon their microscopic morphology and, to a lesser extent, upon their gross

64

MICROBIOLOGY AND BIOCHEMISTRY

colonial morphology. The importance of conidium development in defining the numerous genera of dematiaceous fungi makes it essential to determine how a particular fungus forms its conidia. For this reason, slide culture preparations with potato dextrose agar or cornmeal agar are ideal for identification purposes.

Figure 3.8 : Exophiala jeanselmei. Taken by phase-contrast microscopy after 2 weeks on potato dextrose agar. Bar equals 10 I'm.

Figure 3.9 : Exophiala moniliae. Taken by phase-contrast microscopy after ;2 weeks on potato dextrose agar. Bar equals 10 I'm.

Our new understanding of conidium development has resulted in the redefinition of many genera of medically important fungi. Terms such as spore and conidium (plural, conidia) are no longer used interchangeably. Many mycologists consider spores to be propagules that arise either from meiosis (ascospores, basidio-spores, oospores,

MICROBIOLOGY OF FUNGI

65

or zygospores) or by mitosis within a sporangium (sporangiospores). All other asexual, nonmotile propagules are considered conidia. Conidia usually occur on specialized hyphae or hyphal branches called conidiophores. The actual cells that give rise to the conidia are referred to as conidiogenous cells. The distinction between the various kinds of conidiogenous cells is important for the identification of species of dematiaceous fungi. Phialides usually are flask shaped to cylindrical and have an apex that neither increases in length nor changes in diameter as the phialoconidia are formed. A cup-shaped structure called a collarette may be present at the apex of the phialide. In contrast to phialides, the apices of annellides increase in length, become narrower, and have apical rings called annellations. The annellations result when the annelloconidia separate from the apex of the annellide. Some fungi produce conidia by a blowing-out process. Such conidia are called blastoconidia. They may occur individually or in chains. The term acropetal is used when the conidia at the apex of the chain are the youngest. The term basipetal refers to the condition of a chain of conidia when the youngest conidium is at the base of the chain. A number of the medically important dematia-ceous fungi produce conidiophores that are sympodial. In this type of development, a conidium is formed at the apex of the conidiophore. The conidiophore then increases in length by the formation of a new growing point just below and to one side of the conidium. At the apex of this new growth, a second conidium develops. The entire process is repeated, which often results in a conidiophore that has the appearance of a series of bent knees, which is said to be geniculate. The term anamorph is used to characterize an asexual reproductive structure or form produced by fungi. Occasionally, some fungi seen in the clinical laboratory produce more than one asexual form, or anamorph. An example of this polymorphic nature is Fonsecaea pedrosoi, which may form a sympodiaJ anamorph (RhinocladieUa form), a phial ide anamorph (Phialoph-ora form), and an anamorph consisting of branched chains of blastoconidia (Cladosporium form). When a single fungus produces more than one anamorph, the term synanamorph can be used to designate any of these concurrently existing forms. The Scytalidium form is often associated with the pycnidial fungus Hendersonula toruloidae. This form can be referred to as a synanamorph associated with H. toruloidae. The Rhinocladiella, Phialophora, and Cladosporium forms are all synanamorphs of F. pedrosoi.

66

MICROBIOLOGY AND BIOCHEMISTRY

Figure 3.10 : Exophiala spinifera. Taken by phase-contrast microscopy after 2 weeks on potato dextrose agar. Bar equals 10 /Lm.

A number of medically important fungi have the ability to produce sexual forms. The sexual form of a fungus is referred to as a teleomorph. Pseudallescheria boydii is characterized by the formation of cleistothecia; hence, it is a teleomorph. P. boyd;; also

Figure 3.11 : Exophiala werneckli. Taken by phase-contrast microscopy after 2 weeks on potato dextrose agar. Bar equals 10 /Lm.

may produce two anamorphs, that is, Scedosporium and Graphium forms. The term holomorph is used to encompass the whole fungus.

MICROBIOLOGY OF FUNGI

67

In this example, the whole fungus consists of the Pseudallescheria, Scedosporium, and Graphium fom1S. Because fungi are classified by

sexual structures, the name used for the teleomorph also is used for the whole fungus. Problems occasionally arise with anamorphteleomorph connections because a teleomorph may have more than one anamorph and a single anamorph may be produced by several different teleomorphs. For example, Scedosporium apiospermum is one anamorph that is produced by more than one Pseudallescheria species. The black yeasts at times are extremely difficult and frustrating to identify. Black yeasts typically represent one growth form or anamorph of polymorphic fungi. The genus Phaeococcomyces was established to accommodate isolates that consisted of black, budding yeasts with occasional short elements of pseudohyphae, or toruloid hyphae. The assumption that a black yeast, regardless of whether the colony is initially dematiaceous or not, should be identified as Aureobasidium pullulans is incorrect. One of the most frequently isolated black yeasts in the clinical laboratory is the Phaeococcomyces synanamorph of Exophiala jeanselmei. When fresh isolates of this fungus are transferred from Sabouraud dextrose agar to potato dextrose agar or cornmeal agar, the typical conidiogenous cells and conidia of E. jeanselmei rapidly become evident. Dematiaceous, yeastlike co lonies may be formed by such fungi as A. pullulans, E. jeanselmei, and many other species as well.

Figure 3.12 : Fonsecaea IJedrosoi. Taken by phase-contrast microscopy after 2 weeks on potato dextrose agar. Bar equals 10 Wm.

68

MICROBIOLOGY AND BIOCHEMISTRY

Figure 3.13 : Fonsecaea compacta. Taken by phase-contrast microscopy after 2 weeks on potato dextrose agar. Bar equals 10 I'm.

Sterile isolates represent a second group of medically important fungi that are especially difficult to identify. They commonly are referred to as members of the form-order Mycelia Sterilia. These fungi have been shown to cause phaeohyphomycosis and mycetoma. When sterile fungi are isolated, they should be exposed to near-UV radiation from a black light (310 to 410 nm) for several days in a 12-h-light, 12-h-dark cycle, incubated at both low and high temperatures, and subcultured onto media such as 2% water agar, hay infusion agar, soil extract agar, cereal agar, potato dextrose agar, cornmeal agar, and sterile, moist, wooden applicator sticks or filter paper. These media may help stimulate the production of conidia or fruiting bodies. The cultures should be kept for several weeks before they are discarded. With time, some of these fungi may develop structures that produce spores or conidia, such as ascocarps, pycnidia, or synnemata, either in the agar or at the colony surface. Alternaria spp. Members of the genus Alternaria occasionally are implicated as agents of phaeohyphomycosis. These fungi have been associated with infections involving bone, cutaneous tissue, ears, eyes, and the urinary tract. An ALternaria sp. and Alternaria aLtemata (synonym, A. tenuis) are the only well-documented human pathogens in this genus. The Alternaria anamorph of Pleospora infectoria has been reported to be a pathogen of humans, but this report has not been convincingly documented.

MICROBIOLOGY OF FUNGI

. 69

Alternaria colonies are rapid growing, cottony, and gray to black. The erect conidiophores are dematiaceous, simple or branched, and usually solitary, but they occasionally occur in small groups. The conidia of Alternaria spp. develop at the apex of the conidiophore in branching chains, with the youngest conidium at the apex of each chain. The conidia are dematiaceous, muriform, smooth or rough, tapering toward the distal end, and. typically with a short cylindrical beak at their apices. Alternaria isolates are difficult to identify beyond the generic level. If an isolate is recovered that must be identified to species, it should be sent to a specialist.

Figure 3.14 : Phaeococcomyces exophialae. Taken by phasecontrast microscopy after 2 weeks on potato dextrose agar- Bar equals 10 /LID.

Figure 3.15 : Phialophora parasitIca. Taken by phase-contrast microscopy after 2 weeks on potato dextrose agar. Bar equals 10 /Lm.

70

MICROBIOLOGY AND BIOCHEMISTRY

3.5.1 Aureobasidium Spp. Aureobasidium pullulans has been implicated as an agent of phaeohyphomycosis in humans and other animals. This hyphom-ycete is capable of causing opportunistic infections and has been reported from skin, nail, subcutaneous, and deeper tissues. Colonies of A. pullulans are smooth, moist, and yellow, white, cream, light pink, or light brown, fmally becoming black due to the development of arthroconidia. The conidiogenous cells are undifferentiated from the vegetative hyphae and may be intercalary, terminal, or arising as short lateral branches from the hyphae. The conidia are hyaline, one celled, smooth, ellipsoidal, and variable in shape and size. The conidia develop in a synchronous manner from the conidiogenous cells. Blastoconidia commonly are produced from the conidia that arise from the undifferentiated hyphal cells. A Scytalidium anamorph consisting of dematiaceous arthroconidia typically is present. Hormonema species occasionally are confused with Aureoba-sidium spp. In the genus Hormonema, the conidia arise in a basipetal succession from either hyaline or dematiaceous hyphalike conidiogenous cells. In contrast, A. pullulans produces its conidia in a synchronous manner. Because of the confusion which has surrounded these two genera, some of the reported cases of infection ascribed to A. pullulans may have been caused by misidentified isolates of Hormonema spp. This speculation is based upon the fact that several authors have illustrated Hormonema spp. under the name Aureobasidium. 3.5.2 Cladosporium Spp. Cladosporium bantianum (synonym, c. trichoides) and C. carrionii are the most important pathogenic members of the genus Cladosporium. C. bantianum is the most frequently reported etiologic agent of cerebral phaeohyphomycosis, whereas C. carrionii occasionally is recovered from patients with chromoblas-tomycosis. C. cladosporioides is of some interest since it was the etiobgic agent of a pulmonary fungus ball in one patient. This species has been unconvincingly implicated as a pathogen in eye and nail infections. Occasionally, other Cladosporium spp. are reported from cutaneous, eye, and nail infections. Because pathogenesis of C. bantianum other than cerebral involvement has not been well defined, it would be appropriate to handle this organism in a safety cabinet. Cladosporium isolates are rapid growing, velvety or cottony, and usually some shade of olive gray to olive brown or black. From the

MICROBIOLOGY OF FUNGI

71

mycelium, erect, tall, dematiaceous conidiophores arise. At the apex of the branching conidiophore, acropetally branching chains consisting of one- to several-celled, smooth or rough, dematiaceous blastoconidia form; the conidia have a dark hilum (basal scar). The conidia at the bottom of the chains tend to have the appearance of and are commonly referred to as shield cells.

Figure 3.16 : PhIalophora repens. Taken by phase-contrast mIcroscopy after 2 weeks on potato dextrose agar. Bar equals 10 I'm.

C. bantianum and C. carrionii are morphologically similar. C. carrionii can be distinguished from C. bantianum by its slower growth rate, shorter conidia (2 to 3 by 4 to 5 JLm versus 2 to 2.5 by 4 to 7 JLm, with some being 3 by 15 to 20 JLm), dermotropic nature in contrast to the neurotropic nature of C. bantianum, and maximum growth temperature of 35 to 36°C compared with 42 to 43°C for C. bantianum. Both of thef\e species may form long chains of blastoconidia. C. carrionii can hydrolyze casein and starch, whereas C. bantianum apparently does not have this ability.

3.5.3 Curvularia Spp. Curvularia geniculata, C. lunata, C. pallescens, C. senegalensis. and C. verruculosa have been implicated in a number of opportunistic infections. Members of this genus have caused endocarditis, eye infections, mycetoma, and pulmonary phaeohyphomycosis.

72

MICROBIOLOGY AND BIOCHEMISTRY

Curvularia colonies are rapid growing, woolly, and gray to grayish black or brown. The conidiophores are dernatiaceous, solitary or in groups, simple or branched, septate, and typically geniculate. The conidia are two to several celled, usually curved, dark with pale ends, solitary, and typically with a dark hilum. The conidia develop from a sympodial conidiophore. Works by Ellis should be consulted if a Curvularia isolate must be identified to species. 3.5.4 Drechslera Spp. Several Drechslera species have caused opportunistic infections in humans, including meningitis and cutaneous, eye" nasal, and pulmonary infections. The presently recognized pathogenic members of this genus include an unidentified Drechslera sp., Drechslera hawaiiensis, D. longirostrata, D. rostrata, and D. spici!fera. Alcorn has suggested that some of these species should be classified in the genera Bipolaris and Exserohilum. Additional study is necessary before this issue can be adequately resolved. Drechslera species form rapid-growing, woolly, gray-to-black colonies. The conidiophores are dernatiaceous, solitary or in groups, simple or branched, septate, and geniculate. The dematiaceous, oblong-tocylindrical conidia are multicelled and develop from a sympodial conidiophore.

Figure 3.17 : Phialophora richardsiae. Taken by phase-contrast Illicroscopy after 2 weeks on potato dextrose agar. Bar equals 10 "m.

MICROBIOLOGY OF FUNGI

73

Some medical microbiologists have confused Helminthosp-orium spp. with Drechslera spp. The conidiophores of Helmintho-sporium spp. are straight, and they stop-lengthening when the terminal conidium is formed. The conidia develop along the conidiophore; hence, the conidiophore is not sympodial. Helminth-osporium spp. are rarely, if ever, isolated in the clinical laboratory, and members of this genus have not caused phaeohy-phomycosis in humans. If it is necessary to identify Drechslera isolates, either the works of Ellis or a specialist in this genus should be consulted.

Figure 3.18 : Phialophora verrucosa. Taken by phase-contrast microscopy after 2 weeks on potato dextrose agar. Bar equals 10 /Lm.

Figure 3.19 : Lecythophora mutabilis. Taken by phase-contrast microscopy after 2 weeks on potato dextrose agar. Bar equals 10 /Lm.

74

MICROBIOLOGY AND BIOCHEMISTRY

3.5.5 Exophiala Spp. Exophiala jeanselmei, previously known as Phialophora jeanselmei or Phialophora gougerotii is a relatively common etiologic agent of mycotic subcutaneous abscesses. This demati-aceous hyphomycete may cause either mycetoma or phaeohypho-mycosis. E. moniliae and E. spiniJera also have been reported as agents of phaeohyphomycosis, in which they caused subcutaneous cysts. The last member of this genus known to be pathogenic for humans is E. werneckii (synonym, Cladosporium werneckii), which causes superficial phaeohyphomycosis (synonym, tinea nigra).

Figure 3.20 : Lecytbophora mutabilis. Note the presence of chlamydoconidia. Taken by phase-contrast microscopy after 2 weeks on potato dextrose agar. Bar equals 10 I'm.

The colonial morphology of the members of the genus Exophiala is varied. The colonies are slow to rapid growing, often moist and yeastlike at first, becoming woolly with age, and gray to black. Some isolates of E. wemeckii and the Phaeococcomyces synanamorph of E. jeanselmei may remain black and yeastlike. Conidiophores are dematiaceous, simple, or hyphalike. The conidiogenous cells are annellides. In E. jeanselmei, the annellides are lageniform to cylindrical, tapering to a narrow apex; in E. moniliae, they are inflated to elliptical, tapering to a very long, narrow apex; in E. spini lera, they are lageniform to cylindrical, tapering to a narrow; i

MICROBIOLOGY OF FUNGI

75

apex, and they arise from distinct spinelike conidiophores; and in E. werneckii, either they are hyphalike or they consist of two-celled yeast cells that are clavate. The conidia are one-celled in most species and accumulate in balls at the apices of the annellides. With careful study utilizing the oil immersion objective, annellations (rings) usually can be seen at the apices of the annellides.

Figure 3.21': Lecythophora hoffmannll Taken by phase-contrast rrucroscopy after 2 weeks on potato dextrose agar. Bar equals 10 /Lm.

E. jeanselmei was incorrectly believed by some to belong to the genus Phialophora. However, when it was discovered that the conidiogenous cells of E. jeanselmei were annellides and not phialides, the fungus was transferred to the genus Exophiala. An identical situation occurred when it was discovered that the conidiogenous cells of E. spini fera were annellides and not phialides. E. werneckii produces one- to two-celled conidia from annellides that exist as either yeast cells or intercalary conidio-genous cells incorporated within the hyphae. The fungUs originally described as Sporotrichum gougerotii was considered a morphological variant of Sporothrix schenckii. The name S. gougerotii is best considered a nomen dubium.fungi that are currently identified as either S. gou~erotii or P":',i,?~gerotii are

76

MICROBIOLOGY AND BIOCHEMISTRY

typically misidentified isolates of E. jeanselmei. At one time, these two supposedly different fungi were erroneously distinguished from each other by their tissue morphology. In the sense of some contemporary medical mycologists, P. gougerotii is a misapplied name for isolates of E. jeanselmei that do not form granules in tissue.

3.5.6 Fonsecaea spp. The genus Fonsecaea contains two species, Fonsecaea compacta (incorrectly spelled compactum by some) and F. pedrosoi. Both of these species are agents of chromoblastomycosis.

Figure 3.22 ; RhinocIadiella aquaspersa. Taken by phase-contrast microscopy after 2 weeks on potato dextrose agar. Bar equals 10 pm.

Fonsecaea colonies are slow growing, velvety to woolly, and olive to black. Fonsecaea isolates are extremely polymorphic. They are charac"terized by the development of one-celled primary conidia that form on erect, dark, sympodial conidiophores. The primary conidia in turn become conidiogenous cells and form secondary onecelled conidia. This form of development has been incorrectly called Acrotheca-like by some. It is actually more similar to the form seen in the genus Rhinocladiella. Some of the conidia occur as branching chains of blastoconidia, identical to those found in the genus Cladosporium. Fonsecaea spp. may produce phialides with the collarettes bearing the balls of one-celled conidia that are typical of the genus Phialophora. F. pedrosoi and F. compacta are morphologically distinct. F. pedrosoi is differentiated from F. compacta by its elongate conidia

MICROBIOLOGY OF FUNGI

77

Figure 3.23 : Scedosporium apIOspermum. Taken by phase-contrast microscopy after 2 weeks on potato dextrose agar. Bar equals 10 I'm.

that occur in loose heads, in contrast to the rounded conidia in compact heads produced by F. compacta. As a result of their polymorphic nature, F. pedrosoi and F. compacta have been placed inappropriately in the genera Phialophora and Rhinocladielia by some mycologists. 3.5.7 Phaeococcomyces Spp. Phaeococcomyces is a genus that contains black yeasts. Black yeasts are often synanamorphs associated with several of the medically important polymorphic dematiaceous hyphomycetes such as E. jeanselmei and Wangielia dermatitidis. The genus Phaeococcomyces, which was originally named Phaeococcus, contains four species that are distinguished from each other primarily on morphological criteria. Phaeococcomyces exophialae forms slimy, mucoid, slow-growing, smooth colonies that are grayish black. Budding yeast cells which are at first subhyaline are abundant. With age, some of the cells become darker, with thickened cell walls. Some pseudohyphae usually are present. Hyphal development may become dominant in some isolates of this species with subsequent subculture. Black yeasts occasionally are isolated in the clinical laboratory. They are recognized by their black, mucoid, yeastlike colonies. When grown on cornmeal agar or potato dextrose agar, many black yeasts will rapidly produce the hyphae and conidiogenous cells typical of genera such as Exophiala and Wangielia. Based upon conidiogenesis, other genera of black yeasts probably will be needed in the future to accommodate this group of fungi.

78

MICROBIOLOGY AND BIOCHEMISTRY

Figure 3.24 : Scytalidium Iignacola. Taken by phase-<:ontrast microscopy after 2 weeks on potato dextrose agar. Bar equals 10 I'm.

3.5.8 Phialophora Spp. Members of the genus Phialophora are well-recognized etiologic agents of phaeohyphomycosis and chromoblastomycosis. In addition to cutaneous and subcutaneous tissue invasion, some species have caused endocarditis and mycotic keratitis. The pathogenic species of Phialophora include Phialophora bubakii, P. parasitica, P. repens, P. richardsiae, and P. verrucosa. Phialophora colonies are rapid growing, cottony to woolly, and usually some shade of olive gray. When conidiophores are present, they usually are short. The conidiogenous cells are hyaline to dematiaceous phialides that are cylindrical to flask shaped. At the apices of the phial ides , distinct collarettes are present. The conidia are one celled and usually hyaline, and they occur in balls that may occasionally slip down along the phialides in some species. Some species commonly produce intercalary phialides; a yeast form may occur in some isolates. P. mutabilis and P. hojjmannii have been considered species of the genus Phialophora for a number of years. Gams and McGinnis recently have reclassified these species in the genus Lecythophora. This reclassification was necessary because these fungi produce intercalary phialides with short, lateral, cylindrical necks, which bear the conidia in balls at their apices. Collarettes are present at the tips of the phialides.

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3.5.9 Rhinocladiella Spp. Rhinocladiella aquaspersa, previously known as Acrotheca aquaspersa, is a rare etiologic agent of chromoblastomycosis. Human cases of chromoblastomycosis caused by R. aquaspersa have occurred in Brazil and Mexico. Colonies of R. aquaspersa are rapid growing, velvety, slightly elevated, and olive black. The conidiophores are sympodial, usually darker than the vegetative hyphae, unbranched, and erect. Conidia are one celled, rarely two celled, fusiform, elliptical or obovate, smooth, light brown, and with a dark basal scar. Annellides like those of Exophiala spp. and phialides like those of Wangiella spp. may be present.

Figure. 3.25 : ScytahdlUm hyahnum. Taken by phase-contrast Il1Icroscopy after 2 weeks on potato dextrose agar Bar equals 10 j!m.

3.5.10 Scedosporium Spp. Scedosporium apiospermunl, previously known as MOlloSporiuni apiospermunl, is an anamorph of Pseudallescheria boydii, a fungus once classified as Petriellidium boydii and Allescheria boydii. The fungus may cause mycetoma, as well as infections involving the lungs and brain, where the fungus grows in the form of hyphae that look

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like those produced by Aspergillus spp. S. apiospermum rapidly produces colonies that are cottony and smoky gray to dark brown. One-celled conidia may occur singly along the hyphae or in clusters at the apices of annellides. The conidia are obovate, truncate, and subhyaline to light black. S. apiospermum occasionally has a Graphium synanamorph present. Several members of the genera Pseudallescheria and Petri ella may produce a S. apiospermum anamorph. Therefore, without having the teleomorph present, it is not possible to determine if an isolate of S. apiospermum was produced by Pseu dallescheria boydii. 3.5.11 Scytalidium Spp. Members of the genus Scytalidium have been well documented as opportunistic fungal pathogens of nail, skin, and subcutaneous tissue. The Scytalidium synanamorph associated with the pycnidial fungus H. toruloidea, as well as Scytalidium lignicola and S. hyalinum, all have caused disease. S. hyalinum, because of its hyaline nature, would best be classified in a genus other than Scytalidium. Scytalidium spp. produce rapid-growing colonies that are at first white, becoming dark gray with age. In S. lignicola, arthroconidia of two types are formed. In the first type, the arthroconidia are cylindrical, one celled, and hyaline. In the second type, they are thick walled, yellowish brown, and one or two celled. The Scytalidium synanamorph of H. toruloidea forms arthroconidia that are brown, cylindrical at first, becoming rounded, barrel shaped or subglobose, and one or two celled. The monograph on Malbranchae spp. by Sigler and Carmichael should be consulted for the identification of Scytalidium species and similar hyphomycetes that produce arthroconidia. 3.5.12 Sporothrix Spp. S. schenckii, a dimorphic fungus, is considered the only pathogenic member of the genus Sporothrix. Recently, a new species, Sporothrix cyanescens, was added to the genus. Some of the isolates upon which the species description was based were isolated from patients with mycosis of human skin. Whether or not S. cyanescens is another etiologic agent of sporotrichosis remains to be proven. Colonies of S. schenckii are rapid growing and at first moist, flat, and yeastlike, later developing aerial hyphae. They are initially white, becoming brown to black with age. The conidia are of two kinds in most fresh isolates. Hyaline, one-celled conidia develop solitarily upon denticles along the hyphae; laterally from sympodial,

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slender, tapering, erect conidiophores; and tenninally in clusters at the apices of swollen conidiophores. The second type of conidia are one celled, thick walled, and black. These conidia develop along the hyphae. At 37°C on enriched media, the mold form of S. schenckii converts to a yeast form.

Figure 3.26 : Sporothrix schenckii. Taken by phase-contrast microscopy after 2 weeks on potato dextrose agar. Bar equals 10 /tm.

The etiologic agent of sporotrichosis originally was described as S. schenckii. Later, the fungus erroneously was transferred to the

genus Sporotrichum. Members of the genus Sporotrichum are characterized by the formation of large hyphae with clamp connections and large, one-celled, thick-walled, golden conidia. They are neither dimorphic nor pathogenic for humans and other animals. Wangiella sp. W. dermatitidis is an agent of phaeohyphomycosis that typically causes infections involving cutaneous and subcutaneous tissue. The fungus most frequently has been seen in patients living in Japan. The colonies of W. dermatitidis are moist and at first yeastlike, developing some aerial hyphae with age. They are olive to black. Distinct conidiophores are absent. The conidiogenous cells are phialides which do not have collarettes. Some conidiogenous cells appear to possess a group of slightly raised, truncate denticles at their apices that occur as a result of sympodial development. Rare annellides may be produced by isolates of this fungus. The one-celled,

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lightto-dark, smooth phialoconidia form in balls at the apices of the phialides and then slide down their sides. The phial ides develop from conidiophores that are indistinguishable from the hyphae. Most isolates produce an abundant yeast form and large amounts of toruloid hyphae.

Figure 3.27 : Wangiella dermatitidis. Taken by phase-contrast microscopy after 2 weeks on potato dextrose agar. Bar equals 10 JLm.

The genus Wangiella was established to accommodate date the fungus known as either Hormiscium dermatitidis or Phialophora dermatitidis. The new genus Wangiella was necessary because the phial ides without coUarettes that are typical of W. dermatitidis could not be accommodated in any known genus. W. dermatitidis can be recognized by its ability to grow at 40°C, whereas similar dematiaceous hyphomycetes do not grow at that temperature.

4

Microbiology of Bacteria Bacteria belong to the class of organisms known as the Schizomycetes (schizo, fission, and mycetes, fungi). The organisms are single-celled and reproduce normally by transverse or binary fission. The class Schizomycetes is divided into ten orders. The largest order is the Eubacteriales; it includes most of the common bacterial species. Bacteria are typically unicellular plants, the cells being usually small, sometimes ultrarnicroscopic. They are frequently motile. By means of modem techniques, a true nucleus has been demonstrated in bacterial cells. Individual cells may be spherical or straight, curved or spiral rods. Cells may occur in regular or irregular masses, or even in cysts. Where they remain attached to each other after cell division, l~ey may form chains or even definite trichomes. The latter may show some differentiation into holdfast cells and into motile or nonmotile reproductive cells. Some grow as branching mycelial threads whose diameter is not greater than that of ordinary bacterial cells, i.e., about Ill. Some species produce pigments. The true purple and green bacteria possess photosynthetic pigments much like or related to the true chlorophylls of higher p~ants. The phycocyanin found in blue-green algae does not occur in the Schizomycetes. Multiplication is typically by cell division. Endospores are formed by some species of Eubacteriales. Sporocysts are found in Myxobacteriales. Bacteria are free-living, saprophytic, parasitic, or even pathogenic. The latter types cause diseases of either plants or animals. Filament Formation. Cells that reproduce and divide in a normal manner may be induced to grow in filaments by changing the 83

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conditions of the medium. According to Webb, "the division of the bacterial cell follows a complex sequence, which in many respects, . resembles that occurring in the cellular reproduction of higher forms. It is now known, for example, that bacterial cell division entails division of the nuclear element, division of the cytoplasm, secretion of new cell wall material, and the separation of the daughter cells. Some or all of the events of this sequence are readily thrown out of balance, or even completely inhibited. Thus bacteria, particularly the rod-shaped organisms, may be induced to elongate into filaments by various treatments which apparently inhibit cell division but which do not inhibit growth. Such an effect is produced by various chemical substances, by sub-bacteriostatic concentration of certain antibacterial agents, as, for example, methyl violet, sulfonamides, m-cresol, penicillin, irradiation, and higher temperatures of incubation. These changes in morphology induced by chemical substances are usually temporary, since reversion to normal form occurs promptly when the filamentous bacteria are subcultured in the absence of the inhibitory agents. Irradiation, on the other band, may give rise to a temporary or permanent induction of filamentous cells. From ob~.!rvations such as these the concept has arisen that bacterial growth, in the sense of an irreversible increase in cell substance or volume. and cell division may be considered to some extent as separate anu independent processes; at least, in so far as growth may occur either with or without the operation of the cell division mechanism." Variation in the magnesium (Mg) content of the medium may exert a marked effect on cell division of some bacteria. In a Mgdeficient medium, Gram-positive rods grow in the form of long filaments. Such filaments revert to normal forms when transferred to the same medium supplemented with suitable concentrations of Mg. Filament formation is enhanced by the addition of zinc and cobalt. Inhibition of cell division occurs also in media supplemented with an excess of Mg. Deibel et aI. produced filamentous Lactobacillus leichmannii in the absence of vitamin Bn' Reversion to the normal cell form occurred on the addition of either vitamin B12 to a medium lacking the growth factor or of an excess of the desoxyriboside thymidine.

4.1

SHAPE OF BACTERIA Bacteria exhibit three fundamental shapes: (1) spherical, (2) rod,

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and (3) spiral or curved rod. All bacteria exhibit pleomorphism in more or less degree under normal or other conditions, but a bacterial species is still generally associated with a definite cell form when grown on a standard medium under controlled conditions. The spherical bacteria (singular, coccus; plural, cocci) divide in one, two, or three planes, producing pairs or chains, clusters, or packets of cells. Some are apparently perfect spheres; others are slightly elongated or ellipsoidal in shape. The streptococci divide in only one plane. They grow normally in pairs or chains. Depending upon the species, the distal ends of each pair may be lancet-shaped, or flattened at the adjacent sides to resemble a coffee bean. The staphylococci divide in two planes, producing pairs, tetrads, or clusters of bacteria, the latter resembling bunches of grapes. The sarcinae divide in three planes, producing regular packets. These are cubicle masses with one layer of bacteria atop another. The rod forms also show considerable variation. A rod is usually considered to be a cylinder with the ends more or less rounded. Some rod forms are definitely ellipsoidal in shape. The ends of rods also show considerable variation. Some species are markedly rounded; others exhibit flat ends perpendicular to the sides. Gradations between these two forms may be seen. Rods may show marked variation in their length/width ratio. Some rods are very long in comparison to their width; others are so short they may be confused with the spherical forms. The shape of an organism may also vary depending upon certain environmental factors, such as temperature of incubation, age of the culture, concentration of the substrate, and composition of the medium. Bacteria usually exhibit their characteristic morphology in young cultures and on media possessing favorable conditions for growth. Young cells are, in general, larger than old organisms of the same species. As a culture ages, the cells become progressively larger until a maximum is reached, after which the reverse effect occurs. Bacterial variations resulting from changes in age are only temporary; the original forms reappear when the organisms are transferred to fresh medium. 4.1.1 Size of Bacteria Bacteria vary greatly in size according to the species. Some are

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so small they approach the limit of visibility when viewed with the light microsco~. Others are so large they are almost visible with the normal eye. However, the sizes of the majority of bacteria occupy a range intermediate between these two extremes. Regardless of size, none can be clearly seen without the aid of a microscope. A spherical form is measured by its diameter; a rod or spiral form by its length and width. Calculation of the length of a spiral organism by this method gives only the apparent length, not the true length. The true length may be computed by actually measuring the length of each turn of the spiral. Mathematical expressions have been formulated for making such computations. The method employed for fixing and staining bacteria may make a difference in their size. The bacterial cell shrinks considerably during drying and fixing. This will vary somewhat depending upon the type of medium employed for their cultivation. Shrinkage generally averages about one-third of the length of the cel! as compared to an unstained hanging-drop preparation. Young cells of Bacillus megaterium may shrink from 15 to 25 per cent when transferred from nutrient broth to the same medium containing sodium chloride in 2 M concentration. Measurements show some variation depending upon the staining solution used and the method of application. In dried and fixed SD'pars, the cell wall and slime layer do not stain with weakly staining dyes such as methylene blue but do stain with the intensely staining pararosaniline, new fuchsin, crystal violet, and methyl violet. The great majority of bacteria have been measured in fixed and stained preparations. In some instances dried, negatively stained smears have been used. Therefore, the method employed should be specified when measurements of bacteria are reported; otherwise the results will be of doubtful value. The unit for measuring bacteria is the micron. It is expressed by the symbol p.. It is 0.001 mm. or 0.0001 cm. A millimicron is 0.001 p. or 0.000001 mm. It is expressed by the symbol m. p Some bacteria measure as large as 80 p. in length; others as small as 0.2 p.. However, the majority of the commonly encountered bacteria, including the disease producers, measure about 0.5 p. in diameter for the spherical cells and 0.5 by 2 to 3 p. for the rod forms. Bacteria producing spores are generally larger than the nonspore-producing species. The sizes of some common species in dried and stained smears are as follows: Escherichia coli, 0.5 by 1 to 3

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p.; Proteus vulgaris, 0.5 to 1 by 1 to 3 p.; Salmonella typhosa, 0.6 to 0.7 by 2 to 3 p.; Streptococcus lactis, 0.5 to 1 p. in diameter; S. pyogenes, 0.6 to 1 u in diameter; Staphylococcus aureus, 0.8 to 1 u in diameter; Lactobacillus acidophilus, 0.6 to 0.9 by 1.5 to 6 p.; Bacillus subtilis rods, 0.7 to 0.8 by 2 to 3 p., spores, 0.6 to 0.9 by 1 to 1.5 p.; B. megaterium rods, 0.9 to 2.2 by 1 to 5 p., spores, 1 ·to 1.2 by 1.5 to 2 p.; B. anthracis rods, 1 to 1.3 by 3 to 10 p.. spores, 0.8 to 1 by 1.3 to l.5 p.. The most commonly employed method for measuring bacteria is by means of an ocular micrometer. Measurements may also be made by using a camera-Iucida attachment and drawing oculars, or by projecting the real image on a screen and measuring the bacteria. The same factors that cause variations in the shape of bacteria also affect their size. With few exceptions, young cells are much larger than old or mature forms. Cells of B. subtilis from a 4-hr. culture measure five to seven times longer than cells from a 24-hr. culture. Variations in width are less pronounced. The organism Corynebacterium diphtheriae is a notable exception to the rule of decreasing cell size with age. Variations in cell size with age are due to a variety of factors. The major causes appear to be changes in the environment with the accumulation of waste products. An increase in the osmotic pressure of the medium will also cause a decrease in cell size and may very well be the most important factor.

4.2 THE BACTERIAL CELL Bacteria do not show the same morphological picture. Differences in structure exist between species. It is generally agreed that a bacterial cell consists of a compound membrane enclosing cytoplasm and nuclear material and often containing various granules, fat globules, and one or more vacuoles. In addition, some species contain resistant bodies known as spores, and some have one or more organs of locomotion called flagella. The term protoplasm is used to indicate the thick viscous semifluid or almost jelly-like colorless, transparent material which makes up the essential substance of both the cell body and the nucleus, including the cytoplasmic membrane but not the cell wall. It contains a high percentage of water and holds fine granules in suspension. 4.2.1 CytopIasmic Membrane This membrane appears in young cells as an interfacial fluid film,

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becoming thicker and denser as surface-active material accumulates. It is finally converted into a firm structure. The membrane is believed to be composed mainly of lipide and protein. Polysaccharide has not been demonstrated as a component. The membrane is acid in reaction because of its content of ribonucleic acid. It stains deeply with basic and neutral dyes over a wide range of pH. The membrane stains Gram-positive in Gram-positive;: bacteria and acid-fast in acid-fast organisms. It is a semipermeable membrane and is principally responsible for the Gram and acid-fast reactions. When a cell is plasmolyzed by immersion in a hypertonic solution, this membrane is drawn in with the cytoplasmic constituents. The thickness of the membrane varies even in a single cell. Measurements on a strain of Bacillus cereus at various stages of development ranged from 5 to 10 mp. in thickness.

4.2.2 Cell Wall The cell wall is a more rigid structure and is responsible for the form of the bacterial body. It behaves as a selectively permeable membrane and apparently plays a fundamental role in the life activities of the cell. The cell wall has a low affinity for dyes, which means that it is probably not stained in some of the usual staining procedures. It is lightly stained by certain basic dyes such as basic fuchsin and the methyl violets. Where deep staining of the wall is desired, the use of a mordant, such as tannic acid, is necessary. The mordant not only increases the affmity of the cell for dye, but it may increase the thickness of the wall. The cell wall accounts for an average of about 20 per cent of the dry weight of bacteria and represents .the major structural component. In thickness, it ranges from 10 to 23 mp., depending upon the species. According to Saltori, chemical analyses of cell walls have revealed differences in Gram-positive and Gram-negative bacteria. Cell walls of Gram-positive bacteria are lacking in aromatic and certain sulfurcontaining amino acids, arginine, and proline. On the other hand, cell walls of Gram-negative bacteria show the presence of aromatic and sulfur-containing amino acids, arginine, and proline. Gram-negative cell walls are generally richer in lipides than Grampositive bacteria. Cell walls of a number of Gram-positive and negative bacteria contain the amino acid diaminopimelic acid. Polysaccharides have been detected in both Gram-positive and

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Gramnegative bacteria. The polysaccharide is determined as reducing substances after acid hydrolysis. Some polysaccharides yielded only one reducing sugar; others yielded two or more sugars. Gram-positive organisms gave rhamnose, galactose, and glucose; glucose only; rhamnose only; arabinose, galactose, and mannose. Gram-negative bacteria yielded galactose and glucose; galactose, glucose, mannose, and rhamnose. In addition, all organisms studied contained an amino sugar or hexosamine. Generally, the walls of Gram-positive bacteria are richer in hexosamine than the Gram-negative forms. Work pointed to the existence in Gram-positive bacteria of a common basal structure containing the following constituents: a hexosamine component comprising glucosamine and muramic acid and sometimes also galactosMIlind; a peptide component made up of alanine, glutamic acid, and either diaminopimelic acid or lysine with sometimes also glycine, aspartic acid, or serine; and usually a polysaccharide containing not more than four different sugar residues. Other substances may also be attached to the walls, as for example the protein antigens. Smithies, Gibbons, and Bayley reported a relatively hig}} nitrogen content in the walls of several halophilic bacteria which indicated that the cell material was predominantly protein. They contained only small amounts of lipides. The cell walls were lipoprotein. Barkulis and Jones found that approximately one-third of streptococcal cell walls was made up of rhamnose and hexosamine. The remaining two-thirds was protein in nature. For more information see Cummins and Harris (1956), Graziosi and Tecce (1957), Hayashi and Barkulis (1959), Kakutani (1957), Salton (1956), Strange (1959), Tanaka (1957), Tomcsik and Grace (1955), Trucco and Pardee (1958), Yoshida et al. (1956).

4.2.3 Capsules Extracellular material of a "Hmy or gelatinous nature is formed by many bacteria, especially those producing mucoid growths. This material may remain firmly adherent as a discrete covering layer on each cell, or it may part freely from the cells. In the former case it is known as a capsule; in the latter, as free slime or gum. Capsules and slime are believed to be distinct from the morphological and biochemical point of view. The capsule is a part of the cell, the slime a secretion. According to Klieneberger-Nobel, capsules

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are of definite shape, of more or less definite density throughout, and of defmite outline, whereas slime envelopes are amorphous and can be drawn out into manifold structures, are most concentrated in the vicinity of the bacterial cell~, and decrease in density with increasing distance from the cell. Broth cultures of capsule-producing organisms are usually stringy in texture, and agar colonies exhibit a very moist, glistening surface which is described as mucoid. Capsule formation is dependent upon the composition of the medium but especially the variant phase of the organism. Some disease-producing organisms form large capsules in culture media rich in animal fluids. Others produce prominent dapsules when cultures are incubated at low temperatures (4 to 20°C.). Chemical analyses of capsular material from a number of bacteria show wide differences in composition. For this reason it is impossible to make statements which apply to all bacteria. In some organisms the capsular material appears to be a glycoprotein; in others, a proteinpolysaccharide complex; in still others, a polysaccharide framework with the spaces filled in by a larger amount of glutamyl polypeptide. Capsular material is difficult to distinguish from those gums which flow away from the cells as they are formed. Organisms producing gums do so when grown in sugar solutions. Some organisms produce gums only in the presence of a specific sugar; others produce gums in the presence of anyone of several sugars. In the absence of sugar, usually very little, if any, gum is formed. Organisms producing gums of this type are the cause of considerable losses in the sugar industry. The increased viscosity produced by the gum interferes with the filtration of the sugar solution. The species commonly encountered in sugar-cane juice is Leuconostoc esenteroides. The cells are surrounded by a thick, . gelatinous, colorless polysaccharide consisting of dextran (glucose poiymer). The formation of gums is of common occurrence by soil bacteria. From 5 to 16 per cent of such forms have been shown to be capable of synthesizing gums from sugars. When the cell wall is damaged, the protoplasm usually disintegrates. However, methods are available for removing the cell membranes without destroying the vital nature of protoplasm. The term protoplast is used to indicate living protoplasm exclusive of the cell membranes.

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Action of Lysozyme. Some bacteria are rapidly lysed or dissolved by the action of lysozyme. Weibull reported that lysozyme possessed a specific depolymerizing action on- the cell wall and that this appeared to be the only portion of the cell that was affected by such treatment. As a result of the destruction of the wall, the -protoplasts were liberated. Protoplasmic structures are not very stable. Consequently, if protective agents were not employed, destruction of the walls was accompanied by a rapid lysis of the protoplasts, followed by the liberation of most of the cell protein and nucleic acid in soluble form. This could be prevented by employment of the enzyme in a 0.2 M solution of sucrose or can~ sugar. After digestion of the cell walls, the living protoplasts rounded up into spheres. Spiegelman, Aronson, and Fitz-James found that digestion of protoplasts of Bacillus megaterium led to the liberation of nuclear bodies of the protoplasts. Such bodies were collected by centrifugation for 5 min. at 10,000 x g. Properties of Protoplasts. The difficulty in handling and studying protoplasts is their extreme fragility and sensitivity to osmotic shock, shaking, centrifugation, and aeration. Removal of the cell wall does not change the structure and capabilities of the protoplasm. Permeability, respiration, and spore formation appear to be the same for protoplasts and intact cells. Also both can support the development· of bacteriophages. Under special conditions the protoplasts grow and probably divide like intact cells. However, there is no evidence that protoplasts form colonies.) The metabolism of protoplasts and intact cells appear to be very similar but probably not identical. Filterability of Protoplasts. Sinkovics reported the spontaneous occurrence of units in aged cultures of Escherichia coli which conformed to the description of artificially induced bacterial protoplasts. The disintegration of cells from aged cultures was preceded by swelling of the cell and rupture of the rigid cell wall. Centrifugation of the culture gave a supernate which, after filtration, contained units capable of regeneration when placed in fresh medium. The smallest units capable of regeneration measured about 350 mls in diameter. The units underwent fusion before cell-wall formation occurred. The results supported the assumption that aged E. coli cultures could survive in the form of units having no cell walls and which, under adequate conditions, regenerated into vegetative forms.

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4.2.4 Polysaccharide Structures P'Olysaccharides occur (1) in cell walls, (2) extracellularly in capsules and gums, and (3) inside 'Of bacterial cells. The first tW'O have already been discussed. Penningt'On (1949) revealed the presence 'Of polysaccharides by treating bacteria with sodium metaperiodate f'Oll'Owed by staining with sulfitedec'OI'Orized basic fuchsin. In Bacillus cereus the polysaccharide was c'Oncentrated in the cyt'Oplasmic membrane as well as in the cell wall. Selective staining 'Of P'Olysaccharide in the cell is said t'O depend uP'On the 'Oxidizing acti'On 'Of periodate 'On such chemical c'Onfigurati'Ons as a, 13 glyc'Ols and a-hydroxyket'Ones. P'Olyaldehydes generated by this selective 'Oxidation react with sulfite-dec'OI'Orized fuchsin. P'Olysaccharide areas in the cell are c'OI'Ored red by the stain.

4.2.5 Nucleus The questi'On 'Of the presence 'Of a well-defined nucleus in bacteria has been the subject 'Of investigati'Ons by bacteri'OI'Ogists almost from the beginning 'Of bacteri'OI'Ogy.

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Some 'Of the earlier cyt'OI'Ogists maintained that bacteria were very primitive 'Organisms dev'Oid 'Of nuclei and c'Onsisting simply 'Of cyt'Oplasm, granules, and vacu'Oles. This view was based 'On their failure t'O 'Observe a nucleus in a bacterial cell. Others held the view that the nuclear material was present in a diffuse f'Orm thr'Ough'Out the cyt'Oplasm. Still 'Others believed that the wh'Ole cell sh'Ould be regarded

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as a "naked nucleus," corresponding to the nucleus of higher organisms. The naked nucleus is regarded as a primitive form of living matter. Since bacteria have the structural and physiological attributes of true cells, this concept cannot apply to these organisms. Much of the confusion was caused by the inadequacy of the staining procedures. By means of the HCI-Giemsa staining technique of Piekarski, many observations have been reported demonstrating the presence of chromatinic structures in bacteria. Robinow prepared wet smears of Escherichia coli. Slides were fixed in osmic acid vapor, dried, and immersed in normal HCl for about 9 min. at 53 to 55°C., then washed and stained in 1:20 Giemsa solution for 10 to 60 min., depending on the staining properties of the specimen. The chromatinic structures in E. coli from old cultures were too small to be resolved accurately. After transfer to fresh medium the chromatinic structures increased in size and gave rise to short, often dumbbell-shaped rods or chromosomes, which multiplied by splitting leQgthwise in a plane more or less parallel to the short axis of the cell. A single cell of E. coli contained one chromatinic body or one or two pairs of these representing primary and secondary division products. The Smith technique consisted of fixing the smear in osmium tetroxide vapor, immersion in HCI, mordanting in dilute formaldehyde, and staining with aqueous basic fuchsin. The method was said to possess certain advantages over the procedure of Robinow. Another cause of confusion in the recognition of nuclear structures in bacteria was the lack of appreciation for the ages of the cultures. At certain times nuclear structures cannot be seen. In general, most of the early observations of tidy, intelligible "nuclei" were made on preparations from very young cultures, whereas haphazardly scattered, unintelligible granules of chromatin were persistently observed in preparations made from cultures of the same bacteria beyond the logarithmic growth phase. Recognition of the fact that the configuration of nuclear material in bacteria might change with age removed one of the chief causes of confusion. As Dobell said (1911), "My own belief is that the nucleus in bacteria may display not one but many forms during the whole life cycle. Many of the nuclear structures which have been shown to exist in these organisms shOUld, I think, be regarded as temporary states rather than aspermanent conditions. The different '.

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results which have been reached by different workers when working, apparently, upon the same species, may to some extent find an explanation in this circumstance.» The chromatin bodies, according to Robinow, appear to have the following properties: They are simple structures of relatively low density, not markedly basophilic but reacting positively in the Feulgen test. Normally they lie separately in the cytoplasm, and all those in one bacterium are homologous. Changes in the balance of ions in the cytoplasm may cause the aggregation of several chromatin bodies into a single continuous structure. This effect is reversible. Growth and division of chromatin bodies are attended by changes of form only, not by visible changes of texture. In the simplest type of body, that which in profile looks like a bar or dumbbell, division begins at one end and causes the successive appearance of V-, U-, and H-shaped phases. The chromatin structures of certain bacteria are netlike or spongelike, and their mode of division is not easily imagined. Direct division of the chromatin bodies of E. coli has been demonstrated by Mason and Powelson in a series of remarkable phasecontrast photomicrographs. These observations were made on living bacteria. The nuclear areas in the dividing cells appeared to be as clearly defined as the areas in fixed, hydrolyzed, and stained cells. Vacuoles. Vacuoles have been identified in young bacteria. They are cavities in the protoplasm and contain a fluid known as cell sap. As the cells approach maturity, some of the water-soluble reserve food materials manufactured by the cell dissolve in the vacuoles. Insoluble constituents precipitate out as cytoplasmic inclusion bodies.

4.2.6 Metachromatic Granules The best-known inclusion bodies in bacterial cells are known as volutin or metachromatic granules. The granules are small in young cells and become larger with the age of the culture. They are believed to originate in the cytoplasm of young cells and to localize in the vacuoles of mature forms. The granules show a "Strong afrmity for basic dyes, indicating that they are acid in character. They are usually considered to be a reserve source of food. Grula and Hartsell found the granules to be composed of metaphosphate or another form of inorganic phosphate, some fat, and possibly small amounts of protein. Their presence and size in cells were related to the phosphate concentration of the growth

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medium in the presence of an energy source and specific divalent ions (Mn and Zn). Older cells possessed larger granules. Their basophilic nature did not depend on either ribonucleic acid (RNA) or desoxyribonucleic acid. On the other hand, Widra found metachromatic granules to contain protein-bound lipide, RNA, and polyphosphates. 4.2.7 Fat Globules Bacteria are capable of storing fat in the form of globules. Fat globules may be demonstrated in 24-hr. cultures and usually reach a maximum in about 48 hr. Some cells may contain only one large globule; others may show the presence of a number of small, scattered globules.

Figure 4.2 : Composite representation of a metachromatic granule.

It is generally believed that fat is stored as reserve food material. Globules usually cannot be demonstrated in young, vigorously growing cells. As cells age and slow down in activity, fat globules appear in the cytoplasm and may be recognized by appropriate staining. 4.2.8 Motility Bacterial motion is generally associated with the presence of organs of locomotion known as flagella (singular, flagellum). They were first observed in stained preparations by Cohn. The presence of flagella does not mean necessarily that the organisms are always motile, but it indicates a potential power to move. Independent bacterial motion is a true movement of translation and must be distinguished from the quivering or back-and-forth motion exhibited by very small particles suspended in a liquid. This latter type of motion is called Brownian movement and is caused by the bombardment of the bacteria by the molecules of the suspending fluid.

4.2.8.1 Properties of Flagella Flagella are very delicate organs and easily detached from the

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cell. In the stained condition they are long, slender, undulating organs. They are directed backward to the direction of motion at an angle of about 45 0 • Reversal of direction occurs by swinging the flagella through an angle of about 90°. Turning movements take place, by swinging the flagella forward on one side only. They propel the organism by a spiral or corkscrew motion. The thickness of flagella varies from species to species. In Proteus vuLgaris they measure about 12 mJL. This figure is considerably below the shortest wave length of visible light and explains why flagella cannot be seen in hanging-drop preparations or in smears stained by the usual simple procedures. When special staining methods are employed, sufficient dye becomes deposited on the flagella to make their diameters greater than the wave length of visible light. They may then be seen under a light microscope.

4.2.8.2 Chemistry of Flagella Flagella and bacterial bodies differ in composition. Flagella break up on boiling or when exposed to pH values below 4 or above 11. Their composition is largely protein, having a molecular weight of about 41,000. Weibull found the flagella of P. vuLgaris to be composed of 98 per cent protein, traces of carbohydrate and fat, and no phosphorus. The protein contained only 14 known amino acids. It is an incomplete protein, lacking in some of the essential amino acids. It is well established that the H antigens of bacteria are associated with the flagella and the 0 antigens with the bodies. Purified flagella are agglutinated by H antiserum but not by 0 antiserum; 0 antigens are not agglutinated by H antiserum. This is another indication that flagella and bacterial bodies differ in composition. 4.2.8.3 Origin of Flagella Some believe flagella originate from the cell wall; others believe they traverse the cell wall into the protoplasm. Flagella differ chemically both from the cell wall and the protoplasm. Two observations have been made relative to the site of origin of flagella. Electron micrographs by van Iterson and others show the flagella penetrating the faint outer zones and extending into the cytoplasm. If the outer zone is the cell wall, then the flagella have their origin in the cytoplasm.

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Weibull showed that removal of the cell wall of some bacteria by means of lysozyme produces a spherical protoplast which still retains the flagella of the treated cell. The obvious conclusion is that flagella have their origin in some cell structure deeper than the cell wall.

4.2.8.4 Number and Arrangement of FlageUa The number and arrangement of flagella vary with different bacteria, but they are generally constant for each species. Some have only one flagellum; others have two or more flagella. In rod-shaped cells the flagella arise either at one or both poles, or are distributed laterally with the poles being generally bare. In some species flagella are located both laterally and at the poles. A species may show considerable variation in the number and arrangement of flagella. Single Alcaligenes cultures may contain forms with a polar flagellum only; some with several lateral flagella; and some with both lateral and polar flagella. Sometimes a species may show cells which are flagellated in one environment and nonflagt'llated in another. Leifson, Carhart, and Fulton reported the presence (\f four definite types of curvature in the flagella of Proteus vulg;lris. Individual organisms may have more than one type of flagella, and individual flagella may have one or two types of curves. Environmental factors, particularly pH, may change the curvature of the flagella on some strains, but not on all strains. In acid media the curly curvature tends to predominate; in alkaline media the normal predominates. Organisms have been classified on the basis of the number and arrangement of flagella as follows: Monotrichous-a single flagellum at one end of the cell. Lophotrichous-two or more flagella at one end or both ends of the cell. Amphitrichous-one flagellum at each end. Peritrichous-flagella surrounding the cell.

4.2.8.5 Staining of Flagella The staining of flagella is a difficult technique, especially in the hands of the beginner. For this reason many methods have been proposed. Regardless of the method employed, the film must first be treated with a mordant to make the flagella take the stain heavily. Mordants consist usually of a mixture of tannic acid and some metallic

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salt. In some methods the mordant and stain are applied separately; in others they are combined in one solution. Boltjes came to the following conclusions on the staining of bacterial flagella: Four factors at least influence the results of staining-the skill of the investigator, the organism studied, the culture medium on which the organism was grown, and the staining method. Of these the first is perhaps the most and the last the least important. The importance of skill is shown by the repeated failure of students to stain flagella although their teacher has no difficulty in demonstrating flagella at the same time and with the same suspension. Further, one usually has success with a new formula only after a number of trials; first attempts to stain an unknown bacterium are often a failure. Another point is that as a rule flagella can be clearly seen only in a rather small part of the preparation. The different colours of the stained bacteria show clearly that during the staining process conditions are not everywhere alike, and since many flagella are tom off during drying, we need not wonder that in most cases only a few bacteria are successfully stained. Notwithstanding all this, it is certain that when one has had some experience with the staining technique, the results are so consistent that there will never be any confusion between bacteria with true polar flagella, such, for example, a, Pseudomonas fluorescens or Vibrio comma, and peritrichous bacteria like Proteus mirabilis and Salmonella typhosa. I therefore consider flagella staining to be a reliable procedure. Photomicrographs of the arrangement of flagella on bacteria, according to Leifson.

4.2.8.6 The Pijper Theory of Motility In a series of investigations Pijper et al. questioned the belief that flagella are responsible for motility. He added methyl cellulose to a culture of a motile organism to increase the viscosity of the medium. This treatment decreased the motility of the cells. Under these conditions the cells exhibited a gyratory undulating movement like other aquatic creatures. He concluded that flagella were not organs of locomotion but only artifacts-useless appendages, polysaccharide twirls-the result, not the cause, of bacterial motility. To quote, "That motile bacteria always exhibit a gyratory undulating movement was confirmed by making a slow motion cinemicrographic film of fast-swimming bacteria in br9th, and also by examining the same bacteria at lower temperatures, which reduced their speed.

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This spirillar motion of bacteria is sufficient to propel them, and there is no need to invoke special motor organs like flagella. There is no evidence to show that the flagella-like appendages of bacteria act as motile organs-in fact all the evidence when critically examined points the other way. Analysis of the structure of bacteria excludes the possibility that tails, "flagella,» or the thin wavy threads are live organs, or that they are in direct communication with the living parts of the cell. There is no evidence from either electron pictures or stained preparations that it is otherwise. Not only does the visible gyratory undulating movement of motile bacteria satisfy all requirements for locomotion, but it is possible for bacteria grown under special conditions to swim in this fashion without showing tails or other supposed motor organs.» Notwithstanding the findings and conclusions of Pijper, evidence at present appears to be overwhelmingly in favor of flagella as organs of locomotion. As has already been stated, Weibull reported the flagella of P. vulgaris to be composed of 98 per cent protein. Thif. contradicts Pijper's statement that flagella are formed from the carbohydrate slime layer that is peeled off into a number of thin, wavy threads. Several investigators, among them Labaw and Mosley, by means of electron microscopy, demonstrated the presence on Brucella bronchiseptica of uniform flagella having an external contour of a counterclockwise or left-handed triple helix. The average periodicity along the length of the flagella waG 19 mIL, with an average diameter of 13.9 mIL. 4.2.9 Motion of Colonies Several organisms have been described which exhibit colonial motility when grown on a solid medium. Shinn prepared lapse-time motion pictures of individual colonies of Bacillus alvei grown on agar plates and measured their velocities. The linear motions of colonies measuring 0.2 to 0.5 mm. in diameter averaged about 14 mm. per hr. Comparing this figure with the speed of individual cells of other species of motile bacteria gave the following results: Salmonella typhosa 65 mm. per hr. Bacillus megaterium 27 mm. per hr. B. alvei (colonies) 14 mm. per hr.

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The colonies eXhibited not only linear motion but also a slow rotary movement. The direction of rotation of 200 to 300 colonies observed was counterclockwise, with the exception of two colonies in which it was clockwise. Turner and Eales reported that the rotation of an aerobe occurred very early during growth. The cells segregated in small groups and aligned themselves concentrically around a common center to form disk-like plaques one or a few cells thick. The rate of rotation was greater in smaller groups. As multiplication continued, successive layers were gradually built up in terrace fashion and th colony grew in height. Teh colonies then began to migrate. When a colony migrated, it left a peculiar "track" on the surface of the agar. A small number of cells were left behind, mostly at the edges of the track, which formed two parallel lines separated by the width of the moving colony.

F"JgUI"e 4.3 : Sketch of convoluted track of a wandering coony. showing two series of clockwise spirals followed by a final counterclockwise spiral. The colony had increased considerably in size after coming to rest and showed curved radial markings indicating rotation. The total length of the track was about 2 cm.

Typical migrating colonies pursued curved or spiral paths which were often very elaborate and of relatively great length, even 2 or 3 -em.. The direction of rotation was either clockwise or counter clockwise. After wandering for a variable distance, a colony approached the center of its spiral path with rapidly shortening radius, ceased to migrate, began to rotate around its center, lost its elongated shape, and increased in size to several times the width of the track at the end of which it was formed. Endospores. Endospores are bodies produced within the cells of a considerable number of bacterial species. They are more resistant to unfavorable environmental conditions, such as heat, cold, desiccation, osmosis, and chemicals, than the vegetative cells producing them. However, it is debatable if such extreme conditions -actually occur in nature. For instance, the resistance of spores to high temperatures is a laboratory phenomenon and probably never occurs in a natural environment.

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The bulk of evidence indicates the existence of a close relationship between spore formation and the exhaustion of nutrients essential for continued vegetative growth. Sporulation is a defense mechanism to protect the cell when the occasion arises. Spore formation is limited almost entirely to two genera of rodshaped bacteria: Bacillus (aerobic or facultatively anaerobic), and Clostridium (anaerobic or aerotolerant). With one possible exception, the common spherical bacteria do not sporulate. Some spore-bearing species can be made to lose their ability to produce spores. When the ability to produce spores is once lost, it is seldom regained. SporMation is not a process to increase bacterial numbers because a cell rarely produces more than one spore.

4.2.9.1 Morphology of Spores Spores may be spherical, ellipsoidal, or cylindrical in shape. The position of the spore in a cell may be central, subterminal, or terminal. A fully grown spore may have a diameter greater than that of the vegetative cell. This causes a bulging of the cell. The resulting forms are known as clostridium if central, and plectridium if terminal. As a rule, each species has its own characteristic size, shape, and position of the spore, but this is subject to variation under different environmental conditions. Franklin and Bradley, by means of electron microscopy of carbon replicas, reported that the spores of a majority of species of Bacillus and Clostridium are readily distinguished by surface patterns. The surfaces may be smooth or ribbed, with the ribs usually longitudinal.

Figure 4.4 : Surface structure of a spore of Bacillus polymyxa. From left to right: side vIew; same rotated a quarter turn from right to left; same rotated a further quarter turn; view,of a pole.

The sculpturing consists of a single endless ridge in the form of two loops, similar to the marking on a tennis ball, together with two other separate ridges terminating within the loops. An electron micrograph of ultrathin sections of such spores, by van den Hooff and Aninga. The spore coat consists of an outer and an inner layer separated by a space. The outer layer is sometimes called the exine and the inner layer the intine. The intine faintly follows the surface

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relief. The central core is separated from the intine by a regular nonosmophilic space. A peripheral spot may be observed in the core which probably represents nuclear material. 4.2.9.2 J»arasporal lrodies When sporulation of Bacillus laterosporus is complete, the spores are cradled in canoe-shaped bodies. According to Hannay (1957): "On sporulation the slender vegetative rods swell and fonn larger spindle-shaped cells in which the spores are fonned. When the spores mature they lie in a lateral position cradled in canoe-shaped parasporal bodies which are highly basophilic and can be differentiated from the surrounding vegetative cell cytoplasm with dilute basic dyes. On completion of sporulation the vegetative cell protopla~m and the cell wall lyse, leaving the spore cradled in its parasporal body. This attachment continues indefinitely on the usual culture medium and even persists after the spores have germinated. In thin sections of sporing cells the bodies are differentiated from the cell protoplasm by differences in structure. Whereas the protoplasm has a granular appearance, in both longitudinal and cross-sections the parasporal body comprises electrondense lamellae running parallel with the membranes of the spore coat and less electrondense material in the interstices of the lamellae. The inner surface of the body is contiguous with that of the spore coat as if it were part of the spore, rather than a separate body attached to the spore. The staining reactions of the parasporal body are not consistent with those of any substance descred in bal'ieria. " 4.2.9.3 Composition of Spores Ross and Billing, by means of refractive index measurements on spores and vegetative cells of B. cereus, B. cereus var. mycoides, and B. megaterium, found the values to be very high and comparable with that of dehydrated protein. This suggested that they contained much less water than the vegetative cells. Strange and Dark demonstrated the presence of a hexosamine containing peptide in the spore coats of B. megaterillm and B. subtilis. The breakdown of an insoluble peptide complex might well be one of the first steps of the germination process. It was believed that the release of the hexosamine-amino acid complex was the result of the action of lysozyme present in the spores. 4.2.9.4 En~mes of Spores The presence of enzyme systems in Bacillus spores has been

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reported. Some of these are an inorganic pyrophosphatase that requires manganese for activation, adenine ribosidase that hydrolyzes adenosine, an enzyme that functions possibly to lyse the sporangium and free the spore during germination, highly active alanine racemase that catalyzes the conversion of L-alanine to n-alanine, several glucose dehydrogenases, and an aldolase. 4.2.9.5 Sporulation Process Conditions necessary for sporulation in one species do not necessarily apply to another. The subject appears to be in such a state of confusion that it is impossible to discuss sporulation in terms of generalities. The conditions which have been reported as favoring sporulation include addition of salts of metals such as manganese, chromium, nickel, etc., to the medium; shaking a culture of vegetative cells of sporing aerobes with distilled water at 37°C.; addition of tomato juice to a medium; incubating the cultures at an appropriate temperature; addition of calcium carbonate to a carbohydrate medium to prevent excessive accumulation of acid, and to maintain the pH at 5.5 or above; the necessity c ygen; addition to the medium of certain amino acids; etc.

4.2.9.6 Gen-..ination of Sp?res With the exception of some constituents such as high concentrations of calcium, dipicolinic aci1, and in Bacillus sphaericus, a, Ediaminopimelic acid, sI- ..,re_ are similar to the vegetative cells in composit:on. When a spore prepares itself for germination, it loses its refractility, \"nich coincides with an imbibition of water. This stage is associated with a loss in heat resistance, stainability, and dry weigl.,t. Later the spore coat breaks, followed by the emergence from the spore case of a new germ cell which eventually matures into a vegetative cell. Spore germination has been defined in vaiiolJs ways. According to Campbell, "Spore germinaticn may be regarded as the change from a heat resistant spore to a heat labile entity which may not necessarily be a true vegetative cell." Later development, leading eventually to the formation of a mature vegetative cell, is called outgrowth. Some conditions which stimulate germination are as follows: (1) Treatment at 90 to 100°C. for 1 to 2 min. stimulates germination.

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(2) Spores which fail to genninate overcome this dormancy when activated by heat. (3) The use of certain agents such as alanine, glucose, and adenosine stimulates spore germination in most sporing species. In some species other amino acids may be substituted for the alanine. The same applies to glucose. (4) Yeast extract and mixtures of vitamin-free amino acids have also been shown to stimulate germination. Spores genninate in a variety of ways. There is a considerable degree of constancy in the method of spore gennination for each species. Lamanna classified the modes of gennination as follows: I. Spore gennination by shedding of spore coat. Characteristics of this method. are A. Spore does not expand greatly in volume previous to the germ cell breaking through the spore coat. The limit of volume increase of the spore may be considered to be twice its original volume. B. Spore coat does not lose all its refractive property previous to germination. C. After the second division of the germ cell, giving a chain of three organisms, the original spore coat, remaining attached to the cells, is visible for a long time after gennination. 1. Equatorial germination. 2. Polar germination 3. Comma-shaped expansion.

o 0 0 --G-o%0~ c=dJrfP~ 123

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4 5 4 5 Figure 4.5 : Methods of spore germination. From left to right: equatorial germination without splitting along transverse axis; equatorial germination with splitting along transverse axis; polar germination; spore germination by comma-shaped expansion.

II.

Spore germination by absorption of the spore coat. Characteristics of this method are A. The spore expands greatly during germination. A tripling or greater increase of the original volume occurs. B. The spore loses its characteristic refractiveness during germination, so that it is difficult to say when the sore

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has disappeared and the genn cell appeared. C. After the second division of the genn cell even if a thin capsule originally remains, all traces of the spore coat are gone.

o o

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~c

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c=capsule Figure 4.6 : Spore germination by absorption.

Some strains germinating by absorption regularly show a thin capsule remaining about one end of the growing cell. This would appear as a polar germination. In other cases, equatorial capsules are seen. Yet, in all instances, the spore is considered to genninate by absorption inasmuch as the three characteristics of the method are still adhered to.

5 Microbiology of Viruses A virus is a genetic element containing either DNA or RNA that can alternate between two distinct states, intracellular and extracellular. In the extracellular state, a virus is a submicroscopic particle containing nucleic acid surrounded by protein and occasionally containing other components. In this extracellular state, the virus particle, also called the virion, is metabolic ally inert and does not carry out respiratory or biosynthetic functions. The virion is the structure by which the virus genome is carried from the cell in which the virion has been produced to another cell where the viral nucleic acid can be introduced and the intracellular state initiated. In the intracellular state, virus reproduction occurs: the virus genome is produced and the components which make up the virus coat are synthesized. When a virus genome is introduced into a cell and reproduces, the process is called infection. The cell that a virus can infect and in which it can replicate is called a host. The virus redirects preexisting host machinery and metabolic functions necessary for virus replication. Viruses may thus be considered in two ways: as agents of disease and as agents of heredity. As agents of disease, viruses can enter cells and cause harmful changes in these cells, leading to disrupted function or death. As agents of heredity, viruses can enter cells and initiate pennanent, genetic changes that are usually not harmful and may even be beneficial. In many cases, whether a virus causes disease or hereditary change depends upon the host cell and on the enviromnental conditions. Viruses are smaller than cells, ranging in size from 0.02 1Lm to 0.3 1Lm. A common unit of measure for viruses is the nanometer (abbreviated nm), which is 1000 times smaller than a 1Lm and one million times smaller than a millimeter. 106

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MICROBIOLOGY OF VIRUSES

Viruses are classified initially on the basis of the hosts they infect. Thus we have animal viruses, plant viruses, and bacterial viruses. Bacterial viruses, sometimes called bacteriophages (or phage for short, from the Greek phago meaning to eat), have been studied primarily as convenient model systems for research on the molecular biology and genetics of virus reproduction. Many of the basic concepts of virus particle

I

~ infection

I disease

t

cell (host)

I

cell harmed (disease or death)

I heredity

t cell altered genetically (harm or benefit)

Figure 5.1 : Virus infection: the two-fold path.

virology were first worked out with bacterial viruses and subsequently applied to viruses of higher organisms. Because of their frequent medical importance, animal viruses have been extensively studied. The two groups of animal viruses most studied are those infecting insects and those infecting warm-blooded animals. Plant viruses are often important in agriculture but have been less studied than animal viruses. Although viruses are known which infect eucaryotic microorganisms, they have been little studied. In the present chapter, we discuss the structure, replication, and genetics of viruses infecting bacteria and warm-blooded animals. In a nutshell: 1. The virus genome consists of either RNA or DNA. The genome is surrounded by a coat of protein (and occasionally other material). When the virus genome is inside the coat it is called a virus particle or virion. 2. Viruses lack independent metabolism. They multiply only inside living cells, using the host cell metabolic machinery. Some virus particles do contain enzymes, however, that are under the genetic control of the virus genome. Such enzymes are only produced during the infection cycle.

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smallpox, 200 nm

rabies, 100 x 200

_

n~

influenza, 100 nm

_

adenovirus, 70 nm

•

polio, 28 nm

Figurf' S.2 : Relative sizes of some common viruses infecting humans. DNA viruses are green, RNA viruses are red.

3.

S.!

When a virus multiplies, the genome becomes released from the coat. This process occurs during the infection process. The present chapter is divided into three parts. The first part deals with basic concepts of virus structure and function. The second part deals with the nature and manner of multiplication of the bacterial viruses (bacteriophages). In this part we introduce the basic molecular biology of virus multiplication. The third part deals with important groups of animal viruses, with emphasis on molecular aspects of animal virus multiplication.

THE NATURE OF THE VIRUS PARTICLE

Virus particles vary widely in size and shape. As we have stated, some viruses contain RNA, others DNA. We have discussed nucleic acids in previous chapters and have noted that the DNA of the cell genome is in the double-stranded form. Some viruses have doublestranded DNA whereas others have single-stranded DNA (Figure 6.3). We have also noted in Section 5.8 that the RNA of the cell is generally in the single-stranded configuration. Interestingly, although single-stranded RNA viruses are more common, viruses are known in which the RNA is in the double-stranded form. The structures of virions (virus particles) are quite diverse. Viruses vary widely in size, shape, and chemical composition. The

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nucleic acid of the virion is always located within the particle, surrounded by a protein coat called the capsid. The terms coat, shell, and capsid are often used interchangeably to refer to this outer layer. The protein coat is always formed of a number of individual protein molecules, called protein subunits, (sometimes called capsomeres) which are arranged in a precise and highly repetitive pattern around the nucleic acid. A few viruses have only a single kind of protein subunit, but most viruses have several chemically distinct kinds of protein subunits which are themselves associated in specific ways to form larger assemblies called morphological units. It is the morphological unit which is seen with the electron microscope. Genetic economydictates that the variety of virus proteins be kept small, since virus genomes do not have sufficient genetic information to code for a large number of different kinds of proteins.

,

viruses

f1

1Ar l all SS

other

Irs l ss

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retro- ss viruses

ss

ds

fungi. protozoa. etc.

~ ss ds

Figure 5.3 : Diversity of viruses. ss: single stranded; ds: double stranded.

The information for proper aggregation of the protein subunits into the morphological units is contained within the structure of the subunits themselves, and the overall process of assembly is thus called self-assembly. A single virion generally has a large number of morphological units. The complete complex of nucleic acid and protein, packaged in the virus particle, is called the virus nucleocapsid. Although the virus structure just described is frequently the total structure of a virus particle, a number of animal viruses (and a few bacterial viruses) have more complex structures. These viruses are enveloped viruses, in which the nucleocapsid is enclosed in a membrane. Virus membranes are generally lipid bilayer membranes, but associated with these membranes are often virus-specific proteins. Inside the virion are often one or more virus-specific enzymes. Such enzymes usually play roles during the infection and replication process.

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virus RNA

\

protein subunits (capsomeres)

~

;;

(a)

Figure 5.4 : Structure and manner of assembly of a simple virus. tobacco mosaic virus. (a) Electron micrograph at high resolution of a portion of the virus particle. (b) Assembly of the tobacco mosaic virion. The RNA assumes a helical configuration surrounded by the protein capsomeres. The center of the particle is hollow.

Virus symmetry The nucleocapsids of viruses are constructed in highly symmetrical ways. Symmetry refers to the way in which the protein morphological units are arranged in the virus shell. When a symmetrical structure is rotated around an axis, the same form is seen again after a certain number of degrees of rotation. Two kinds of symmetry are recognized in viruses which correspond to the two primary shapes, rod and spherical. Rod-shaped viruses have helical symmetry and spherical viruses have icosahedral symmetry. A typical virus with helical symmetry is the tobacco mosaic virus (TMV). This is an RNA virus in which the 2130 identical protein subunits (each 158 amino acids in length) are arranged in a helix. In TMV, the helix has 16 112 subunits per tum and the overall dimensions of the virus particle are 18 x 300 nm. The lengths of helical viruses are determined by the length of the nucleic acid, but the width of the helical virus particle is determined by the size and packing of the protein subunits.

MICROBIOLOGY OF VIRUSES

111 envelope

caps~,me,e ~~::id~r~,. ~~~~~.

8

~

...-capsid

1

aCid

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naked virus

enveloped virus

Figure 5.5 : Comparison of naked and enveloped virus. two basic types of virus particles.

An icosahedron is a symmetrical structure roughly spherical in shape which has 20 faces. Icosahedral symmetry is the most efficient arrangement for subunits in a closed shell because it uses the smallest number of units to build a shell. The simplest arrangement of morphological units is 3 per face, for a total of 60 units per virus particle. The three un~ts at each face can be either identical or different. Most viruses have more nucleic acid than can be packed into a shell made of just 60 morphological units. The next possible structure which permits close packing contains 180 units and many viruses have shells with this configuration. Other known configurations involve 240 units and 420 units. When discussing symmetry, one speaks of axes of rotation. A flat triangle shape, for instance, has one three-fold axis of symmetry, since there are three possible rotations that will lead to the exact configuration seen originally. Three dimensional objects such as viruses can have more than one axis of symmetry. An icosahedron, for instance, has three different axes of symmetry, two-fold, threefold, and fivefold. When a rod is placed through the two-fold axis of symmetry (one of the edges) in the model, the model can be turned once around this axis (1/2 way or 180) to obtain the same configuration again. When the rod is placed through one of the threefold axes of symmetry (one of the faces), the model can be turned three times, and if the rod is placed through one of the five-fold axes of symmetry (one of the vertices) the model can be turned five times. In all cases, the characteristic structure of the virus is determined by the structure of the protein subunits of which it is constructed. Self-assembly leads to the fmal virus particle.

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nucleic acid

Figure 5.6 : A simple icosahedral virus. Each face has three subunits. A single subunit consists of one or more proteins. (a) Whole virus particle. (b) Virus particle opened up; nucleic acid released.

Enveloped viruses Many viruses have complex membranous structures surrounding the nucleocapsid. Enveloped viruses are common in the animal world (for example, influenza virus), but some enveloped bacterial viruses are also known. The virus envelope consists of a lipid bilayer wi$ proteins, usually glycoproteins, embedded in it. Although the glycoproteins of the virus membrane are encoded by the virus, the lipids. are derived from the membranes of the host cell. The symmetry of enveloped viruses is expressed not in terms of the virion as a whole but in terms of the nucleocapsid present inside the virus membrane.

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113

Figure 5.7 : Demonstration of icosahedral symmetry.

What is the function of the membrane in a virus particle? We will discuss this in detail later but note that because of its location in the virion, the membrane is the structural component of the virus particle that interacts first with the cell. The specificity of virus infection, and some aspects of virus penetration, are controlled in part by characteristics of virus membranes. Complex viruses Some virions are even more complex, being composed of several separate parts, with separate shapes and symmetries. The most complicated viruses in terms of structure are some of the bacterial viruses, which possess not only icosahedral heads but helical tails. In some bacterial viruses, such as the T4 virus of Escherichia coli, the tail itself is a complex structure. For instance, T4 has almost 20 separate proteins in the tail, and the T4 head has several more proteins. In such complex viruses, assembly is also complex. For instance, in T4 the complete tail is formed as a subassembly, and then the tail is added to the DNA-containing head. Finally, tail fibers formed from another protein are added to make the mature, infectious virus particle.

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The virus genome We have stated that the virus genome consists of either DNA or RNA, never both. Viruses differ in size, amount, and character of their nucleic acid. Both single-stranded and doublestranded nucleic acid is found in viruses, and the amount of nucleic acid per virion may vary greatly from one virus type to another. In general, in enveloped viruses the nucleic acid constitutes only a small part of the mass of the virus particle (1-2 percent), whereas in nonenveloped viruses the percent of the particle which is nucleic acid is much larger, often 25-50 percent. Interestingly, the nucleic acid in some viruses is not present in a single molecule, the genome being segmented into several or many molecules. For instance, retroviruses-causal agents of some cancers and AIDS, among other diseases-have two identical RNA molecules, influenza virus has 8 RNA molecules of sizes varying over about three-fold, and some other animal viruses have even more RNA molecules. The manner in which these various pieces of nucleic acid are replicated in the cell and then assembled into mature virions is of considerable interest-how do all these nucleic acid pieces end up together in one particle? Enzymes in viruses We have stated that virus particles do not carry out metabolic processes. Outside of a host cell, a virus particle is metabolically inert. However, some viruses do contain enzymes which play roles in the infectious process. For instance, many viruses contain their own nucleic acid polymerases which transcribe the viral nucleic acid into messenger RNA once the infection process has begun. The retroviruses are RNA viruses which replicate inside the cell as DNA intermediates. These viruses possess an enzyme, an RNA-dependent DNA popo called reverse transcriptase, which transcribes the information in the incoming RNA into a DNA intermediate. It should be noted that reverse transcriptase is unique to the retroviruses and is not found in any other viruses or in cells. A number of viruses contain enzymes which aid in release of the virus from the host cells in the final stages of the infectious process. One group of such enzymes, called neuraminadases, break down glycosidic bonds of glycoproteins and glycolipids of the connective tissue of animal cells, thus aiding in the liberation of the virus. Virions infecting some bacteria possess an enzyme, lysozyme , which hydrolyzes the cell wall, causing lysis of the host cell and release of the virus particles. We will discuss some of these enzymes in more detail later.

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5.2 THE CLASSIFICATION OF VIRUSES As we have noted, viruses can be classified into broad groups depending on their hosts. For instance, there are plant viruses, animal viruses, and bacterial viruses. A number of viruses infecting insects are also known and although viruses are known for fungi, protozoa, and algae, these viruses have been so little studied that no classification has been developed. In the present chapter, we discuss only the animal (primarily mammalian) and bacterial viruses, and we discuss here briefly how these two groups of viruses are classified. Classification of bacterial viruses In the bacterial viruses, a formal classification scheme is rarely used. Rather, each bacterial virus is designated in terms of its principal bacterial host, followed by an arbitrary alphanumeric. Thus, we speak of T4 virus of Eschericbia coli or P22 virus of Salmonella typhimurium. An overview of some of the major types of bacterial viruses is given later. We should note, however, that although a bacterial virus may be designated in reference to its principal host, the actual host range of the virus may be broader. Thus, bacteriophage Mu, generally studied with Eschericbia coli, also infects Citrobacter and Salmonella. Classification of animal viruses We should note first that classification of animal viruses presents some major differences from the classification of organisms. The conventional approach to classification of organisms, involving hierarchical categories such as species, genera, families, etc., has been applied only to animal viruses. Even here, the higher levels of classification are not used. The highest level of animal virus classification is the virus family. Virus families are designated by terms ending in -viridae. Thus, the group of poxviruses is called the Poxviridae and the herpesviruses are called the Herpesviridae. Note that the major criteria used in classifying animal viruses are the type of nucleic acid, the presence or absence of an envelope, and, for certain families, the manner of replication. Virus genera are designated by terms ending in - virus. Thus, among the Poxviridae those poxviruses which infect fowl are called by the genus name Avipoxvirus. Note that frequently in the animal viruses, the genus is defmed based on the host which the virus infects. Except in a few cases, virus species have not been formally designated, but would refer to specific virus entities that have been recognized. At present, virus species are only designated by common names, such as mumps virus, poliovirus 1, and smallpox virus. For

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instance, in the virus genus Onhopoxvirus two virus species currently recognized are vaccinia and smallpox, but are not given Latin names. At present, it does not appear useful to use Latin names for virus species. When contemplating the problem of virus classification, we can be truly impressed with the enormous diversity of viruses. Undoubtedly, many new viruses are awaiting discovery, although most undiscovered viruses will probably be considered members of existing virus families.

5.3 THE VIRUS HOST Because viruses only replicate inside living cells, research on viruses requires use of appropriate hosts. For the study of bacterial viruses, pure cultures are used either in liquid or on semi-solid (agar) medium. Because bacteria are so easy to culture, it is quite easy to study bacterial viruses and this is why such detailed knowledge of bacterial virus reproduction is available. With animal viruses, the initial host may be a whole animal which is susceptible to the virus, but for research purposes it is desirable to have a more convenient host. Many animal viruses can be cultivated in tissue or cell cultures, and the use of such cultures has enormously facilitated research on animal viruses. Cell cultures A cell culture is obtained by enabling growth of cells taken from an organ of the experimental animal. Cell cultures are generally obtained by aseptically removing pieces of the tissue in question, dissociating the cells by treatment with an enzyme which breaks apart the intercellular cement, and spreading the resulting suspension out on the bottom of a flat surface, such as a bottle or petri dish. The cells generally produce glycoprotein-like materials that permit them to adhere to glass surfaces. The thin layer of cells adhering to the glass or plastic dish, called a monolayer, is then overlayed with a suitable culture medium and the culture incubated. The culture media used for cell cultures are generally quite complex, employing a number of amino acids and vitamins, salts, glucose, and a bicarbonate buffer system. To obtain best growth, addition of a small amount of blood serum is usually necessary, and several antibiotics are generally added to prevent bacterial contamination. Some cell cultures prepared in this way will grow indefmitely, and can be established as permanent cell lines. Such cell cultures are most convenient for virus research because continuously available cell material can be available for research purposes. In other cases,

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indefinite growth does not occur but the culture may remain alive for a number of days. Such cultures, called primary cell cultures, may still be useful for virus research, although of course new cultures will have to be prepared from fresh sources from time to time. Cancer Cancer is a cellular phenomenon of uncontrolled growth that is sometimes induced by virus infection. Most cells in a mature animal, although alive, do not divide extensively, apparently because of the presence of growth-inhibiting factors which prevent them from initiating cell division. Under a variety of pathological conditions, among which is included infection by certain viruses, growth inhibition is overcome and the cells begin to divide uncontrollably. Under some conditions, this extensive cellular growth is so excessive that the animal body is virtually consumed by cancer cells: the animal dies. Cancerous growth is thus due to a derangement in the control of cellular growth, and is of great medical as well as theoretical interest. The tumorigenic or cancer-causing ability of viruses can often be detected by observing the induction in cell cultures of uncontrolled growth. In cell cultures, the general arrangement of the cells is as a monolayer, arising because growth generally ceases when the cells, as a result of growth, come in contact with each other. Cancer cells have altered growth requirements and continue to grow, pilmg up to form a small focus of growth. By observing for the induction of such foci of growth from virus infection, it is possible to observe the tumorigenic properties of viruses. In some cases, cell culture monolayers can not be obtained but whole organs, or pieces of organs, can be cultured. Such organ cultures may still be useful in virus research, since they permit growth of viruses under more or less controlled laboratory conditions.

5.4 QUANTIFICATION OF VIRUSES In order to obtain any significant understanding of the nature of viruses and virus replication, it is necessary to be able to quantify the number of virus particles. Virus particles are almost always too small to be seen under the light microscope. Although virus particles can be observed under the electron microscope, the use of this instrument is cumbersome for routine study. In general, viruses are quantified by measuring their effects on the host cells which they infect. It is common to speak of a viru..~ infectious unit, which is the. smallest unit that causes a detectable effect when placed with a susceptible host. By determining the number of infectious units per

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volume of fluid, a measure of virus quantity can be obtained. We discuss here several approaches to assessment of the virus infectious unit. Plaque assay When a virus particle initiates an infection upon a layer or lawn of host cells which is growing spread out on a flat surface, a zone of lysis or growth inhibition may occur which results in a clearing of the c!!ll growth. This clearing is called a plaque, and it is assumed that each plaque has originated from one virus particle. Plaques are essentially "windows" in the lawn of confluent cell growth. With bacterial viruses, plaques may be obtained when virus particles are mixed into a thin layer of host bacteria which is spread out as an agar overlay on the surface of an agar medium. During incubation of the culture, the bacteria grow and form a turbid layer which is visible to the naked eye. However, wherever a successful virus infection has been initiated, lysis of the cells occurs, resulting in the formation of a clear zone, called a plaque. The plaque procedure also permits the isolation of pure virus strains, since if a plaque has arisen from one virus particle, all the virus particles in this plaque are probably genetically identical. Some of the particles from this plaque can be picked and inoculated into a fresh bacterial culture to establish a pure virus line. The development of the plaque assay technique was as important for the advance of virology as was Koch's development of solid media for bacteriology. Plaques may be obtained for animal viruses by using animal cellculture systems as hosts. A monolayer of cultured ani.JIals cells is prepared on a plate or flat bottle and the virus suspension overlayed. Plaques are revealed by zones of destruction of the animal cells. In some cases, the virus may not actually destroy the cells, but cause changes in morphology or growth rate which can be recognized. For instance, tumor viruses may not destroy cells but cause the cells to grow faster than uninfected cells, a phenomenon called transfonnation. As we have noted, in a tissue culture monolayer, these transformed cells gradually develop into a recognizable cluster of cells called a focus of infection. By counting foci of infection, a quantitative measure of virus may be obtained. Efficiency of plating One important concept in quantitative virology involves the idea of efficiency of plating. Counts made by plaque assay are always lower than counts made with the electron

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119

bacterial cells

top agar

pour mixture onto agar plate

nutrient agar plate

sandwich of top agar and nutrient agar

incubate

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phage plaques

~~~5~~~~- lawn of host cells

Figure 5.8 : Quantification of bacterial virus by plaque assay using the agar overlay technique.

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microscope. The efficiency with which virus particles infect host cells is almost never 100 percent and may often be considerably less. This does not mean that the virus particles which have not caused infection are inactive. It may merely mean that under the conditions used, successful infection with these particles has not occurred. Although with bacterial viruses, efficiency of plating is often higher than 50 percent, with many animal viruses it may be very low, 0.1 or 1 percent. Why virus particles vary in infectivity is not well understood. It is possible that the conditions used for quantification are not optimal. Because it is technically difficult to count virus particles with the electron microscope, it is difficult to assess the actual efficiency of plating, but the concept is important in both research and medical practice. Because the efficiency of plating is rarely close to 100 percent, when the plaque method is used to quantify virus, it is accurate to express the titer of the virus suspension not as the number of virion units, but as the number of plaque forming units. Animal infectivity methods Some viruses do not cause recognizable effects in cell cultures but cause death in the whole animal. In such cases, quantification can only be done by some sort of titration in infected animals. The general procedure is to carry out a serial dilution of the unknown sample, generally at ten-fold dilutions, and samples of each dilution are injected into numbers of sensitive animals. After a suitable incubation period, the fraction of dead and live animals at each dilution is tabulated and an end point dilution is calculated. This is the dilution at which, for example, half of the injected animals die. Although such serial dilution methods are much more cumbersome and much less accurate than cell culture methods, they may be essential for the study of certain types of viruses.

5.5 GENERAL FEATURES OF VIRUS REPRODUCTION The basic problem of virus replication can be simply put; the virus must somehow induce a living host cell to synthesize all of the essential components needed to make more virus particles. These components must then be assembled into the proper structure and the new virus particles must escape from the cell and infect other cells. The various phases of this replication process in a bacteriophage can be categorized in seven steps: 1. Attachment (adsorption) of the virion to a susceptible host cell; 2. Penetration (injection) into the cell by the virion or by its

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Figure 5.9 : The replication cycle of a bacterial virus. The general stages of virus replication are indicated.

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nucleic acid; Early steps in replication of the virus nucleic acid, in which the host cell biosynthetic machinery is altered as a prelude to virus nucleic acid synthesis. Virus-specific enzymes may be made; 4. Replication of the virus nucleic acid; 5. Synthesis of 'protein subunits of the virus coat; 6. Assembly of nucleic acid and protein subunits (and membrane components in enveloped viruses) into new virus particles; 7. Release of mature virus particles from the cell (lysis). These stages in virus replication are recognized when virus particles .infect cells in culture and are illustrated in Figure 6.13, which exhibits what is called a one-step growth curve. In the tirst few minutes after infection, a period called the eclipse occurs, in which the virus nucleic acid has become separated from its protein coat so that the virus particle no longer exists as an infectious entity. Although virus nucleic acid may be infectious, the infectivity of virus nucleic acid is many times lower than that of whole virus particles because the machinery for bringing the virus genome into the cell is lacking. Also, outside the virion the nucleic acid is no longer protected from deleterious activities of the environment as it was when it was inside the protein coat. 3.

maturation

eclipse adsorption period relative virus count (plaqueforming units)

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30

60

time Figure 1.10 : The one-step growth curve of virus replication. This graph displays the results of a single round of viral multiphcation in a population of cells.

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The eclipse is the period during which the stages of virus multiplication occur. This is called the latent period, because no infectious virus particles are evident. Finally, maturation begins as the newly synthesized nucleic acid molecules become assembled inside protein coats. During the maturation phase, the titer of active virus particles inside the cell rises dramatically. At the end of maturation, release of mature virus particles occurs, either as a result of cell lysis or because of some budding or excretion process. The number of virus particles released, called the burst size, will vary with the particular virus and the particular host cell, and can range from a few to a few thousand. The timing of this overall virus replication. cycle varies from 20-30 minutes in many bacterial viruses to 8-40 hours in most animal viruses. We now consider each of the steps of the virus multiplication cycle in more detail.

5.6 EARLY EVENTS OF VIRUS MULTIPLICATION In order to discuss the stages of virus multiplication, we must return briefly to a consideration of the virus genome. As we have noted, virus genomes consist of either DNA or RNA, and both singlestranded and double-stranded forms of each of these nucleic acids is known to occur in different viruses. In the case of DNA viruses, the nucleic acid may be in either a linear or a circular form. The nucleic acid of RNA viruses is always in a linear form. Some virus nucleic acids also contain covalently linked polypeptides or amino acids which play roles in replication. In addition, in some RNA viruses the genome is not present in a single molecule, but may be divided over two or many nucleic acid molecules. Even more complicated, once inside the cell, the genetic information present in the virus genome may be transfered to another nucleic acid molecule. To avoid confusion, we restrict the term virus genome to the nucleic acid found in the virion (virus particle). As we have noted, the outcome of a virus infection is the synthesis of viral nucleic acid and viral protein coats. In effect, the virus takes over the biosynthetic machinery of the host and uses it for its own synthesis. A few enzymes needed for virus replication may be present in the virus particle and may be introduced into the cell during the infection process, but the host supplies everything else: energy-generating system, ribosomes, amino-acid activating enzymes, transfer RNA (with a few exceptions), and all soluble factors. The virus genome codes for all new proteins. Such proteins would include the coat protein subunits (of which there are generally more than one kind) plus any new virus-specific enzymes.

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Attachment There is a high specificity in the interaction between virus and host. The most common basis for host specificity involves the attachment process. The virus particle itself has one or more proteins on the outside which interact with specific cell surface comp<)nents called receptors. The receptors on the cell surface are normal surface components of the host, such as proteins, polysaccharides, or lipoprotein-polysaccharide complexes, to which the virus particle attaches. In the absence of the receptor site, the virus cannot adsorb, and hence cannot infect. If the receptor site is altered, the host may become resistant to virus infection. However, mutants of the virus can also arise which are able to adsorb to resistant hosts. In general, virus receptors carry out normal functions in the cell. For example, in bacteria some phage receptors are pili or flagella, others are cell-envelope components, and others are transport binding proteins. The receptor for influenza virus is a glycoprotein found on red blood cells and on cells of the mucous membrane of susceptible animals, whereas the receptor site of poliovirus is a lipoprotein. However, many animal and plant viruses do not have specific attachment sites at all and the virus enters passively as a result of phagocytosis or some other endocytotic_ process. Penetratio~ The means by which the virus penetrates into the cell depends on the nature of the host cell, especially on its surface structures. Cells with cell walls, such as bacteria, are infected in a different manner from animal cells, which lack a cell wall. The most complicated penetration mechanisms have been found in viruses that infect bacteria. The bacteriophage T4, which infects E. coli, can be used as an example. The particle has a head, within which the viral DNA is folded, and a long, fairly complex tail, at the end of which is a series of tail fibers. During the attachment process, the virus particles first attach to the cells by means of the tail fibers. These tail fibers then contract, and the core of the tail makes contact with the cell envelope of the bacterium. The action of a lysozyme-like enzyme results in the formation of a small hole. The tail sheath contracts and the DNA of the virus passes into the cell through a hole in the tip of the tail, the majority of the coat protein remaining outside. The DNA of T4 has a total length of about 50 JLm, whereas the dimensions of the head of the T4 particle are 0.095 Am by 0.065 JLm. This means that the DNA must be highly folded and packed very tightly within the head.

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With animal cells, the whole virus particle penetrates the cell, being carried inside by endocytosis (phagocytosis or pinocytosis), an active cellular process. We describe some of these processes in detail later in this chapter. Virus restriction and modification by the host We have already seen that one form of host resistance to virus arises when there is no receptor site on the cell surface to which the virus can attach. Another and more specific kind of host resistance involves destruction of the viral nucleic acid after it has been injected. This destruction is brought about by host enzymes that cleave the viral DNA at one or several places, thus preventing its replication. This phenomenon is called restriction, and is part of a general host mechanism to prevent the invasion of foreign nucleic acid. Restriction enzymes are highly specific, attacking only certain sequences (generally four or six base pairs). The host protects' its own DNA from the action of restriction enzymes by modifying its

Figure S.11 : Attachment of T4 bacteriophage particle to the cell wall of E. coli and injection of DNA: (a) Unattached particle. (b) Attachment to the wall by the long tail fibers. (c) Contact of cell wall by the tail pin. (d) Contraction of the tail sheath and injection of the DNA.

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DNA at the sites where the restriction enzymes will act. Modification of host DNA is brought about by methylation of purine or pyrimidine bases. Viruses can overcome host restriction mechanisms by modifications of their nucleic acids so that they are no longer subject to enzymatic attack. Two kinds of chemical modifications of viral DNA have been recognized, glucosylation and methylation. The bacteriophages T2, T4, and T6 have their DNA glucosylated to varying degrees, and the glucosylation prevents or greatly reduces nuclease attack. In bacteriophage lambda the amino groups of adenine and cytosine bases are methylated by an enzyme- that uses S-adenosylmethionine as methyl donor. Many other viral nucleic acids have been found to be modified by methylation but glucosylation has been found only in the T-even bacteriophages .(bacteriophages T2, T4, and T6). It should be emphasized that modification of viral nucleic acid occurs after replication has occurred and the modified bases are not copied directly. The enzymes for methylation are actually present in the host before infection, and hence are not virus induced functions. These host modification enzymes probably have as their main role the modification of host DNA so that it can be transferred without inactivation into other cells during genetic recombination. The ability to modify nucleic acid is not found in all strains that support the growth of a given virus. Thus, when bacteriophage lambda is grown on E. coli strain C it is not modified (E. coli strain C lacks both the lambda modification and restriction enzymes), and nucleic acid of virus grown on strain C is destroyed when it enters E. coli strain K- 12, which does have the restriction enzyme. However, strain K12 also has the modification enzyme, and, if lambda is grown on K- 12, its nucleic acid is modified and it will infect both strains K-12 and C equally well. However, if lambda is grown on a K-12 strain made methionine deficient, methylation cannot occur and the phage particles' released are unable to replicate in K12. In the case of the T-even phages, glucosylation requires uridine diphosphoglucose (UDPG), and if a T-even phage is grown on a host deficient in UDPG its nucleic acid is not glucosylated and it is unable to replicate in susceptible cells. A knowledge of modification and restriction systems is of considerable practical utility in studying DNA chemistry. So far, no evidence exists that either modification or restriction occurs in eucaryotic organisms.

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Virus messenger RNA In order for the new virus-specific proteins to be made from the virus genome, it is necessary for new virus-specific RNA molecules to be made. Exactly how the virus brings about new mRNA synthesis depends upon the type of virus, and especially upon whether its genetic material is RNA or DNA, and whether it is single-stranded or double-stranded. Which copy is read into mRNA depends upon the location of the appropriate pr~moter, since the promoter points the direction that the RNA polymerase will follow. In cells (uninfected with virus) all mRNA is made on the DNA template, but with RNA viruses the situation is obviously different. A virus-specific RNA RNA polymerase is needed, since the cell RNA polymerase will generally not copy double-stranded RNA (and ribosomes are not able to translate double-stranded RNA either). A wide variety of modes of viral mRNA synthesis are outlined in Figure. By convention, the chemical sense of the mRNA is considered to be of the plus (+) configuration. The sense of the viral genome nucleic acid is then indicated by a plus if it is the same as the mRNA and a minus if it is of oppposite sense. If the virus has double-stranded DNA (ds DNA), then mRNA synthesis can proceed directly as in uninfected cells. However, if the virus has a singlestranded DNA (ss DNA), then it is first converted to ds DNA and the latter serves as the template for mRNA synthesis with the cell RNA polymerase. If the virus has double-stranded RNA (ds RNA), this RNA serves as a template in a manner analogous to DNA. There are three classes of viruses with ss RNA and they differ in the mechanism by which mRNA is synthesized. In the simplest case, the incoming viral RNA is the plus sense and hence serves directly as mRNA, and copies of this viral RNA are also copied to make further mRNA molecules. In another class, the viral RNA has a minus (-) sense. In such viruses a copy is made (Plus sense) and this copy becomes the mRNA. In the case of the retroviruses (causal agents of certain kinds of cancers and AIDS), a new phenomenon called reverse transcription is seen, in which virion ss RNA is copied to a double-stranded DNA (through a ss DNA intermediate) and the ds DNA then serves as the template for mRNA synthesis (thus: ss RNA ss DNA ds DNA). Retrovirus replication is of unusual interest and complexity. Viral proteins Once mRNA is made, viral proteins (for example, enzymes, capsomers) can be synthesized. The proteins synthesized as a result of virus infection can be grouped into two broad categories,

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the enzymes synthesized soon after infection, called the early enzymes, which are necessary for the replication of virus nucleic acid, and the proteins synthesized later, called the late proteins, which include the proteins of the virus coat. Generally, both the time of appearance and the amount of these two groups of virus proteins are regulated. The early proteins are enzymes which, because they act catalytically, are synthesized in smaller amounts and the late proteins, often structural, are made in much larger amounts. Virus infection obviously upsets the regulatory mechanisms of the host, since there is a marked overproduction of nucleic acid and protein in tht; infected cell. In some cases, virus infection causes a complete shutdown of host macromolecular synthesis while in other cases host synthesis proceeds concurrently with virus synthesis. In either case, the regulation of virus synthesis is under the control of the virus rather than the host. There are several elements of this control which are similar to the host regulatory mechanisms, but there are also some uniquely viral regulatory mechanisms. We discuss various regulatory mechanisms when we consider the individual viruses later in this chapter.

5.7 VIRAL GENETICS Viruses exhibit genetic phenomena similar to those of cells. Studies of viral genetics have played a significant role in understanding many aspects of genetics at the molecular level. In addition, knowledge of the basic phenomena of viral genetics has increased our understanding of processes involved in virus replication. Understanding these processes has also led to some practical developments, especially in the isolation of viruses which are of use in immunization procedures. Most of the detailed work on viral genetics has been carried out with bacteriophages, because of the convenience of working with these viruses. We mention here briefly some of the types of genetic phenomena of viruses. Mutations Much of our knowledge of viral reproduction and how it is regulated has depended on the isolation and characterization of virus mutants. Several kinds of mutants have been studied in viruses: host-range mutants, plaque-type mutants, temperature-sensitive mutants, nonsense mutants, transposons, and inversions. Host-range mutations are those that change the range of hosts that the virus can infect. Host resistance to phage infection can be due to an alteration in receptor sites on the surface of the host cell, so that the virus can no longer attach, and host-range mutations of

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the virus can then be recognized as virus strains able to attach to. and infect these virus-resistant hosts. Other host-range mutants may involve changes in the viral and host enzymes involved in replication, or in the restriction and modification systems. Plaque-morphology mutations are recognized as changes in the characteristics of the plaques formed when a phage infects cells in the conventional agarplate technique. Characteristics of the plaque, such as whether it is clear or turbid, and its size, are under genetic control. The underlying basis of plaque morphology lies in processes taking place during the virus multiplication cycle, such as the rate of replication and the rate of lysis. Under appropriate experimental conditions plaque morphology can bea highly reproducible characteristic of the virus. The advantage of plaque mutants for genetic studies is that they can be easily recognized on the agar plate, but a disadvantage is that there is no convenient way of selecting for them among the large background of normal particles. Temperature-sensitive mutations are those which allow a virus to replicate at one temperature and not at another, due to a mutational alteration in a virus protein that renders the protein unstable at moderately high temperatures. For instance, temperature-sensitive mutants are known in which the phage will not be replicated in the host at 43°C but will at 25°C, although the host functions at both temperatures. Such mutations are called conditionally lethal, since the virus is unable to reproduce at the higher temperature, but replicates at the lower temperature. Nonsense mutations change normal codons into nonsense codons. In viruses, nonsense mutations are recognized because hosts are available that contain suppressors able to read nonsense codons. The virus mutant will be able to grow in the host containing the suppressor, but not in the normal host. Transpositions several viruses are known which act essentially as transposons and transposition events involving viruses can lead to their genetic change. Genetic recombination in viruses The availability of virus mutants makes possible the investigation of genetic recombination. If two virus particles infect the same cell, there is a possibility for genetic exchange between the two virus genomes during the replication process. If recombination does occur, the progeny of such a mixed infection should include not only the parental types, but recombinant types as well. With appropriate mutants, it is possible to recognize

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both the parental types and the recombinants and to study the events involved in the recombination process. Genetic recombination in viruses is an extremely complex process to analyze because recombination does not occur as a single discrete event during mixed infection, but may occur over and over again during the replication cycle. It has been calculated that the T-even bacteriophages undergo, on the average, four or five rounds of recombination during a single infection cycle. By detailed and careful analysis of a wide variety of virus crosses, it has been possible to construct genetic maps of a number of bacterial viruses. Such maps have provided important information about the genetic structure of viruses. We present a few genetic maps when discussing specific viruses later in this chapter. Genetic recombination arises by exchange of homologous segments of DNA between viral genomes, most often during the replication process. The enzymes involved in recombination are DNA polymerases, endonucleases, and ligases, which also playa role in DNA repair and synthesis processes. Phenotypic mixing During studies on genetic recombination between viruses, another phenomenon was discovered which superficially resembles recombination but has a quite different basis. Phenotypic mixing occurs when the DNA of one virus is incorporated inside the protein coat of a different virus. For phenotypic mixing to occur, the two viruses must be closely related, so that the protein coat is of proper construction of the packaging of either viral DNA. As an example of phenotypic mixing, in phage T2 of E. coli there is a gene called the h gene which controls host specificity through modification of the tail fibers of the phage. If a mixed infection is set up with two T2 phages, mutant T2h and wild-type T2h+, tail fibers of b specificity may be incorporated onto the particles containing DNA of h+ specificity. Since it is the h function of the tail fibers that affects attachment, these mixed particles will show b specificity during the next round of infection, even though they contain h+ DNA, but the particles resulting from this second round of infection will phenotypically become b+, because the DNA has been unchanged.

5.8 GENERAL OVERVIEW OF BACTERIAL VIRUSES Most of the bacterial viruses which have been studied in any detail infect bacteria of the enteric group, such as Escherichia coli and Salmonella typhimurium. However, viruses are known that infect a variety of procaryotes, both eubacteria and archaebacteria. A few bacterial viruses have lipid envelope~ but most do not. However,

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many bacterial viruses are structurally complex, with head and complex tail structures. The tail is involved in the injection of the nucleic acid into the cell. We now discuss some of the bacterial viruses for which molecular details of the multiplication process are known. Although these bacterial viruses were first studied as model systems for understanding general features of virus multiplication many of them now serve as convenient tools for genetic engineering. Thus, the information on bacterial viruses is not only valuable as background for the discussion of animal viruses, but is essential for the material presented in the next two chapters on microbial genetics and genetic engineering. It should already be clear from what has been stated that a great diversity of viruses exist. It should therefore not be surprising that there is also a great diversity in the manner by which virus multiplication occurs. Interestingly, many viruses have special features of their nucleic acid and protein synthesis processes that are not fomid in cells. In the present chapter, we are only able to present some of the major types of virus replication patterns, and must skip some of the interesting exceptional cases.

5.9 RNA BACTERIOPHAGES A number of bacterial viruses have RNA genomes. The bestknown bacterial RNA viruses have single-stranded RNA. Interestingly, the bacterial RNA viruses known in the enteric bacteria group infect only bacterial cells which behave as gene donors (males) in genetic recombination. This restriction to male bacterial cells arises because these viruses infect bacteria by attaching to male-specific pili. Since such pili are absent on female cells, these RNA viruses are unable to attach to the females, and hence do not initiate infection in females. The bacterial RNA viruses are all of quite small size, about 26 nm in size, and they are all icosahedral, with 180 copies of coat protein per virus particle. The complete nucleotide sequence of several RNA phages are known. In the RNA phage MS2, which infects Eschericbia coli, the viral RNA is 3,569 nucleotides long. The virus RNA, although single stranded, has extensive regions of secondary and tertiary structure. The RNA strand in the virion has the plus ( +) sense, acting directly as mRNA upon entry into the cell. The genetic map is shown and the flow of events of MS2 multiplication. The infecting RNA goes to the host ribosome, where it is translated into four (or more) proteins. The four proteins that have been recognized are maturation protein (A-protein; present in

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ss 0 MS2, Q13

dsO <1>6

ss DNA

o

~

X 174

=======~==

~

ffdd" M M1133

•

ds DNA T3, T7

lambda, T5

Mu

T2,T4

Figure 5.12 : Schematic representations of the main types of bacterial viruses. Those discussed in detail are fd, M 13, X 174, MS2, T4, lambda, T 7, and Mu. Sizes are to approximate scale.

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133

Figure 5.13 : The RNA of bacteriophage MS2. The molecule is single stranded but there are extensive regions of complementary bases, so that pairing within the strand leads to the secondary structure shown. Note that the start sites for three coding regions are in the same part of the folded molecule.

the mature virus particle as a single copy), coat protein, lysis protein (involved in the lysis process which results in release of mature virus particles), and RNA replicase, the enzyme which brings about the replication of the viral RNA. Interestingly, the RNA replicase is a composite protein, composed partly of a virus-encoded polypeptide and partly of host polypeptides. The host proteins involved in the formation of active viral replicase are ribosomal protein Sf (one of the subunits of the 30S ribosome), and elongation factors Tu and Ts, involved in the translation process. Thus, the virus appears to co-opt host proteins that normally have entirely distinct functions and make them become part of active viral replicase. As noted, the viral RNA is of the plus (+) sense. Replicase synthesizes RNA of minus (-) sense using the infecting RNA as template. After minus RNA has been synthesized, plus RNA is made from this minus RNA. The newly mad~ plus RNA strands now serve as messengers for virus protein synthesis. The gene for the maturation protein is at the 5' end of the RNA. Translation of the gene coding for the maturation protein (needed in only one copy per virus . particle), occurs only from the newly formed plus-strand RNA as

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the replication process occurs. In this way, the amount of maturation protein needed is limited. As the virus RNA· is made, it folds into a complex form with extensive secondary and tertiary structure. Of the four AUG start sites, the most accessible to the translation process is that for the coat protein. As coat protein molecules increase in number in the cell, they combine with the RNA around the AUG start site for the replicase protein, effectively turning off synthesis of replicase. Thus, the major virus protein synthesized is coat protein, which is needed in 180 copies per RNA molecule. Another interesting feature of MS2 RNA virus is that the fourth virus protein, the lysis protein, is coded by a gene which overlaps with both the coat protein gene and the replicase gene. The start of this lysis gene is not directly accessible to ribosomes. As the ribosome passes over the coat protein gene, a frame shift occasionally occurs, resulting in reading of the lysis gene. By restricting the efficiency of translation in this way, premature lysis of the cell is probably avoided. Only after sufficient coat protein is available for the assembly of mature virus particles, does lysis commence. (In another RNA phage, QP, the maturation protein itself also functions as a lysis protein, and a separate lysis gene as such is not present.) Ultimately, assembly occurs and release of virions from the cell occurs as a result of cell lysis. The features of replication of these simple RNA viruses are themselves fairly simple. The viral RNA itself functions as an mRNA and regulation occurs primarily by way of controlling access of ribosomes to the appropriate start sites on the viral RNA.

5.10 SINGLE-STRANDED ICOSAHEDRAL DNA BACTERIOPHAGES A number of small bacterial viruses have genomes consisting of single-stranded DNA in circular configuration. These viruses are very small, about 25 nm in diameter, and the principle building block of the protein coat is a single protein present in 60 copies (the minimum number of protein subunits possible in an icosahedral virus), to which are attached at the vertices of the icosahedron several other proteins which make up spike-like structures. In contrast to the RNA viruses, much of the enzymatic machinery for the replication of DNA already exists in the cell. These small DNA viruses possess only a limited amount of genetic information in their genomes, and the host cell DNA replication machinery is used in the replication of virus DNA. The most extensively studied virus of this group is the phage

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135

designated cjlX174, which infects Escherichia coli. cjlX174 is of special interest because it was the first genetic element shown to have overlapping genes. The genomes of cells are organized in linear fashion, with the gene coding for each protein separate from that for all other genes. In very small viruses such as cjlX 174 there is insufficient DNA to code for all virus-specific pfjteins. cjlX174 has solved this problem by the use of overlapping genes. Thus, parts of certain nucleotide sequences are read twice, in different directions and in different reading frames. It should be noted that although the use of overlapping genes makes possible more efficient use of genetic information, it seriously complicates the evolution process, since a mutation in a region of gene overlap may affect two genes simultaneously. As seen in the genetic map, the sequences of genes D and E overlap each other, gene E being contained completely within gene D. In addition, the termination codon of gene D overlaps the initiation codon of gene J by one nucleotide. The reading frame of gene E is therefore in a different phase (starting point) from that of gene D. Obviously, any mutation in gene E will also lead to an alteration in the sequence of gene D, but whether a given mutation affects one or both proteins will depend on the exact nature of the alteration (because the genetic code is degenerate). Other instances of gene overlap through use of overlapping reading frames in cjlX 174 DNA are genes AlB, K/B, KlC, KIA, AIC, and DIE. Additionally, a small gene A protein, called A * protein, is formed by reinitiation of translation (not transcription) within gene A mRNA, with A' protein being read and terminated from the same mRNA reading frame as A protein. The DNA of OX174 consists of a circular singlestranded molecule of 5386 nucleotide residues. The DNA of cjlX174 was the first DNA to be completely sequenced, a remarkable achievement when it was accomplished by Sanger and colleagues in 1977. Now, DNA sequencing is a routine procedure. The replication process of such a circular singlestranded DNA molecule is of considerable general interest, since cellular DNA replicates always in the double-stranded configuration. The DNA strand in the virion is referred to as the plus (+) strand and the complementary strand the minus (-) strand. Upon infection, the viral plus strand becomes separated from the protein coat; entrance into the cell is accompanied by the conversion of this singlestranded DNA into a double-stranded form called the replicative form (RF) DNA. Cell-coded proteins involved in the

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MICROBIOLOGY AND BIOCHEMISTRY

conversion of viral DNA into RF consist of the enzyme RNA primase and DNA polymerase. ligase. and gyrase. No virus-coded proteins are involved in the conversion of single-stranded DNA to RF. The RF is a closed, double-stranded, circular DNA which has extensive supercoiling. DNA replication differs between the leading strand and the lagging strand of the DNA double helix. In cells, replication Of the lagging strand involves the formation of short RNA primers by action of an enzyme called RNA primase (or primase for short). Such RNA primers are made at intervals on the lagging strand and are then removed and replaced with DNA by DNA polymerase. In 4>X174, however, replication begins with a single stranded closed circle, a rather atypical situation. First, primase brings about the synthesis of a short RNA primer, beginning at one or more specific initiation sites on the DNA. Once priming of DNA synthesis has been carried out, the RNA primer is replaced with DNA through action of DNA polymerase. Continuation of DNA replication around the closed circle leads to the formation of the complete double-stranded RF. Once the complete second strand has been formed, its circle is closed with DNA ligase and a DNA gyrase introduces twists that result in supercoiling. DNA gyrase introduces supercoils by cutting one of the two strands of the DNA double helix, holding the two ends apart without rotation, passing a distant region of the circle through the cut, and resealing the ends. The degree of supercoiling is determined by the number of twists that have been introduced into the DNA. One result of supercoiling is that it converts the DNA into a more compact form where it takes up less room in the cell or virion. Once the RF is formed, nucleic acid replication occurs by conventional semiconservative replication, resulting in the formation of new RF molecules. As in general DNA synthesis, initiation of the formation of a new strand begins at a unique site on the DNA, the origin of replication. In 4>X174, the origin of replication is at residue 4395. Formation of single-stranded viral progeny begins with a single-stranded cleavage of the viral (Plus) strand_ of the RF at the origin of replication. Cleavage is brought about by a protein called gene A protein; this protein also makes a covalent bond to the 5' P of the viral strand. Asymmetric replication by the rolling circle mechanism results in the formation of single-stranded molecules that will become the virus progeny. When the growing viral strand reaches

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MICROBIOLOGY OF VIRUSES

unit length (5386 residues for cl>X174), gene A protein cleaves and then ligates the two ends of the newly synthesized single strand to give a viral single-stranded DNA. Early in infection, viral singlestranded progeny DNA are rapidly converted into RF by the mechanism already described above. Later in infection, when coat protein has accumulated, the single-stranded DNA is packaged into -virions. . RNA primer rNA prtmer

replaced with DNA .... ' "

o-o=o-o-~ (a)

virion DNA

supercoiled RF

replicating form (RF)

~-~-~TOning Circle replication

(b)

ma,:::::::. •

000 00

o

mature virus particle

capSid proteins

Figure 5.14: Events in the replication of ~X174 DNA. (a) Formation of the replicating double-stranded DNA (RF). (b) Rolling circle replication leads to the formation of virus progeny.

Viral mRNA synthesis is directed by the RF. Synthesis of mRNA begins at several major promoters on the RF, and terminates at a number of sites. The polycistronic mRNA molecules are than translated into the various phage proteins. The strengths of the several promoters vary, so that some proteins are made in smaller amounts than others. Each promoter activates the transcription of a functionally related set of genes. As we have noted, protein A and protein A' are both made from the same gene, protein A' arising as a result of translation of a secondary initiation site internal to the A mRNA. Further, as we have noted, several proteins are made from mRNA transcripts formed from different reading frames from the same DNA sequences. One can truly be impressed by the efficiency with which such a small genome as that of cl>X174 can have multiple uses. Ultimately, assembly of mature virus particles occurs. Release

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MICROBIOLOGY AND BIOCHEMISTRY

of virions from the cell occurs as a result of cell lysis, which involves the participation of gene E protein.

5.11 SINGLE-STRANDED FILAMENTOUS DNA BACTERIOPHAGES Quite distinct from q,X174 are the filamentous DNA phages, which have helical rather than icosahedral symmetry. 'ijJ.e most studied member of this group is phage M13, which infects Escherichia coli. but related phages include f1 and fd. As with the small RNA viruses, these filamentous DNA phages only infect male cells, entering after attachment to the male-specific pilus. Interestingly, even though these phages are linear (filamentous) they possess circular DNA. The DNA is not self-complementary, however, so that the two adjacent halves of the molecule which run up and down the virus particle form loops at the ends but exhibit very little if any base pairing. Phage M13 has found extensive use as a cloning vector and DNA sequencing vehicle in genetic engineering. The virion of M 13 is only 6 run in diameter but is 860 run long. These phages have the additional unique property of being released from the cell without killing the host cell. Thus, a cell infected with phage M13 or fd can continue to grow, . all the while releasing virus particles. Virus infection causes a slowing of cell growth, but otherwise a cell is able to coexist with its virus. Plaques are thus seen only as areas of thinner cell growth in the bacterial lawn. Many aspects of DNA replication in filamentous phages are similar to that of q,X 174. The unique property, release without cell killing, can be briefly discussed. The release from the cell occurs by a budding process in which the virus particle is always released from the cell with the end containing the A protein first. Interestingly, the orientation of the virus particle across the cell membrane is the same for its entry and exit from the cell. There is no accumulation of intracellular virus particles; the assembly of mature virus particles occurs on the inner cell membrane and virus assembly is coupled with the budding process. Several features of these phages make them useful as cloning and DNA sequencing vehicles. First, they pave single-stranded DNA, which means that sequencing can be carried out by the Sanger dideoxynucleotide method. Second, as long as infected cells are kept in the growing state they can be maintained indefinitely with cloned DNA, so that a continuous source of the cloned DNA is available. Third, there is an intergenic space which does not code for protein and can' ~ teplaced by variable amounts of foreign DNA.

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MICROBIOLOGY OF VIRUSES A protein

B protein

~~. D protein

Gene 3: A protein Gene 8: B protein Genes 7,9: C protein Gene 6: D protein

intergenic space

Figure 5.15 : The filamentous single-stranded DNA bacteriophage fd. Orientation of the proteins and genes in the virion. Note the intergenic space which contams the origin of DNA synthesis. Gene cloning is done in this intergenic space.

cell wall

cell membrane coat proteins embedded in cell membrane

phage DNA: ss circle

environment

cytoplasm

Figure 5.16 : Illustration of the manner by which the virion of a filamentous singlestranded phage (such as M13 or fd) leaves an infected cell without lysis. The A protein passes first through the membrane at a site on the membrane where coat protein molecules have first become imbedded. The intracellular circular DNA is coated with dimers of another protein, gp5, \\'hich is displaced by coat protein as the DNA passes through the intact membrane.

5.12 DOUBLE-STRANDED DNA BACTERIOPHAGES Many· bacterial viruses have genomes containing double-stranded DNA. Such viruses were the first bacterial viruses discovered, and have been the most extensively studied. With such a range of doublestranded DNA viruses, a wide variety of replication systems are present. In the present section, we discuss the best studied and most representative of the group, T4 and 17. The simpler, 17, will be discussed first.

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MICROBIOLOGY AND BIOCHEMISTRY

Bacteriophage 1'7 Bacteriophage 17 and its close relative T3 are relatively small DNA viruses that infect Escherichia coli. (Some strains of Shigella and Pasteurella are also hosts for phage 17.) The virus particle has an icosahedral head and a very small tail. The virus particle is fairly complex, with 5 different proteins in the head and 3-6 different proteins in the tail. One tail protein, the tail fiber protein, is the means by which the virus particle attaches to the bacterial cell surface. Only female cells of Escherichia coli can be infected with 17; male cells can be infected but the multiplication process is terminated during the latent period. The nucleic acid of the 17 genome is a linear double-stranded molecule of 39,936 base pairs. The complete genome has been sequenced, and the sequence information has permitted discernment of gene structure and features of gene regulation. About 92 percent of the DNA of 17 codes for proteins. At least 25 separate genes have been characterized, but not all genes are separately coded on the DNA. Gene overlap occurs for several genes. through translation in different reading frames and through internal reinitiation with one or more genes in the same reading frame. Further genetic economy is achieved by internal frame shifts within certain genes to yield longer proteins. When the phage particle attaches to the bacterial cell, the DNA is injected in a linear fashion, with the genes at the "left end" of the genetic map entering the cell first. Several genes at the left end of the DNA are transcribed immediately by a cell RNA polymerase, using three closely spaced promoters, generating a set of overlapping polycistronic mRNA molecules. These mRNA molecules are then cleaved by a specific RNase of the cell at 5 sites, thus generating smaller mRNA molecules which code for one to four proteins each. One of these proteins is an RNA polymerase that copies doublestranded DNA. Two other early mRNA molecules code for proteins which stop the action of host RNA polymerase, thus turning off the transcription of the early genes as well as the transcription of host genes. Thus, a" host RNA polymerase is used just to copy the first few genes and to make the mRNA for the phage-specific RNA polymerase, and this phage specific RNA polymerase is then involved in the major RNA transcription processes of the phage. This 17 RNA polymerase uses a new set of promoters that are distributed along the left-center and center portions of the genome. It is thus seen that regulation of 17 has both negative and positive control: negative, by means of the formation of proteins that stop host RNA polymerase

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MICROBIOLOGY OF VIRUSES

and thus shut off transcription of the early T7 genes that are recognized by this enzyme, and positive, by means of the formation of the new gene disignation

function

left end Early promoters - _ 0.3 _

Overcomes host restriction

0.7 •

Protein kinase

1

RNA polymerase

•

1.1

UnknOwn --Stert of DNA replication DNA ligase

Promoters - - - - 1.3 1.7 _

Nonessential

2 _ 3 _ 3.5 -

Inactivate host RNA polymerase Endonuclease Lysozyme

4

Hellcase. prlmase

11

I ____ 8_._ _ 5

promoter

Early transcription

DNA polymerase Exonuclease

7 8

•

Protein in phage particle Head protein

9

•

Head assembly protein

promoter - - - - _

Protein for DNA replication

Major head protein Tall protein Tall protein

promoter - - - - - .

promoter

1 14 _

Protein in phage particle Head protein

15

Head protein

11

_____161

Phage maturation

Head protein

17

Tail protein

18 •

DNA maturation

19 _

DNA maturation

Right end

Figure 5.17 : Genetic map of phage T 7, showing gene numbers, approximate sizes, and functions of the gene products.

RNA polymerase which recognizes the rest of the T7 promoters. We also note that T7 is an example of a virus which strongly affects host transcription and translation processes, by producing proteins

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MICROBIOLOGY AND BIOCHEMISTRY

which turn off transcription of host genes. The virus also has genes coding for enzymes which degrade host cell DNA, and nucleotides from such degraded DNA end up in ViI'Us.'progeny. Obviously, such a virus has profound pathological effects on its host cell. As seen in the genetic map, the genes after gene 1.1, transcribed by the T7 RNA polymerase, code for proteins that are involved in 17 DNA synthesis, the formation of virus coat proteins, and assembly. Three classes of T7 proteins are formed: class I, made 4-8 minutes after infection, which use the cell RNA polymerase; class II, made 6-15 minutes after infection, which are made from T7 RNA polymerase and are involved in DNA metabolism; class III, made from 6 minutes to lysis, which are transcribed by T7 RNA polymerase and which code for phage assembly and coat protein. This sort of sequential pattern, commonly seen in many large double-stranded DNA phages, results in an efficient channeling of host resources, first toward DNA metabolism and replication, then on to formation of virus particles and release of virus by cell lysis. DNA replication in T7 begins at an origfn of replication at which DNA synthesis is initiated, and DNA synthesis proceeds bidirectionally from this origin. In both directions, an RNA primer is involved, but the enzyme involved in the synthesis of this primer is different for primer synthesis in the leftward and rightward direction. In the rightward direction, the RNA primer is synthesized by 17 RNA polymerase. whereas in the leftward direction, a virus-specific enzyme, T7 primase (gene 4 protein) is used. Both primers are then elongated by T7 polymerase. Replicating molecules of T7 DNA can be recognized under the electron microscope by their characteristic structures. Because the origin .of replication is near the left end, Y-shaped molecules are frequently seen, and earlier in replication, bubble-shaped molecules appear. . A structural feature of the T7 DNA which is important in DNA replication is that there is a direct tenninal repeat of 160 base pairs at the ends of the molecule. In order to replicate DNA near the 5' terminus, RNA primer molecules have to be removed before replication is complete. There is thus an unreplicated portion of the T7 DNA at the 5' terminus of each strand. The opposite single 3' strands on two separate DNA molecules, being complementary, can pair with these 5' strands, forming a DNA molecule twice as long as the original T7 DNA. The unreplicated portions of this end-toend bimolecular structure are then completed through the action of

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MICROBIOLOGY OF VIRUSES

DNA polymerase and DNA ligase, resulting in a linear bimolecule, called a concatamer. Continued replication can lead to concatamers of considerable length, but ultimately a cutting enzyme slices each concatamer at a specific site, resulting in the formation of virussized linear molecules with repetitious ends. We thus see that 17 has a much more complex replication scheme than that seen for the other bacterial viruses discussed earlier.

5.13 LARGE DOUBLE-STRANDED DNA BACTERIOPHAGES One of the most extensively -studied groups of DNA viruses is the group sometimes called the T-even phages, which include the phages T2, T4, and T6. These phages are among the most complicated in terms of both structure and manner of multiplication replication. In the present section, we will discuss primarily bacteriophage T4, the phage of this group for which the most information is available. The virus particle of phage T4 is structurally complex. It consists of an icosahedral head which is elongated by the addition of one or two extra bands of protein hexamers, the overall dimensions of the .

N~

~\~C~~Ylation--HOH,c6o I

H

Figure 1.18 : The unique base in the DNA of the T-even bacteriophages, 5-hydroxymethylcytosine. The site of glucosylation is shown.

head being 85 x 110 nm. To this head is attached a complex tail consisting of a helical tube (25 x 110 nm) to which are connected a sheath, a connecting "neck" with "collar" and "whiskers," and a complex base plate with pins, to which are attached long jointed tail fibers. All together, the virus particle has over 25 distinct types of proteins. As we noted, the DNA of T4 has a total length about 650 times longer than the dimension of the head. This means that the DNA is highly folded and packed very tightly within the head. The genome structure of T4 is quite complex. The DNA is large, with a molecular weight of about 120 x 10, and is chemically distinct from cell DNA, having a unique base, 5-hydroxymethylcytosine instead of cytosine. The hydroxyl groups of the 5-hydroxymethylcytosine are

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MICROBIOLOGY AND BIOCHEMISTRY

modified by addition of glucosyl residues. This glucosylated DNA is resistant to virtually all restriction endonucleases of the host. Thus, this virus-specific DNA modification plays an important role in the ability of the virus to attack a host cell. Over 160 separate genes have been recognized in T4, of which the functions of 120 are known. These genes code not only for the complex array of coat proteins, but for a variety of enzymes and other proteins involved in the replication process itself.

Figure 5.19 : Simplified genetic map of T4. Late genes with morphogenetic functions (coat proteins and assembly), and genes with functions in DNA replication are identified. Note that although the genetic map is represented as a circle, the DNA itself is actually linear.

The genetic map of T4 is generally represented as a circle, even though the DNA itself is linear. This "genetic circularity" arises because the DNA of the phage exhibits a phenomenon called circular permutation. This arises because in different T4 phage particles, the sequence of bases at each end differs (although for a given molecule the same base sequence occurs at both ends). This structure, a consequence of the way the T4 DNA replicates (see later), results in

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145

an appearance of genetic circularity even though the DNA itself is lineat. mRNA synthesis and regulation in bacteriophage T4 In bacteriophage T4, the details of regulation of replication are more complex than those of T7, but involve primarily positive control. T4 is a much larger phage .than T7 and has many more genes and phage functions. In addition, the DNA of T4 contains the unusual base, 5hydroxymethylcytosine and some of the OH groups of this base are glucosylated. Thus enzymes for the synthesis of this unusual base and for its glucosylation must be formed after phage infection, as well as formation of an enzyme that breaks down the normal DNA precursor deoxycytidine triphosphate. In addition, T4 codes for a number of enzymes that have functions similar to those host enzymes in DNA replication, but are formed in larger amounts, thus permitting faster synthesis of T4-specific DNA. In all, T4 codes for over 20 new proteins that are synthesized early after in fection. It also codes for the synthesis of several new tRNAs, whose function is presumably to read more efficiently T4 mRNA. Overall, the T4 genes can be divided into three groups, for early, middle, and late proteins. The early proteins are the enzymes involved in DNA replication. The middle proteins are also involved in DNA replication. For instance, a DNA unwinding protein (DNA gyrase) is formed which destabilizes the DNA double helix, forming short single-stranded regions at which DNA synthesis can be initiated. The late proteins are the head and tail proteins and the enzymes involved in liberating the mature phage particles from the cell. In T4, there is no evidence for a new phage-specific RNA polymerase, as in T7. The control of T4 mRNA synthesis involves the production of proteins that modify the specificity of the host RNA polymerase so that ·it recognizes different phage promoters. The early promoter, present at the beginning of the T4 genome, is read directly by the host RNA polymerase, and involves the function of host sigma factor. Host RNA polymerase m~ves down the chain until it reaches a stop signal. One of the early proteins blocks host sigma factor action. The early protein combines with the RNA polymerase core enzyme, and when this protein builds up, initiation of early phage genes is stopped. The RNA polymerase cores are now available to combine with new phagespecific activators, which control the transcription of the middle and late genes. The middle genes are generally transcribed along the same DNA strand as the early genes, but the late genes are transcribed along the opposite strand.

MICROBIOLOGY AND BIOCHEMISTRY

146

head host

-==

! ®

I

tail endplate IiiiiJ

l l !

®

Iiiiil

head precursor

~

IiiiiI

endplate joined to core

DMA packaged into head

j

~ t t

l

-1 ~l·':· 1~ -1 j~ sheath protein added

1

~.=~ head and tail joined

stabilized tail

tail fibers added complete infective particle

fr,'.

tsJrj" host

\~

~ ::\~\~\..J L

-I-i-

Figure 5.20 : Steps in the assembly of a T4 bacterial virus.

MICROBIOLOGY OF VIRUSES

147

DNA replication The process of DNA replication in T4 is similar to that in TI, but in T4, the cutting enzyme which forms virus-sized fragments does not recognize specific locations on the long molecule, but rather cuts off head-full packages of DNA irrespective of the sequence. Thus each virus DNA molecule not only contains repetitious ends, but the nucleotide sequences at the ends of different molecules are different, although each molecule contains the complete sequence of viral genes. As shown, the cutting process results in the formation of DNA molecules with permuted sequences at the ends. Assembly and lysis In the case of T4, assembly of heads and tails occur on independent pathways. DNA is packaged into the assembled head and the tail and tail fibers are added subsequently. Somehow, the DNA is packed tightly and inserted into the empty phage head. Exit of the virus from the cell occurs as a result of cell lysis. The phage codes for a lytic enzyme, the T4 lysozyme, which causes an attack on the peptidoglycan of the host cell. The burst size of the virus (the average number of phage partides per cell) depends upon how rapidly lysis occurs. If lysis occurs early. then a smaller burst size occurs, whereas slower lysis leads to a higher burst size. The wild type phage exhibits the phenomenon of lysis inhibition, and therefore has a large burst si,?:e, but rapid lysis mutants, in which lysis occurs early, show smaller burst sizes.

5.14 TEMPERATE BACTERIAL VIRUSES: LYSOGENY Most of the bacterial viruses described above are called virulent viruses, since they usually kill the cells they infect. However, many other viruses, although also often able to kill cells, frequently have more subtle effects. Such viruses are called temperate. Their genetic material can become integrated into the host genome and is thus duplicated along with the host material at the time of cell division, being passed from one generation of bacteria to the next. Under certain conditions these bacteria can spontaneously produce virions of the temperate virus, which can be detected by their ability to infect a closely related strain of bacteria. Such bacteria, which appear uninfected but have the hereditary ability to produce phage, are called lysogenic. With most temperate phages, if the host simply makes a copy of the viral DNA, lysis does not occur; but if complete virion pruticles are produced, then the host cell lyses. In a lysogenic bacterial culture at anyone time, a small fraction of the cells, 0.1 to 0.0001 percent,

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MICROBIOLOGY AND BIOCHEMISTRY

produce virus and lyse, while the majority of the cells do not produce virus and do not lyse. Although only rarely do cells of a lysogenic strain actually produce virus, every cell has the potentiality. Lysogeny can thus be considered a genetic trait of a bacterial strain. The temperate virus does not exist in its mature, infectious state inside the cell, but rather in a latent form, called the provirus or prophage state. In considering virulent viruses we learned tha~ the DNA of the virulent virus contains information for the synthesis of a number of enzymes and other proteins essential to virus reproduction. The prophage of the temperate virus carries similar information, but in the lysogenic cell this information remains dormant because the expression of the virus genes is blocked through the action of a specific repressor coded for by the virus. As a result of a genetic switch, the repressor is inactivated, virus reproduction occurs, the cell lyses, and virus particles are released. A lysogenic culture can be treated so that most or all of the cells produce virus and lyse. Such treatment, called induction, usually involves the use of agents such as ultraviolet radiation, nitrogen mustards, or X rays, known to damage DNA and activate the SOS system. However, not all prophages are inducible; in some temperate viruses, prophage expression occurs only by natural events. Although a lysogenic bacterium may be susceptible to infection by other viruses, it cannot be infected by virus particles of the type for which it is lysogenic. This immunity, which is characteristic of lysogenized cells, is conferred by the intracellular repression mechanism under the control of virus genes. It is sometimes possible to eliminate the lysogenic virus (to "cure" the strain) by heavy irradiation or treatment with nitrogen mustards. Among the few survivors may be some cells that have been cured. Presumably the treatment causes the prophage to detach from the host chromosome and be lost during subsequent cell growth. Such a cured strain is no longer immune to the virus and can serve as a suitable host for study of virus replication. How is it possible to determine whether a strain is lysogenic? A sensitive host is necessary-that is, a strain closely related to the presumed lysogenic strain but not infected with the prophage. In practice, a large number of related strains are obtained, either from nature or from culture collections. The presumed lysogenic strain is cultured in a suitable medium where it grows, infrequently releasing phage. The titer of phage particles in an induced lysogenic culture

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MICROBIOLOGY OF VIRUSES

~

~altachment \ cell (host)

Injection

lytic infection

~I!I ~. ~

!

#\

\ : ~ '" Q ~.J coat proteIns synthesized; virus particles assembled

lysogenic cycle

viral DNA rephcates

lysogenIC induction

normal cell growth

Figure 5.21 : Consequences of infection by a temperate bacteriophage. The alternatives upon infection are integration of the virus DNA into the host DNA (lysogenization) or replication and release of mature VIruS (lysis). The lysogenic cell can also be induced to produce mature virus and lyse.

is typically lO'-lO$/ml. Irradiation can be used to attempt to induce the prophage to replicate. After further incubation, the culture is filtered to remove live bacteria, and the filtrate is tested against the various test strains using the agar overlay technique described for use in assaying virus particles. If plaques are seen, it can be assumed that virus particles are present and that the strain is lysogenic. Sometimes a large number of strains must be tested to fmd a sensitive host. Most bacteria isolated from nature are lysogenic for one or more viruses. Consequences of temperate virus infection What happens when a temperate virus infects a nonlysogenic organism? The virus may

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MICROBIOLOGY AND BIOCHEMISTRY

inject its DNA and initiate a reproductive cycle similar to that described for virulent viruses, with the infected cell lysing and releasing more virus particles. Alternatively, when the virus injects its DNA, lysogenization may occur instead: the viral DNA becomes incorporated into the bacterial genetic material and the host bacterium is converted into a lysogenic bacterium. In lysogenization the infected cell thus becomes genetically changed. Sensitive cells can undergo either lysis or lysogenization; which of these occurs is often determined by the action of a complex repression system, as will be described below. We thus see that the temperate virus can have a dual existence. Under one set of conditions, it is an independent entity able to control its own replication, but when its DNA is integrated into the host genetic material, replication is then under the control of the host.

\(4'\ I": ~

"': ~ .:,

~.:~;,;-?r~ ~W1~I~ ~ ~\:;:.~ :~ :N~:'?:f;~;'·" : .•

Figure 5.22 : Electron micrograph by negative staining of a lambda bacteriophage . particle.

Regulation of lambda reproduction One of the best studied temperate phages is lambda, which infects E. coli, and our knowledge of the molecular mechanisms involved in lysogenization and lytic processes in this phage is very adQnced. Morphologically, lambda particles iook like those of many other bacteriophages. The virus particle has an icosahedral head 64 nm in diameter, and a tail 150 nm which has helical symmetry. Attached to the tail is a single 23 nm long fiber. In addition to the major proteins of the coat, there are a number of minor coat proteins. The nucleic acid of lambda consists of a linear double-stranded

MICROBIOLOGY OF VIRUSES

151

molecule of 31 x 1()6 molecular weight. At the 5' terminus of each of the single-strands is a single-stranded tail of 12 nucleotides in length which are complementary (the ends of the DNA are'said to be cohesive). Thus, when the two ends of the DNA are free in the host cell, they associate and form a double-stranded circle. In the circular form the DNA contains 48,502 base pairs, and its complete sequence is known. Lysis or lysogenization? If lysogeny occurs, then the phage genes are maintained stably in the lysogenic state until a switch occurs and they are converted with high efficiency into a second state in which lytic growth occurs. This process is called lysogenic induction. How does the genetic switch from lysogeny to lysis occur? The lambda genome has two sets of genes, one controlling lytic growth, the other lysogenic growth. Upon infection, genes promoting both lytic growth and lysogenic integration are expressed. Which pathway succeeds is determined by the competing action of these early gene products and by the influence of host factors. To understand how this works, we need to present the genetic map of lambda. The genetic map, although actually linear, can thus be oriented as a circle (because of the cyclization via the cohesive ends mentioned above). The lambda map consists of several operons, each of which controls a set of related functions. Some of the phage genes are transcribed by RNA polymerase from one strand of the double helix, and others are transcribed from the other strand. Upon injection, transcription of the phage genes which code for the lambda repressor occurs, and if repressor builds up before lytic functions are expressed, lytic reproduction is blocked. The repressor protein blocks the transcription of all later lambda genes, thus preventing expression of the genes involved in the lytic cycle. In a lysogenic cell, a single phage gene is expressed continuously, the gene which codes for the lambda repressor protein. This repressor protein, which is coded for by a gene called cI, binds to two operators on the lambda DNA and thereby turns off the transcription of all the other genes of the phage genome. This is the negative control function of the lambda repressor. In addition, the lambda repressor turns on its own synthesis. This is the positive control function of the lambda repressor. Thus, by promoting its own synthesis, the lambda repressor ensures that no other genes except the gene coding for itself is made. In a lysogenic cell, there will usually be only one copy of the lambda genome, but about 100 active molecules of

152

MICROBIOLOGY AND BIOCHEMISTRY "

repressor protein. Therefore, there is almost always excess repressor to bind to lambda DNA and prevent the transcription of the genes necessary for lambda growth and reproduction.

L1

~

§ E

.5

5'G VIrion

i

3'

DNA ends

Figure 5.23 : Genetic and molecular map of lambda.

Lytic growth of lambda How, then, does lambda virus multiplication occur? In a lysogenic cell, multiplication of lambda occurs only after the repressor is inactivated. As we have noted, agents which induce lysogenic cells to produce phage are agents which damage DNA, such as ultraviolet irradiation, X rays, or DNAdamaging chemicals such as the nitrogen mustards. Upon DNA damage, a host defense mechanism called the SOS response is brought into play. An array of 10-20 bacterial genes is turned on, some of which help the bacterium survive radiation. However, one result of DNA damage is that a bacterial protein called RecA (normally involved in genetic recombination) is turned into a special kind of protease which participates in the destruction of the lambda repressor. With lambda repressor destroyed, the inhibition of expression of

MICROBIOLOGY OF VIRUSES

153

lambda lytic genes is abolished. We should note that the protease activity of RecA, brought about by DNA damage, nonnally plays an important role in the cell's response to DNA damaging agents, by participating in the breakdown of a host protein, LexA, which represses a set of host genes involved in DNA repair. Induction of bacteriophage lambda is thus an indirect consequence of the SOS response. Once the lambda repressor has been inactivated, the positive and negative control exerted by this repressor are abolished, and new transcriptional events can be initiated. The most important transcriptional event is that involved in the synthesis of another lambda protein called Cro. coded by a gene called ero. The gene ero is located almost adjacent to the gene cl which codes for lambda repressor. The key to the genetic switch lies in the close proximity of the regulator genes for repressor and cro protein. These two genes are transcribed in opposite directions, beginning at different start points. DNA damage

~ 1I I I IiI .-~

____

_

host genome

activation of SOS response

!

activation of host RecA protein; conversion of RecA protein to a protease

protease cleavage of lambda repressor protein (cl)

lytic response Figure 5.24 : Activation of the host SOS response leads to lysis of a lysogenic cell.

In the region separating these two genes are two kinds of sites, promoters and operators, to which each of the proteins of the switch can bind. When lambda repressor is bound to its operator, it covers

MICROBIOLOGY AND BIOCHEMISTRY

154

leftward transcription -

rightward transcription

C~RM r-,--O-R----.I PRcro~

Figure 5.25 : Two back-to-back promoters in the region of cI and cro control the genetic switch. When cI is present, it activates its own synthesis and blocks transcription of cro. When cI is inactivated, transcription of cro can occur, resulting in the lytic cycle. The cI (repressor) protein combines with the operator, OR'

the ero promoter, whereas when Cro is bound, it covers one of the cl promoters. As we have noted, the direction in which transcription occurs on a DNA double-strand (and hence which of the two strands is read) depends upon the promoter. A promoter essentially points the RNA polymerase in the proper direction. In the case of lambda, the cl promoter points RNA polymerase "leftward," whereas ero promoter points the polymerase "rightward." The lambda system provides one of the best studied examples of a genetic switch, in which one or the other of two competing genetic functions occurs. Which of the two genetic functions gets the upper hand will depend initially on chance events, but once one of the two functions has become established, it prevents the action of the other function. Only under unusual circumstances, such as when induction occurs, would the dominant genetic function be superseded. Integration Integration of lambda DNA into the host chromosome occurs at a unique site on the E. coli genome. Integration occurs by insertion of the virus DNA into the host genome (thus effectively lengthening the host genome by the length of the virus DNA). The cohesive ends of the linear lambda molecule find each other and form a circle, and it is this circular DNA which becomes integrated into the host genome. To establish lysogeny, genes cl and int must be expressed. As we have noted, the cI gene product is a protein which represses early transcription and thus shuts off transcription of all later genes. The integration process requires the product of the int gene, which is a site-specific topoisomerase catalyzing recombination of the phage and bacterial attachment sites. During cell growth, the lambda repression system prevents the expression of the integrated lambda genes except for the gene c/, which codes for the lambda repressor. During host DNA replication, the integrated lambda DNA is replicated along with the rest of the host genome, and transmitted to progeny cells. When release from repression occurs, the lambda productive cycle occurs. '

MICROBIOLOGY OF VIRUSES

155 m'

att

m

lambda DNA

l

m'

cyclizes at cohesive ends

m.m'

l

m'

--- ---

site-specific nuclease creates staggered ends of . / phage and host gal phr . / bio ch/A ---·----==~::::::JI _~====-

":=::==.=-,==

I t

gal

phr

,==

-_:::::::.:.-_-=----------

integration of lambda DNA and closing of gaps m' by DNA ligase

m

i

bio

ch/A

i

Figure 5.26 : Integration of lambda DNA into the host. Integration always occurs at a specific site on the host DNA, involving a specific attachment site (att) on the phage. Some of the host genes near the attachment site are given. A sitespecific enzyme (integrase) is involved, and specific pairing of the complementary ends results in integration of phage DNA.

Replication Replication of lambda DNA occurs in two distinct fashions during different parts of the phage production cycle. Initially, liberation of lambda DNA from the host results in replication of a circular DNA, but subsequently linear concatamers are formed, which replicate in a different way. Replication is initiated at a site close to gene 0 and from there proceeds in opposite directions (bidirectional

156

MICROBIOLOGY AND BIOCHEMISTRY

symmetrical replication), tenninating when the two replication forks meet. In the second stage, generation of long linear concatamers occurs, and replication occurs in an asymmetric way by rolling circle replication. In this mechanism, replication proceeds in one direction only, and can result in very long chains of replicated DNA. This mechanism is efficient in permitting extensive, rapid, relatively uncontrolled DNA replication; thus it is of value in the later stages of the phage replication cycle when large amounts of DNA are needed to form mature virions. The long concatamers formed are then cut into virus-sized lengths by a DNA cutting enzyme. In the case of lambda, the cutting enzyme makes staggered breaks at specific sites on the two strands, twelve nucleotides apart, which provide the cohesive ends involved in the cyclization process. Lambda is one of the agents of choice for use as a cloning vector for artificial construction of DNA hybrids with restriction enzymes. It has several features that make it an excellent system for genetic engineering. One feature of lambda that makes it of special use for cloning is that there is a long region of DNA, between genes Jand au, which does not seem to have any essential functions for replication, and can be replaced with foreign DNA. Temperate viruses as plasmids Another class of temperate viruses have quite a different mechanism for maintenance of the prophage state. In this group, viruses resemble plasmids. They do not actually become integrated into the host chromosome, but instead replicate in the cytoplasm as circular DNA molecules. Among such viruses is bacteriophage PI of Escherichia coli. Although in broad features, such viruses resemble the temperate viruses such as lambda just discussed, at the level of virus replication they are, of course, quite different. Interestingly, although the plasmid prophage is not physically connected to the host DNA, phage DNA replication is closely coordinated with cell division, since only one copy of the prophage is present per host chromosome.' The phage repressor is somehow involved in this regulation process.

5.15 A TRANSPOSABLE PHAGE: BACTERIOPHAGE MU One of the more interesting bacteriophages is that called Mu, which has the unusual property of replicating as a transposable element. This phage is called Mu because it is a mutator phage, inducing mutations in a host into which it becomes integrated. This mutagenic property of Mu arises because the genome of the virus

MICROBIOLOGY OF VIRUSES

157

can become inserted into the middle of host genes, causing these genes to become inactive (and hence the host which has become infected with Mu behaves as a mutant). Mu is a useful phage because it can be used to generate a wide variety of mutants very easily. Mu can be used in genetic engineering. A transposable element is a piece of DNA which has the ability to move from one site to another as a discrete element. Transposons are found in both procaryotes and eucaryotes, and play important roles in genetic variation. There are three types of transposable elements: insenion elements, transposons, and viruses like Mu. (Also the retroviruses discussed later, are a group of animal viruses which have features of transposable elements). An insenion element is the simplest type of transposable element: it carries no detectable genes and simply moves itself around. A transposon is a genetic element with a unique piece of DNA, usually coding for one or more proteins, to which is attached, at each end, an insertion element. The insertion element at each end of the transposon is identical, although the DNA sequence may be either a direct repeat or an inverted repeat. Mu is a very large transposable element, carrying insertion elements and a number of Mu genes involved in Mu multiplication. Structurally, bacteriophage Mu is a large doublestranded DNA virus, with an icosahedral head, a helical tail, and 6 tail fibers. The DNA of Mu is arranged inside the virus head as a linear doublestranded molecule. It has a molecular weight of 25 X 106 (3839 kilobases). The genetic map of Mu is shown in Figure 6.37a. It can be seen that the bulk of the genetic information is involved in the synthesis of the head and tail proteins, but that important genes at each end are involved in replication and immunity. At the left end of the Mu DNA are 50-150 base pairs of host DNA and at the right end are 500-3000 base pairs of host DNA. These host DNA sequences are not unique and represent DNA adjacent to the location where Mu had become inserted into the host genome. When a Mu phage particle is formed, a length of DNA containing the Mu genome just large enough to fill the phage head is cut out of the host, beginning at the left end. The DNA is rolled in until the head is full but the place at the right end where the DNA is cut varies from one phage particle to another. For that reason, as shown on the genetic map, there is a variable sequence of host DNA at the righthand end of the phage (right of the attR site) which represents the host DNA that has become packaged into the phage head. Each phage

158

MICROBIOLOGY AND BIOCHEMISTRY

particle arising from a single infected cell will have a different amount of host DNA, and the host DNA base sequence of each particle from the same cell will be different. In some cases, completely empty Mu heads become filled with purely host DNA. Such particles can transfer host genes from one cell to another, a process called transduction. posrtive acbvat90r of late mANA synthesIS Immunity

invertible G segment (host range)

lysis

I

head and tail genes

Integration replication host DNA

,

lie

I

variable and

rL,

(host DNA)

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C

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(a)

l

I - - - - , I G C C G A A G C A G C G T T G~_ _ _--1 .CGGCTTCGTCGCAAC. L---I

1-----41

GC CGA

L -_ _ _~CGGCTTCGTC

I----,IG C C G A A G C A G CGGCTTCGTC

'-------'

•

1

L---I

A G C A G C G T T GI..._ _ _-I GCAAC

'-----I

A G C A G C G T T G~_ _ _--1 TCGTCGCAAC '-------' repair of DNA leads to formation of S-base·pair duplication

Figure 5.27 : Bacteriophage Mu. (a) Genetic map of Mu. (Confusingly, there are two G's, the G gene and the invertible G segment. These are different O's.) (b) Integration of Mu into the host DNA. showing the generation of a five-base-pair duplication of host DNA.

MICROBIOLOGY OF VIRUSES

159

A~ shown in the genetic map, a specific segment of the Mu genome called G (distinct from the G gene) is invertible, being present in either the orientation designated SU, or in the inverted orientation U'S'. The orientation of this segment determines the kind of tail fibers that are made for the phage. Since adsorption to the host cell is controlled by the specificity of the tail fibers, the host range of Mu is determined by which orientation of this invertible segment is present in the phage. If the G segment is in the orientation designated G+· then the phage particle will infect Escherichia coli strain K12. If the G segment is in the G- orientation, then the phage particle will infect Escherichia coli strain C or several other species of enteric bacteria. The two tail fiber proteins are coded on opposite strands within this small G segment. Left of the G segment is a promoter that directs transcription into the G segment. In the orientation G +, the promoter directing transcription of S and U is active, whereas in the orientation G-, a different promoter directs transcription of genes S' and U' on the opposite strand. Upon infection of a host cell by Mu, the DNA is injected. In contrast with lambda, integration into the host genome of Mu is essential for both lytic and lysogenic growth. Integration requires the activity of the A gene product, which is a transposase enzyme. At the site where the Mu DNA becomes integrated, a 5 base pair duplication of the host DNA arises at the target site. This host DNA duplication arises because staggered cuts are made in the host DNA at the point Mu becomes inserted, and the resulting single-stranded segments are converted into the double-stranded form as part of the integration process. Lytic growth of Mu can occur either upon initial infection, if the c gene repressor is not formed, or by induction of a lysogen. In either case, replication of Mu DNA involves repeated transposition of Mu to multiple sites on the host genome. Initially, transcription of only the early genes of Mu occurs, but after gene C protein, a positive activator of late RNA synthesis, is expressed, the synthesis of the Mu head and tail proteins occurs. Eventually, expression of the lytic function occurs and mature phage particles are released. Because Mu integrates at a wide variety of host sites, it can be used to induce mutants at many locations. Also, Mu can be used to carry into the ceil genes that have been derived from other host cells, a form of in vivo genetic engineering. In addition, modified Mu phage have been made artificially in which some of the harmful functions

160

MICROBIOLOGY AND BIOCHEMISTRY

of Mu have been deleted. These phages, called Mini-Mu, are deleted for significant portions of Mu but have the ends of the phage in - nonnal orientation. Mini-Mu phages are usually defective, unable to form plaques, and their presence must be ascertained by the presence of other genes which they carry. One set of Mini-Mu phages containing the [3-galactosidase gene of the host (called Mudlac, d for defective) can be detected in 'the integrated state if the lac gene is oriented properly in relation to a host promoter. Under these conditions, the hOst cell will form the enzyme [3-galactosidase, which can be detected in colonies by a special color indicator. f3galactosidase-positive colonies from a agalactosidase-negative host are thus an indication that Mud-lac infection has occurred. We thus see that phage Mu provides a useful tool for geneticists, as well as being an interesting bacteriophage in its own right.

5.16 GENERAL OVERVIEW OF ANIMAL VIRUSES We have discussed in a general way the nature of animal viruses in the first part of this chapter. Now we discuss in some detail the structure and molecular biology of a number of important animal viruses. Viruses will be discussed which illustrate different ways of replicating, and both RNA and DNA viruses will be covered. One group of animal viruses, those called the retroviruses, have both an RNA and a DNA phase of replication. Retroviruses are especially interesting not only because of their unusual mode of replication, but because retroviruses cause such important diseases as certain cancers and acquired immunodeficiency syndrome (AIDS). Before begilming our discussion of the manner of replication of animal viruses, we should mention first the important differences which exist between animal and bacterial cells. Since virus replication makes use of the biosynthetic machinery of the host, these differences i1:t cellular organization and function imply differences in the way the viruses themselves replicate. Bacteria, being procaryotic, do not show compartmentation of the biosynthetic processes. The genome of a bacterium relates directly to the cytoplasm of the cell. Transcription into mRNA can lead directly to translation, and the processes of transcription and translation are not carried out in separate organelles. Animal cells, being eucaryotic, show compartmentation of the transcription and translation processes. Transcription of the genome into mRNA occurs in the nucleus, whereas translation occurs in the cytoplasm. The messenger RNA in the eucaryote is usually modified by adding to it

MICROBIOLOGY OF VIRUSES

promoters

161

B

c

~A t:.::::~p:.;::L_ _ _..lI_ _ _ _

....L_ _ _ _..........1 bacterial

genome

cistrons (genes)l

operator

A

c

B

polycistronic mRNA

formation of polyribosome and direct translation into protein (a) procaryote intron

"

L-_ _ _...J®t..::...;:"a._ _ _ _... ~;;:.~.;:",;: ......_ _ _~1

eucaryotic genome

(~----+::"::'+-----+::"::'~~----J) f§SJ ,

primary RNA transcript

,, ,,

~ ,

I , I , I ,I

,

l l

I I I I I I , I

I

I

I

I

k "~-....1...------L..------4(

,

poly AI tail

J ---------

5'cap

• •

RNA processing removes introns

capping and polyadenylation nuclear membrane transfer from nucleus to cytoplasm

monocistronic mRNA translated

(b) eucaryote

Figure 5.28 : Comparison of protein synthesis processes in procaryote and eucaryote. (a) Procaryote. (b) Eucaryote.

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MICROBIOLOGY AND 6IOCHEMISTRY

a poly (A) tail of 100 to 200 adenylic residues at the 3' end and a methylated guanosine triphosphate, called the cap .. at the 5' end. The cap is required for binding of the mRNA to the ribosome and the poly A tail may be involved in subsequent RNA processing and transfer of the mature mR:NA 'from the nucleus to the cytoplasm. All of the protein-synthesizfqg,machinery of the eucaryotic cell, the ribosomes, tR"NA molecules, and accessory components, is in the cytoplasm, "~nd the mature mRNA associates with the proteinsynthesizing apparatus once it leaves the nucleus. The genes of eucaryotes are often split, with noncoding regions called introns separating coding regions (exons). TraPscription of both the coding and noncoding regions of a gene occurs and an RNA, called the primary RNA transcript, is formed and is subsequently converted into the mature mRNA by a mechanism called RNA processing, in which the noncoding regions are excised (the cap and tail remain after RNA processing is complete). After processing, the mature mRNA is translated into protein. One important distinction between eucaryotic and procaryotic mRNA is that procaryotic mRNA is generally polycistronic, with more than one coding region present in a single mRNA molecule, whereas eucaryotic mRNA· is monocistronic. In procaryotes, during the translation process the ribosomal machinery moves down the mRNA past a stop site and initiates translation of another gene without ever leaving the mRNA. A-lthough eucaryotic mRNA is usually monocistronic, this does not mean that only a single type of protein molecule results from the translation of a eucaryotic mRNA. Frequently, the eucaryotic mRNA codes for a single, large multifunctional protein, called a poly-protein, which may subsequently be cleaved by a specific protease into several distinct enzymes. In other cases, the polyprotein may remain as a single multifunctional polypeptide. We might also note another important difference between animal and bacterial cells. Bacterial cells have rigid cell walls containing peptidoglycan and associated substances. Animal cells, on the other hand, lack cell walls. This difference is important for the way by which the v:rus genome enters and exits the cell. In bacteria, the protein coat of the virus remains on the outside of the cell and only the nucleic acid enters. In animal viruses, on the other hand, uptake of the virus often occurs by endocytosis (pinocytosis or phagocytosis), processes which are characteristic of animal cells, so that the whole virus particle enters the cell. The separation of animal virus genomes from their protein coats then occurs inside the cell.

MICROBIOLOGY OF VIRUSES

163

Classification of animal viruses Most of the animal viruses which have been studied in any detail have been those which have been amenable to cultivation in cell cultures. As seen, animal viruses are known with either single-stranded or doublesttanded DNA or RNA. Some animal viruses are enveloped, others are naked. Size varies greatly, from those large enough to be just visible in the light microscope, to those so tiny that they are hard to see well even in the electron microscope. In the following sections, we will discuss characteristics and manner of multiplication of some of the mqst important and best-studied animal viruses.

@) ~

~

(al

whole virus particle (en\I8Ioped)

uptake into animal cell

by e~OSls

and loss of

.

f:i;\

~

viral envelope nucleocapsid

-

uncoating of capsid

/

processes of

............ virus multiplication

virus

nucleic acid

(bl

Figure 5.29 : Uptake of an enveloped virus particle by an animal cell. (a) The process by which the viral nucleocapsid is separated from its envelope. (b) Electron micrograph of adenovirus particles entering a cell. Each particle is about 70 om in diameter.

Consequences of virus infection in animal cells Viruses can have varied effects on cells. Lytic infection results in the destruction of the host cell. However, there are several other possible effects following viral infectioA of animal cells. In the case of enveloped viruses, release of the viral particles, which occurs by a kind of h¢dingprocess, may be s10w and the host cell may not be lysed. The cell may remain alive and continue (0 produce virus over a long /period of time. Such infections are referred to as persistent infections.

164

MICROBIOLOGY AND BIOCHEMISTRY

Viruses may also cause latent infection of a host. In a latent infection, there is a delay between infection by the virus and the appearance of symptoms. Fever blisters (cold sores), caused by the herpes simplex virus, result from a latent viral infection; the symptoms reappear sporadically as the virus emerges from latency. The latent stage in viral infection of an animal cell is generally not due to the integration of the viral genome into the genome of the animal cell, as is the case with latent infections by temperate bacteriophages. Viruses and cancer A number of animal viruses have the potential to change a cell from a normal cell to a cancer cell. This process, called transformation, can be induced by infections of animal cells with certain kinds of viruses. One of the key differences between normal cells and cancer cells is that the latter have different requirements for growth factors . Rapidly growing cells pile up into accumulations that are visible in culture as foci of infection. Because cancerous cells in the animal body have fewer growth requirements, they grow profusely, leading to the formation of large masses of cells, called tumors. The term neoplasm is often used in the medical literature to describe malignant tumors. Not all tumors are seriously harmful. The body is able to wall off some tumors so that they do not spread; such noninvasive tumors are said to be benign. Other tumors, called malignant, invade the body and destroy normal body tissues and organs. In advanced stages of cancer, malignant tumors may develop the ability to spread to other parts of the body and initiate new tumors, a process called metastasis. How does a normal cell become cancerous? The process can be broken down into several stages. In the first step, initiation, genetic changes in the cell occur. This step may be induced by certain chemicals, called carcinogens, or by physical stimuli, . such as ultraviolet radiation or X rays. Certain viruses also bring about the genetic change that results in initiation of tumor formation. Once initiation has occurred, the potentially cancerous cell may remain dormant, but under certain conditions, generally involving some environmenta1 alteration, the cell may become converted into a tumor cell, a process called promotion. Once a cell has been promoted to the cancerous condition, continued cell division can result in the formation of a tumor. Although the ability of viruses to cause tumors in animals has been proved for many years, the relationship of viruses to cancer in

MICROBIOLOGY OF VIRUSES nonenveoped

165 enveloped

o

ssDNA

paNOllirus

~

dsDNA

papovavirus

dsDNA

poxvirus

adenovirus

dsDNA herpesvirus

iridovirus ~

(a) DNA viruses

-

-o

100nm

ssANA

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.... ANA

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100rwn

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ANA_

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•

coronaYOrus

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Figure 5.30 : The shapes and relative sizes of vertebrate viruses of the major taxonomic 100 DDl. groups. Bar

=

MICROBIOLOGY AND BIOCHEMISTRY

166

,' . ' . .•.

transfonnatlon

.

adsorptiOn

' .

_r/'.'-",)

. .

penetration

.,

transformatJon

. " of normal celts ."~,' 10 turnor cells

_(!).". _®-'. _.

Vi~

.

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,nlO tumor c e U /

cell

$

.(; :::=ncell~ @ \

...

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death of cell ~-. _ ._ and releasa __

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.' (i) ---""."'" of virus

.-.:.:~, .persistent infection

slow releasa ',' '~'-""::':. of virus _

without cell death

-

tih -~ ~rnection \iJ!!!B ~ v'rus present but nol caUSing harm 10 cell: later emerges in lytic infection

Figure 5.31 : Possible effects that animal viruses may have on ceIls they infect.

humans has, in most cases, been uncertain. It is difficult to prove the viral origin of a human cancer because of the difficulties of carrying out the necessary experimentation. However, it is now well established that certain specific kinds of human tumors do have a viral origin. A summary of some of the human cancers with definite viral origins is given in Table. TABLE 5.1 : SOME HUMAN CANCERS WmCH MAY BE CAUSED BY VIRUSES Cancer Adult T-cell leukemia (type 1) Burkitt's lymphoma Nasopharyngeal carcinoma Hepatocellular carcinoma (liver cancer)

Family

Genome

Retrovirus

RNA

Epstein-Barr virus

Herpes

DNA

Epstein-Barr virus

Herpes

DNA

Hepatitis B virus

Not yet classified

DNA

Virus

Human T-ceU leukemia virus

MICROBIOLOGY OF VIRUSES

167 Table Contd.

Cancer Cervical cancers (?) Skin and cervical cancers

Virus

Family

Genome

Herpes simplex type 2 virus (?)

Herpes

DNA

Papilloma virus

Papova

DNA

10oor-------------------------~

I"

100 number of infectious 10 virus particles per cell

,I

nucleic acid

,I

/

I

,/

/

/

,

hou'rs ....eclipse period-+- rise period---4 ..early period+--Iate period----i ........ period necessary to encapsidate viral genome Figure 5.32 : One-step growth curve of animal viruses.

Replication cycle of animal viruses One-step growth curves are also obtained for animal viruses. Such curves exhibit the same overall features that are found in the bacterial viruses, but the rate of multiplication is much slower. A typical one-step growth curve of an animal virus is given in Figure. This figure also presents data on the length of the eclipse period and the total duration of the multiplication cycle for several well-studied animal viruses. Note that the multiplication cycles of animal viruses range from around eight hours to as long as 48 hours.

6

General Metabolism 6.1

CHARACTERIZATION OF METABOLISM

The vital activity of any living organism is determined by the specific organization of biological structures, metabolic processes, energy metabolic processes, energy metabolism, genetic information transfer, and regulatory mechanism. Damage of any of these links develops a pathological process and a disease in the organism. An understanding of the molecular mechanisms involved in the vital activity or malfunction of the organism constitutes the basis for the search and clinical applications of biological medicinal preparations. In the overall metabolism of the living organism distinguished are: exogenous metabolism, which comprises extracellular transformations of materials on the way to their uptake and excretion by the cells, and intermediary metabolism, which occurs in the cells. Tht' intermediary metabolism is conceived as the sum total of chemical reactions that occur in the living cell. Functionally, metabolism encompasses the following major processes: (I) accumulation of energy from decomposition of compounds or supplied by light; (2) utilization of energy for synthesis of essential molecular components (monomers, macromolecules) and the performance of work (osmotic, electric, mechanical); (3) decomposition of renewable structural components of the cell; (4) synthesis and decomposition of specialized biological molecules (hormones, mediators, hormonoids, cofactors, etc.). The sequences of chemical reactions involved form metabolic pathWays, or cycles, each of these performing a defmite function. 168

GENERAL METABOLISM

169

Conventionally, central and special metabolic pathways are distinguished. Central pathways are common to the decomposition and synthesis of major macromolecules. Actually, they are much alike in all representatives of the living world. Special cycles are characteristic of the synthesis and decomposition of individual monomers, macromolecules, cofactors, etc. Special cycles are extremely diversified, especially in the plant kingdom. For this reason, the plant metabolism is conventionally classified into primary and secondary metabolisms. The primary metabolism includes the classical processes of synthesis and deeradation of major macromolecules (proteins, carbohydrates, lipids, nucleic acids, etc.), while the secondary metabolism ensuing from the primary one includes the conversions of special biomolecules (for example, alkaloids, terpenes, etc.) that perform regulatory or other functions, or simply are metabolic end byproducts. In the metabolism, two opp·ositely directed processes, or phases, are commonly distinguished: catabolism and anabolism. Catabolism is the sum of degradative processes leading to the cleavage of large molecules into smaller ones. Anabolism is the sum 'of metabolic processes leading to the synthesis of complex molecules from simpler ones. Catabolism is accompanied by a release of energy that can be stored as energy-rich ATP. Anabolic processes proceed through consumption of ATP and decomposition of the latter into ADP and H,P04. Therefore, ATP may be said to be a coupling energetic link between the two metabolic pathways. However, ATP is not the only linking component shared by catabolism and anabolism. Other simple metabolites are also formed by the catabolic pathway from macromolecules and monomers to be used as starting materials for the subsequent synthesis of monomers and macromolecules, i.e. in the process of anabolism. This linking pathway, or cycle, unifying degradative and synthetic routes, is called the amphibolic pathway. This signifies that the catabolic and anabolic pathways are coupled not only via the energetic ATP-ADP system, but also through their common metabolites, which renders the metabolism more versatile and economical. At need, simple intermediates can be utilized in the biosynthesis, without the necessity of their supply from the exterior. The amphibolic pathways are associated with the terminal, or ultimate, system of oxidation of the materials involved, as the latter are degraded to the end products, CO 2 and HP, with a release of a large amount of energy. Apart from them, urea and uric acid, which are produced by specific metabolic reactions of amino acids and nucleotides, are also end products of metabolism.

MICROBIOLOGY AND BIOCHEMISTRY

170

carbohydrat•• Proteins. Lipids, Polynucleotida

Uric acid

Urea

H 20

Figure 6.1 : Scheme for catabolic and anabolic pathways (shown is their interrelation through ATP-ADP system and amphibolic metabolite cycle)

During catabolic and anabolic processes, a renovation of the molecular cellular components takes place. It should be emphasized that the catabolic and anabolic pathways are independent of each other. Be these pathways coincident and differing in the cycle direction only, the metabolism would have been side-tracked to the so-called useless, or futile, cycles. Such cycles arise in pathology, where a useless turnover of metabolites may occur. To avoid this undesirable contingency, the synthetic and degradative routes in the cell are most commonly separated in space. For example, the oxidation of fatty acids occurs in the mitochondria, while the synthesis thereof proceeds extrarnitochondrially, in the microsomes.

6.2 ENERGY CYCLES IN ANIMATE NATURE Nutrients and energy are supplied to the living organisms from various sources. As far as the nutritional sources are concerned, living organisms are classified into two large groups, autotrophs (from the Greek autos, self, and trophos, food) capable of assimilating CO2 as a startiQg nutrient for the buildup of other carbon-containing materials,

GENERAL METABOLISM

171

and heterotrophs (heteros, other) which utilize diverse organic compounds as synthetized by other organisms. In a sense, the autotrophs are primary to the heterotrophs. Reduced organic compounds (for example, gIu cose) synthetized by the autotrophs from CO2 contain a larger amount of energy as compared with that in the consumed carbon dioxide. In regard to the energy sources, living organisms are divided into phototrophs, for which the sun light is. a source of energy, and chemotrophs, which utilize the energy of reduction-oxidation (redox) reactions. In redox reactions, the energy that has been acquired by the cell is released on transport of the electrons from a donor to an acceptor (or oxidant). In these processes, the donor and the acceptor act as partners and constitute a donor-acceptor pair, or a redox pair. If the redox pair is made up of organic compounds, the living organisms involved are called chemoorga 'notrophs, and if it consists of inorganic compounds, chemolithotrophs. They are further diversified by their relationship to oxygen as an electron acceptor. The organism cells that utilize oxygen are referred to as aerobic ones, those capable of dispensing with oxygen are called anaerobic. Most commonly, the cells of higher organisms and bacteria possess both types, anaerobic and aerobic, of energetics. For this reason, such cells and organisms are calledfacultative anaerobes, although the degree of faculty, or the dependence on oxygen supply, in them may vary. For example, higher organisms are incapable of subsisting without oxygen for longer periods of time. There are obligate anaerobes, in particular, microorganisms, which do not need oxygen altogether and even defy it as poisonous. The green plants possess a combined type of energetics, phototrophic and'chemoorganotrophic (respiratory and glycolytic), which enables them to absorb the energy of sun light at different periods of their development; in the absence of sun light, the plants make use of chemical energy. Microorganisms are especially remarkable for the diversity of energetics types and combinations thereof. The energetics types occurring in a number of representatives of the animate nature are listed in Table. Owing to the diversity of nutrition forms and energy consumption, the living organisms are in nature closely related. _The interrelation between nutrition and utilization of energy sources may be conceived of from the standpoint of specific cycles operative in the animate nature. Major participants of a global cycle are the Sun as a source of energy, autophototrophs capable of acquiring the solar energy and of synthetizing carbohydrates and other org~c materials from CO2 ,

.....

Table 6.1 Energetics Types Encountered in Living Organisms. Source of energy

Energetics type

Representatives Photosynthetic organs of higher plants, algae, bacteria Animal and bacterial .cells, nonpho

1.

Light

Phototrophic

2.

Redox reactions of chemical compounds (donor-acDonor: organic compounds; acceptor: 02 Donor and acceptor: organic compounds Donor: inorganic compounds; acceptor: 02 Donor and acceptor: inorganic compounds Donor: organic compounds; acceptor: of mixed type (organic compounds and 02) Combined type: light and redox reactions Light and redox reactions of organic compounds Light and redox reactions of inorganic compounds

ChemCltrophic tosynthetic plant cells ceptc r) Chemoolrganotrophic (aerobic} ChemooJrganotrophic (anaerobic) Chemol ithotrophic (aerobic:) Chemolithotrophic (anaerobic) Chemoorga otrophic (facultativ anaerobic)

3.

;j

Photochemotropl. .c

Idem Idem

~

Bacteria Idem CellS of higher animals; bacteria

Photosynthetic plant cells at differ ent (light and dark) phases

Photochemoorganotroph- Idem ie

("') ~

~

§ -< > ~

g= f§

~

C l)

Photochemolithotrophic

Photosynthetic bacteria

o-l

~

GENERAL METABOLISM

173

and animals which consume organic materials and oxygen generated by phototrophs. Energy losses associated with the vital activity of all organisms on the Earth are being compensated for by the energy of solar radiation. It should be noted that the cells of man and animals utilize highly reduced, i.e. hydrogen-containing, compounds (carbohydrates, lipids, proteins, etc.) as energetic materials. Hydrogen is an energetically valuable material. Its energy in a transformed form is stored in the ATP chemical bonds in the cells of heterotrophic organisms.

6.3 ENERGETICS OF BIOCHEMICAL REACTIONS To get a deeper insight into the metabolic and energetic processes, knowledge of general principles of chemical energetics appears to be of help. In the living cell, all chemical reactions contributing to the metabolism obey the laws of energetics. The first law of energy conservation states that the energy of a chemical reaction can be neither annihilated nor generated from nothing, it can merely be converted from one form into another. In terms of this law, it becomes possible to defme the energy balance of a chemical process. Spontaneous chemical processes can proceed only in one direction towards a state of equilibrium; the state of equilibrium having been reached, the process is brought to termination. The second law of thermodynamics (energetics) enables one to predict the direction of biochemical processes. According to this law, any spontaneous process takes the route corresponding to a maximum of entropy under the given conditions until an equilibrium state for the reaction is reached. Entropy conveys a measure of the disorder in a system. An increase in entropy in the course of a reaction prevents the return of the reaction to the initial state, since for that to occur a diminution in entropy would be required. A spontaneously disordered system is never capable of turning into an ordered one. Therefore, all reactions that proceed with a concomitant increase in entropy are irreversible. For their reversal, an additional energy is needed to be spent to compensate for the losses in entropy change, i.e. to bring the system from the disordered into the ordered state. However, from the pract~cal standpoint it appears expedient to use the so-called free energy which, in contrast to entropy, is amenable to measurement in the course of a reaction. The free energy defmes a portion of the total energy of a system that can be converted to work at pressure and temperature kept constant. The free energy is

174

MICROBIOLOGY AND BIOCHEMISTRY

denoted AG. The portion of total energy of a chemical process that cannot be converted to work at pressure and temperature kept constant is called the bound energy and is expressed as the product TAS, where T is the absolute temperature and AS is the entropy change of a system under the given chemical reaction conditions. The sum of the changes in free and bound energies is called enthalpy (internal energy of a system) or heat energy content of a system and is denoted OH. Enthalpy can be measured experimentally and is equal to the amount of heat released in the given process. The enthaply change is defined by the equation MI = /lG + T /l S Otherwise stated, /lG=MI - T /l S The energetic state of any system, including that of a cell and an organism, can be defmed in terms of this very important equation. The free energy is expressed in kilojoules per mole of substance, kJ/ mol. The free energy is a very convenient parameter for defining the spontaneity or nonspontaneity of a chemical process. For spontaneous processes, the free energy is seen to decrease, i.e. /lG has a negative value. Such reactions are called exergonic, i.e. proceeding with a release of energy. These reactions supply energy to the cells. The processes for which the free energy is increased (i.e. /lG has a positive value) cannot proceed spontaneously. They require energy supply from the outside. Such processes are referred to as endergonic, i.e. proceeding with an expenditure of energy. In a state of equilibrium, /lG = O. The free energy of chemical reactions may be estimated both under the standard conditions and under real, or physiological, conditions. The standard free energy, /lGo , of a biochemical reaction is defined as a free energy change under the standard conditions, i.e. at the concentration of reactants I mol/litre, temperature 25°C < 298 X), and pH 7. If water is involved either as a starting compound or as a reaction product, its concentration is taken equal to 1.0 mol/litre, although the true concentration of water in dilute aqueous solutions is close to 55 mol/litre. The standard free energy is found as a difference between the sum values of free energy for the end products and the initial reactants. The value of /l Gp for a biochemical reaction proceeding under the

175

GENERAL METABOLISM

physiological conditions is estimated with allowance for the actual concentrations of components involved. With reference to the free energy as a characteristic of metabolism one may say that catabolic reactions proceed with a release of energy and anabolic ones, with a consumption of energy' The anabolic reactions can proceed only as closely coupled to the catabolic reactions. High-energy, or macroergic,compounds act as energetic mediators between these two types of reactions.

6.4 HIGHT-ENEGRY AND LOW-ENERGY PHOSPHATES: GENERAL CONSIDERATIONS Commonly, all compounds are classified into high-energy and lowenergy ones. As a conventional borderline between these two classes, the free energy of about 20 kJ/mol for the phosphate bond hydrolysis has been taken. This value used for the purpose of characterization of biochemical processes should not be confused with the bond energy which is conceived as an energy required for disruption of the bond between two neighbouring atoms in any molecule. Cleavage of the phosphate bond results, alongside energy release, in the formation of inorganic phosphates. Energetic characteristics for some of the compounds of interest are listed in the following Table. TABLE 8.2 Standard Free Energies, AGo, for Hydrolysis of Some High-energy and Low-energy Compounds, and Free Energies for Hydrolysis of Compounds Under Physiological Conditions, AGp. Compound

- AGpI

kJ/mol

High-energy compounds Phosphoenol pyruvate l,3-Diphosphoglycerate Creatine phosphate ATP Acetyl-CoA

61.7 49.2 42.5 30.4 30.4

66.7 41.7 50.0 (Table 8.2 Contd.)

Compound

ADP Pyrophosphate (H4P20,)

_ AGO,

- AGpI

kJ/mol

kJ/mol

28.3 28.3

50.0 50.0

176

UDP-glucose Glucose I-phosphate Fructose 6-phosphate AMP

Glucose 6-phosphate a-Glycerol phosphate

MICROBIOLOGY AND BIOCHEMISTRY

24.2 20.8 Low-energy compounds 15.8 14.1 13.8

23.8

9.2

It shouij be emphasized that ATP, the key mediator in energy metabolism, is not the most energy-rich species. ATP is found in the middle of. energy scale. The most frequently occurring route is cleavage of the end phosphate from ATP: ATP ~ ADP + Hl04 (1) The end phosphate adds water and is transferred onto another compound, causing thereby the phosphorylation of the latter. An alternative route for the phosphate bond energy release is exemplified by pyrophosphate cleavage of ATP: ATP ~ AMP + H4 Pp? (2) This type of reaction is less frequent in biological processes. Its distinctive feature is the formation of pyrophosphate, which ranks among energy-rich materials. Hydrolysis of pyrophosphate (3) releases roughly as much energy as hydrolysis of the ATP end phosphate bonds. In biological processes, energy-rich pyrophosphate bonds are seldom used for synthesis of other compounds because of the heat energy released by pyrophosphate hydrolysis. ADP may be used as a high-energy reactant in biochemical processes. The cleavage of the ADP end phosphate bond (4) releases the same amount of energy as that generated by splitting the end phosphate bond in ATP. At first glance, it may appear that ATP can be adequately replaced by ADP in chemical reactions, in particular in the phosphoryla9 ion of other compounds. However, as evidenced by the available experimental data, such a possibility has never been realized in biological processes. At least such reactions are as yet unknown. It is known, however, that ADP can be hydrolyzed to lowenergy AMP and phosphate with heat release. For a long time, there , have been difficulties in determining the free energy for the ATP phosphate bond. The standard free energy for hydrolysis of the ATP phosphate bond, AGo, is equal to about -30.4 kJ/mol. This value has

GENERAL METABOLISM

177

been derived under standard conditions, i.e. at concentrations I M for initial reactants and end products, pH 7.0, temperature 37°C, and excess of Mg2+ ions. It stands to reason that in the cell under physiological conditions, the concentrations of initial reactants, end products, and Mg" ions are substantially inferior to the standard valueS; variations in pH are also possible. Therefore, the real free energy for hydrolysis of the end phosphate bond in ATP and ADP, and of the phosphate bonds in pyrophosphate is close to -50.0 kJ/mol. To be noted, the values of ~Go for other compounds differ from the standard value, but not necessarily towards higher energies.

6.5 ENERGY TRANSFER IN BIOCHEMICAL PROCESSES The biological activity of the cell is closely associated with the continuous redistribution of the energy delivered by the compounds that enter the cell. The storage of energy in the specific phosphate bonds of ATP constitutes the basis for the energy transfer mechanism in the living cell. The living cell is a nonequilibrated chemical systemthe circumstance that permits storing the energy, produced by catabolic reactions of nutrients, in the ATP phosphate bonds. The ATP energy in the cell can be converted, via tree major routes, to energy of chemical bonds, to thermal energy, and to energy for performing work. We now consider in general terms the transfer of chemical bond energy. The chemical reaction is associated with the generation of ATP energy. If the reaction is in a state of equilibrium, the energy transfer is accomplished to a 100% efficiency. Nonetheless, no ATP energy will accumulate, since at equilibrium, the free energy release is zero. Therefore, in order to provide for energy storage in the ATP phosphate bonds, the reaction must be a nonequilibrium one. This is accomplished as a portion of the chemical reaction energy is lost as heat. The remaining energy is transferred onto the ATP phosphate bonds to be stored therein. It follows, therefore, that biological generators of energy cannot attain the 100% efficiency. The third route of energy conversion leads to the performance of work. The chemical energy of ATP phosphate bonds can be spent on osmotic, electric, mechanical, and other types of work. In doing so, not all of the ATP energy is used for performing work; a portion of it is dissipated as heat. Especially much heat is produced by muscle contraction, which represents a mechanochemical mechanism for heat generation in the living organisms. Heat is of little use for performing

178

MICROBIOLOGY AND BIOCHEMISTRY

work in the living system. It is only in wann-blooded animals and man that the heat is consumed for warming and maintaining a constant temperature of the body, equal to about 37°C. All chemical processes in the living organism can proceed only with the involvement of enzymes. For this reason, prior to consider the mechanism of energy extraction and various metabolic pathways, it appears worthwhile to discuss enzymes and their functions.

7

Metalbolism of Saccharides Carbohydrate metabolism in the organism tissues encompasses enzymic processes leading either to the breakdown of carbohydrates (catabolic -pathways), or to the synthesis thereof (anabolic pathways). Carbohydrate breakdown leads to energy release or intermediary products that are necessary for other biochemical processes. The carbohydrate synthesis serves for replenishment of polysaccharide reserve or for renewal of structural carbohydrates. The effectiveness of various routes of carbohydrate metabolism in tissues and organs is defined by the availability of appropriate enzymes in them.

7.1

CARBOHYDRAGE CATABOLISM IN TISSUES

A number of routes for carbohydrate catabolism in tissues are known. They include glycolysis and its variant, glycogenolysis, which are auxiliary pathways to energy production, respectively, by breakdown of glucose (or other monosaccharides) and glycogen to lactate (under anaerobic conditions) or to CO2 and Hp (under aerobic conditions). The involvement of glycolysis and glycogenolysis in the energetic function has been discussed in detail in the foregoing section 'Bioenergetics" . There is known one more catabolic route for carbohydrates commonly referred to as the pentose phosphate cycle (also called hexose mono phosphate shunt, or phosphogluconate pathway). As a tribute to the biochemists who have played a decisive role in its investigation, the pentose phosphate cycle is also referred to as the Warburg-Dickens-Horecker pathway. The pentose phosphate cycle represents a mUltienzyme system in 179

MICROBIOLOGY AND BIOCHEMISTRY

180

which the important intermediates are, as the name implies, pentose phosphates. This cycle may be regarded as a branching, or shunt, at the glucose 6-phosphate step in the overall glycolysis. 7.1.1 Pentose Phosphate Cycle To provide for all steps of the pentose phosphate cycle, at least three glucose 6-phosphate molecules are required. Let us consider separate reactions of this cycle. 1. Dehydrogenation o( g1ueose 6-phosphate is the reaction that directs- glucose 6-phosphate via the pentose phosphate pathway; it is catalyzed by glucose-6phosphate dehydrogenase (in the schemes below, for a fuller description of the cyclic process, three glucose 6-phosphate molecules are used)

,/

H

OH

~-oH 3 HO-C-H

I

6

GI~hate

~-oHI

dehydrogenaa

·7"'3NADP +

3NADP·H+H+

H-C

I

H,C-oPO)H, glucose 6-phosphate

Glucose-6-phosphate dehydrogenase is a dimer with a molecular mass of about 135 000. Up to eight electrophoretic ally separable isoenzymes for this enzyme are known. A specific feature of the above reaction is the formation of NADP • Hr The reaction equilibrium is strongly shifted to the right, since the lactone formed is liable to hydrolysis, which is spontaneous or lactonase-assisted. 2. Hydrolysis of 6-phosphoglueonate lactone to 6-phosphoglueonate:

o C"

H-~-OH 1

3 HO-C-H

I I

H-b-OH

Lactonaae I 0 ~ 3 HO-C-H

H - t - OH H-C------!

1-

COOH

H-~-OH

H-C-OH

1

HaC-OPOaH,

H.C-OPOaH.

6-phoapbocl uconate lactone

8-pbOllphoglueonate

181

METABOLISM OF SACCHARIDES

3. Dehydrogenation of 6-phosphogluconate to ribulose 5phosphate. This reaction is catalyzed by 6-phosphogluconate dehydrogenase according to the scheme: COOH

I

3

"-r-O "o-y-"

H

"-y-o".'

. /"

""

.

6-phosphoglUCOlllte dehydrogenaR

3NADP'

+lC01

H-C-OH

.1

H2C-oP0 3H1 tJ.phosphog!uconate

D-ribulose S-phosphate

The reaction equilibrium is shifted to the right. 6-Phosphogluconate dehydrogenase is a dimer with a molecular mass of about 100 000. Several isoenzymes are known for this dehydrogenase. A specific feature of this reaction is that dehydrogenation leads to an unstable intermediate which is immediately decarboxylated on the surface of the enzyme. This is the second oxidation reaction in the pentose phosphate cycle that leads to NADP • H2 ; therefore, the conversion of glucose 6-phosphate to ribulose 5-phosphate is commonly referred to as the oxidative phase of the pentose phosphate cycle. The sequence of reactions starting from ribulose 5-phosphate to the formation of initial glucose 6-phosphate is called the nonoxidative, or anaerobic, phase of this cycle. 4. Interconversion or isomerization of pentose phosphates. Ribulose 5-phosphate is capable of a reversible isomerization to other pentose phosphates-xylulose 5-phosphate and ribose 5-phosphate. These reactions are catalyzed by two respective enzymes, viz., pentosephosphate epimerase and pentose-phosphate isomerase, according to the scheme below: H.C-OH

H.C-OH

I

c=o I

2 HO-C-H

I

H-C-OH

I

HsC-OPO,H. ]).:1,1111_

~pllOl)lllate

Pmt_pllOlphate eplmeralll

I I

C= 0

~===-~ 3 H-C-OH

I

H-C-OH

I

IItC-OPO.H. ])'rlllulOlO

5-p.~OI)IIIate

H-C=O

-

I

Pentooe-pllOlp/late H-C-OH

-

Isomerue

I

H-C-OH

I

H-C-OH

I

H.C-OPO.H. ]).r\IIoIe

5-pII01)111ate

Two other pentose phosphates (ribose 5-phosphate and xylulose 5-phosphate), which are derived from ribulose 5~phosphate, are important for the subsequent reaction of the cycle. Two molecules of

182

MICROBIOLOGY AND BIOCHEMISTRY

xylulose 5-phosphate and one molecule of ribose 5-phosphate are required for this. S. Transfer of glycolic aldehyde from xylulose S-phosphate onto ribose S-phosphate or the first transketolase reaction. The next reaction, which is catalyzed by transketolase, involves the pentose phosphates produced by the foregoing reaction (the transferable moiety is shown in the box): I

c=o I

2 HO-C-H

I

H-C=O

H.C-OH

HoC-OH Pent_pboopbate eplmerue

I

I

Pent __pboopbate H-C-OH

C= 0

I

.........I _~===-:: H-C-OH I

~====-:: 3 H-C-OH I

H-C-OH

H-C-OH

H-C-OH

I

I

I

HaC-OPOIHa

HoC-OPOIH.

BsC-OPOIH.

J>rtbal_

J>syl.l_

$-p:toopbate

$-pboopbate

A ribose 5-phosphate molecule and one of the two xylulose 5phosphate molecules are used during the first transketolase reaction. The other xylulose 5-phosphate molecule is consumed later, in the second transketolase reaction. Transketolase is a dimer with a molecular mass of 140000. Its coenzyme is thiamine bisphosphate. Mgt+ ions are required for the reaction. Both transketolase reaction products are used as substrates at the next step of the cycle. 6. Transfer of dihydroxyacetone moiety from sedoheptulose 7-phosphate onto glyceraldehyde 3-phosphate. This reaction is reversible and is catalyzed by transaldolase according to the scheme:

[H2C:'o'Hi

r-----'

I

I

I

C.O ... -,--_...1

I H,C-OH I I I I IL __c-o 1___ .-JI

I

.

Transketolase

HO-C-H

I

I

HO-C-H

I H-C-OH

I H-C-OH

H-C-OH

I

I

H-C-O

+

I H-C-OH

I H 2C-OPO)H2

H 2C- OPO)H2

H-C- OH

D·xylulote &-phosphate

H 2 C- OPO)H2 D·sedoheptulose D.gIycerilldehyde 711hotphate 3·pllosphllte

I

D·ribose S.phosphllte

Transaldolase is a dimer with a molecular mass of about 70 000. The fructose 6phosphate molecule produced by this reaction enters the glycolysis, while erythrose 4-phosphate is used as a substrate at the subsequent steps of the cycle.

METABOLISM OF SACCHARIDES

183

7. Transfer of glycolic aldehyde from xylulose S-phosphate onto erythrose 4-phosphate or the second transketloase reaction. This reaction is related to the first transketolase reaction and is catalyzed by the same enzyme. The only distinction is that erythrose 4-phosphate acts as an acceptor for glycolic aldehyde:

r-:-----' I H,C-OH I I IL

I

I

__C-O 1___ -JI

HO-C-H

I

H-C-OH

I

HzC- OPOJH Z

O·xylulOll 5-phOlPhlte

fH2C:' O"H; I

I

H-C-O

I

I

H-C-OH

I + H-C-OH I

H-C-OH

I

HO-C-H Transketolase ' I , H- c- OH M.H I H-C-OH

H ZC-OPO JH2

O·ribose 5-phosphate

I

C.O

I

... -I---.J

I

H-C .. O

+

II

H-1- 0H H~-OPOJH2

H-C- OH

I

HzC- OPOJH Z O-sedoheptulOll O-glyceraldehyde 7 -phosphite 3·pnosphate

Fructose 6-phosphate and glyceraldehyde 3-phosphate also enter the glycolysis. Thus, in the course of reactions catalyzed by the intrinsic enzymes of the pentose phosphate cycle, two fructose 6-phosphate molecules, one glyceraldehyde 3-phosphate molecule, and three carbon dioxide molecules are produced from three glucose 6-phosphate molecules. In addition, six NADP -H2 molecules are formed. The overall scheme for the pentose phosphate cycle is: 3 Glucose 6-phosphate-6NADP+ ~ 2 Fructose 6-phosphate + Glyceraldehyde 3-phosphate + 6NADPeH2+ 3C02 Interrelation of the Pentose Phosphate Cycle and Glycolysis These two pathways for carbohydrate conversion are closely related. The products of the pentose phosphate route-fructose 6phosphate and glyceral-dehyde 3-phosphate-are likewise glycolysis metabolites; for this reason, they are involved in glycolysis and undergo conversion by glycolytic enzymes. Two molecules of fructose 6-phosphate can regenerate to two glucose 6-phosphate molecules through the agency of the glycolytic enzyme glucose-phosphate isomerase. Here, the pentose phosphate pathway functions as a cycle. The other product, glyceraldehype 3-phosphate, enters the glycolysis to be either converted to lactate (under anaerobic conditions) or oxidized to CO2 and Hp (under aerobic conditions). As can easily be estimated, the conversion of glyceraldehyde 3-phosphate to lactate 7.1.2

MICROBIOLOGY AND BIOCHEMISTRY

184 Glucose

I

Glucose 6-pLsphate

~

G

L Fructose 6-phosphate y ' F ructose 6-phosphate

e o

Fructose 1.6-bisphosphate

,

Synthesis of nucleotides and nu" Ieosides. nucleoS Dihyd-~Glyceraldehyde 3-phosphate L~r--"-.tide coenzymes. I roxyaceI ~-.;....--' polynucleotid8s. Stone I and histidine I phosphate + • Pyruvate

Ly

r

i

U

Lactate Figure 7_1 Scheme for integration of pentose phosphate shunt and glycolysis.

leads to the formation of two ATP molecules, while the combustion to CO2 and Hp produces 20 ATP molecules. It follows therefore that under physiological conditions, when the pentose phosphate pathway for carbohydrate conversion is included in the glycolysis, the overall process of glucose 6-phosphate conversion may be expressed via the pentose phosphate cycle. Under anaerobic conditions: 3 Glucose 6-phosphate+6NADP+ + 2P l ~ 2 Glucose 6-phosphate + Lactate + 2ATP + 6NADP . H2 + 3C02 Under aerobic conditions: 3 Glucose 6-phosphate + 6NADP+ + 20ADP + 20Pl ~ 2 Glucose 6-phosphate + 6NADP.H2+ 6C0 2 + 6Hp + 20ATP At, first glance, the energetic value of this conversion of glucose 6-phosphate via the pentose phosphate cycle appears to be inferior to that of the aerobic glycolysis pathway, the latter providing a maximum of 38 ATP molecules. However, it should be borne in mind that a major portion of energy is stored in NADP - H2, and 6 NADP - H2 molecules are energetically equivalent to 18 A TP molecules. Consequently, the energetic effect remains the same.

7.1.3 The Biological Function of the Pentose Phosphate Cycle The biological function of the pentose phosphate cycle involves the production of two compounds: NADP -H 2, which is a "reductive force" in the synthesis of various materials, and the metabolite ribose

METABOLISM OF SACCHARIDES

185

5-phosphate, which is used as a building material in the synthesis of various specieiS. The major functions of the pentose phosph~te cycle ~e:

(1) amphibolic function: the cycle is a route to degradation of

carbohydrates and, simultaneously, to the supply of materials used in synthetic reactions (NADP • Hz and ribose 5phosphate); (2) energetic function, since the involvement of pentose phosphate cycle products (glyceraldehyde 3-phosphate) in the glycolysis produces energy; (3) synthetic function, as a major function associated with the use of NADP • Hz and ribose 5-phosphate. NADP • ~ is used: (1) in the detoxification of drugs and poisons in the monooxygenase oxidation chain of the endoplasmic reticulum of the liver; (2) in the synthesis of fatty acids and other structural and reserve lipids; (3) in the synthesis of cholesterol and its derivatives-bile acids, steroid hormones (corticosteroids, female and male sex hormones), and vitamins D; and (4) in the neutralization of ammonia under reductive amination. Ribose 5-phosphate is used in the synthesis of histidine, nucleosides and nucleotides (nucleotide mono-, di-, and triphosphates), nucleotide coenzymes (NAD, NADP, FAD, and CoA), and polymeric nucleotide derivatives (DNA, RNA, and short-chain oligonucleotides). The pentose phosphate pathway for carbohydrate conversion is primarily operative in the organs and tissues in which an intensive utilization of NADP • H2 is needed for reactions of reductive synthesis and for reactions involving ribose 5phosphate in the synthesis of nucleotides and nucleic acids. For this reason, a high activity of this pathway is observed in fat tissue, liver, mammary gland tissue (especially during lactation, since the milk fat synthesis is essential in this case), adrenal glands, gonadal glands, marrow, and lymphoid tissue. Relatively high is the activity of pentose phosphate shunt dehydrogenases in the erythrocytes. A low activity of the pentose phosphate pathway is observed in muscular tissue (heart and skeletal muscle).

MICROBIOLOGY AND BIOCHEMISTRY

186

7.2 BIOSYNTHESIS OF CARBOHYDRATES IN TISSUES In the human and animal tissues and organs, synthesis of carbohydrates occurs. Since glucose is the starting structural unit for producing other monosaccharides and for assembling polysaccharides, it is expedient to consider potential routes for the glucose synthesis in tissues and organs. Formation of glucose from nonccu:bohydrate materials is attested by the fact that, under prolonged starv'ation (in an extreme contingency or as applied in therapy), the polysaccharide carbohydrate reserves are rapidly consumed, while the glucose level in the circulating blood is maintained to supply tissues, especially brain, with energy. 7.2.1 Gluconeogenesis The synthesis of glucose from noncarbohydrate sources is referred to as the gluconeogenesis. It is feasible only in certain organism tissues. The major site for gluconeogenesis is the liver. To a lesser extent, the kidneys and intestinal mucosa are involved in this process. Mechanism for Gluconeogenesis. Since the glycolysis involves three energetically irreversible steps at the pyruvate kinase, phosphofructokinase, and hexokinase levels, the production of glucose from simple noncarbohydrate materials, for example, pyruvate or lactate, by a reversal of glycolysis ("from bottom upwards") is impossible. Therefore, indirect reaction routes are to be sought for. The Jirst indirect route in glucose synthesis involves the formation of phosphoenolpyruvate from pyruvate without the intervention of pyruvate kinase. This route is catalyzed by two enzymes. At first, pyruvate is converted into oxaloacetate. This reaction occurs in the mitochondria as the pyruvate molecules enter them, and is catalyzed IJy pyruvate carboxylase according to the scheme Pyruvate .arOOsyl. .

CH.-C-COOH+HCO.+ATP

II

°

.... HOOC-CH.-C-COOH+ADP+H.PO.

II

o

onlo_tate

This enzyme, similar to all CO2 assimilating enzymes, contains

biotin for a cofactor. Oxaloacetate is released from the mitochondria into the cytoplasm to enter gluconeogenesis. In the cytoplasm, oxaloacetate converts to phosphoenolpyruvate via a reaction catalyzed by phosphoenolpyruvate carboxylase:

187

METABOLISM OF SACCHARIDES ..._ _ _lpfnl...te e..~ ...

HOOC-CH.-C"":COOH+GTP(ATP)

-

~

oulo_late

... CHs=C-COOH+GDP(ADPHCO.

6-POaHs

pboopboonolpfnlY.te

The reaction equilibrium is shifted to the right. The major supplier of phosphate groups is GTP, but for this purpose, ATP may also be available. All of the glycolysis reactions ranging from phosphoenolpyruvate to fructose 1,6-bisphosphate are reversible, and the phosphoenolpyruvate IOOlecules fonned are consumed for producing fructose 1,6-bisphosphate by making use of the same glycolysis enzymes. The second indirect route involves the formation of fructose 6phosphate from fructose 1,6-bisphosphate without the intervention of phosphofructokinase reaction. This route is catalyzed by fructose bisphosphatase: Fructose 1,6-bisphosphate

+ H20

Fructosebispoosphatase)

Fructose 6-phosphatc

+ H2 PO4

The reaction is irreversibly shifted to the right. Fructose 6phosphate is isomerized to glucose 6-phosphate by glucose-phosphate isomerase. The third indirect route involves the formation of free glucose from glucose 6phosphate by circumventing the hexokinase reaction. This route is catalyzed by Glucose 6-phosphate

+ H2 0

Glucose 6-poosphatase

)

Glucose + H 2 PO 4

The free glucose produced by this reaction is supplied to the blood from the tissues. As exemplified by gluconeogenesis, one may easily envision the economical organization of these metabolic routes, since, apart from four special gluconeogenesis enzymes-pyruvate carboxylase, phosphopyruvate carboxylase, fructose bisphosphatase, and glucose 6phosphatase-individual glycolytic enzymes are also used in the gluconeogenesis. Noncarbohydrate Sources for Gluconeogenesis. In addition to pyruvate and lactate, which are delivered to the liver and kidneys, other noncarbohydrate compounds serve as substrates for glucose synthesis. In accordance with the gluconeogenesis scheme, it may be anticipated that all materials of noncarbohydrate nature that are

MICROBIOLOGY AND BIOCHEMISTRY

188

t.

Cl

rr-

e (") ... ru~,......

-<

o z

(")

o

m

r

o Cl

m

z m

i Figure 7.2 Schematic representation of gluconeogenesis.

amenable to conversion to a glucolysis metabolite (first group of materials), to pyruvate (second group), or to oxaloacetate (third group), can serve as potential sources for glucose synthesis. For example, glycerol may be included in the first group of materials, since this triol, which can convert to dihydroxyacetone phosphate, can further take up, depending on the reaction conditions, either gluconeogenesis route, or glycolysis route. The involvement of glycerol in gluconeogenesis proceeds according to the scheme: Glycerol phosphokinase

(11

Glycerol

7 '\

ATP (2)

·

Cl-Glycerol phosphate

AOP CI-GIYtero/ phosphate dehydrogenase

Cl -Glycerol phosphate --"';"'7"'~'-';;"~~---..

"

NAO+

NAO-H+tf

O·hydr I

oxyecetOne phosphate

METABOLISM OF SACCHARIDES

189

Subsequently, dihydroxyacetone phosphate is used in glucose synthesis. The Krebs cycle acids convertible to oxaloacetate (third group materials) may also be assigned to gluconeogenesis substrates. However, amino acids that can convert the major source for gluconeogenesis to both pyruvate and oxaloacetate and, consequently, to glucose are the major source for gluconeogenesis. Amino acids involved in the gluconeogenesis are referred to as glycogenic amino acids. They encompass all of the protein amino acids, barring leucine. 7.2.2 Biosynthesis of Glycogen (Glycogenogenesis) Synthesis of glycogen is carried out in all the cells of organism (the erythrocytes, perhaps, being the only exception), but this process is especially active in the skeletal muscles and in the liver. The reaction of glycogen breakdown, which is catalyzed by glycogen phosphorylase, is nearly irreversible; for this reason, this enzyme takes no part in the synthesis of glycogen. Two routes for glycogen synthesis are possible. One route involves a successive addition of glucose units to the extant glycogen moiety (glycogen primer); the other one originates in glucose molecules. The source of glucose residues durinr; glycogen synthesis is the active form of glucose, uridine diphosphate glucose (UDP-glucose), which is produced from glucose I-phosphate and uridine triphosphate (UTP) through the agency of the enzyme glucoseI-phosphate uridyltransferase according to the scheme: Glucose I-phosphates-UTP p UDP-glucose+H4pp7 The next step involves a transfer of the glucose residue from UDP-glucose onto the glycogen primer through the aid of the enzyme

glycogen synthetase: UDP-glucose + (Glucose) ~ UDP +(Glucose). + I To be noted, the glycogen synthetase catalyzes the formation of aI -.4-glycoside bonds only. The "branching" enzyme, amylo-(a-I,4 -a-I,6)-transglycolysase, transf~rs short fragments (two or three 'glucose residues) from one portion of the glycogen molecule onto another and forms a-I -.6-glycosidic bonds (branch points). The alternating action of these two enzymes results in the lengthening of the glycogen molecule. If the synthesis starts from glucose molecules, then the initial step is the transfer of glucose residues from UDP-glucose onto an intermediary acceptor-tiolichol phosphate (membrane-bound polyprenol phosphate). Dolichol phosphate assists in the synthesis of an

MICROBIOLOGY AND BIOCHEMISTRY

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oligosaccharide which is then transferred onto the protein. The successive addition of oligosaccharide chains to the glycogen molecule proceeds in the same manner as in the former route. The feasibility of the latter route is substantiated by the fact that glycogen is always bound with some protein. Interrelation of Glycogen Synthesis and Degradation. Glycogen synthetase exists in two interconvertible forms. The phosphorylated, or inactive, form is called glycogen synthetase D; the nonphosphorylated, or active, form is referred to as glycogen synthetase I. The transition from one form to the other is accomplished by two enzymes, glycogen synthetase kinase (1) and glycogen synthetase phosphatase (2) according to the scheme: ATP

ADP

Glycogen synthetase I -==='>~1~L==~. Glycogen synthetase 0 (nonphosphorylated .• 7" 2 (phosphorylated enzyme)

,

enzyme)

")P0 4

The processes of glycogen synthesis and degradation in the cells are controlled via phosphorylation mechanisms involving the key enzymes of glycogen metabolismglycogen synthetase and glycogen phosphorylase. The activation of adenylate cyclase (for example, with adrenal in or glucagon) leads to the production of CAMP which triggers the "cascade" mechanism of phosphorylation of glycogen synthetase and glycogen phosphorylase, with the resultant formation of the inactive (phosphorylated) glycogen synthetase D and active glycogen synthetase I. This favours glycogen synthesis. 7.2.3

Biosynthesis of Other Oligosaccharides and Polysaccharides Homo- and heteropolysaccharides are carbohydrate components of plasmic and structural glycoproteins in the mammals. These carbohydrates are made up of a small set of monosaccharides: galactose, mannose, N-acetylglycosamine, N-acetylgalactosamine, fucose, and sialic acid. The connective tissue polysaccharides contain glucuronic acid, iduronic acid, xylose, and sulphated derivatives of Nacetylglucosamine or N-acetylgalactosamine. These homo- and heteroglycans can be assembled by activated monosaccharide forms, such as nucleoside phosphate derivatives of monosaccharides. UDP-derivatives of glucose, galactose, N-acetylglucosamine, N-acetylgalactosamine, glucuronic acid and xylose; GDP-derivatives of mannose and fucose; and CMPderivatives of sialic acid are used in the synthesis. Most nucleoside phosphate saccharides are produced through reaction of monosaccharides

191

METABOLISM OF SACCHARIDES

or their derivatives with the corresponding nucleoside triphosphates. Occasionally, certain monosaccharides convert to other monosaccharides as constitutive components of nucleoside diphosphate saccharides. For example, UUPglucuronic acid and UDP-xylose are formed from UDPglucose:

"'.

UDPlIlucuronic acid - -.... \ ......~ UDP-xylose

CO 2

2NAD.~

GDP-fucose is formed from GDP-mannose: GDP-mannose

--7"-=--,,0::::::---" NADP+

GDP-fucose

NADP'~

For example, it is to be noted that UDP-glucuronic acid which is formed in the tissues, is used not only for polysaccharide synthesis, but also for neutralization and removal of toxic and useless materials or foreign compounds from the organism. Synthesis of oligosaccharides is carried out with the participation of specific glyc'Osyltransjerases whose diversity and functional specialization in each cell provide for the production of various types of homo- and heteroglycans. In the cell, glycosyltransferases are bound with the membranes of endoplasmic reticulum or 'Golgi apparatus, i.e. the organelles primarily responsible for assembling oligosaccharides. It may be presumed that initially the assembly is made on the polyprenol phosphate molecule onto which the monosaccharide residues from nucleoside phosphate saccharides are successively transferred by glycosyltransferases. Subsequently, the synthetized oligosaccharide is transferred onto proteins to form glycoproteins. Simultaneous transfer of glycosyl residues and assembly of proteinbased oligo- and polysaccharides are also possible.

7.3 CARBOHYDRATE METABOLISM CONTROL IN THE ORGANISM The carbohydrate metabolic routes in various tissues of the organism discussed above differ in intensity, which is defined by metabolic features specific of each tissue and organ. However, from the standpoint of activity of the whole organism, certain specializations of the carbohydrate metabolic routes in individual tissues are profitably complementary. For example, strenuous muscular exertion requires energy which is initially supplied by the breakdown ·of glycogen to lactic acid. The latter compound is excreted into the blood to be supplied

192

MICROBIOLOGY AND BIOCHEMISTRY

to the hepatic tissue, where it is used for the synthesis of glucose during gluconeogenesis. From the liver, glucose is delivered in the blood to the skeletal muscles to be consumed for energy generation and to be deposited as glycogen. This intertissue (or interorgan) cycle in the carbohydrate metabolism is referred to as the Cori cycle (called also glucoselactate cycle): Liver

Blood

Glucose _Glucose _

t

Muscle Glucose ...............

~

Glycogen

Lactate 4 - - Lactate - - Lactate""""""'-

The maintenance of a constant glucose level in the blood is of primary importance for the organism, since glucose is the major energy substrate for the nervous tissue. The normal glucose content in the blood is 3.3 to 4.0 mmoIllitre. An increased concentration of glucose in blood is known as hyperglycemia. If hyperglycemia reaches as high as 9 to 10 mmoIllitre, the glucose excess is released into the urine, i.e. glucosuria sets in. On the contrary, a decreased glucose percentage in the blood is known as hypoglycemia. Hypoglycemia as low as about 1.5 mmoIllitre leads to the syncopal state, while a still lower glucose concentration results in high excitability of the nervous system and ultimately leads to convulsions and coma. To gain a better understanding of the mechanism that controls the glucose level in the blood, it is important to examine processes that contribute to an increased or lowered glucose concentration. Processes leading to hyperglycemia: (1) absorption of glucose from ·the intestine (alimentary hyperglycemia); (2) breakdown of glycogen to glucose (commonly, in liver); (3) gluconeogenesis (in liver and kidney). Processes leading to hypoglycemia: (1) transport of glucose from the blood to tissues followed by glucose oxidation to end products; (2) synthesis of glycogen from glucose in liver and skeletal muscles; (3) production of triacylglycerol from glucose in fat tissue. The dietary intake of carbohydrates leads to a short-term (within 1 or 2 hours) hyperglycemia and, occasionally, glucosuria. Starvation stimulates the consumption of glycogen reserves in liver

METABOLISM OF SACCHARIDES

193

and skeletal muscles, which prevents the development of hypoglycemia, but within the space of a few hours only. Then, under lasting starvation, the glucose level is maintained solely owing to the gluconeogenesis, primarily at the expense of proteinic amino acids which suffer degradation in the tissues. In point of fact, the potential ability to sustain starvation is determined by the protein reserves available for glucose production. Blood Glycogen breakdown Absorption from intestive ---tl-i

Gluconeogenesis

Glycogen (liver, skeletal muscle)

C10+ H1 0 (numerous tissues) Triacylglycerides (adipose tissue)

The glucose level in the blood is monitored by neurohormonal mechanisms. Excitation of the sympathetic portion of the autonomic nervous system increases the glucose level in the blood, while excitation of the parasympathetic portion produces a reverse effect. The only hormone capable of reducing the glucose content is insulin. It stimulates all of the three processes of glucose assimilation (intracellular transport and degradation of glucose, synthesis of glycogen, and synthesis of triglyceride from glucose in fat tissue). All other hormones make the glucose level increase; for this reason, they are occasionally referred to as contrainsular hormones. These include adrenalin, glucagon, thyroxin and triidothyronin, somatotropin (which stimulate glycogen degradation), and glucocorticoids (which stimulate gluconeogenesis).

8 Metabolism of Fats and Glycerides Lipids are continually renewed in the organism tissues. The major part of lipids in the human body is represented by triacylglycerides which occur as in-clusions in most tissues; the fat tissue, which consists nearly totally of triacyl-glycerides, is especially lipid-rich. Since triacylglycerides play an important role in the encrgetics of the organism, the processes of their renewal (the conversion half-time for triacylglycerides in different organs varies from 2 to 18 days) involve both mobilization and deposition during energy production. Compound lipids (phospholipids, sphingolipids, glycolipids, and cholesterol and its esters) that make part of the biomembrane are subject to a less active renew-al as compared with triacylglycerides. Their renewal is associated either with the restoration of an impaired portion of the membrane, or with the replacement of a "defective" molecule by a new one. The renewal of tissue lipids involves their preliminary intracellular hydrolysis by enzymes.

8.1 DEGRADATION OF LIPIDS IN TISSUES 8.1.1 Intracellular Hydrolysis of Lipids Hydrolysis of triacylglycerides in tissues is effected by a tissue enzyme, tri-acylglyceride lipase. which hydrolyzes triacylglycerides to glycerol and free fatty acids. There are a variety of tissue lipases that differ primarily in their optimum pH and their location in the cell. The acidic lipase is contained in lysosomes; the basic lipase, in microsomes; and the neutral lipase, in cytoplasm. A specific feature of the tissue lipase is its sensitivity to hormones which, by activating adenylate cyclase, elicit the transition of the inactive tissue lipase to its active 194

METABOLISM OF FATS AND GLYCERIDES

195

fonn via phosphorylation with protein kinase. This mechanism bears resemblance to the activation of phosphorylase B. Lipases mobilize triacylglycerides. This process is also known as the tissue lipolysis. The cell membrane phosphoglycerides are hydrolyzed with phospholipases AI' A2 , C, and D, which are located chiefly in lysosomes. However, certain phospholi-pases also occur in other cell organelles. Hydrolysis of phosphoglycerides yields glycerol, fatty acids, nitrogenous alcohols, and inorganic phosphate. There are also known specific enzymes for hydrolysis of sphingolipids and glycolipids; the enzymes are involved in the renewal of these lipids. As is known, hydrolysis of intracellular lipids does not lead to a storage of glycerol and fatty acids. This indicates that the hydrolysis rate for the lipids is balanced against the rate of their intracellular oxidation. In the adipose tissue, glycerol and fatty acids as produced by triacylglyceride hydrolysis are not subject to oxidation and are released into the blood to be supplied to other organs.

8.1.2 Oxidation of Glycerol The glycerol metabolism is closely related to the glycolysis involving glycerol metabolites according to the following scheme:

At first, glycerol is converted to a-glycerol phosphate through the agency of glycerol phosphokinase. a-Glycerol phosphate, by the action of NAD-dependent a-glycerol-phosphate dehydrogenase, is converted to dihydroxyacetone phosphate, which, as a common glycolysis metabolite, enters glycolysis to be reduced by enzymes to lactate under anaerobic conditions, or to CO2 and Hp under aerobic conditions. Conversion of one glycerol molecule yields one ATP molecule under anaerobic, and 19 ATP molecules, under aerobic conditions. Glycerol is a profitable energy sub-strate and is used as an energy source practically by all organs and tissues.

8.1.3 Oxidation of Fatty Acids Oxidation of higher fatty acids was first studied in 1904 by Knoop who fed animals with phenyl-substituted fatty acids and analyzed the products in the urine. He showed that the fatty acid oxidation results in the successive cleavage of two carbon moieties from the carboxyl end. Knoop coined the fatty acid oxidation mechanism as n-oxidation. As has been established by Kennedy and Lehninger in 1948-1949, oxidation of fatty acids o~curs in the mitochondria only. Lynen and coworkers

MICROBIOLOGY AND BIOCHEMISTRY

196

have outlined major enzymic processes in fatty acid oxida-tion. At the present time, the p-oxidation of fatty acids is referred to as the Knoop-Lynen cycle. The fatty acids, as produced by intracellular hydrolysis of triacylglycerides or supplied to the cell from the blood, must be brought into a state of activation. Their activation is effected in the cytoplasm with the participation of acyl-CoA synthetase according to the scheme: fIIIt+

CH.-(CH.),,-CH.-CH.-COOH+ATP+CoASH

acyl-COA IJIltbet":

o

-+

CH,-(CH.J,,-CH.-CH.-~ -

SCoA+AMP+H.P.O,

Since the activation process is effected extramitochondrially, transport of acyls across the membrane into the mitochondria is necessary. The transport is accomplished with the participation of camitine, which takes up the acyl from acyl-CoA on the outer membrane side. Acylcarnitine assisted by carnitine translocase diffuses to the inner side of the membrane to give its acyl to the CoA located in the matrix. The process of reversible acyl transfer between CoA and carnitine on the outer and inner sides.of the membrane is effected by the. enzyme acyl-CoA -camitine transferase. Cytoplasm

Membrane

Mitochondrion

?

R-C-SC

Matrix

o

I R-C-SCoA

CoASH

Figure 8.1 Transport of fatty acids across the mitochondrial membrane.

The oxidation of fatty acids within the Knoop-Lynen cycle occurs in the matrix. The Knoop-Lynen cycle includes four enzymes that act successively on acetyl-CoA. These are: acyl-CoA dehydrogenase (FADdependent enzyme), enoyl-CoA hydratase, 3-hydroxyacyl-CoA dehydrogenase (NAD-dependent enzyme), and acetyl-CoA acyltranslerase. Each turn, or revolution, of the "fatty acid spiral" produces

METABOLISM OF FATS AND GLYCERIDES

197

an acetic acid residue split from the fatty acid as acetyl-CoA to yield one FAD • H2 molecule and one NAD • H2 molecule. The cycle turns are then repeated until the fatty acid chain becomes shortened to a four-carbon fra~ent, i.e. butyryl-CoA. In the last turn, butyrylCoA splits apart, and two, rather than one, acetyl-CoA molecules are formed.

Figure 8.2 Scheme for oxida-tion of fatty acids in the Knoop-Ly-nen cycle.

The oxidation products of an even-numbered fatty acid are acetylCoA, FAD • H2 and NAD • H2 Subsequently, acetyl-CoA enters the Krebs cycle, and FAD • H2 and NAD • H2 are directly supplied to the respiratory chain. The specific behaviour of odd-numbered fatty acids under o

MICROBIOLOGY AND BIOCHEMISTRY

198

oxidation is that one propionyl-CoA molecule (qH3-C~-CO-SCoA) per oxidized fatty acid mole-cule, alongside the products acetyl-CoA, FAD • H2 , and NAD • H2 (common to even-numbered fatty acids), is formed. Propionyl-CoA converts to succinyl-CoA:

fOOH

COz CHJ-CHr-CO-SCOA

, ) " . CHJ-CH-CO-SCoA All'

ADP+'

melhylmllonyl
HOOC-CHrCHz-CO-SCoA SUCCinyf..CoA

Carboxylation of propionyl-CoA is accomplished by propionylCoA carboxylase (biotin, which is the carboxyl group carrier, serves as a coenzyme for this enzyme); the presence of ATP is also required. The methylmalonyl-CoA formed is converted by methylmalonyl-CoA mutase (whose coenzyme, deoxyadenosylcobalamin, is a derivative of vitamin B12) to succinyl-CoA; the latter enters the Krebs cycle. The specific behaviour of unsaturated fatty acids under oxidation is determined by the position and the number of double bonds in the fatty acid molecule. The stepwise oxidation of an unsaturated acid to the position of a double bond in it proceeds in a manner similar to that of saturated acid oxidation. If the double bond retains the same configuration (trans-configuration) and position (~2, 3) as those of the enoyl-CoA, which is produced during the oxidation of saturated fatty acids, the subsequent oxidation proceeds via conventional route. Otherwise, the oxidation reaction proceeds with the involvement of an accessory enzyme, ~3, 4-CiS-~2, 3Jrans-enoyl-CoA isomerase; this facilitates the translocation of the double bond to an appropriate position and alters the double-bond configuration from cis to trans. The unsaturated fatty acid oxidation proceeds at a rate higher over that for saturated acids. For example, if the oxidation rate for saturated stearic acid is taken as a reference value, the oxidation rate for oleic acid is 11 times, linolic acid, 114 times, linolenic acid, 170 times, and arachidonic acid, nearly 200 times as high as that for stearic acid. In addition to p-oxidation, two other oxidation routes are known for fatty acids, referred to as a- and ro-oxidation. However, they exhibit a lower activity and initially involve the formation of a- and o-hydroxy acids, with subsequent con-versions thereof. These oxidation routes are of inferior energetic value as compared with p-oxidation; presumably, they are implicated in special functions of the cell. Energy Balance of Fatty Acid Oxidation. The energetic value of an even-numbered fatty acid is estimated in the following manner. Complete oxidation of a fatty acid composed of 2n carbon atoms yields

METABOLISM OF FATS A)'J1) GLYCERIDES

199

n acetyl-eoA molecules (each acetyl con-taining two carbon atoms) and n-I FAD -Hz and NAP -~ molecules (since the last tura of the "fatty acid spiral"-yields two acetyl-eoA molecules, one FAD -H2 molecule and one NAD -H2 molecule). Oxidation of FAD -H 2 gives two ATP mole-cules, and oxidation of NAD -H1i three ATP molecules, i.e. a total of 5 ATP mole-cules, or, in the general case, 5 (n-I) ATP molecules. As has been noted above, the complete oxidation of one acetyl-eoA molecule results in the formation of 12 ATP molecules, while n acetyl-eoA molecules provide 12n ATP molecules. One ATP molecule being used for the fatty acid activation, 12n-1 ATP molecules remain. Now, the ATP balance for the complete oxidation of an even-numbered fatty acid may be expressed by the formula 5 (n - I) + 12n - I = (17n - 6) ATP molecules where n is equal to half the number of carbon atoms in a given fatty acid. For example, the palmitic acid molecule, which contains 16 carbon atoms, produces 130 ATP molecules. The energetic value of fatty acids is superior, for example, to that of glucose. For example, the complete oxidation of capronic acid (whose molecule contains the same number of carbon atoms as glucose) yields 45 ATP molecules as compared with 38 molecules which can ,be derived from gluco~e. However, the acetyl-eoA molecules as produced by P-oxidation require a sufficient amount of oxaloacetate to be degraded by the Krebs cycle. In this resp(!ct, carbohydrates have an advantage over fatty acids, since the breakdown of the former species leads to pyruvate serving as a source for both acetyl-eoA and oxaloacetate (pyruvate-carboxylase reaction), i.e. the acetyl-eoA conversion within the Krebs cycle is thus facilitated. It is not without reason that in the older biochemical literature the notion that "fats burn down in the carbohydrate flame" was popular, since the ATP from glycol-ysis can be used for the cytoplasmic activation of fatty acids, while the pyruvate-derived oxaloacetate facilitates the insertion of fatty acid acetyl residues into the Krebs cycle. Importance of Fatty Acids as Energy Substrates for Various Organs and Tissues. The organism tissues differ in the extent of utilization of fatty acids and their intermediary oxidation products, the so-called ketone bodies, as energy substrates. Fatty acids are actively consumed in the heart as well as in the kidneys and skeletal muscles (under prolonged physical exertion). In these organs, the ketone bodies undergo oxidation to yield additional energy. In the

200

MICROBIOLOGY AND BIOCHEMISTRY

nervous tissue, the con-sumed amount of fatty acids and ketone bodies as sources of energy is insignifi-cant.

8.2 BIOSYNTHESIS OF LIPIDS IN TISSUES 8.2.1 Biosynthesis of Fatty Acids In the organism tissues, fatty acids are continually renewed in order to provide not only for the energy requirements, but also for the synthesis of multicomponent lipids (triacylglycerides, phospholipids, etc.). In the organism cells, fatty acids are resynthetized from simpler compounds through the aid of a supramolecular multienzyme complex referred to as fatty acid synthetase. At the Lynen laboratory, this synthetase was first isolated from yeast and then from the liver of birds and mammals. Since in mammals palmitic acid in this process is a major product, this multienzyme complex is also called palmitate synthetase. Biosynthesis of fatty acids exhibits a number of ~ific features: (1) fatty acid biosynthesis, as distinct from oxidation, is localized in the endo-plasmic reticulum; (2) the !;ource for the synthesis is malonyl-CoA, which is produced from acetyl-CoA; (3) acetyl-CoA is involved in the synthetic reactions as a primer only; (4) NADP .H 2 is used to reduce fatty acid biosynthesis intermediates; (5) all the steps of malonyl-CoA fatty acid biosynthesis are cyclic processes that occur on the surface of palmitate synthetase. Production of Malonyl-CoA for the Fatty Acid Biosynthesis. Acetyl-CoA serves as a substrate in the production of malonyl-CoA. There are several routes by which acetyl-CoA is supplied to the cytoplasm. One route is the transfer of acetyl residues from the mitochondrial matrix across the mitochondrial membrane into the cyto-plasm. This process resembles a fatty acid transport and is likewise effected with the participation of carnitine and the enzyme acetyl-CoA-carnitine transferase. Another route is the production of acetyl-CoA from citrate. Citrate is delivered from the mitochondria and udergoes cleavage in the cytoplasm by the action of the enzyme ATP-citrate lyase: Citrate + ATP + CoA ~ Acetyl-CoA + OXaloacetate + ADP + PI The reaction is practically irreversible, and is shifted to the right.

METABOLISM OF FATS AND GLYCERIDES

201

The acetyl-CoA supplied to the cytoplasm via the above routes is used for the synthesis of malonyl-CoA:

CH,

COOH

1-

1

,,1+

~ = 0+ HCO.+ATP -(B--bl-ot-In"~

CHi I

+ ATP+H.PO.,

C=O

SCoA

I

acetyloCoA

SCoA maionyloCoA

The reaction is catalyzed by the biotin,enzyme acetyl-CoA carboxylase (E-biotin) assisted by Mg2+ ions. This enzyme is a tetramer with a molecular mass of 400 000-500000. Steps of Fatty Acid Biosynthesis Assisted by Palmitate Synthetase. Palmitate synthetase is composed of seven enzymes; of these, each is assigned a defmite func-tion. The acyl carrier protein (ACP) is located at the centre of the multienzyme complex; the other six enzymes occupy peripheral positions. ACP acts both as an acceptor and a distributor of acyl groups. ACP contains a covalently bound 4phos-phopantethein bearing a free SH group for accepting an acyl moiety. In addition to this central SH group, palmitate synthetase has a peripheral SH group. Both SH groups participate as acyl acceptors in the synthesis of fatty acids on the sur-face of the multienzyme complex. The cyclic process of fatty acid synthesis may be represented by a series of consecutive reactions (hereafter palmitate synthetase is designated by the symbol (

SH) • SH

1. Transfer of acetyl moiety from acetyl-CoA onto the synthetase:

o SH

CHI

II

S - C-CHa

+ ~=o- { SH SH ~SCoA

FI

"

"

+CoASIl

This reaction is carried out by the first enzyme of palmitate synthetase-acetyl-transacylase, which possesses an SH group. At this stage of the synthesis, the acetyl acts as a primer. 2. Transfer of malonyl moiety from malonyl-CoA onto the synthetase:

202

MICROBIOLOGY AND BIOCHEMISTRY

o

o

COOH

II S-C-CH.

g'

~H. + ~=O

II S-C-CH.

,

~

"

SH

0

+

CoASH

" S - C-CH.-COOH II

SCoA

The reaction is effected by the second synthetase ellzymemalonyltransacylase.

3. Acetyl-malonyl condensation and decarboxylation of the product fonned:

o II S-C-CH.

/

E

SH

0

-+

'0

0

"°11 II S - C-CH.-C-CH.

" S - C-CH.-COOH \I

+00.

The reaction is catalyzed by the third synthetase enzyme-3-ketoacyl synthetase. An acetoacetyl, which is bound to synthetase, is fonned at this stage . .4. The first reduction of the intennediate with the involvement of NADP -H 2 :

I

SH 0

E's _

_~,....-~_.

~-CH-CH-CHl ~H+

k+

\

/SH

0

5 - !-CH1-CH1-CH l

The reaction is catalyzed by the fourth synthetase enzyme- Pketoacyl reductase, to yield intennediary hydroxybutyryl.

5. Dehydration of the intermediate: 8H

/

E

8H 0

OH

..... II I 8,.., C-CH,-CH-CH.

-+

/

E

"

0

+

H,O

1\ 8,.., C-CH=CH-CH.

The reaction is catalyzed by the fifth synthetase enzymehydroxyacyl hydratase, to produce crotonyl. 6. The second reduction of the intennediary product with the involvement of

203

METABOLISM OF FATS AND GLYCERIDES

The reaction is catalyzed by the sixth synthetase enzymeenoylreductase, to form an enzyme-bound butyryl. The butyryl thus synthetized is transferred, through the mediacy of the first synthetase enzyme, acetyltransacylase, onto the SH group (the upper one in the Scheme) initially bound to the acetyl primer. The SH group (the lower one in the Scheme), thus freed, accepts a new malonyl residue:

o II

SII ./ ,

S --- C-CH,-CH,-CH.

0 II

S --- C-CH,-CH,-CH.

- I'-.

SH

The synthetic cycle is thus repeated. Seven cycles are implicated in palmitic acid biosynthesis and, accordingly, seven malonyl residues and one acetyl are required. Acetyl is the end moiety in fatty acid biosynthesis. The palmitic acid thus synthetized is either transferred onto the outer CoA to produce acyl-CoA, or, more commonly, is hydrolyzed by the specific palmitate deacylase to yield a free fatty acid. . Fatty Acid Chain Elongation., The mitochondria and endoplasmic reticulum provide the conditions for an eventual chain elongation of the cell-synthetized or dietary fatty acids. This process is different from the fatty acid biosynthesis in the proper sense of the term. In the mitochondria, the chain elongation is achieved through the aid of an enzyme complex by adding acetyl residues from acetyl-CoA. In the endoplasmic reticulum, the chain elongation is accomplished by an enzyme complex through making use of malonyl-CoA. Biosynthesis of Unsaturated Fatty Acids. In the mammalian tissues, the forma-tion of monoene fatty acids is only possible. Oleic acid is derived from stearic acid, and palmitooleic acid, from palmitic acid. This synthesis is carried out in the endoplasmic reticulum of the liver cells via the monooxigenase oxidation chain. Any other unsaturated fatty acids are not produced in the human organism and must be supplied in vegetable food (plants are capable of generating polyene fatty acids). Polyene fatty acids are essential food factors for mammals. 8.2.2 Biosynthesis of Triglycerides Triglyceride biosynthesis proceeds with the involvement of the lipids deposited in fat tissue or· in other tissues of the organism. This process is localized in the hyaloplasm of cells.

204

MICROBIOLOGY AND BIOCHEMISTRY·

a-Glycerol phosphate and acyl-CoA, rather than corresponding free glycerol and free fatty acid, are utilized in the direct synthesis of triglycerides. a-Glycerol phosphate is produced either by phosphorylating the glycerol supplied to the tissue, or by reducing dibydroxyacetone phosphate as an intermediary product of glycolysis. The first step of triglyceride biosynthesis is the formation of phosphatidic acid with the involvement of glycerophosphate acyltransferase:

i I i H-y-o-C-R' H2C-O-C-R

Hi~-OH

:;:::>'"

H-l:-OH

R-CO-SCoA

HJ-o P0 3H 2

cc::::::: 2 CoA SH

R!...CO-SCoA

H 1C-OPO l H 2

a - glycerol phosphite

. 'Pholphltid~ ICid

Further, the phosphatidic acid is subject to an attack by phosphatidate phosphatase to yield diglyceride:

o 11

J=:=i_:. H.bPQ,A pbolpllaUtUe actd

dlllJC8l'lde .

The third acyl residue is transferred onto diglyceride by means of diglyceride acyltransferase

W

HIC,-O-CR

9

HC-o-~-R' HJ-oH diaCY'g'yceride

~ R"-CO-SCoA

""

•

~

H2C- O- -R

CoA SH

0

H -o-!R' H

f-o-~R"

t~lCYIglyceride

, The triacylglyceride thus synthetized is stored as fat inclusions in the cell cyto-plasm. 8.2.3 Phospholipid Biosynthesis Biosynthesis of phospholipids is associated with the renewal of

205

METABOLISM OF FATS AND GLYCERIDES .~ of p/IoIpIIagIycer c2nd pethwrf)

.'oflIIY"theIis

tri.cylgl~

.'aeynthells

of phOlPhoglycericllll C11t~yl---

PhoopFhat:;osito, I.-itol

.

j

CH,O-C_

LoLR' I

CH,o--CDP CDP-diecylgtycerol

O-CHr::HCNH')cOOH

~,of

eMP

.

R

H

II

-",.

HQ-C-R'

j

CH,o-r-OCH'fHCOOH OH

NH,

PhoophatidylMrl...

co'1

Phoophatidylethano...... i ...

~ S-adenosylmethionine ~ 5-lIdenosylhomocystel... Phoophatldylchollne

Figure 8.3 Two pathways for the synthesis of certain phospholipids.

membranes, This process is accomplished in the, tissue hyaloplasm. The fIrst steps of phospholipid and trig~yceride biosyntheses coincide; subsequently, these routes diverge at the level of phosphatidic acid and diglyceride. Two routes to phospholipid biosynthesis are known; in either, the participation of CTP is necessary. The first route involves phosphatidic acid in phosphoglyceride biosynthesis. Phosphatidic acid reacts with CTP to yield CDP-diglyceride which, as a coenzyme, can participate in the transfer of diglyceride onto serine (or inositol) to produce phosphatidylserine (or phosphatidylinositol). Serine phosphatides are liable to decarboxylation (pyridoxal phosphate acting

MICROBIOLOGY AND BIOCHEMISTRY

206

as a coenzyme) to yield ethanolamine phosphatides. The latter species are subject to methylation by S-adenosylmethionine (which donates three methyl groups), tetrahydrofolic acid and methylcobalamin acting as methyl group carriers. The second synthetic route involves activation of an alcohol (for example, choline) to produce CDP-choline. The latter participates in the transfer of choline onto diglyceride to form phosphatidylcholine. The phospholipids thus obtained are transported by lipid-carrier cytoplasmic proteins to the membranes (cellular or intracellular) to replace the used or impaired phospholipid molecules. Because of the competition between the phospholipid and triglyceride synthetic routes for common substrates, all substances that favour the former route impede the tissue deposition of triglycerides. Such substances are referred to as lipotropic factors. They include: choline, inositol, and serine, as structural components of phospholipids; pyridoxal phosphate, as an agent facilitating the decarboxylation of serine phosphatides; methionine, as a donor of methyl groups; and folic acid and cyanocobalamin, involved in the formation of methyl group transfer coenzymes (tetrahydrofolic acid and methylcobalamin). They may be used as drugs preven-tins excessive deposition of triglycerides in tissues (the so-called fatty infiltra-tion). 8.2.4 Biosynthesis of Ketone Bodies Three compounds: acetoacetate, P-hydroxybutyrate, and acetone, are known as ketone bodies. They are suboxidized metabolic intermediates, chiefly those of fatty acids and of the carbon skeletons of the so-called ketogenic amino acids (leucine, isoleucine, lysine, phenylalanine, tyrosine, and tryptophan). The ketone body production, or ketogenesis, is effected in the hepatic mitochondria (in other tissues, ketogenesis is inoperative). Two pathways are possible for ketogenesis. The more active of the two is the hydroxymethyl glUJarate cycle which is named after the key intermediate involved in this cycle. The other one is the deacylase cycle. In activity, this cycle is inferior to the former one. Acetyl-CoA is the starting compound for the biosynthesis of ketone bodies. Hydroxymethyl Glutarate Cycle. At the first step of this cycle, condensation of two acetyl-CoA molecules takes place, with the participation of acetyl CoA acetyltransferase: CH.-C..., SCoA+CH.-C..., SCoA .... CH.-C-CHa-c"" SCoA+CoASH

~

acetyl-CoA

~

~

~

METABOLISM OF FATS AND GLYCERIDES

207

Further, acetoacetyl-CoA becomes coupled once more to an acetylCoA molecule through the assistance of hydroxymethylglutaryl-CoA

synthase: CH.

II

CH.-C-CH.-C - SCoA+CH.-C - SCoA _ HOOC-CH.-C-CH.-C _ SCoA+CoASH

II

II

o

II

0

I

0

~-Hydroxy-~-methylglutaryl-CoA

OH

is split by hydroxymethylglutaryl-

CoA lyase into acetyl-CoA and acetoacetate: CH.

I HOOC-CH.-C-CH.-C - SCoA ..... CH.-C- SCoA+HOOC-CH.-C-CH.

I

OH

II

II

0

II

0

0

Acetyl-CoA is again used at the fIrst step and closes thereby the whole process into a cycle. Acetoacetate, as a representative of the ketone body family, is the end product of the hydroxymethyl- glutarate cycle. The other ketone bodies are derived from acetoacetate: Phydroxybutyrate, by reduction with the involvement of NAD-dependent hydroxyburyrate dehydrogenase, and acetone, by decarboxylation of acetoacetate with the participation of aceto-acetate decarboxylase:

i

7":C::::;..

HOOC-C H Z-C-CH3

~coz

IIIAD'H+H+

NAD+

OH H'OOC-CH -bH-CH

z

3

11- hydroxybutyrlte

H 3C-C-CH 3

~

acetone

The Deacylase Pathway for Ketogenesis is feasible after the formation of acetoace-tyl-CoA which is subject to hydrolysis to acetoacetate in the liver with the involvement of acetoacetyl-CoA

hydrolase,

or deacylase.

In the liver, the ketone bodies suffer no transformation, and are excreted into the blood. The normal contents of ketone bodies (as acetoacetate or ~-hydroxy-butyrate) amount to mere 0.1-0.6 mmol/ litre). Other tissues and organs (heart, lung, kidney, muscle, and nervous tissue), as distinct from the liver, utilize the ketone bodies as energy substrates. In the cells of these tissues, acetoacetate and 1hydroxybutyrate enter ultimately the Krebs cycle and "burn down" to CO 2 and H,O to release energy.

208

MICROBIOLOGY AND BIOCHEMISTRY

8.2.5 Biosynthesis of Cholesterol In the experiments with acetic acid labelled radioisotopically and fed to ani-mals, it has been established that the cholesterol carbon framework is made up entirely of the acetic acid carbon. Biosynthesis of cholesterol from acetyl-CoA proceeds, assisted by the enzymes of endoplasmic reticulum and hyaloplasm, in many tissues and organs. This pro-cess is especially active in the liver of adult humans. Cholesterol biosynthesis is a multistage process; in general, it may be divided into three steps: (1) production of mevalonic acid from acetyl-CoA; (2) synthesis of an "active isoprene" from mevalonic acid followed by the con-densation of the former to squalene; (3) conversion of squalene to cholesterol. The initial reactions in the first step, prior to the formation of P-hydroxy-p-methylglutaryl-CoA from acetyl-CoA, resemble those involved in ketogenesis with the only distinction that ketogenesis occurs in the mitochondria, while cho-lesterol biosynthesis is carried out extrarnitochondrially: 2 Acetyl-CoA ~ Acetoacetyl-CoA + Acetyl-CoA ~ I3-Hydroxy-l3-methylglutaryl-CoA Further, l3-hydroxy-l3-methylglutaryl-CoA is converted with hydroxymethylgluta-ryl-CoA reductase to mevalonic acid:

fH

CH l

l

HOOC-CHI-C-CHI-C-SCoA

bH

A

7' 2NADP·H+H+

•

~p.

I I

HOOC-CHIC-CHl-CHIOH

+ CoASH

OH

This reaction is irreversible and is a rate-limiting stage of the overall cholesterol biosynthesis. An alternative route to mevalonic acid is also possible, which differs from the former one in that the formation of l3-hydroxy-l3methylglutaryl residue occurs on the surface of an acyl carrier protein (like in fatty acid biosynthesis). The intermediary product in this route, P-hydroxy-p-methylglutaryl-S-ACP, is re-duced by another enzyme to mevalonic acid. During the second step, mevalonic acid is implicated in a number of enzymic reactions involving ATP, and is converted to isopentyl pyrophosphate and to its isomer 3,3-dimethylaUyl pyrophosphate. Actually, the two compounds constitute the "active isoprene", which

METABOLISM OF FATS AND GLYCERIDES

209

is consumed in the production of squalene. During the third step, cholesterol is generated from squalene: Squalene ~ Lanosterol ~ Cholesterol The steroid ring hydroxylation proceeds with the involvement of the monooxygen-ase chain of endoplasmic reticulum membranes. Cholesterol esters are produced by transferring an acyl moiety from acyl-CoA or from phosphatidylcholine onto the cholesterol hydroxyl group. The latter process is catalyzed by phosphatidylcholine cholesterol acyltransferase: Phosphatidylcholine + Cholesterol ~ Lysophosphatidylcholine + Cholesterol ester Cholesterol esters are produced especially actively in the intestinal mucosa and in the liver. Thus, the tissue cholesterol can be synthetized from any materials whose break-down leads to acetyl-CoA. These include carbohydrates, amino acids, fatty acids, and glycerol. The liver plays a decisive role in the cholesterol metabolism. The liver accounts for 90% of the overall endogenic cholesterol and its esters; the liver is also impli-cated in the biliary secretion of cholesterol and in the distribution of cholesterol among other organs, since the liver is responsible for the synthesis of apoproteins for pre-~­ lipoproteins, a-lipoproteins, and l3-lipoproteins which transport the secreted cholesterol in the blood. In part, cholesterol is decomposed by intestinal micro-flora; however, its major part is reduced to coprostanol and cholestanol which, together with a small amount of nonconverted cholesterol, are excreted in the feces. Cholesterol, mostly esterified, is utiliZed in the buildup of cell biomembranes. Besides, cholesterol is a precursor to biologically important steroid compounds: bile acids (in liver), steroid hormones (in adrenal cortex, male and female sexual glands, and placenta), and vitamin D3 , or cholecalciferol (in skin).

8.3 REGULATION OF LIPID METABOLISM IN THE ORGANISM The rate of lipid metabolism in the organism tissues is dependent on the dietary supply of lipids and on the neurohormonal regulation. An excessive intake of high-calory food (carbohydrates and triglycerides) impedes the consumption of endogenic triglyceride reserves stored in the fat tissues. Moreover, carbohydrates provide a very favourable basis for the neogenesis of various lipids; for this reason, a large

210

MICROBIOLOGY AND BIOCHEMISTRY

dietary intake of carbohydrate-rich food exerts a significant influence on the production of triglycerides and cholesterol in the organism. Synthesis of endogenic cholesterol is also controlled by exogenous cholesterol supplied in food: the more dietary cholesterol is digested, the less endogenic cho-Iesterol is produced in the liver. Exogenous cholesterol inhibits the activity of hydroxymethylglutaryl-CoA reductase and the cyclization of squalene to lanosterol. The dietary ratio of vru:ious lipids plays an important role in the lipid metab-olism in the organism. The available amounts of polyene fatty acids and phospho-lipids acting as solvents for fat-soluble vitamins affect not only the absorption of the latter species, but also the solubility and stability of cholesterol in the organism fluids (blood plasma and lymph) and biliary ducts. Vegetable oils with a high percentage of phospholipids and polyene fatty acids impede an excessive accumulation of cholesterol and its deposition in blood vessels and other tissues, and facilitate the removal of cholesterol excess from the organism. These processes are most markedly affected by corn oil, safflower oil, cottonseed oil, and sunflower oil. The consumption of unsaturated fatty acids contained in vegetable oils pro-duces a favourable effect on the synthesis of endogenic phospholipids (for which these acids are substrates); polyene fatty acids are also needed in the production of other materials, for example, prostaglandins. Unsaturated fatty acids act as uncouplers for the oxidative phosphorylation and thus accelerate oxidation processes in the mitochondria and control thereby an excessive triglyceride deposition in the tissues. TIle lipotropic factors exercise a marked effect on the biosynthesis of phospho-lipids and triglycerides. As has been mentioned above, they facilitate the phospho-lipid synthesis. The dietary deficiency of lipotropic factors favours the triglyceride production in the organism. Starvation elicits mobilization of triglycerides from the adipose tissue and inhibits the endogenic cholesterol synthesis owing to the low activity of hydroxy-methylglutaryl-CoA reductase. The latter process provides the possibility for the active production of ketone bodies in the liver. The neurohormonal control of lipid metabolism chiefly affects the mobilization and synthesis of triglycerides in the fat tissue. The lipolysis in tissues is dependent upon the activity of triglyceride lipase. All the regulators that favour the conversion of the inactive (nonphosphorylated) lipase to the active (phosphorylated) one, stimulate the lipolysis and the release of fatty acids into the blood. Adrenalin

METABOUSM OF FATS AND GLYCERIDES

211

and noradrenalin (secreted in the sympathetic nerve endings), honnones (glucagon, adrenalin, tbyroxin, triiodothyronine, somatotropin, 3lipotropin, corticotropin, etc.), tissue hormones, including biogenic amines (histamine, serptonin, etc.) act as stimulators for this process. Jnsulin, on the contrary, inhibits the adenyl ate cyclase activity, preventing thereby the formation of active lipase in the fat tissue, i.e. retards the lipolysis. In addition, insulin favours the neogenesis of triacyl-glycerides from carbohydrates, which, on the whole, provides for lipid deposition in the fat tissues as well as for the cholesterol production in other tissues. The thyroid hormones thyroxin and triiodothyronine assist in the oxidation of the cholesterol side chain and in the biliary excretion of cholesterol in the intestine.

8.4 PATHOLOGY OF LIPID METABOLISM Most commonly, the lipid metabolism pathology is manifest as hyperlipenaia (elevated concentration of lipids in blood) and tissue lipidoses (excessive lipid de-position in tissues). Normally, the lipid contents in the blood plasma are: total lipids, 4-8 g/litre; triglycerides, 0.5-2.1 mmolflitre; total phospholipids, 2.0-3.5 mmolflitre; total cholesterol, 4.0-8.0 mmol/litre (esterified cholesterol accounts for 2/3 of total cholesterol). Hyperlipemia may manifest itself by an increased concentration of lipids, or certain groups thereof. For example, hypercholesterolemia and hypertriglyceri-demia may be mentioned in this connectiori. Since practically all the blood plasma lipids make part of lipoproteins, hyperlipemias may be reduced to one of the hyper-lipoproteinemia forms which differ in the varied ratios of plasma lipoproteins of different groups. Distinguished are exogenous, or alimentary, hyperlipemias which are actually associated with a normally increased blood lipid concentration after the intake of a food high in fat, and endogenic hyperlipemias caused by impaired lipid metab-olism. The endogenic hyperlipemia may be due to a primary hereditary defect in apoproteins or in a lipid metabolism enzyme. However, of more frequent occur-rence are hyperlipemias attributable to secondary causes, for example, to regulatory disturbances of the lipid metabolism or to unfavourable environmental factors. Five types of primary hyperlipoproteinemias are distinguished. Hyperlipoproteinemia, Type I, is characterized by the enhanced content of chylo-microns in the blood plasma; simultaneously, the percentage of u- and f3-lipopro-teins may be lowered. The triglyceride

212

MICROBIOLOGY AND BIOCHEMISTRY

content is 8-10 times above the norm, while cholesterol does not exceed the normal level. Presumably, Type I is associated with a defective lipoprotein lipase that destroys chylomicrons. Hyperlipoproteinemia, Type II. is characterized by an increased I-lipoprotein content in the blood plasma and, respectively, by a 1.52-fold higher, against the norm, cholesterol concentration. The familial form of hyperlipoproteinemia, Type Ila, is also known, which manifests itself in the occurrence of a defective apoprotein for Plipoproteins, and in a slower breakdown of these materials in the tissues. Hyperlipoproteinemia, Type III, is a rare hereditary disease (also called familial dysbetalipoproteinemia) manifested by the occurrence of an uncommon P-lipo-protein form. Cholesterol and triglyceride contents in the patients may occasion-ally be 2-5 times superior to the norm. Hyperlipoproteinemia, Type IV, is characterized by increased contents of pre-p-lipoproteins and triglycerides (2-5 fold) in the blood plasma. Its incidence rate is higher in aged patients. Hereditary forms of this disease (called also familial hyperprebetalipoproteinemia) have been described. Hyperlipoproteinemia, Type V. This pathology is manifested by increased con-tents of chylomicrons, pre-p-lipoproteins, triglycerides, and cholesterol in the patients' blood plasma. Secondary hyperlipoproteinemias, which arise from a disordered lipid tissue metabolism or its impaired control, are observed in diabetes mellitus, thyroid gland hypofunction, alcoholism, etc. Tissue Lipidoses. Hyperlipoproteinemias may lead to tissue lipidoses. Lipidoses can also arise from hereditary defects of the enzymes involved in the synthesis and breakdown of lipids in the tissues. We now discuss certain instances of tissue lipidoses. Atherosclerosis is a wide-spread pathology, manifested chiefly by the deposition of cholesterol in arterial walls, which results in the formation of lipid plaques (atheromas). Lipid plaques are specific foreign bodies around which the connective tissue develops abnormally (this process is called sclerosis). This leads to the cal-cification of the impaired site of a blood vessel. The blood vessels become inelastic and compact, the blood supply through the vessels is impeded, and the plaques may develop into thrombi. Atherosclerosis results from hyperlipoproteinemia. All of the lipoproteins, ex-cepting chylomicrons, are capable of penetrating the

METABOLISM OF FATS AND GLYCERIDES

213

vessel wall. However, a-lipo-proteins, which are rich in proteins and phospholipids, are liable to an easy break-down within the vessel Wall, or are apt to leave it because of their small size. ~-Lipoproteins and, partly, pre-~-lipoproteins containing much cholesterol exhibit atherogenic properties. Elevated concentrations of lipids of these groups and an increased vessel wall permeability are conducive to deposition of atherogenic lipoproteins within the walls, with the subsequent development of atherosclerosis. Fatty infiltration of the liver. In this pathology, the triglyceride concentration in the liver is lO-fold superior to the norm.' The accumulation of fat in the cyto-plasm of hepatic cells leads to an impaired liver function. The causes of this pathol-ogy are numerous; one of these may be a deficiency in lipotropic factors and the associated therewith synthesis of excess triglycerides. Ketosis is a pathologic state produced by an excess, of ketone bodies in the organism. However, ketosis may be regarded as a lipid metabolism pathology with a certain reserve, since excessive biosynthesis of ketone bodies in the liver is sequent upon an intensive hepatic oxidation not only of fatty acids, but also of keto-genic amino acids. The breakdown of the carbon frameworks of these amino acids leads to the formation of acetyl-CoA and acetoacetyl-CoA, which are used in ketogenesis. The ketosis is accompanied by ketonemia and ketonuria. which is manifested by the increased concentration of ketone bodies in blood and their ex-cretion in the urine. In an aggravated form of ketosis, the ketone body concentra-tion in blood may be as high as 10-20 mmolllitre. The ketone bodies are normally present in the daily urine in trace amounts, while in pathology, 1 to 10 g (or even more) of ketone bodies per day is excreted in the urine. Most commonly, ketonemia and ketonuria are observed in diabetes mellitus (the manifest ketosis symptoms are dependent on the extent of diabetes mellitus), as well as in prolonged starvation or in "steroid" diabetes.

8.5 APPLICA TIONS OF LIPIDS AND THEIR COMPONENTS IN PHARMACOTHERAPY Fat-emulgated preparations for parenteral administration have been elaborated for clinical applications. Since these are administered to the patients intravenously, the size of fat emulsion particles should not exceed the size of the largest naturally occurring lipoproteinschylomicrons, i.e. about I JLm. Fat emulsions on the basis of corn oil (preparation lipomaize), cottonseed oil (lipofundin, lipomo!),

214

MICROBIOLOGY AND BIOCHEMISTRY

soybean oil (intralipid) have been proposed. These preparations are composed of lipids (10 to 20%), emulsifying agents (phosphatides and other materials) and, occasion-ally, glycerol. They are prescribed to asthenic patients for increasing energy re-sources of the organism. In addition, lipotropic preparations (methionine, choline, and inositol), which make part of natural phospholipids, are used in the prophy-Iaxis of fatty infiltration of the liver.

9 Metabolism of Nucleic Acid The discovery of the base-paired, double-helical structure of deoxyribonucleic acid (DNA) provides the theoretic framework for determining how the information coded into DNA sequences is replicated and how these sequences direct the synthesis of ribonucleic acid (RNA) and proteins. Already clinical medicine has taken advantage of many of these discoveries, and the future promises much more. For example, the biochemistry of the nucleic acids is central to an understanding of virus-induced diseases, the immune re-sponse, the mechanism of action of drugs and antibiotics, and the spectrum of inherited diseases. In approaching the study of the molecular mechanisms of heredity, this chapter first discusses the structural and functional roles of the genetic material, DNA. This includes an analysis of its replication and susceptibility to mutation. The health-related aspects of the use of recombinant DNA techniques are considered, and examples of their use in the analysis of several human genetic diseases are used to illustrate the biochemical side of genetics.

9.1 FUNCTIONAL ROLES OF DNA 9.1.1 DNA as the Genetic Material The nucleic acids were recognized as chemical substances more than 70 years before DNA was found to be responsible for the transmission of inherited characteristics. Later it was suspected that DNA might be the genetic material because of its high concentration in chromosomes and in some viruses. The premise was complicated, however, because the concentration of protein in these structures was 215

216

MICROBIOLOGY AND BIOCHEMISTRY

also high. Furthermore, RNA but not DNA was found in some viruses. Indirect evidence pointed to a role for nucleic acids as the transmitters of biologic information; the wavelengths of light in the ultraviolet region that are the 'most mutagenic are the same wavelengths at which nucleic acids absorb the most light energy. 9.1.1.1 Constancy of DNA concentration One property expected of the genetic material is a constancy of amount in every cell of the body under every environmental situation. DNA, not RNA or protein, fulfills this expectation. Its content per nucleus is the same in every cell except the germ cells, which have exactly half that found in the somatic cells. Again, this is expected if progeny obtain half their characteristics from each parent. This constancy is so dependable that the measurement of the DNA concentration in a tissue can be used to calculate the number of nuclei and thus the number of cells. This works well for diploid cells such as those of the kidney" but corrections must be made for polyploid mammalian liver or cancer cells. 9.1.1.2 Transformation of ceUs with DNA The best evidence that exogenous DNA can produce permanent changes in cells came from the experiments of Avery et al. DNA from one strain of bacterial cells was used to transform a different strain of cells so that they came to resemble the strain from which the DNA was derived. In the original experiment, DNA was isolated from' cells of a strain of Diplococcus pneumoniae that contained a characteristic complex polysaccharide on their surfaces. This polysaccharide made the cells pathogenic for mice and gave a glistening, smooth appearance to colonies formed by these cells on nutrient agar. When the polysaccharide was missing, as it was in some other strains of the microorganism, the colonies were rough in appearance and the cells were harmless when injected into mice. When DNA from the smooth cells was added to rough cells, the DNA entered some of the cells and became a permanent part~,f their genetic apparatus; subsequent generations were permanently changed to pathogenic cells that formed smooth colonies. This process is called bacterial transformation. Subsequently, similar experiments were done with viral nucleic acids. The pure viral nucleic acid, when added to cells, led to the synthesis of complete virus particles; the protein coat was not required. This process is called transfection. More recently, DNA has been used in cell-free extracts to program the synthesis of RNA that functions as the template for the synthesis of proteins characteristic of the DNA

217

METABOLISM OF NUCLEIC ACID

template. Considering all this evidence, DNA undoubtedly is a carrier of genetic infonnation.

9.1.2 Cellular Location of DNA Most of the DNA of animal cells is found in the nucleus, where DNA is the major constituent of the chromosomes. On the other hand, most of the RNA is located in the cytoplasm. Nuclear DNA exists as a thin, double helix only 2 nm wide. The double helix is folded and complexed with protein to form chromosomal strands approxim-ately 100 to 200 nm in diameter. Each chromosome contains a single DNA duplex. The human chromosomes vary in size; the smallest contains approximately 4.6 x 10' base pairs of DNA, and the largest 2.4 x 10' base pairs. In contrast, the Escherichia coli chromosome has 4.5 x H)6 base pairs. The DNA of the chromosomes is tightly packed and associated with both histone and nonhistone proteins. The amount of genomic DNA in a particular organism is roughly proportional to the c.,omplexity of the organism. Table shows the content of DNA in the genomes of several widely different organisms. The data are normalized to a haploid set of chro-mosomes, since some cells listed are haploid and others are diploid. The DNA content TABLE 9.1 : DNA CONTENT OF SOME CELLS AND VIRUSES. Source of DNA Viruses SV40 Papilloma (wart) Adenoviruses Herpesviruses Poxviruses Cells

Haploid size of genome, base pairs 5 8 2.1 1.56 2.4

x x x x x

1()3 1()3 100 l()5 l()5

4.5 x 1()6 Yeast 1.3 x 10' 1.6 x loa Drosophila 3.2 X 109 Human 1.5 x lOA Animal mitochondria of a few viruses is given for comparison. The size of DNA is often measured in base pairs, since cellular DNA is all double stranded and base paired. Thus the number of base pairs in a DNA molecule is a precise measure of the number of mononucleotides that comprise the Escherichia coli

218

MICROBIOLOGY AND BIOCHEMISTRY

polynucleotide chain. On the average, one base pair represents about 600 daltons; thus, to estimate the molecular weight of a DNA, the number of base pairs is multiplied by 600.

9.1.2.1 Histones and chromosome strocture The chromosome structure is visible only during the mitotic portion of the cell cycle. The constituent parts of the chromosomes· are nucleoprotein fibers called chromatin. When condensed, chromatin fonns a microscop-ically visible chromosome-like structure. The chromosomes are composed of DNA, RNA, and proteins. The relative amounts of the three vary, but chromatin is primarily protein and DNA. 9.1.2.1.1 IVucleosomes The his tones are the major proteins associated with the chromosomes. These small, basic proteins can be separated into five groups by polyacrylamide gel electrophoresis. All five histone groups are found in every eukaryotic cell. These groups are called HI, H2A, H2B, H3, and H4. Each histone is present in equimolar amounts except for HI, which is present in approximately half the concentration of the others. Furthermore, the HI electrophoretic band is composed of many similar but slightly different proteins. In this respect the HI group differs from the other histone groups, which are each single proteins. The histone groups differ in their relative content of lysine and arginine residues. Table lists some of the properties of these chromosomal proteins. The sequences of histones H2A, H2B, H3, and H4 are greatly conserved between species, even though an organism might have several genes for the same histone. This virtual sequence identity testifies to a very similar and essential function for the four histones in all eukaryotic species. A clue to this function is the ability of these histones to associate at high ionic strength to form an octomer containing two copies of each of the four histone groups. 9.1.2.1.2 Histones of the nucleosome core The four histone groups that are composed of ho-mogeneous proteins, H2A, H2B, H3, and H4, make up the nucleosome core. Each core consists of two copies of the four histones. The double-stranded DNA is wrapped twice around each core in a left-handed superhelix. A superhelix is the name given to the additional helix made by the doublestranded, helical DNA as it is wrapped around the nucleosome core. A familiar superhelix in everyday life is a twisted spiral telephone cord. The nucleosome core of histones do not recognize specific DNA structures; rather, they can bind to any stretch of DNA as long as it is not too close to a neighboring nucleosome. The order of contact of histones to the DNA is as follows:

219

METABOLISM OF NUCLEIC ACID

TABLE 9.2 : PROPERTIES OF ANIMAL mSTONES. Electrophoretic group HI H2A H2B H3 H4

Mass (kilo-

daltons)

Lysine (%)

21 14.5

Z7 11

13.8 15.2 11.3

16 10 10

Arginine (%) 2 9 6 15 14

Protein-protein interactions between the histone subunits are undoubtedly important in promoting formation of a nucleosome in which 146 base pairs of DNA are coiled around the outside of the histone core. One molecule of histone HI binds to an exterior region of each nucleosome, but histone Hi is not needed to determine nucleo-some structure. The distance between nucleosomes is approximately 200 base pairs; consequently, in electron micrographs, nucleosomes resemble evenly spaced beads on a string of DNA. Neutron and x-ray diffraction data are also consistent with this structure. The histone core protects the DNA bound to the nucleosome from digestion by pan-creatic deoxyribonuclease (DNase) I or micrococcal nuclease. Nucleases, however, will cleave the linker DNA that connects the nucleosome subunits to one another. Nucleosornes can be reconstructed in the laboratory from DNA and pure histones. Histone HI is not necessary for the reconstruction, which further shows that HI is an accessory protein and not a major structural part of the nucleosome subunit. The primary function of the nucleosomes is to condense DNA. Further condensation of nucleosome DNA requires nonhistone nuclear proteins. These proteins make up a scaffoldlike structure around an additional helix consisting of coiled nucleosomes. This produces a structure that resembles a solenoid, with six nucleosome subunits per turn. The solenoid structure can form large loops that give additional structure to the incipient chromosome.

~

9.1.2.1.3 1kuuied chromosomes Although it is not known how the characteristic banded structure of

220

MICROBIOLOGY AND BIOCHEMISTRY

a chromosome is related to its function, the DNA of a single chromosome probably consists of a single DNA duplex running from one end of the chromosome to the other. These bands, which can be seen microscopically after staining with fluorescent dyes such as Giemsa or quinacrine, are believed to represent regions of heterochromatin complexed with histones and nonhistone proteins. 9.1.2.1.4 Nonhistone proteins The nucleus contains a large number of proteins other than histones. These so-called nonhistone proteins mayor may not be tightly associated with the chromosomes. For example, the nucleus contains enzymes associated with the synthesis of RNA and DNA; these are nonhistone proteins, but they are not part of the structure of chromosomes. One group of nonhistone proteins are the high mobility group (HMG) proteins, named for their rapid movement on polyacryl-amide gel electrophoresis. The HMG proteins, but not histone HI, are ~sociated with the chromatin that is most active in RNA synthesis. 9.1.2.1.5 Mitochondrial nucleic acid Not all the cellular DNA is in the nucleus; some is found in the mitochondria. In addition, mitochondria contain RNA as well as several enzymes used for protein synthesis. Interestingly, mitochond-rial RNA and DNA bear a closer resemblance to the nucleic acid of bacterial cells than they do to animal cells. For example, the rather small DNA molecule of the mitochondrion is circular and does not form nucleosomes. Its information is contained in approximately 16,500 nucleotides that func-tion in the synthesis of two ribosomal and 22 transfer RNAs (tRNAs). In addition, mitochondrial DNA codes for the synthesis of 13 proteins, all components of the respiratory chain and the oxidative phosphorylation system. Still, mitochondrial DNA does not contain sufficient information for the synthesis of all mitochondrial proteins; most are coded by nuclear genes. Most mitochondrial proteins are synthesized in the cytosol from nuclearderived messenger RNAs (mRNAs) and then transported into the mito-chondria, where they contribute to both the structural and the functional elements of this organelle. Because mitochondria are inherited cytoplasmically, an individual does not necessarily receive mitochondrial nucleic acid equally from each parent. In fact, mito-chondria are inherited maternally.

9.1.3 Clinical Comment

9.1.3.1 Leber's Hereditary Optic Myopathy This disease is one of several myopathies caused by defects in the

221

METABOLISM OF NUCLEIC ACID

mitochondrial genome; thus they are called mitochondrial myopathies. Leber's hereditary optic neuropathy causes blindness in young males more often than females, even though the disease is transmitted maternally. The myopathy is caused by a single base mutation at position 11,778 of the mitochondrial DNA that changes an arginine codon to a histidine codon in subunit four of NADH-coenzyme Q oxidoreductase. This enzyme is part of complex I of the respiratory chain. 9.1.4 Other Conformations of DNA A -DNA The Watson-Crick model of DNA is based on the x-ray diffraction patterns of B-DNA. Most DNA is B-DNA; however, DNA may take on two other conformations, A-DNA and Z-DNA. These conformations are greatly favored by the base sequence or by bound proteins. When B-DNA is slightly dehydrated in the laboratory, it takes on the A conformation. A-DNA is very similar to B-DNA except that the base pairs are not stacked perpendicular to the helix axis; rather, they are tilted because the deoxyribose moiety "puckers" differently. An A-DNA helix is wider and shorter than the B-DNA helix.

o

o n

11

-O-P-O-CH

I

0-

-O-P-O-CH

•0

I

·0

00-

o-P~O-

U

Syn

o

0-

o-P~O-

n o Anti

Z-DNA This conformation differs more radically. It is a left-handed helix instead of the right-handed conformation of A-DNA and B-DNA. The Z-DNA conformation exists only along a string of alternating purines and pyrimidines, especially several guanine-cytosine residues in a row. An alternating dinucleotide sequence results where the external phosphate groups zigzag, thus Z-DNA. This structure results from alternating anti and syn conformations of the glycosidic bonds. In A- and B-DNA the conformations of the glycosidic bonds are all anti.

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MICROBIOLOGY AND BIOCHEMISTRY

In Z-DNA, guanine residues are syn, whereas cytosine and thymine residues are anti. Eukaryotic DNA contains several alternating purinepyrimidine sequences consistent with the Z-DNA confonnation; however, the biologic significance of Z-DNA is still unclear.

9.1.5 The "Central Dogma" DNA has two broad functions: replication and expression. First, DNA must be able to replicate itself so that the information coded into its primary structure is transmitted faithfully to progeny cells. Second, this information must be expressed in some useful way. The method for this expression is through RNA intermediaries, which in tum act as templates for the synthesis of every protein in the body. The relationships of DNA to RNA and to protein are often expressed in a graphic syllogism called the "central dogma.» The concept was proposed by Crick in 1958 and was revised in 1970 to accommodate the discovery of the RNA-dependent DNA polymerase. Crick's original theory suggested that the flow of information was always from RNA to protein and could not be reversed, yet it allowed for the possibility of DNA synthesis from RNA.

,,

, \ ~

RNA - - - - - - - Protein

Figure 9.1 : The "central dogma."

Genetic expression involves the transfer of information by the processes of transcription and translation. Transcription is the process that transfers information using the same four-letter language of the nucleic acids; that is, one strand of DNA serves as a template for the synthesis of an RNA strand, the sequence of which is analogous to one DNA strand and complementary to the other. Transcription is "reversible" in a few cases. The dashed line in Figure represents the synthesis of DNA from information contained in the RNA of certain tumor viruses. Information flow in the direction of RNA to protein is termed translation, since the four-letter language of the nucleic acids must be converted to the different 20-letter language of the amino acids that make up proteins. The process of translation is always unidirectional. Single-stranded DNA templates can be translated in the laboratory, but

METABOLISM OF NUCLEIC ACID

223

no evidence exists for such a function in vivo; consequently the line between DNA and protein in Figure is dashed.

9.1.5.1 DNA synthesis Knowing that DNA was the hereditary material gave no clues as to how the molecule might reproduce itself until Watson and Crick proposed their model for the structure of DNA. In this model the DNA strands are arranged in an antiparallel fashion and are base paired along their entire length in the form of a double helix. The molar concentration of adenine equals that of thymine, and the cytosine concen-tration is the same as that of guanine. Base pairing of adenine with thymine and of cytosine with guanine yields a structure in which the sequence of one strand can be automatically determined if the sequence of the other strand is known. The importance of this concept in the replication of DNA and in the synthesis of RNA strands of com-plementary sequences was recognized immediately, but several years were required for the enzymatic studies that gave unequivocal proof.

:" ~ . S~~) S +

...... . . . . . ,..'

""

4

Semiconservative replication

S~Conservativa~ S +

':-:'. ,'~-:::

...

replicatiOn

"

Maternal strands

Double-strandad DNA

Daughter strands

9.1.6 Strand Separation I1nplicit in the functioning of the Watson-Crick DNA model is the idea that the strands of a DNA molecule must separate and new daughter strands must be synthesized in response to the sequence of bases in the mother strand. This is called semiconservative replication. Still, conservative replication, in which both strands of a daughter molecule are newly synthesized, could not be ruled out by consideration of the structure of DNA alone. The experiments of Meselson and Stabl proved replication to be semiconservative. These consisted of growing E. coli cells in a medium containing 'sNH 4Cl, so that the nitrogen atoms of the purine and pyrimidine bases of the DNA were heavily labeled. Cells were then transferred to a medium containing the usualligbt 14NH4Cl and grown for one or more generations. The DNA was then prepared and separated by density-gradient equilibrium centrifugation in a solution of cesium

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MICROBIOLOGY AND BIOCHEMISTRY

chloride. After one generation the progeny DNA separated in such a way that all of it appeared at a position midway between the very heavy parental DNA and the light DNA of a control culture. Because all the DNA existed as a hybrid that contained one heavy strand and one light strand, DNA clearly was replicated by a semiconservative mechanism. This presented a more difficult problem: How do the double-helical strands separate during DNA synthesis? In a rapidly growing cell such as E. coli it has been calculated that if the strands separate by untwisting, the molecule would have to rotate at 10,000 rpm, a rate that is highly improbable. The answer to this problem lies in an understanding of the mechanism of DNA replication at the enzyme level. We will return to this subject after first considering the enzymes involved in DNA synthesis. 9.1.7 DNA Polymerases DNA synthesis is more complex than originally thought. One reason is that DNA rep-lication requires many different enzymes, not just DNA polymerase. For example, rep-lication requires enzymes that coordinate the growth of cell membranes with DNA syn-thesis. Other enzymes and protems initiate the synthesis of small RNA primers that bind to single-stranded DNA. Additional enzymes are needed to remove the RNA primers from the growing deoxyribonucleotide chain, fill in the small regions vacated by the RNA primers, seal the strands together, and aid in the untwisting of the DNA helix. More than one enzyme may be required for each of these functions, and this list of functions is not meant to be complete. 9.1.8 Bacterial DNA Polymerases The mechanisms involved in DNA synthesis are most easily understood by considering the DNA polymerases. The most extensively studied are the three DNA polymerases, I, II, and III, from E. coli. Some ambiguity still exists about the essentiality of the specific roles played by each of the polymerases. One complicating feature is that it _ is difficult to distinguish the polymerases from enzymes that function exclusively to repair damaged DNA, primarily because some of the processes that occur during DNA replication are identical to events necessary for DNA repair. However, all the DNA polymerasees require a DNA template and all four of the de-oxyribonucleoside triphosphates. Synthesis proceeds from the 5' to the 3' end of the growing polynucleotide, arid inorganic 'pyrophosphate (PP) is a product of the reaction. Polynucleotides formed using radioactive deoxyribonucleoside

I

225

METABOLISM OF. NUCLEIC ACID

triphosphates have sequences identical to those of one strand of the DNA template and complementary to sequences of the other strand. Both strands are labeled in vitro. DNA polymerase I is a nonessential enzyme, since viable E. coli mutants lack it (pol A). This conclusion is complicated, however, since the enzyme catalyzes three separate chemical reactions. It polymerizes deoxyribonucleoside triphosphates, and it has two exonucleolytic activities, a 3' to 5' activity and a 5' to 3' activity. The pol A - mutants lack only the polymerization activity. Other mutants lacking both the polymerase and the 5' to 3' exonuclease activity are lethal. Thlis the exonuclease function is the more important one. This fits with the role of this enzyme in removing damaged DNA segments (DNA repair) and in removing covalently attached RNA from DNA chains. We will later see that small RNAs serve as primers of DNA synthesis. dATP dCTP

DNA polymerase I

---------~..

dGTP

DNA template Mg++

DNAPoIymer + PPj

dTTP I Figure 9.2 : Reaction catalyzed by DNA polymerase I. dATP

DNA polymerase I has been purified to homogeneity. When the pure enzyme is treated with subtilisin, a proteolytic enzyme from Bacillus subtilis. the polymerase is cleaved into two pieces. The small fragment retains the 5' to 3' nuclease activity, whereas the larger piece, called ,a Klenow fragment, has both polymerase activity and the 3' to 5' exonuclease activity. The Klenow fragment is sold commercially for use in labeling DNA for use in detecting recombinant DNA. DNA polymerase II is more likely needed for the repair synthesis of DNA. Repair synthesis requires excision of the damaged DNA, the synthesis of a fresh replacement segment complementary to the remaining single strand, and the sealing of the replacement segment to the larger polynucleotide chain. DNA polymerase II does not have 5' to 3' exonuclease activity. Mutants deficient in DNA polymerase II activity, as determined by in vitro assay, grow well; therefore the enzyme does not seem to have an indispensable function in the cell. DNA polymerase III has all the enzymatic activities of DNA polymerase I. A subunit of the enzyme is the product of the dna E gene. Temperature-sensitive mutations of this gene testify to the importance

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MICROBIOLOGY AND BIOCHEMISTRY -

of DNA polymerase III. A temperature-sensitive mutant is one that grows at 30· C but fails to grow at 42° C, a temperature not lethal for wild-type E. coli. The failure to grow at the higher temperature is caused by a mutation in the gene for DNA polymerase III so' that a very heat-labile enzyme is produced. Since this appears to be the only mutation in this strain, DNA polymerase III is the only enzyme inactivated at 42° C; thus the enzyme is essential to the organism. 9.1.8.1 Template and primer At this point it is necessary to make a distinction between the meanings of template and primer. The word template refers to the structural sequence of the polymerized monomeric units of a macromolecule that provides the pattern for the synthesis of another macromolecule with a complementary or characteristic sequence. The word primer, on the other hand, refers to a polymeric molecule that contains the growing point for the further addition of monomeric units. Glycogen is an example of a primer to which glucose units are added; however, glycogen has no template activity. Under certain circumstances DNA has both primer and template activities. For example, the addition of mononucleotides is to the 3' end of the growing DNA primer. This presents a problem with regard to how the other strand is synthesized. Biochemists have looked hard but unsuccessfully for an enzyme that can add deoxyribonucleotides onto the 5' end of DNA primers. Such a primer should contain a triphosphate on the hydroxyl group of the 5' end. Although a very active 5'-exonuclease, actually part of DNA polymerase I, has made the search for such an activated 5' end extremely difficult, investigators conclude that a polymerase able to use such a primer probably does not exist. On the contrary, good evidence suggests that the synthesis of both strands is by the known DNA poly-merases. 9.1.9 Stages of DNA Synthesis 9.1.9.1 Origin of Replication In E. coli cells, DNA replication starts at a specific site called oriC. The oriC locus contains only 245 base pairs. Similar sequences are responsible for initiating the synthesis of plasmid and bacteriophage DNA. The oriC nucleotide sequence binds several units of the tetrameric form of the dnaA protein. This protein is named for the gene that encodes it. The dnaB and dnaC proteins then bind to the complex. As a result of binding these proteins, a portion of the helical DNA is unwound. This forces the rest of the DNA into a left-handed double helix that wraps around the proteins to give a structure

METABOLISM OF NUCLEIC ACID

227

resembling the histone-containing nucleosome of eukaryotic cells. The exposed single-stranded DNA is stabilized by the binding of a 74 kDa, single-stranded DNA-~inding protein called SSB.

9.1.9.2 RNA primers All DNA polymerases add mononucleotides to the 3'end of an existing primer. Consequently a special primer is needed for DNA to replicate in its entirety. RNA polymerases can initiate polymer synthesis without a primer; thus short RNA primers are used to initiate DNA synthesis. The RNA oligonucleotides are complementary to a sequence on one of the strands of the DNA template and base pair with a portion of the DNA molecule. Subsequently, deoxyribonuc1eotides are covalently attached to the RNA primer. The synthesis of the primer itself is catalyzed by a special RNA polymerase called primase. Similar RNA polymerase-like enzymes are used to prime the synthesis of certain viral DNAs and eUkaryotic DNA. The dnaB and dnaC proteins, as well as at least a few other accessory proteins, are required for the primase to initiate RNA synthesis from the DNA template. This enzyme complex, called a primasome. is very large, almost as large as the DNA polymerase III holoenzyme (800 kDa) , which joins the primasome and catalyzes the addition of mono-deoxyribonuc1eotides to both growing strands. No problem exists in visualizing the ad-dition of oligonucleotide monomers to the RNA primer at the 5' end of the continuous strand, since this DNA polymerase is a highly accurate, processive enzyme. Processivity means that the polymerase can rapidly add many mononucleotides to the primer, more than 1000 per second, before dissociating from it. On the other strand, DNA synthesis must proceed away from the replicative fork; however, if the template strand is looped back toward the replicative fork, subunits of the DNA polymerase could add nucleotides to both growing strands. Addition to an RNA primer would continue until synthesis was blocked by the previously made primer and its attached oligodeoxyribonuc1eotide. This stalling might trigger the synthesis of a new RNA primer and the addition of deoxynucleotides to it. The mechanism of DNA synthesis is known in considerable detail so the steps illustrated in Figure are very much simplified. 9.1.10 Bidirectional Synthesis DNA synthesis occurs in both directions at each of the rep-licating forks. Once a DNA strand has been primed, synthesis toward the replicating fork can be visualized as continuous. Growth of the opposite,

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MICROBIOLOGY AND BIOCHEMISTRY

New DNA, continuous strand Figure 9.3 : A single unit of DNA polymerase m complex synthesizes both new strands of DNA. one continuously and the other in short pieces. Deoxynucleotide additon to the daughter strands is indicated by vertical lines across the strands.

lagging strand occurs in dis-continuous bursts, each burst primed by a short RNA segment. DNA synthesis visualized by electron microscopy gives the appearance of an "eye" or several eyes along a DNA template and resembles this: Eyes are thought to be regions of DNA where recent synthesis has been initiated. Synthesis from an eye is bidirectional. Thus as the eye enlarges, DNA synthesis along either new strand may be considered continuous where the DNA polymerase is close to and moving toward the replicating fork, and it may be considered discontinuous where the DNA polymerase is close to a replicating fork but moving away from the fork. Consequently, as an eye enlarges, DNA synthesis is more or less continuous at one end of a growing strand but discontinuous at the other end of the same strand.

9.1.10.1 Removal of the RNA primer and ligation of the DNA fragments The end result is newly synthesized DNA that is interspersed with segments of RNA and that is discon-tinuous but base paired with an intact parental strand. Subsequently, the 5' exonucleolytic activity of DNA polymerase I removes the RNA segment, and either DNA polymerase I or II fills the gap vacated by the RNA. DNA ligase (sometimes called polynucleotide ligase) is required to join these short pieces into phosphodiester linkage. The ligation reaction shown in the following diagram requires that energy be supplied from ATP. This enzyme also occurs in animal cells.

229

METABOLISM OF NUCLEIC ACID ATP + Ugase ~ Ligase - AMP + PP!

H

LigaIIe - AMP +

:',-'-""T'~' 0

PO:

O"",,-,,---r-,-r,:: __ I

Ugase+

O· O-P-O

AMP +

I ~~I..... 1

11

0

I

"'r---I-r--T""I-'-1

DNA ligase is not only important in DNA replication; it is also used to seal deoxyri-bonucleotide segments in the crossover events during gene recombination. The enzyme also functions to close breaks in segments of DNA undergoing repair and is required to join theends of mitochondrial DNA to form their characteristic circular structure.

9.1.10.2 Topoisomerases Armed with this information, the unwinding problem menfioned ear-lier can be reconsidered. By the alternating action of endonucleolytic and ligase activities, the unwinding of DNA could be reduced to an untwisting of only a small part of the double helix at any given time. Both activities are part of the enzyme called topo-isomerase 1.

9.i.1O.2.1 Topoisomerase 1 This enzyme releases the torque developed during the unwinding required for replication. This torque introduces superhelices into DNA. A superhelix can be visualized as a helix on top of the basic DNA helix. The enzyme first introduces a single-strand break in the superhelix. This is not a hydrolytic cleavage but rather a transesterifica-tion of the 5' phosphoryl at one end of the broken strand to a tyrosine hydroxyl group, thus conserving the energy of the phosphodiester bond. The single-strand break relieves the torque as the broken strand with the enzyme still attached rotates about the unbroken strand. When the strain on the double helix is relaxed, the enzyme transfers the 5' phosphorylated end back to the polynucleotide chain and dissociates from the DNA duplex. Actually the broken strand need only pass through the neighboring intact strand and reseal to remove one superhelical turn. To relieve the tension of several superhelical turns, as occurs during DNA replication, the topoisomerase catalyzes several "nicking-closing" reactions. Topoisomerase I recognizes either positively or negatively supercolIed DNA. Topoisomerase I activity has also been found in the nuclei of animal cells.

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MICROBIOLOGY AND BIOCHEMISTRY

9.1.10.2.2 Topoisomerase II Another enzyme, called topoisomerase II, or DNA gyrase, also plays a role in the unwinding of replicating DNA. Although topoisomerase I can relieve the positive superhelical torsion introduced into DNA as a result of unwinding, topoisomerase II can introduce negative superhelices ahead of the replicating fork. This relieves the twisting pressure of DNA replication before it can develop. The enzymes also differ in that topoisomerase I does not require high energy in the form of adenosine S'-triphosphate (ATP), whereas topoisomerase II does, since energy is required to make negatively supercoiled DNA. This torsional energy is conserved in the negative superhelices found in most naturally occurring DNA, such as the DNA of nucleosomes. The topoisomerases also differ in that enzyme I cleaves only one DNA strand, whereas enzyme II cleaves both. Approximately 200 base pairs of DNA coil about topoisomerase II, much as occurs with the DNA in a nucIeosome. Both strands are opened, and the 5' phosphoryl groups are linked to tyrosine hydroxyl groups on the enzyme. A DNA segment is passed through both the anchored but cleaved ends. This passage is always in the same direction, so that only a negative superhelix forms when the strands are resealed. A DNA gyrase-like activity has been isolated from animal cells.

9.2 DNA SYNTHESIS IN ANIMAL CELLS The replication process in animal cells is necessarily more complex than in bacteria because several chromosomes must be replicated. DNA syniliesis in animal cells also differs in that several origins of replication occur within a single chromosome rather than the single site in E. coli. This speeds up the duplication of the animal genome, which is approximately 1000 times larger than that of bacteria. The eukaryotic origins of replication have a high affinity for the nuclear matrix, the nucleoprotein material that remains after nuclei have been washed with a high concentration of salt. DNA polymerases from several different animal cells have been isolated and studied. The three DNA polymerases of animal cells, called a, /3, and y, can be distinguished by their molecular weights, template specificity, and sensitivity to sulfhydryl reagents. Table 14.3 compares the three in regard to these differences. DNA polymerase a is probably the most important for DNA replication. This enzyme shares many functional properties with DNA polymerase III of E. coli: 9.2.1 DNA Polymerase a. Even though DNA polymerase a and its associated subunits have

METABOLISM OF NUCLEIC ACID

231

not been purified to homogeneity, much is known about the function of this enzyme complex. The concentration of DNA polymerase a is higher than that of the other two polymerases. One of the associated subunits has primase activity capable of making short RNA primers. At frrst this enzyme was thought to be present in the cytoplasm of cells, but with ~pecial precautions it can be isolated from the nuclei. Unlike E. coli DNA polymerase I, polymerase a has no associated nuclease acitivity. It is membrane-bound: however, and fractionates with ribonucleotide reductase, dTMP synthase, and thymidylate kinase, enzymes important in the synthesis of DNA precursors. The synthesis of poly-merase a increases greatly in regenerating liver and in other rapidly dividing cells.

9.2.2 DNA Polymerase J3 This is a smaller, stable enzyme that has been highly purified. It is immunologically distinct from the other polymerases, indicating that it is not merely a subunit of the larger polymerases. Polymerase 13 is undoubtedly a repair enzyme. 9~

DNA Polymerase y This is the enzyme responsible for the synthesis of mitochondrial DNA and the DNA of some viruses, such as adenoviruses. Polymerase -y is very large and consists of a tetramer of identical oligomers, each having a molecular weight of 47,000. Synthetic ribonucleotides are very effective· templates in the laboratory, but this mitochondrial enzyme differs from reverse transcriptase in that natural RNAs are poor templates.

9.2.4 Reverse Transcriptase This enzyme is associ.ated with the virions of RNA tumor viruse& such as the ROllS sarcoma virus (RSV). The enzyme has remarkable enzymatic activity in that it can catalyze several seemingly diverse steps in the synthesis of double-stranded DNA from the single-stranded RNA viral genome. The enzyme uses a tRNA for tryp-tophan as a , primer to make a copy of DNA that is complementary to the viral RNA. The resulting RNA-DNA hybrid is converted to a double-stranded DNA molecule by ribon-uclease (RNase)H and DNA-dependent DNA polymerase activities that are intrinsic to reverse transcriptase.

9.2.4.1 Replication of linear eukaryotic chromosome Eukaryotic chromosomes, unlike their bacterial counterparts, are linear rather than· circular. Since RNA oligonucleotides prime both prokaryotic and eukaryotic DNA synthesis, the 5' termini of the daughter

TABLE 9.3: COMPARISON OF PROPERTIES OF DNA POLYMERASES FROM ANIMAL TISSUES. ~

Property

DNA polymerase a

DNA polymerase

DNA polymerase 'Y

Molecular weight

155,000, plus three other subunits

43,000

193,000 (four oligomers)

Template specificity

Nicked DNA template, RNA primer

Nicked DNA template, DNA primer

Ribonucleotide template and DNA primer

Deoxyribonuc1eoside triphosphate dependence

All four required

All four work, but single nucleotide will incorporate

All four

Inhibition by sulfhydryl reagents

Sensitive

Less sensitive

Sensitive

METABOLISM OF NUCLEIC ACID

233

strands are incomplete in that they lack the DNA sequences that correspond to the RNA primers. S'

---rRNA\------ --

3'~

In protozoa this problem is solved by the addition of preexisting oligodeoxynucleotide blocks to the 3' ends of DNA. These blocks are composed of tanderoly repeated units of (T2G4)n or (TP4)n' where n is approximately 50. The enzyme that adds these polymers requires a primer but not a template. These oligonucleotide block polymers are called telomers. Another DNA polymerase,

(G-G-G-G- T- T - T - 1)" -3'

which is template dependent, copies the G4T4 units, synthesizing a complementary loop of C4A4 . This looping back allows the 5' end of the genomic daughter strand to be finished.

DNA ligase joins both ends of the telomere to the daughter strand, but the loop is subsequently cleaved to give flush-ended telomeres that consist of one strand of G4T4 and another of C4A4.

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MICROBIOLOGY AND BIOCHEMISTRY

9.2.5 Nucleosome Formation· The synthesis of DNA and of histone proteins is coordinated. Duplication of a genome requires doubling the amount of histone proteins. During DNA synthesis the parental histones remain associated with only the growing strand made continuously at the replicative fork. The DNA of this new strand immediately hybridizes to one parental strand; thus the "parental" histones tend to stay associated with the DNA structure that remains essentially double stranded throughout the replication· process. The other daughter strand is made in bits and pieces, and at anyone time during replication it might contain considerable amounts of single-stranded DNA. As the segments on the lagging strand are fmished and ligated together, the structure now binds newly synthesized "daughter" histones. The association of histones to one or the other of the strands can be distinguished using electron microscopy of material from cells grown under conditions where protein (i.e. histone) synthesis is inhibited but DNA synthesis is not inhibited.

9.2.5.1 Information stored in eukaryotic genes Most of the E. coli genome codes for mRNAs that are translated into proteins, but this is not the case for the animal genome. Since animals are more complex, they probably require 50 to 100 times more protein than bacteria, but their genomes are more than 500 times larger. For example, the single chromosome of E. coli contains 4.5 million base pairs, whereas the 23 haploid human chromosomes contain 2400 million base pairs. The animal genome does have duplicate genes for many proteins, and the genes for ribosomal RNA and for some tRNAs are repeated many times, but the function of most animal DNA is still unknown. Even the genes that code for proteins are more complex in vertebrates than in bacteria. Most, but not all, are expressed as long RNA molecules that are reduced in size by splicing together the coding segments. This yields a continuous template RNA that is sequentially decoded by protein-synthesizing enzymes. DNA that has no apparent function as a template for cellular RNAs is sometimes called non genetic DNA. Nongenetic DNA includes the pseudogenes. Pseudo genes are genes that cannot be expressed because they lack sequences necessary for RNA modification or protein synthesis initiation or because they contain protein synthesis "stop" signals in the middle of coding sequences. Nongenetic DNA also includes much repetitive DNA; about 30% of human DNA is repetitive. An

METABOLISM OF NUCLEIC ACID

235

example is the human Alu sequence. This 300-base pair sequence is repeated almost a million times at many places throughout the genome. Mouse satellite DNA is composed of a repeated sequence of similar size and number, but its sequence is tandemly repeated. The Alu sequence is named for the restriction endonuclease that cleaves at a single site within each repeated segment to yield many, almost identical copies of 300 base pairs each. Individual Alu sequences are homologous to one another by about 85 %. Alu sequences are sometimes transcribed into RNA. A small cytoplasmic RNA, called 7SL RNA, that functions as part of a protein-secreting system is homologous with the Alu sequences at its 3' and 5' end; consequently the Alu sequences originally may have been derived from the 7SL RNA gene. What role, if any, they now play is unknown.

9.2.6 Transposable Genetic Elements Mobile genetic elements further complicate the or-ganization of the chromosome. Mobile genetic elements are relatively small pieces of DNA that have characteristic sequences at either end. These pieces of DNA can move from one gene, or larger piece of DNA, to other locations, even on a different chromosome. The short sequences that flank the genetic elements are cleaved by an endonuclease to give staggered ends that base pair with complementary strands, a result of the nucleolytic cleavage of the target DNA by the same or a similar enzyme. A recombinational event (i.e., a crossing-over reaction) serves to transfer the small genetic element to its new location. Many examples of mobile elements are found in bacteria, where they are called transpo-sons. Bacterial transposons have terminal repeat sequences that both code for the enzymes catalyzing the process of transposition (transposases) and physically interact with these enzymes to bring them to the DNA target site. At this site the DNA-bound transposase presumably ca~alyzes the endonucleolytic cleavage of the terminal repeat sequence of the trahsposon and also catalyzes a similar sequence in the target DNA. Perhaps the best examples of genetic transposition in animal cells are the integration and subsequent removal of DNA programmed by RNA retroviruses to and from any number of sites on eukaryotic chromosomal DNA. The single-stranded RNA retrovirus uses the enzyme reverse transcriptase to make a complementary DNA copy of itself. The RNA present in the DNA-RNA hybrid is rapidly degraded, leaving a single-stranded DNA, which integrates into the host genome when copied. Integration is random in respect to the host DNA. Transcription

236

•

MICROBIOLOGY AND BIOCHEMISTRY

of the integrated viral genome produces many copies of viral RNA, which can be packaged into virus particles; the whole process is repeated in other cells or other organisms (horizontal transmission). Sometimes the pro-virus is carried in germ line cells, where the sequences might be transmitted to new generations (vertical transmission). In the case of the retroviruses, the enzymes needed for the movement of the proviral DNA are coded within the transposon, not in the repeated terminal sequences, as they are in bacteria. The animal transposons, as with other transposons, can carry along pieces of the host DNA. For example, cellular oncogenes (abbreviated c-onc) are sometimes carried along with the proviral DNA when it is excised. These host sequences are maintained and carried, as RNA, in the retrovirus, where over time and after many passages they are extensively modified. Oncogenes isolated from retroviruses are abbreviated v-onc. More than 20 different v-onc genes have been found in the retroviruses of different experimental animals. These oncogenes code for proteins that lead to the transformation of normal cells to cancer cells. The various oncogene proteins have diverse functions and may be found in different parts of the cell. Several are tyrosine-specific protein kinases or other protein kinases, some bind guanine nucleotides and have guanosine triphosphatase (GTPase) activity, whereas others may be derivatives of normal hormone receptors or protein growth factors.

9.3 MOLECULAR BASIS OF MUTATION On rare occasions a base may be changed or modified in the DNA sequence. When protein synthesis is considered, such a change in the structural gene for a protein could lead to the insertion of the wrong amino acid. If changed at a crucial position, the resulting protein will be unable to function. If the amino acid replacement occurs at a less important position, activity may be diminished or not affected at all. Mutations are responsible for dozens of known genetic diseases and undoubtedly for many more yet to be discovered. Usually these changes are subtle so they cannot be detected cytologically at the level of the chromosome. Gross chromosomal abnormalities do occur and are very important in the health sciences, but generally they are not inherited in the classic mendelian way. Rather, most are caused by nondisjunction, that is, a failure of either the egg or the sperm to receive an exact set of haploid chromosomes or of a mitotic cell to receive an exact diploid set early in development. Many others are caused by translocations that are also difficult to predict.

METABOLISM OF NUCLEIC ACID

237

Mutations are caused by both chemical and physical agents, although the action of even the physical agents, (e.g., ionizing radiation) can usually be explained by a chemical mechanism. Regardless of the agent used to produce a mutation, none is selective in the sense that it can specifically mutate one gene and not another. Because all genes are composed of only four different types of purine or pyrimidine bases, an agent that may react specifically with only one of the four could potentially cause mutations in every gene. Mutations are essentially random events. During our evolution the selective pressures of nature eliminated an astronomic number of deleterious mutations. The smaller number of beneficial mutations gave primitive life a survival advantage over competitors and allowed for the eventual emergence of intelligent beings. Consequently, in a highly evolved species· such as humans, most mutations produce deleterious effects.

9.3.1 Mutagens 9.3.1.1 Purine and pyrimidine analogues Mutations may be produced in many ways. Bases may be deleted or new ones may be inserted; more frequently an existing base may be chemically modified so that on replication, improper base pairing will cause a different base to appear at the modified position. The latter type of mutation is called a replacement. When a purine is replaced by another purine or a pyrimidine by a different pyrimidine, the change is called a transition. A transversion is a change from pyrimidine to purine or purine to pyrimidine. Many of the mutations caused by artificially produced base analogues are transitions. Mutations are produced by base analogues in one of two different ways. On entering the cell, a base analogue is converted to a nucleoside triphosphate that base pairs, perhaps incorrectly, with a DNA template and is inserted into the nucleotide chain. This is one way in which the mutation can be produced. The other requires an additional round of replication so that an improper base pair forms as a result of the previously incorporated analogue. The result in both cases is a permanently modified DNA. As might be expected, base analogues can also inhibit DNA synthesis and cell multiplication. It is this feature that has stimulated organic chemists to create hundreds of different base analogues in the hope that some may be useful for inhibiting rapidly proliferating cancer cells. Examples of base analogues that have some usefulness in cancer chemotherapy and that are also mutagenic are 6-mercaptopurine and 2-aminopurine.

238

MICROBIOLOGY AND BIOCHEMISTRY SH

0:) N

H

6-Mercaptopurine

2-Amlnopurlne

Not all analogues become active against cancer cells through incorporation into nucleic acid. Some analogues block the synthesis of normal purine and pyrimidine nucleotides; for example, 8-azaguanine blocks guanosine monophosphate (GMP) synthesis and 6-mercaptopurine inhibits adenosine monophosphate (AMP) syn-thesis.

9.3.1.2 Alkylating agents Alkylating agents are also mutagenic substances that have been used in cancer chemotherapy. Alkylating agents such as nitrogen or

o

N .... CH,CH.CI

p .... ,

C

I ~o CH,CH,CI NH

?!

~

o

~

CH, -fl-O-(CH,J. -O-S-CH,

Busulfan

Cyclophosphamide

sulfur mustards chiefly cause transversions. Bifunctional compounds such as those shown next produce cross-links between DNA strands or between a DNA strand and any other reactive group in the vicinity. The mechanism of action of alkylating agents is complex. Adenine and guanine are easily alkylated. Guanine is alkylated primarly at position 7 and adenine at position 3. The reaction produces an exceedingly labile glycosidic bond. Splitting of this bond leads to depurination.

o

A:J= I N>

HN H.N

DNAChain~

~

N

N

OCH.

o ~DNAChain

METABOLISM OF NUCLEIC ACID

239

In .those cases where alkylation does not lead to depurination, it is more likely that the mutation will be of the transition type. However, when depurination does occur, on replication the position opposite the gap might be filled by anyone of the four bases. This accounts for the transversions often caused by these agents.

9.3.1.3 Dyes Acridine dyes such as the antimalarial agent quinacrine (Atabrine) shown next are large planar aromatic compounds that intercalate or sandwich themselves between the stacked bases of the helix. CH.

I

/CH,CH. H, N-CH-CH,CH.N, CH,CH. -::r -::r I ""'" OCH.

a

~ : : :,.

"'N

#

On replication, insenion or deletion of bases may occur. Chain scission and chromosome breaks are also possible. Quinacrine is useful in human cytogenetics, since it intercalates significantly into the heterochromatin of the Y chromosome, making it fluoresce and rendering it identifiable cytologically. Detection of the Y chromosome is important in prenatal sex determination. Other dyes present in our environment are potentially mutagenic. For example, some hair dyes were shown to be mutagenic for E. coli.

9.3.2 Physical Agents Growing tissues are most sensitive to ionizing radiation. DNA synthesis is inhibited, yet the action of x-rays is indirect. They produce free radicals, which in turn react with DNA and thus produce point mutations or chromosomal breaks. Large doses of ultraviolet light can damage DNA. In humans this damage is confmed to the skin, since, unlike x-rays, ultraviolet light is easily absorbed. The chemical lesion in this case is the formation of dimers between adjacent thymine residues on the same DNA strand. Unless corrected or removed, these dimers will stop DNA synthesis. DNA repair Because most mutations are very damaging, even the simplest organisms have enzyme systems that repair DNA. These DNA repair systems are important because genetic defects in them can cause some human diseases. 9.3.3 Excision Repair The excision repair system consists of several enzymes, each involved in several steps. First, the error must be recognized. For example, an

240

MICROBIOLOGY AND BIOCHEMISTRY

endonuclease binds regions of the DNA that contain thymine dimers and cleaves at the 5' sides of the dimers. A DNA polymerase activity replaces that portion of the DNA strand that had contained the thymine dimer. An exonuclease then removes the piece of DNA containing the dimer, and a DNA ligase rejoins the repaired and restored DNA strand. These reactions are diagrammed in Figure 14.5. This form of nucleotide repair also acts on other types of damaged DNA, such as carcinogen-DNA adducts, and removes them by chain scission, patching, and ligation. Some damaged bases, particularly alkylated purine bases, are removed by N-glyco-sylases. The gapped chain is cleaved by apurinic endonucleases and the defective strand patched and ligated. Singlestrand breaks are repaired by analogous excision repair mech-anisffiS. Mitomycin D and platinum complexes used in cancer therapy can cause DNA-DNA cross-links between bases on opposite strands. These crosslinked bases can be excised and repaired, first on one strand and then on the other. The repair is error free unless the drugs have cross-linked directly opposing bases.

9.3.4 Postreplication Repair . Sometimes damaged DNA 'is replicated before it can be repaired. When this happens, the replicating strand stops at the site of damage, skips over the damaged base, and completes synthesis of the new strand. The new daughter and old maternal strands separate, and eventually the missing base is added, postreplicatively. The complemen-tary maternal strand still contains the damaged DNA, so this mechanism is not, strictly speaking, a repair mechanism, even though it allows synthesis of normal DNA. Eventually the damaged DNA is repaired by another mechanism. 9.3.4.1 Photoreactivation This system acts directly 'on DNA damaged by ultraviolet light to restore the damaged base to its original state without actually replacing it. Because this system operates only on ultraviolet light-damaged DNA, it plays a limited role in repairing human DNA. A light-activated DNA photolyase catalyzes the conversion of thymine dimers to monomers. 9.3.4.2 DNA glycosylases The DNA bases that contain amino groups tend to de aminate spontaneously. In particular, cytosine significantly deaminates to uracil, but adenine and guanine can also deaminate to hypoxanthine and xanthine, respectively. If not corrected, the new bases can cause serious mutations

241

METABOLISM OF NUCLEIC ACID

---I

..

Endonuclease

---

(

DNA polymerase I ..

---I

.,

- --', ,, ,,

- --I

- --I

I

New DNA

---I ---.

,

--I

---I

___ I

- - - : 4 ,',

---I -

===~ ---: )

..

DNA ligase

---I

---,,

---, , ___ I

---I

Figure 9.4 : Repair of DNA inactivated by ultraviolet light. Light causes the dimerization of adjacent thymine residues that block DNA replication. The four enzymes shown are involved in removal and replacement of a portion of the DNA that contains the dimer.

on replication. Fortunately, highly specific enzymes recognize these bases as being foreign to DNA, and they catalyze the hydrolysis of the N-glycosyl bonds that connect the bases to the DNA polymer. This produces DNA polymers with a few skipped bases. Another repair enzyme, an endonuclease, recognizes the skipped bases and cleaves the chain to leave a 3' hydroxyl group on the 5' adjacent nucleotide. A DNA polymerase now fills in the missing mononucle-otide, taking its instructions from the intact complementary strand. Defects have been found in these mechanisms that cause various human diseases. For example, patients with the genetic disease xeroderma pigmentosum are especially sensitive to ultraviolet light and develop skin cancer. Skin fibroblasts cultured from these patients have been shown to be defective in DNA repair.

9.4 CHEMICAL CARCINOGENESIS Most human cancer is caused by substances in the environment,

242

MICROBIOLOGY AND BIOCHEMISTRY

chemicals, viruses, or radiation. All these carcinogens affect DNA. Sometimes we can relate a substance to a specific type of cancer; for example, cigarette smoking to lung cancer. In other cases it is more difficult to draw a cause-and-effect relationship. Nevertheless, the evidence is overwhelming that carcinogens cause cancer by interacting with DNA. When the DNA modified by these agents cannot be repaired, cancer often results. Carcinogenesis develops in three stages: initiation, promotion, and progression. Chem-ical substances can act at the initiation stage or at the promotion stage. Moreover, some chemicals have both initiating and promoting activities. 9.4.1 Initiating Agents Initiating agents alter the native molecular structure of DNA. They - may cause an accu-mulation of somatic mutations over the lifetime of an individual. Initiating agents may be physical, biologic, or chemicalfor example, ionizing radiation, tumor viruses, and cyclophos-phamide. Chemical initiating agents have been extensively studied. These substances either interact directly with the DNA or are enzymatically modified to produce a metabolite that interacts with the DNA. The interaction is often covalent, although noncovalent reactions are possible. The resultant action of the initiating agent with DNA causes an irreversible change similar to a mutation; however, an observable mutational event, that is, a phenotype, is not an obligatory step in the initiation process. Furthermore, initiation does not in itself cause cancer. Instead it programs the cell so that subsequent reaction at a later time with a promoting agent starts the formation of cancerous cells. 9.4.2 Promoting Agents Unlike initiating agents, pr6moting agents do not interact directly with DNA, but rather influence the expression of the genetic information coded in DNA. Promoting agents include a variety of substances, such as hormones, protein growth factors, drugs, and plant products. Asbestos, cigarette smoke, alcohol, and phorbol esters are examples of promoting agents. These substances influence genetic expression by binding to receptors on cell surfaces or in the cytoplasm or nucleus. In contrast to initiating agents, .the action of promoting agents is reversible. Some promoting agents, (e.g., estrogen and prolactin) are very specific so that they promote the formation of a Hunor only in their target tissues. Other promoting agents do not act through a receptor mechanism, are nonspecific, and can promote tumor formation in a variety of tissues (e. g., iodoacetate).

243

METABOLISM OF NUCLEIC ACID

Phorbol esters are promoters that interact with cellular receptors and activate protein kinase C. Usually protein kinase C is activated by Ca++ and diacylglycerol, both of which result from the hydrolysis of phosphoinositides catalyzed by phospholipase C. Phospholipase C is normally activated by several different growth factors. Thus phorbOl esters bypass a tightly regulated step in the control of cell growth. Since protein kinase C phosphorylates various proteins, it is not known how this activity participates in establishing a cancerous line of cells. Following promotion, cells go through a stage in carcinogenesis called progression. During this stage, there is a karyotype change from diploid to aneuploid that is associated with metastasis and morphologic changes. 9.4.3 Oncogenes Oncogenes are genes involved in the transformation of normal cells to tumor cells. They do this by affecting cell growth and differentiation. Oncogenes were discovered as part of the genomes of RNA tumor viruses. Recall that RNA tumor viruses are propagated through DNA intermediates that are synthesized in reactions mediated by reverse transcriptase, a RNA-dependent DNA polymerase that is part of the virion. Thus RNA tumor viruses are called retroviruses. Not all retrovinses transform normal cells into tumor cells; for example, the human immunodeficiency virus (HIV) causes acquired immuno-deficiency syndrome (AIDS).

~~o~

Q.e

;

/

/

VI~e~ tumor'

~

\

Q_~ /,""g~tu.....

/

~

~~,-<eJ I

\:J f19 ~

u~

I

Initiating agent Euploid cell population

~

Increasing aneuploidy

•

Figure 9.S : Karyotypic changes during initiation. promotIon. and progression stages of carcinogenesis.

244

MICROBIOLOGY AND BIOCHEMISTRY

The ROllS sarcoma virus (RSV) is a retrovirus that contains an oncogene called v-src. Oncogenes that are part of the genomes of retroviruses are abbreviated v-one whereas oncogenes of cellular genomes are designated c-one. Cellular oncogenes are often called proto-oncogenes to distinguish them from their viral counterparts and to emphasize that the viral oncogenes originated at one time from cellular oncogenes. Evidence for the first oncogene was found when certain mutants of RSV failed to transform cells at high temperature but did transform cells at low temperature. The mutant virus replicated well at both temperatures. This means that the virus contained a tem-perature-sensitive mutation in a gene that coded for a sarcoma-producing protein, tllat is, for v-src. Normal cells also were found to contain src-like genes when their DNA was hybridized with labeled nucleic acid probes from the v-src gene. As with other cellular genes, the c-onc genes were interrupted with introns. v-One genes lack introns. Consequently, in the distant past c-one genes must have been transferred to the retroviruses. If the transfer had been from the virus to the cell, e-one genes probably would not contain introns. The retroviral one gene products have diverse functions, depending on the particular oncogene. A few representative oncogenes and the functions of their protein products. Notice that the oncogene proteins are all involved in some aspect of cellular growth promotion. For example, the tyrosine kinase activity of sre is a property shared by several receptors for hormones (e. g., insulin) and for growth factors (e.g., epidermal growth factor, or EGF). Some oncogenes code for subunits of growth factors; an example is the v-sis protein, which is homologous to a subunit of platelet-derived growth factor (PDGF). The oncogene v-erbB, unlike e-erbB, codes for a shortened form of the EGF receptor protein. Usually the binding of EGF is required to turn on the tyrosine kinase activity of the EGF receptor protein, the one coded by e-erbB. However, the tyrosine kinase activity of the receptor derived from v-erbB is switched on permanently, even in the absence of EGF. The uncontrolled grqwth Cllaracteristic of cancer cells results. Other oncogenes code for proteins that bind guanine nucleotides, and others code for nuclear proteins. The guanine nucleotide-binding proteins, the so-called G proteins, affect several key reactions. Some G proteins are stimulatory, whereas others are inhibitory. For example, they link hormone receptors to adenylate cyclase, they translocate

245

METABOLISM OF NUCLEIC ACID

TABLE 9.4 : EXAMPLES OF PROMOTERS WITH THEIR ASSOCIATED NEOPLASMS IN HUMANS. Agent

Neoplasm

Excessive dietary fat (calories)

General increase in incidence of cancer Oral, liver, and esophageal cancer Bronchogenic carcinoma, esophageal, and bladder cancer Liver adenomas Bronchogenic carcinoma and mesothelioma

Alcoholic beverages Cigarette smoke Synthetic estrogens Asbestos Not yet proved In humans Saccharin Tetradecanoylphorbol acetate

Bladder Skin (a phorbol ester)

molecules in protein synthesis, and they regulate cell proliferation. The func-tions of the nuclear oncogene proteins are not as well characterized, but they also are probably involved in key reactions of gene expression that regulate growth.

TABLE 9.5 : ONCOGENES OF RNA TUMOR VIRUSES. Virus

Oncogene

Oncogene protein product

Abelson murine leukemia virus Rous sarcoma virus Avian erythroblastosis virus

abi src erbB

Simian sarcoma virus

sis

Harvey murine sarcoma virus

Ha-ras

Avian myelocytomatosis virus

myc

Tyrosine kinase Tyrosine kinase Receptor for growth factor; tyrosine kinase activity Growth factor, subunit of PDGF Guanine nucleotidebinding protein Nuclear protein

A unifying theme of the role of oncogene function in cell transformation is that the expression of the proto-oncogenes is normally tightly

246

MICROBIOLOGY AND BIOCHEMISTRY

regulated. Anything that disrupts the regulation of oncogene expression could potentially cause cancer. For example, a chromosome break or a mutation, in either somatic or germinal cells, at or near an oncogene might lead to aberrant expression of the oncogene (see case 5). Similarly, a tumor virus could introduce an unregulated oncogene into cells and also cause cancer.

9.5 DNA SEQUENCE ANALYSIS Some of the chemical reactions that cause mutations by modifying purine and pyrimidine bases can be used in the laboratory for sequencing DNA. One of the most commonly used methods for sequencing DNA is the chemical technique developed by Maxam and Gilbert. Since DNA molecules are extremely large, they are first broken into pieces more easily sequenced. This is done with restriction endonucleases, and the fragments are separated one from the other. A double-stranded DNA fragment is then labeled at its 5' end using ATP (y_32P) and a polynucleotide kinase derived from bacte-riophage T4-infected cells. The DNA fragment is denatured to yield single-stranded pieces, and these are separated from one another by electrophoresis on neutral gels of polyacrylamide: Large DNA

...=e,

DoubIe-stranded (ds) DNA fragments; resolve on polyacrylamide gel

I

T4~

7" _"" dsDNA

dsDNA fragment

ATP (y-32P)

_. \(32Pal5' ends) ADP

5' 3' 32p------dsDNA PoIy~ 11"\ (32P al5' ends) - . , p o -

+ 3' --------~5'32P

Resolved dsONA, labeled al 5' end

Each 5'-labeled single-stranded DNA is then sequenced. The method is based on two principles: (1) polyacrylamide gel electrophoresis in 7 mollL urea is capable of separating oligo-deoxyribonucleotides that differ in size by just a single base. (2) DNA can be chemically reacted so that chain cleavages occur at specific bases. In a typical experiment a portion of the 5' -labeled single-stranded DNA is reacted with dimethylsulfate under conditions where many but not all of the purine bases react. This reagent alkylates the N7 position

METABOLISM OF NUCLEIC ACID

247

of guanine and the N3 position of adenine. Depurination of methylated guanine residues predominates when an aliquot is heated at neutral pH. Methylated adenine bases are preferentially removed by treatment with dilute acid. Both samples are then cleaved at the depurinated sites by alkaline hydrolysis. One now has two samples: one that contains DNA fragments all 3' -terminated at adenine residues and another consisting of DNA fragments all terminated at guanine residues. In practice, however, a few adenines are retOOved with the guanines. The deadenylation reaction is more specific. Since the 32p label is at the 5' end of the DNA and the depurination reactions are not allowed to go to completion, the mixture of labeled oligonucleotides will range in size from the very largest, cleaved at the 3' purine residue most distal to the label, to the smallest, where the purine residue most proximal to the label has been retOOved. An analogous ~ries of reactions is used to produce depyrimidinated DNA fragments. Hydrazine is used in these reactions, since both cytosine and thymine react with hydrazine. The bases are cleaved to yield urea and a pyrazole ring. The deoxyribose moiety is left as a hydrazone. Piperidine, which reacts with the hydrazone, is used to cleave the nucleotide chain. Cytosines react specifically with hydrazine in 5 mol! L NaCI, but no specific reaction eXIsts for thymines. Consequently, one aliquot yields labeled oligonu-cleotides 3'-terminated at cytosines, whereas a second aliquot contains nucleotides cleaved in the absence of NaCI at both cytosine and thymine residues. Each of four aliquots terminated at A, G, C, and C + T are denatured and loaded onto slabs of polyacrylamide gel. After electrophoresis, the radioactively labeled nu-cleotides are visualized by exposing the gel to an x-ray fIlm. The resultant autoradiogram shows the position of each labeled oligonucleotide. The longer polynucleotides are shown at the top of the diagram, and the shorter, faster moving polynucleotides are at the bottom. Because the chemical reactions do not go to completion, the autoradiogram displays a whole spectrum of polynucleotides, some of which have been cleaved at only a single base and others that have been cleaved at several. Each band differs from the adjacent band by one nucleotide. Consequently, the DNA sequence, 5' to 3', can be read starting from the bottom. Figure 14.8 shows only a portion of the gel. Sequences of more than 350 bases can be obtained from some gels. The sequence of the complementary DNA strand is often determined as a check on accuracy of the first analysis. The primary structure of thousands of deoxyribonucleotides can be deduced by the analysis of

248

MICROBIOLOGY AND BIOCHEMISTRY

overlapping DNA fragments generated by different restriction endonu-cleases. This technology is useful in determining the proximity of one gene to another and in studying mechanisms of gene expression.

9.6 RECOMBINANT DNA TECHNOLOGY IN MEDICINE Thirty years ago the thought of sequencing all the genes in the -human genome seemed as impossible as a trip to the moon. Now it is in the realm of possibility, and scientists are seriously talking about sequencing the entire human genome, all 23 chromosomes, amounting to 2,400 million base pairs (2.4 x 1()6 kb). Such plans are not based on the physical chemical separation of thousands of genes from human tissue, but rather on the isolation of genes or short pieces of DNA from a relatively small number of cells. These pieces of DNA can be cleanly separated from one another and then amplified to obtain sufficient material by using the powerful biologic methods of recombinant DNA tech-nology. S'End

3' End

o II 32 -o-p-o - - - - - - - - - - - - - - - - - - - - - O H I

Single-stranded polydeoxyribonucleotide

IDim~thYI rl

-0

sulfate

H,..yd_r_a_Zi_ne--,-_ _...,

Heat. pH 7

Adenines modified

Ad,,'"

SM NaCI

Guanines modified

Cytosines modified

1-Vo::""riO~ if G"",..

~P"rid" --i 0'\,.,...,

OH-

Cytosine

Chain cleaves Chain cleaves at at adenine gaps guanine gaps

Cytoslnes and thymines modified

~ -_.

Chain cleaves al cytosine gaps

f

thymine

Chain cleaves at cytosine and thymidine gaps

Figure 9.6 : Summary of reactions used to prepare oligonucleotides specifically cleaved at a particular base.

9.6.1 Restriction Endonucleases Restriction endonucleases are at the core of recombinar,.t DNA technology. The specificity of some of these enzymes is described in Chapter 13. Fortunately the _restriction endo-nucleases cleave DNA at

METABOLISM OF NUCLEIC ACID

249

relatively few sites. The DNA fragments that result are usually long enough to be useful but short enough to analyze and even sequence. The mixture of DNA fragments following restriction nuclease digestion are resolved by electrophoresis on gels of polyacrylamide or agarose. The dye ethidium bromide is often used to detect the resolved fragments on the gel. For further analysis, individual fragments can be cut out of the gel and separated from the gel matrix and dye. 9.6.2 Restriction Maps Now commercially available are many restriction enzymes, named for the bacterial species from which they are isolated and possessing a W\de range of specificity. Because the enzymes recognize such different nucleotide sequences, they can be used individually and in combination to develop "maps" of a particular DNA. For example, a 12 kb viral DNA might be cut into two pieces of 2 kb and 10 kb by endonuclease "A"; however, endonuclease "B" might cleave it into 5 kb and 7 kb fragments. All these can be resolved from one another by gel electrophoresis. Figure 14.9 illustrates these cleavages and shows that there are two different arrangements for the cleavages catalyzed by each nuclease. When endonuclease "A" and endonuclease "B" are mixed together during the digestion and the products separated by elec~ophoresis, the size of the fragments allows one to determine the relative position of the cleavage sites produced by each of the restriction enzymes. In this example the 2 kb fragment is assumed to be located at the left end of thee original DNA molecule. 9.6.3 Cloning of Recombinant DNA The most useful restriction enzymes cleave both strands of duplex DNA, but the nucleotide breaks are not directly opposite one another; the cleavages occur a few base pairs from a point of symmetry. These staggered ends can base pair to hold the DNA together weakly, or they can base pair with other DNA preparations that have been cut with the same type of nuclease. In the laboratory the broken strands can be covalently joined together by treating them with DNA ligase. In this way foreign DNA can be linked to the DNA of plasmids or bacteriophages (bacterial viruses). Which shows some of the genes and restriction sites of the commonly used plasmid pBR322. ApR is a gene for ampicillin resistance, Tc R is the gene for tetracycline resistance, and on denotes the origin o'f replication sequences necessary for the plasmid to replicate its DNA separately from the bacterial DNA. The bacteriophages or plasmids that have foreign DNA built into them in this manner are called vectors. Usually bacteriophages insert

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MICROBIOLOGY AND BIOCHEMISTRY

DNA very efficiently into their host bacteria. A single bacteriophage DNA can be replicated approximately lOO-fold or more per cell depending on the type of phage and its host. 9.6.3.1 Plasmid vectors Plasmids are small, circular DNAs that range in size from 2 to approximately 100 kb. They also can be amplified to approximately 20 copies per cell. In addition, plasmids can be introduced into bacterial cells, although less efficiently than phage vectors. Some plasmids can be used to introduce recombinant DNA into yeast cells.

9.6.4 Clones One usually tries to adjust experimental conditions so that only one plasmid or phage is introduced into a single cell. When that cell replicates on a plate of nutrient agar, it will produce millions of cells until a colony becomes visible to the naked eye. If cells are sufficiently diluted before they are spread onto the plate, each colony will consist of a clone derived from a single ancestral cell. If each cell originally contained only one copy of the phage or plasmid vector, each colony will contain recombinant DNA that is homogeneous. That is, each colony will contain cloned DNA. 12 kb DNA

Endonuclease 'B'

Endonuclease 'A'

Ll 5kb L ~l

10

kb

7kb

11

R (b)

or

R (aI

LI 7kb LwC!J17 I

R

11

Skb

R (e)

(a,b)

or •

Figure 9.8 : The construction of restriction maps. The relative position of cleavage sites for two hypothetic restriction enzymes is illustrated for a 12 kb piece of DNA.

251

METABOLISM OF NUCLEIC ACID

9.6.4.1 Clone selection using antibiotic resistance Plasmids found in nature often carry genes that code for proteins that make their hosts resistant to antibiotics. These genes are used to good advantage to select bacterial colonies that contain recombinant plasmids. For example, a restriction site may occur within a gene for tetracyline resistance. A Sall site exists within the tetracycline resistance gene of plasmid pBR322. When foreign DNA is inserted at this site, it interrupts the gene for tetracycline resistance so that the cells are now sensitive to tetracycline. Tetracycline sensitivity can be detected by plating the vectorcontaining cells onto agar containing ampicillin but not tetracycline. All colonies on such plates will contain either the original plasmid or plasmids containing recombinant DNA. Colonies are then replicateplated onto agar that contains tetracycline. Colonies that are sensitive to this antibiotic can be determined from the differences in the pattern of colonies appearing on the two plates. The antibiotic-sensitive colonies can be picked off and used for further experiments. These colonies will contain plasmids carrying foreign DNA . ......G-G-A-T-c-c..... . ......C-C-T-A-G-G..... .

BamH endonuclease

......a G-A-T-c-c.... ~ ......c-c-T-... -a a......

G-A-T-o-")_ .... (

0NA1Igaae

G

......O-G-A-T-C-C'

......C-C-T-A-G-G.

ForaisJ'! DNA

I

C-C-T-A-G

I

G-Q-A-T-C-C......

C-C-T-A-G-G._..

Figure 5.9 : The formation of recombinant DNA using the restriction endonuclease BamHI.

252

MICROBIOLOGY AND BIOCHEMISTRY

9.6.4.2 Phage and cosmid vectors

Plasmids usually are able to carry much less foreign DNA than a bacteriophage such as lambda. Thus plasmid vectors mainly are used to obtain pieces of DNA (up to 3 kb in size) suitable for sequencing, to subclone larger DNAs carried by phage vectors, or to clone single expressible genes derived from complementary"DNAs (cDNAs). On the other hand, vectors of bacteriophage lambda can accommodate up to 23 kb of foreign DNA. The amount of DNA that can be inserted depends on the amount of nonessential lambda genes that can be replaced and the total amount of DNA that can be packaged into a particle able to be covered with phage coat proteins. Some eukaryotic genomic genes have such long intervening sequences that their DNA may not fit into one phage head. For cloning these large genes, cosmid vectors are often used. Cosmids can accommodate very large pieces of genomic DNA. They contain only a small part of the lambda DNA, the two cohesive Eco RI

lSa11 DNA ligase

1

Eco RI·

Sal I foreign DNA 4

"--.. One of many different pieces

+Original circular pBR322 plasmid

+Circular fragments of foreign DNA

Figure 12.10 : Some genes and restriction sites on the E. coli plasma pBR322. Eco RI, Pst I, and Sal I are restriction endonucleases and the sites cleaved by these enzymes.

METABOLISM OF NUCLEIC ACID

253

to be packaged into particles in vitro. As with bacterioph~ges, these ends (cos gene) that allow any DNA particles are very efficient vep.icles for inserting DNA into E. coli cells; however, unlike phages; they cannot make infectious particles in vivo. Cosmids can carry up to 40 kb of recombinant DNA. They are engineered to cOIitain an origin of replication, so the recombinant DNA can be amplified, and an antibiotic resistance gene, so they can be easily selected. Once in the cell, cosmids behave more as plasmids do. cDNA is made in the laboratory from mRNAs using the enzyme reverse transcriptase. Unlike eukaryotic genomic DNA, cDNA does not contain intervening sequences. A complete cDNA may contain all the information needed to synthesize a mRNA for a specific protein. If the cDNA or its vector is modified so that the recombinant DNA has all the sequences necessary for RNA and protein synthesis, one can synthesize in bacterial cells the protein encoded in the recombinant cDNA. Such a vector is called an expression vector. Expression vectors can be used to obtain large quantities of a protein that might be very difficult to isolate by conventional techniques. This is possible because bacterial cells contain many copies of the recombinant gene and because RNA and protein synthesis occurs very rapidly in bacteria.

9.6.5 Libraries of Genomic DNA A recombinant gene library is made by digesting cDNA or genomic DNA with one or more restriction enzymes, ligating the fragments to a vector, and introducing the vector into appropriate bacterial or yeast cells so that each cell will contain a single recombinant DNA. A culture of millions of cells may contain fragments of all the cDNAs or all the fragments of a genome. These libraries can be screened to isolate a clone of interest by using a variety of techniques. Genomic DNA is much more complex than cDNA. Since cDNAs are synthesized in the laboratory from mRNAs using reverse transcriptase, they are no more diverse than the number of mRNAs present at the time of isolation. However, only about 2% of the mammalian genome codes for the synthesis of proteins and their mRNAs. Thus genomic DNA libraries are at least 50 times more diverse than cDNA libraries. cDNA libraries are not easier to make, however, both because of the inherent instability of mRNA compared to DNA and because reverse transcriptase prematurely terminates when copying long mRNAs. The otherwise impossible task of analyzing the 4 to 8 X 109 base pairs of human genomic DNA can be simplified. Many of the 23 human chromosomes can be physically separated from each other, their DNA

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isolated, and genomic libraries made from them. Also, a technique called pulsed-field electrophoresis allows the separation of DNAs as large as 1()6 base pairs. This procedure uses short pulses of current at alternating angles to the direction of movement of the DNA. The long DNA molecules take longer to reorient in the agarose gel matrix than the shorter molecules, and thus migrate more slowly. 9.6.6 Detection of Recombinant DNA Recombinant DNA in bacterial colonies or in bacteriophage plaques is most frequently detected using labeled hybridization probes. The hybridization probes may be composed of RNA, DNA, or oligonucleotides that have been labeled. Often a DNA restriction fragment is labeled in vitro using commercially available enzymes and radioisotopes. Smaller deoxyribonucleotides can be synthesized chemically using automated machines called DNA synthesizers. Methods are available for labeling either end of a nucleotide with 32p substrates. Heavily labeled DNA probes can be made using a (l2P)-deoxyribo-nucleoside triphosphates and the Klenow fragment of the E. coli DNA polymerase I. Small, random oligonucleotides are added as primers to the denatured DNA template so that large segments of cDNA are labeled with 32P, not just the ends of the oligonucleotides. A Petri dish containing bacterial colonies is blotted with nitrocellulose paper. This transfers a large portion of each colony to the paper. which is saturated with a solution that lyses (breaks open) the cells. The DNA of the lysed colonies is denatured with alkali. The nitrocellulose paper is neutralized, washed, and the paper either baked in an oven or treated with ultraviolet light to immobilize the denatured DNA. The DNA on the paper is hybridized with the labeled probe of interest, and the excess label is washed off. The dried paper is exposed to photographic film and the film developed. The exposed spots on the film can be matched with the colonies on the master plate and colonies picked off for further study. A very similar protocol can be used to detect a particular DNA or RNA that has been resolved by electrophoresis. E.M. Southern developed a method for detecting individual DNAs that had been resolved by gel electrophoresis. The gel was blotted to nitrocellulose paper and the DNA on the paper hybridized with a labeled DNA probe. This procedure of analyzing DNA resolved by electrophoresis is called Southern blotting. Procedures wefe developed later for transferring by blotting RNAs that had been resolved by electrophoresis. This procedure was called Northern blotting. Western blotting is the analogous transfer

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of electrophoretically rec;olved proteins to nitrocellu-Iose paper. In this case the proteins are usually detected immunolo-gically. 9.6.6.1 Restriction fragment length polymorphism The Southern blotting of genomic DNA is used to detect restriction fragment length polymorphisms (RFLPs). Polymorphism is a genetic term indicating variation of a gene product within a population. Such variation may result in observable phenotypes; if serious, these are called mutations. In humans polymorphisms usually are not associated with an easily identifiable phenotype. Many polymorphisms occur in clinically normal people as part of their natural diversity. It has been estimated that a normal individual may be heterogeneous at 20% of his or her genes. The resulting enzymes are different but normal. Some loci, such as a-haptoglobin, are so diverse that no single allele can serve as the "normal" standard. Usually a single gene product must be represented in less than I % of the cases examined for it to be considered polymorphic. With the advent of electrophoresis, many polymorphisms were detected by the abnormal migration of a protein or enzyme. Many more polymorphisms now can be detected by the gel electrophoresis of DNA restriction fragments followed by Southern blotting. These DNA fragments show different mobilities on the gel, and thus they differ in length. For this reason they are called length polymorphisms. Length differences can result either from a mutation at an existing restriction site so that it is no longer recognized by the enzyme or from a mutation at another position to create a new restriction site. In the former case the restriction fragment would be longer; in the latter case it would be shorter. RFLPs are often a reflection of individual genetic diversity and are not related to a clinical phenotype, but occasionally they can be diagnostic of an inherited disease. This technique is relatively new; yet, it has been applied to the prenatal detection of sickle cell anemia, thalassemia, phenylketonuria, a,-antitrypsin deficiency, Huntington's chorea, Duchenne muscular dystrophy, hemophilia A and B, cystic fibrosis, and several other. diseases.

9.6.7 Clinical Comment 9.6.7.1 Prenatal diagnosis of sickle ceU disease Sickle cell disease is caused by a mutation that results in the substitution of a valine residue for a glutamate residue in the sixth position of the hemoglobin ~-chain. This results from the substitution of a T for an A in the glutamate codon. When (1) DNA from a patient

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with sickle cell disease is digested with the restriction enzyme MstII, (2) the fragments separated by gel electrophoresis, (3) the gel blotted under denaturing conditions to nitrocellulose paper, and (4) the resolved DNA fragments hybridized with radioactive human globin DNA, one finds a new 376-base pair fragment instead of the normal 175-base pair DNA fragment. This indicates that a site usually recognized by the enzyme MstII has been changed as a result of the mutation. MstII requires the following sequence for its endonucleas~-;action: .... C-C T-N-A-G-G .... The letter N indicates that any nucleotide at that position satisfies the specificity of MstII. The nonnaI sequence of the DNA of the 13chain of hemoglobin A near the site of the sickle cell mutation is: .... C-C T-G-A-G-G .... This sequence is cleaved by MstII; however, the mutation to sickle cell anemia results in a change to the sequence: .... C-C-T-G~T-G-G .... This sequence cannot be cleaved by MstII. Restriction analysis can be used to detect sickle cell disea!!e prenatally, since the DNA of all cells, including amniotic cells, carries the mutant DNA. It is much more difficult to obtain fetal blood for the analysis of the mutant hemoglobin A l3-chain. Furthermore, fetal blood is composed mo~tly of fetal hemoglobin, sinu-hemoglobin A is madelaler in development.

9.6.8 Reverse Genetics . Originally this term applied to the modification in vitro of a piece of DNA of unknown function, with the subsequent identification of its function by introducing it baCkmto cells. In human genetics the term is now used to describe the determination of the cause of an inherited disease by starting with the responsible gene and tracing it back to a defective enzyme. In the past, human geneticists first recognized the defective enzyme and then located the responsible gene. Reverse genetics has been applied to diseases such as Duchenne muscular dystrophy and cystic fibrosis, in which the responsible enzymes are unknown and the disease. results from a significant deletion. By combining RFLP analysis with cytogenetics, it has been possible to increasingly narrow the location of the defective genes to small regions on the affected chromosomes. 9.6.9 Polymerase Chain Reaction Polymerase chain reaction (PCR) is a technique for amplifying

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small amounts of DNA when only small amounts of human tissue or cells are available. One needs to know the sequence of the DNA in question. Using the sequence, one chemically synthesizes two oligodeoxyribonucleotides of approximately 20 to 25 residues that are complementary to each of the two ends of the DNA of interest. These are annealed (heated, then cooled slowly) to the ends and serve as primers for the in vitro copying of each DNA strand using a heat-stable DNA polymerase from Thermus aquaticus (Taq polymerase). The chemically synthesized primers are in excess so that when the reaction mixture is heated to 63 0 C, the DNA strands separate and reanneal with more primer on cooling. This process of heating, cooling, and synthesis can be repeated many times, and in the process the DNA fragment of interest is greatly amplified. So much of a particular DNA can be made that it can be detected by procedures that do not require radioisotopes. The poly-merase chain reaction has been applied to the detection of carriers of hemophilia A, to the prenatal diagnosis of sickle cell disease, and to fetal sex determination, among others. 9.6.10 Cloning and Sequencing the Human Genome Molecular biologists around the world have proposed the longterm goal of sequencing every gene on every human chromosome. Such a project will require international co-operation on a grand scale, as well as the development of automated machines to do most of the time-consuming work. At present such a project is theoretically possible, but the necessary machines have not yet been built. The goal is a worthy one because it will provide the framework to analyze every human genetic disease. Literally thousands of DNA probes would be available for screening patients, carriers, and others at potential risk, 'The sequences would also provide the information needed for genetic therapy when such procedures become available.

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