Advances in Microbial Physiology Volume 02

Advances in

MICROBIAL PHYSIOLOGY

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Advances in

MICROBIAL PHYSIOLOGY Edited by A. H. ROSE Department of Microbiology University of Newcastle upon Tyne England

and

J. F. WILKINSON Department of General Microbiology University of Edinburgh Scotland

VOLUME 2

1968

ACADEMIC PRESS

LONDON and NEW YORK

2 0 JUN ?68

PRESS INC. (LONDON) LTD. BERKELEY SQUARE HOUSE BERKELEY SQUARE LONDON, W.1

U.S. Edition published by ACADEMIC PRESS INC. 111 FIFTH AVENUE NEW YORE, NEW YORE

10003

Copyright 0 1968 by ACADEMIC PRESS INC. (LONDON) LTD.

All Rights Reserved No part of this book may be reproduced in any form, by photostat, microfilm, or any other means, without written permission from the Dublishers Library of *Congress Catalog Card Number : 67-19850

PRINTED IN GREAT BRITAIN BY SPOTTISWOODE, BALLANTYNE AND CO. LTD LONDON AND COLCHESTER

Contributors to Volume 2 K. BERAN,Department of Technical Microbiology, Institute of Microbiology, Czechoslovak Academy of Sciences, Prague, Czechoslovakia.

W. X. KELLEY, Department of Microbiology, Tufts University School of Medicine, Boston, Massachusetts, U.S.A.

JUNE LASCELLES, Bacteriology Department, University of California, Los Angeks, California 90024, U.S.A.

B. E.B.MOSELEY, Biology Division, Oak Ridge National Laboratory, Tennessee, U.S.A.

M. H. RICHMOND, Department of Moleculur Biology, University of Edinburgh, West Mains Road, Edinburgh 9, Xcotland.

M. SCRBECHTER,Department of Microbiology, Tufts University School of Medicine, Boston, Massachusetts, U.S.A.

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Contents Contributors t o Volume 2

.

V

The Bacterial Photosynthetic Apparatus. JUNE LASCELLES I. An Introduction to the Photosynthetic Bacteria and their Pigments A. The Photosynthetic Pigments . B. Environmental Effects on Pigment Synthesis . 11. The Structure and Location of Chromatophore Material . A. Discovery and Definition of Chromatophores B. Electron Microscopy of Cells . C. Chromatophore Material and Cell Membrane D. Chlorophyll and Membrane Content . E. Peripheral and Internal Membrane as Sites for Pigments F. The Vesicles of the Green Bacteria . G . Conclusions 111. Isolation and Composition of Purified Chromatophores . A. Isolation Procedures . B. Composition C. Fractionation of Chromatophores . D. Immunological Reactivity IV. Formation of the Photosynthetic Apparatus . A. Differences between Members of the Athiorhodaceae Grown Photosynthetically and Aerobically B. Adaptive Synthesis of the Photosynthetic Apparatus . C. Chlorophyll Synthesis D. Regulation of Chlorophyll Synthesis . References

.

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.

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1 3 6 8 8 8 12 14 16 18 19 19 19 21 24 26 27 27 29 31 34 39

The Plasmids of Staphylococcus aureus and their Relation to Other Extrachromosomal Elements in Bacteria, M. H. RICHMOND I . Introduction . A. Historical . B. Curing C. Stability . D. The Plasmid Location of Penicillinase Genes

.

.

. . . . .

43 43

44 45 48

...

CONTENTS

Vlll

.

11. Types of Staphylococcal Plasmid A. Plasmid-Borne Markers . B. Classification of Plasmids . C. Distribution and Abundance of Plasmid Types . D. Plasmid Nomenclature . 111. Transduction of Plasmids . A. Preparation of Transduciilg Phage B. Transductants Formed in Negative Recipients . C. Transductants Formed in Plasmid-Carrying Recipients . D. Compatibility E. The Role of Attachment in Recombination and Diploid Formation . F. Chromosomal Attachment of Plasmid Markers . G. ThePlasmidMap H. Size of the Penicillinase Plasmids . IV. A Comparison of Penicillinase Plasmids with Other Extrachromosomal Elements in Bacteria V. Acknowledgements . References

51 51 55 58 59 60 60 60 61 71

The “Life Cycle” of Bacterial Ribosomes. WILLIAM S. KELLEY

and

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.

.

.

75 78 80 83 84 86 87

MOSELIO SCHAECHTER

I. Introduction . . A. Problems Particular to the Study of a Ribosomal “Life . Cycle” . . B. Scope of the Review . C. Studies of Ribosome Structure and Composition . . * D. Cellular Localization of Bacterial Ribosomes . . 11. The Role of Ribosomes in the Control of RNA Synthesis . 111. The Production of Ribosomal RNA . . IV. The Production of Ribosomal Proteins . . V. The Assembly of Ribosomal Subunits . . A. Studies on Cells in Balanced Growth . B. Studies of Metabolically Inhibited Cells . C. I n Vitro Experiments on Ribosome Assembly . D. Conclusions . VI. The Formation of Functional Ribosomes . VII. The Participation of Ribosomes in Protein Synthesis . . . VIII. The Release of Ribosomes from Messenger RNA . IX. Conclusions . X. Acknowledgements . . References

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.

. .

89 90 91 91 97 99 101 107 112 112 115 124 126 126 131 133 136 137 137

ix

CONTENTS

Budding of Yeast Cells, Their Scars and Ageing. I . Introduction .

K. BERAN

. 11. Composition and Structure of the Cell Wall of Yeast A. Macromolecular Wall Components . B. Structure of the Wall . 111. Mechanism of the Primulin-Induced Fluorescence . IV. Budding of Yeast Cells and its Mechanism A. Budding of Yeast Cells as Observed in the Optical and Electron Microscopes . B. Examination of Buds and Scars Using the Fluorescence Microscope . C. Mechanism of Budding . D. Some Further Perspectives . . V. Ageing of Cells and Age Distribution in a Population A. Maximum Reproductive Capacity of an Individual Cell B. Age Distribution in a Population . References

.

.

143 145 145 147 148 148 148 149 154 161 162 162 164 169

The Repair of Damaged DNA in Irradiated Bacteria. B. E. B. MOSELEY

.

I . Introduction 11. Evidence that DNA is an Important Target in Radiation Inactivation . 111. Radiation-Induced Chemical and Physical Changes in DNA . A. Changes in DNA Caused by Ultraviolet Irradiation . B. Changes in DNA Caused by Ionizing Irradiation . IV. The Repair of Damaged DNA A. Photoreactivation . B. DarkRepair . V. Summary . References

.

174 176 176 181 183 183 187 191 192

195

Author Index

Subject Index

173

.

203

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=Photosynthetic Apparatus JUNE

LASCELLES

Bacteriology Department, University of Californ$a, Los Angeles, California 90024, U.S.A.

.

1 I. An Introduction to the Photosynthetic Bacteria and their Pigments A. The Photosynthetic Pigments . 3 B. Environmental Effects on Pigment Synthesis . . 6 11. The Structure and Location of Chromatophore Material . . 8 A. Discovery and Definition of Chromatophortttes . . 8 B. Electron Microscopy of Cells . . 8 C. Chromatophore Material and Cell Membrane . . I2 D. Chlorophyll and Membrane Content . . 14 . 16 E. Peripheral and Internal Membrane as Sites for Pigments . F. The Vesicles of the Green Bacteria. . . 18 G. Conclusions . . 19 111. Isolation and Composition of P u r s e d Chromatophores . . 19 A. Isolation Procedures . . 19 B. Composition . . 21 C. Fractionation of Chromatophores . . 24 D. Immunological Reactivity . . 26 IV. Formation of the Photosynthetic Apparatus . . 27 A. Differences between Members of the Athiorhodaceae Grown Photosynthetically and Aerobically . . 27 B. Adaptive Synthesis of the Photosynthetic Apparatus . . 29 C. Chlorophyll Synthesis . 31 D. Regulation of ChIorophyIl Synthesis . . 34 References . . 39

.

1. An Introduction to the Photosynthetic Bacteria and their Pigments

The unique physiological characteristic of the photosynthetic bacteria is their ability to grow anaerobically in the light, a property conferred upon them by their photosynthetic pigment system. Unlike green plant photosynthesis oxygen is not evolved in the bacterial process, and, connected with this, they require an exogenous reductant (Stanier, 1961 ;van Niel, 1962; Vernon, 1964; Gest, 1966). The different genera of photosynthetic bacteria characteristically use either reduced inorganic sulphur compounds, hydrogen, or organic substrates as reductant ;they 1

TABLE1. Some Characteristics of Photosynthetic Bacteria Photosynthetic growth supported by A

Group Green sulphurbacteria Thiorhodaceae Athiorhodaceae

Representative members

Chlorobium spp. Chloropseudomonas ethylicum Chromatium spp. Rhodopseudomonas spp. Rhodospirillum spp.

Sulphur compounds

+ c02

Organic acids alone

Ability to grow aerobically (dark)

Yes

NO

NO

>

Major form of chlorophyll Chlorobium chlorophyll 660 or 650

a 24

M

:: M

u)

Yes No

Yes Yes

NO

Yes (some species)

Bacteriochlorophylla Bacteriochlorophylla or b"

Bacteriochlorophyll 6 is found in a new species, Rps. viridis (Eimhjellenet al., 1963; Drews and Giesbrecht, 1965, 1966).

3

THE BACTERIAL PHOTOSYNTHETIC BPPaRATUS

also vary with respect to their ability to use carbon dioxide as sole carbon source (Table 1). The overall process of bacterial photosynthesis may be represented by Fig. 1. There is strong experimental evidence for this scheme as a general outline though the precise sequence of events is yet to be established (Vernonand Ke, 1966; Gest, 1966).The scheme in Fig. 1 fits observations made largely with Rhodospirillurn rubrum, and variations in detail are likely in other organisms.

Cytochromoid?.

I F

-

Ubiquinone

1

i

Ferredoxin

_____f

I

Flavoprotein

ADP+Pi

NAD

FIG.1. Possible pathway of light-induced electron flow in Rhodospirillurn rubrum.

It has been well established that particulate preparations from the photosynthetic bacteria catalyse anaerobically: (1) light-dependent synthesis of ATP in the absence of an external reducing agent (photophosphorylation), and (2) light-dependent formation of reduced nicotinamide nucleotides in the presence of weak reductants such as succinate or certain reduced dyes (photoreduction). By analogy with the photosynthetic apparatus of the plant chloroplast the bacterial system might be expected to be associated with some type of organized structure and these structures are the concern of this review.”

PIGMENTS A. THE PHOTOSYNTHETIC 1. Chemistry In common with other photosynthetic forms of life the bacteria have both carotenoids and chlorophylls. The variety of carotenoids among the

* The term chromatophorewill be used for the pigmented particles isolated from cell-free extracts. When referring to the putative photosynthetic structure in intact cells the less precise terms “chromatophore material” or “photosynthetic apparatus” will be used.

4 JUNE LASCELLES different species is great (Jensen, 1963; Schmidt et al., 1965). Their probable function is to harvest light at wavelengths which are not absorbed by chlorophyll and also to protect cells from photodynamic oxidation reactions (Stanier, 1960; Dworkin, 1958).

Fk

II

II

CH=CHz

H~C

H3C

N-

H3C +

I CHz I

7 .

H3cx$cH3 CH2

COOH

CH2- CH3

\

c:~cH3 CHz

p< CHz

CH2

CH2

I CH2 I

I

H 'Z

I

COOH Protoporphyrin

HC-C=O

I

COOCHs

COOH Mg-vinylpheoporphyrin

c(5

$533 I

-CHI

p'

I

COO.CH3

COO.CzoHsg Bacteriochlorophyll

FIG.2. Structures of protoporphyrin, Mg-vinylpheopoqhyrh a5 (protochlorophyllide a ) and bacteriochlorophyll.

So far, three types of chlorophyll have been recognized in photosynthetic bacteria (Fig. 2) of which bacteriochlorophyll a is the most widely distributed (Jensen et aZ., 1964; Allen, 1966).Uhlorobium chlorophylls 660 and 660, a nomenclature based on their red absorption maxima in ether, are unique to the green sulphur-bacteria (Holt, 1966).Significant

THE BAUTERLBL PHOTOSYNTRETIC APPARATUS

5

differences in their structure from that of bacteriochlorophyll a are : (a) the Chlorobium pigments have a dihydro- rather than a tetrahydroporphyrin ring structure ; consequently, the red maxima are shifted towards the blue; (b) they lack the -COOCH, grouping on the cyclopentanone ring ; ( c )they are esterifiedwith farnesol rather than with phytol ; (d) the 660 pigment has an alkyl substituent on the 6-methene carbon atom of the porphyrin ring. A recent development is the recognition of bacteriochlorophyll b in a new species, Rhodopseudomonas viridis (Eimhjellen et al., 1963; Drews and Giesbrecht, 1965, 1966). This pigment is characterized by a red maximum in acetone at 795 mp, but its structure is yet to be determined. Its discovery should alert workers to careful scrutiny of new isolates for yet more forms of chlorophyll. 2. Spectrum of Chlorophylls in vivo ; Reaction-Centre Chlorophyll

The early spectroscopic observations of Wassink and of French (see Rabinowitch, 1951)indicated that the bacterial chlorophyllswere bound

Wavelength (mp)

FIG.3. Spectrum of bacteriochlorophyll from R h o d o p s e u d m m qvheroides in &o (-) and in methanol (---).

6

JUNE LASCELLES

in vivo in the form of macromolecular complexes. The red absorption maxima of all forms of chlorophyll found so far in bacteria exhibit a marked shift of at least 100 mp towards the blue when extracted into organic solvents. Such a change can be reasonably attributed to a release of the pigment from a bound form. The in vivo spectra also show several maxima in the red whereas the extracted pigment shows only one red peak (Olson and Stanton, 1966; Fig. 3). The multiple peaks in the in vivo spectra suggest that chlorophyll molecules are in association with different complexes, and the physiological significance of these is now being clarified (Clayton, 1966). The work with preparations from Rhodopseudomoms spheroides suggests that the bulk of the chlorophyll, associated with a complex absorbing at 850 mp (P-850),functions merely to harvest light. The complex absorbing at 870 mp (P-870) accounts for only about 5% of the total pigment but appears to represent the photosynthetic reaction centre. This pigment, but not P-850, is bleached (i.e. oxidized) reversibly upon illumination ;this phenomenon is shown most clearly in preparations which have been treated by methods which preferentially destroy P-850 (Clayton, 1963,1966). The isolation of mutant strains, which have the normal complement of P-850 yet cannot grow photosynthetically since they lack P-870, provides the most convincing evidence for the role of P-870 as the photosynthetic centre (Sistrom and Clayton, 1964).A complex absorbing at 800 mp is closely associated with P-870 (Clayton and Sistrom, 1966). There is evidence for reaction-centre chlorophyll in other photosynthetic bacteria. I n the green sulphurbacteria it seems that bacteriochlorophyll a is the reactive form, though the bulk of the pigment of the cell is Chlorobium chlorophyll (Olson and Romano, 1962; Olson, 1966).

EFFECTS ON PIGMENT SYNTHESIS B. ENVIRONMENTAL Photosynthetic bacteria, in common with plants, have the capacity to regulate chlorophyll synthesis in response to the demands of the environment. I n the bacteria the key factors are light intensity and, in the case of the Athiorhodaceae, oxygen pressure (see Tables 3, p. 15, and 6, p. 28). The elegant studies of Cohen-Bazire et al. (1957) and of Sistrom (1962b) showed that cultures of Rps. spheroides and Rsp. rubrum growing anaerobically respond to an increase or to a decrease in light intensity by suppression or stimulation respectively of synthesis of the photosynthetic pigments. Consequently, cells grown a t high intensities contain less chlorophyll and carotenoid per unit of protein than do those grown with low illumination. Repression of pigment synthesis by oxygen was also observed when this gas was introduced into cultures growing under continuous illumination. Oxygen repression is critically dependent

THE BACTERIAL PHOTOSYNTHETIC APPARATUS

FIG.4. Electron micrograph of section of photosynthetically grown Rhodospirillum rubrum (Vatter and Wolfe, 1958). CW = cell wall; CM= cytoplasmic membrane; Ch = chromatophore. Inset: isolated chromatophores; the line indicates 1 p. Reproduced by kind permission of the authors and the editors of the Journal of Bacteriology.

8

JUNZ LASCELLES

on the oxygen concentration and occurs only under high aeration. Many of the Athiorhodaceae form chlorophylland carotenoids when the oxygen concentration is low and, under such conditions, the specific pigment content may reach that of photosynthetic cultures (Lascelles, 1959 ; 1960a). Formation of the pigments in response to decreased oxygen pressure occurs in the dark; a light-dependent step is therefore not obligatory (or may not even occur) in chlorophyll synthesis by the bacteria.

11. The Structure and Location of Chromatophore Material A. DISCOVERY AND DEFINITION OF CHROMATOPHORES The term LLchromatophore” was coined by Schachman et al. (1952)for the relatively homogeneous, pigmented particles which were prepared by differential centrifugation of extracts of Rsp. rubrum. They contained the entire complement of photosynthetic pigments and were about 600 A in diameter. Particles of this size (about 1908)were not found in extracts of the organism grown aerobically and therefore devoid of photosynthetic pigments. It appeared that the chromatophores represented a specialized structure to house the photosynthetic pigments. Later studies by Frenkel (1956, 1958) showed that chromatophores isolated from Rsp. rubrum catalysed photophosphorylation and the photoreduction of NAD in the presence of succinate, as well as other photoreduction reactions, indicating that they represented the bacterial photosynthetic apparatus.

MICROSCOPY OF CELLS B. ELECTRON Electron microscopy of sectioned cells of photosynthetic bacteria showed them to contain structures similar in size and appearance to isolated chromatophores (Vatter and Wolfe, 1958). Rsp. rubrum and Rps. spheroides exhibited discrete membrane-bound vesicles (in diameter 500-1000 A and 400-800 A respectively) which appeared to be dispersed throughout the cytoplasm (Fig. 4). The association of the vesicles with the photosynthetic pigments was suggested by their absence from non-pigmented cells of Rsp. rubrum grown aerobically. It has since been firmly established with many organisms that the number and extent of the vesicular structures is directly related to the pigment content of the cells (Cohen-Bazire and Kunisawa, 1963; Drews and Giesbrecht, 1965; Gibbs et al., 1965; Holt and Marr, 1965c; Holt et al., 1966b). The vesicular structures found in Rsp. rubrum are not typical of all photosynthetic bacteria (Table 2 ) . Some exhibit stacks of paired

9

T H E BACTERIAL PHOTOSYNTHETIC APPARATUS

TABLE2. Appearance of Chromatophore Material in Electron Micrographs of Cell Sections Organism

Chlorobium thiosulphatophilum Chlorobium limicola Chloropseudomonas ethylicum

Appearance of structure

Oblong vesicles arranged around periphery, immediately under the cytoplasmic membrane ; 1000-1500 i% long, 300-400 i% wide Membrane-bound vesicles throughout cytoplasm

Chromatiuin strain D Chromatium okenii Thiospirillumjenseni Thiopedia sp. Vesicles and large lamellar structure Thiocapsa sp. Membrane-bound vesicles throughout Rhodospirillum rubrum the cytoplasm, 400-1000 d diameter Rhodopseudomoms spheroides Rhodospirillum molischianum Discrete lamellar structures at periRhodospirillum f u l v u m phery Rhodospirillum photometricurn Extensive lamellar structure disposed Rhodopseudomoms palustris around periphery Rhodopseudomonas viridis Rhodomicrobium vannielii

Reference 1 1 2

3 3 3 3 3 3,4, 5 4, 6 7,899 10 10 10 11 12

References: (1) Cohen-Bazire et al. (1964); (2) Holt et al. (1966a); (3) Cohen-Bazire (1963); (4) Vatter and Wolfe (1958); (5) Hickman and Frenkel (1959, 1965b); (6) Drews and Giesbrecht (1963); (7) Giesbrecht and Drews (1962); (8) Gibbs et al. (1965); (9) Hickman and Frenkel (1965a); (10) Cohen-Bazire and Sistrom (1966); (11) Drews and Giesbrecht, 1965; Giesbrecht and Drews, 1966; (12) Vatter et al. (1959).

lamellae, similar to the structures found in blue-green algae, and arranged variously according to the organism. I n Rhodomicrobium vannielii, for instance, the lamellae are arranged concentrically around the periphery of the cell (Vatter et al,, 1959) whereas in Rhodospirillurn rnolischianum (Fig.6#) the lamellae appear as discrete discs a t intervals around the periphery (Drews, 1960). Perhaps the most remarkable structures are he large oblong vesicles found in the green sulphurbacteria (Fig. ; Cohen Bazire et al., 1964; Holt et al., 1966a).These lie immediately under the peripheral membrane. Conclusive proof that these various structures are the site of the photosynthetic pigments is difficult to obtain in the absence of techniques for locating the pigments in cell sections. Indirect evidence has been provided in many cases by observing a correlation between the number and extent of the structures with the pigment content of the cells. Also, the appearance of pigmented fractions isolated from disrupted cells has been shown with some organisms to resemble the structures found in cell sections

a

FIG.5. Electron micrograph of a section of Chlorobium thiosulphatophilum showing the complex cell wall (w) with its rod-shaped extensions (ex), the cell membrane (m) and ellipsoidal vesicles (cv) adjacent to but distinct from the peripheral membrane (vm). Two large mesosomal elemcnts (M) and a granule of polymetaphosphate (p)are also visible. From Cohen-Bazireet at. (1964). Reproduced by kind permission of the authors, and the editors of the Journwl of Cell Biology.

FIG.6. Electron micrograph of a section of Rhodospirillurn rnolischianurnshowing lamellar invaginations o f the peripheral membrane. As the culture ages, the lamellae increase in number and become closely associated into groups ( a t D'). The space (G) within the folds then increases and separates adjacent sides of the folds. The distinct lamellae seen at D average about 160 A in width. From Hickman and Frenkel (1965a). Reproduced by kind permission of the authors, and the editors o f the Journal of Cell Biology.

12

JUNE LASCELLES

(Cohen-Bazire and Sistrom, 1966). Such a correspondence is not always easy to show, possibly because of the fragility of the structures. For instance, isolated chromatophores from Rsp. molischianum appear as welldefined particles which may be subunits of the intracellular lamellar structures (Hickman and Frenkel, 1965a). Recently, Giesbrecht and Drews ( 1966) have succeeded in isolating chromatophores from Rps. viridis in which the lamellar structure is preserved.

C. CHROMATOPHORE MATERIAL AND CELL MEMBRANE Assuming that the structures described so far represent chromatophore material, a problem which arises is whether they exist as discrete entities in the cytoplasm (analogousto chloroplasts and mitochondria) or whether they are an integral part of a continuous membrane system. If the latter is true, the preparations of isolated chromatophores arise presumably by comminution of the large membrane structure into quite homogeneous particles. The evidence is strong that chromatophores from many organisms exist in the cell as part of a membranous continuum. However, there is still controversy over whether or not discrete chromatophores can arise in vivo from such a continuum (Stanier, 1963; Hickman and Frenkel, 1965b; Gibson, 1966). The derivation of chromatophores from large intracellular structures was suggested some years ago by Newton and Newton (1957). Sonic treatment of Chromatiurn for varying periods of time showed that pigmented particles were not released immediately upon cell rupture but appeared at a relatively slow rate. Also, the chlorophyll appearing during the early stages of disruption was distributed equally between small and large particles, but with more prolonged treatment, it was found almost exclusively in the small particle fraction. These observations are consistent with comminution of large membrane structures into small pieces. The method of controlled sonic treatment for demonstrating the locus of an intracellular component (first used by Marr and Cota-Robles, 1957) has since been applied to other photosynthetic bacteria with similar results (Holt and Marr, 1965a; Holt et al., 1966a). The association of chromatophore material with membrane was shown more directly with lysed preparations of Rsp. rubrurnz(Tutt1e and Gest, 1959). The ghost fraction from osmotically lysed spheroplasts, prepared by lysozyme treatment, contained the bulk of the photosynthetic pigments and had photophosphorylation activity. The significanceof this early work was not adequately recognized until later developments in the field of electron microscopy, which suggested that chromatophore material is associated with internal membrane, arising by invagination of the peripheral membrane. Evidence for this

T H E BACTERIAL PHOTOSYNTHETIC APPARATUS

13

was first obtained with osmotically lysed Rsp. molischianum, in which the lamellar structures could be seen to arise by invagination and folding of the peripheral membrane (Giesbrecht and Drews, 1962). A similar origin for the vesicular structures of Rsp. rubrum and Rps. spheroides has been strongly indicated by many observations which may be summarized as follows. (1) The few vesicles to be seen in cells of low pigment content are located exclusively a t the periphery (Cohen-Bazire and Kunisawa, 1963). As the pigment concentration increases, the vesicles become more numerous and extend more deeply into the cytoplasm. I n these studies with Rsp. rubrum, the chlorophyll content was varied by growth a t different light intensities under anaerobic conditions. Progressive extension of the vesicles from the periphery into the cell interior can also be seen in cells from cultures sampled a t various stages of growth at fixed light intensity (Drews and Giesbrecht, 1965; Hickman and Frenkel, 1965b).As the cell population increases, the specific chlorophyll content increases and the solely peripheral distribution of the vesicles gives way to deeper penetration into the cytoplasm. (2) The dimensions and structure of the membrane surrounding the vesicles are similar to the peripheral membrane, both being defined as two dense parallel lines with a total thickness in Rsp. rubrum of 75-85 d (Cohen-Bazire and Kunisawa, 1963; Holt and Marr, 1965a). (3) Continuity of the peripheral membrane and vesicles has been observed (Fig. 7 ; Cohen-Bazire and Kunisawa, 1963; Drews and Giesbrecht, 1965; Hickman and Frenkel, 19658). Such continuity is most clearly seen in cells of low pigment content which show a peripheral distribution of vesicles ; as the pigment content increases, the peripheral membrane exhibits more numerous invaginations, similar in appearance

FIG.7. Electron micrograph of section of Rhodopseudomoms spheroides showing chromatophores arising by invagination of the peripheral membrane (Drews and Giesbrecht, 1965). Reproduced by kind permission of the authors and of the editors of Zentralblatt fur Bakteriologie.

14

JUNE LASCELLES

to the vesicles. Such progressive invagination of the peripheral membrane may be followed in non-pigmented cells (grown with high aeration) as they form pigment upon transfer to semi-anaerobic-dark or anaerobiclight conditions (Cohen-Bazire and Kunisawa, 1963; Giesbrecht and Drews, 1963). Interconnections between vesicles have also been observed (Giesbrecht and Drews, 1963; Holt and Marr, 1965a). Studies with osmotically lysed Rsp. rubrurn have also provided evidence of continuity between an internal membrane system with a vesicular appearance and the peripheral membrane (Boatman, 1964; Holt and Marr, 1965a). (4) Impressive evidence that the internal membrane seen by electron microscopy gives rise to isolated chromatophores has been provided by Holt and Marr (1965b, c). These experiments are discussed in Section I1E (Pa 17).

D. CHLOROPHYLLAND MEMBRANECONTENT Electron microscopy shows a considerable amount of internal membrane in photosynthetic bacteria of high pigment content. This raises the question of how much chromatophore material is represented by the membranes, and this question cannot be answered in the absence of a method for locating chlorophyll by electron microscopy. Various indirect approaches have, however, been used to determine quantitatively the amount of chromatophore material. Gibbs et al. (1965)calculated the volume of membrane by measurement of electron micrographs of sectioned cells of Rsp. rnolischianum, containing varying amounts of pigment. The chlorophyll content was correlated with the amount of total membrane (peripheral and internal) but a constant ratio between pigment and internal membrane was not observed. Direct analysis was also made of the amount of chromatophore protein isolated from cells of differing pigment content, with results which did not accord with those of electron microscopy. I n experiments where recovery of chlorophyll and protein was almost quantitative, the amount of protein in the purified chromatophore fraction did not match the amount of pigment. Thus, in cells with a four-fold difference in chlorophyll content, the amount of protein in the purified chromatophore fraction varied by only 1.2-fold (65 and 54% respectively of the total cell protein). This lack of correlation between chlorophyll concentration in cells and the quantity of isolated chromatophore protein has been a frequent observation (Cohen-Bazire and Kunisawa, 1960; Drews and Giesbrecht, 1965; Bull and Lascelles, 1963). Lipid phosphorus has been used as an index of membrane in photosynthetic bacteria; in these organisms, as in other bacteria, the

15 phospholipids are located predominantly in the membrane fraction (Lascelles and SzilBgyi, 1965). The lipid phosphorus content of Rps. spheroides is higher in pigmented cells than in those grown with high aeration and consequently virtually devoid of photosynthetic pigments (157 and 90 mpmoles lipid phosphorus per mg. protein, respectively). The association of chlorophyll synthesis with an enrichment in phospholipid was also suggested by kinetic experiments which showed a THE BACTERIAL PHOTOSYNTHETIC APPARATUS

TABLE3. Chlorophyll Concentration in Cells and Pursed Chromatophores ~

Organism

Rhodospirillum rubrum

Rhodopseudomonas spheroides Rhodospirillum molischianum Chloropseudomonas ethylicum

~

~

Chlorophyll concentraLight tion (mpmoles/mg. n intensity protein) i during >-’-r growth Crude Purified (foot cell chromatocandles) extract phores 200 2000 18.5 1020 2040 1 100 2400 6000 18.5 1020 2040 70 2000 10 100 1000 10000

29.8 9.9 24 13 9 39.5 11 6 4 27 20 5 111 26 475 178 111 92

55 22.5

}

~~

Reference Cohen-Bazire and Kunizawa (1960) Drews (1963)

17 82

Holt and Mam (1965~) 42

Drews (1963) 9 159 45

I

212 130

J

Gibbs et al. (1965) Holt et al. (1966b)

All organisms were grown photosynthetically at the light intensity shown

parallel increase in pigment and lipid phosphorus in cells transferred from high to low aeration. However, constant proportionality between the concentration of phospholipid and that of chlorophyll has not been found in analyses of photosynthetic bacteria of varying pigment content, with the exception of Rhodopseudomonas palustris (Cohen-Bazire and Sistrom, 1966). I n Rsp. rubrum, for instance, a six-fold difference in chlorophyll concentration was reflected by only a 1-9-fold increase in phospholipid. 3

16

J U N E LASCELLES

Analyses of the specific chlorophyll content of chromatophores prepared from cells containing large and small amounts of pigment has provided conflicting results. If there is proportionality between membrane and pigment, isolated chromatophores should have the same specific chlorophyll content, irrespective of the chlorophyll concentration in the cells. This expectation has not been generally realized; the chlorophyll-to-pigment ratios of most chromatophore preparations reflect the specific pigment content of the cells from which they were isolated (Table 3). The highly purified chromatophores of Holt and Marr (1965b), however, did show a constant ratio in all preparations except those from cells grown at very high light intensity, where destruction of pigment may have occurred (Table 3). On the whole, the analytical results suggest that a considerable increase in pigment can be accommodated without a substantial increase in the amount of the membrane matrix of the chromatophore material. This is not substantiated by electron microscopy which shows the membranes of photosynthetic bacteria to be of constant thickness (70-80 8) in cells of low and high pigment content. A change of chlorophyll concentration of the magnitude observed experimentally could not occur without an increase in the thickness of the membrane (Holt and Marr, 1965~).

E. PERIPHERAL AND INTERNAL MEMBRANE AS SITESFOR PIGMENTS The discrepanciesbetween the analytical data and electron microscopy might stem from an uneven distribution of chromatophore material between the peripheral and internal membranes. It is possible that the pigments are located predominantly in the internal membranes, which may arise by differentiation of the peripheral structure. Chromatophores isolated by the usual procedures may be composed of peripheral membrane, low in pigment, and of inner membrane representing the purest chromatophore material. There is evidence in support of a differentiation of the internal membrane from the peripheral membrane at least in respect to chlorophyll content. Many workers have found at least two pigmented bands (“heavy” and “light” chromatophores) upon density-gradient centrifugation of the particulate fraction from various photosynthetic bacteria (Hickman and Frenkel, 1959; Cohen-Bazire and Kuniaawa, 1960; Worden and Sistrom, 1964; Gibson et aZ., 1962).The specific chlorophyll content of the heavy material is less than that of the light fraction (Table 4), and consequently the latter has usually been designated as the purified chromatophore fraction. The derivation of the light material from internal membrane of Rsp.

17

THE BACTERIAL PHOTOSYNTHETIC APPARATUS

rubram has been shown convincingly by Holt and Marr (1965b). The membrane fraction from cells disrupted by osmotic chock was resolved into three pigmented bands upon sucrose-gradient centrifugation (Table 4).The intermediate band was free of cell envelopes, but showed large membranous structures in the electron microscope ;upon sonic Greatment TABLE 4. Chlorophyll Content of Pigmented Fractiom after SucroseGradient Centrifugation

Method of preparation of extract French press Osmotic shock

Position of pigment on gradient (em. from top)

Chlorophyll content (mpmoles/mg. protein)

yoTotal

1.90 3.60 1.85 2.90 3.60

81 35 59 60 45

23.2 77-5 13.2 12.7 63.5

Data from Holt and Marr (196513). Extracts were prepared from Rhodospirillum rubrum grown at an illumination of 73 foot candles

these were fragmented into material similar in appearance, density and specific chlorophyll content to the typical chromatophores found in the light fraction. It was concluded that the intermediate band represented internal membrane, and that the light chromatophores arose from this by fragmentation. The origin of the pigment in the heavy fraction is more difficult to establish. Electron microscopy has shown a heterogeneous collection of fragments, including fractured cell envelopes filled with large masses of internal membrane (Holt and Marr, 1965b). This fraction contains a major proportion of the total chlorophyll, at least when isolated from French-press extracts of Rsp. rubrum and Rps. spheroides (Worden and Sistrom, 1964; Holt and Marr, 1965b; Table 4). Also, it can give rise to particles similar in density and chlorophyll content to light chromatophores by further passage through the French press or by sonic treatment. This could be interpreted as release and subsequent comminution of internal membrane which was not originally detached from the cell envelope. Another possibility is that the heavy chromatophore fraction is derived from the peripheral membrane. Worden and Sistrom (1964) observed that the amount of protein in this fraction from Rps. spheroides was constant irrespective of the pigment content of the cells, and amounted to about 24% of the total cell protein. The constancy suggested that the

18

JUNE LASCELLES

heavy material originated from a structure present in both pigmented and non-pigmented cells. The chlorophyll-to-carotenoidratio and the chlorophyll absorption spectrum of the heavy chromatophores differed from the light material, suggesting that the pigments in the two fractions were associated with different complexes. There is some evidence for differentiation of internal and peripheral membrane in non-photosynthetic bacteria, at least with respect to components of the respiratory apparatus. I n Azotobacter agilis, 60% of the NADHz oxidase was located in the internal membrane, and was released by osmotic shock followed by ballistic disintegration (Pangborn et al., 1962). The residual oxidase (located in the peripheral membrane?) remained firmly attached to the cell envelope. Succinate dehydrogenase in Bacillus subtilis, located by reduction of a tetraeolium dye combined with electron microscopy, was observed in both peripheral and internal membrane (mesosome) but the activity was more pronounced in the latter structure (Sedar and Burde, 1965).Kashket and Brodie (1963a,b) found a difference in distribution of “succinoxidase” and NADHz oxidase in the large and small particles prepared from Escherichia coli ; both fractions had NADHz oxidase activity, but “succinoxidase” was confined mainly to the large particle fraction.

F. THEVESICLES OF

THE

GREENBACTERIA

The ellipsoidal vesicles seen in electron micrographs of green sulphurbacteria have been shown fairly conclusively to contain chlorophyll by techniques similar to those used by Holt and Marr (1965b) with Rsp. rubrum (Holt et al., 1966a). The vesicles provide the most clear-cut example of a differentiated chromatophore structure distinct from the peripheral membrane. The surrounding membrane of the vesicles is thinner than that of the peripheral structure (50 and 80 A respectively) and electron microscopy has not shown evidence of attachment between the structures (Cohen-Bazireet al., 1964).Differences in the composition of the structures has been indicated by comparative studies of the location of succinate dehydrogenase, a typical component of peripheral membrane, and chlorophyll (Holt et al., 1966a). Sonic treatment of Chloropseudomonas ethylicum released chlorophyll from the cells more rapidly than the dehydrogenase, in accord with the association of the enzyme with the peripheral membrane and the pigment with the more readily released internal structures. Fractionation of the dehydrogenase and the chlorophyll has also been achieved by differential centrifugation of French-press extracts of Chlorobium thiosulphatophilum (Fuller, 1963). The enzyme was found in the fraction containing large fragments of membrane, whereas the pigment was predominantly located in the

THE BACTERIAL PHOTOSYNTHETIC APPARATUS

19

small-particle fraction. Although the composition of the peripheral membrane and the vesicles appears to be quite radically different, the possibility still remains that the two structures may be connected by delicate attachments which are destroyed upon disintegration of the cells.

G. CONCLUSIONS A tentative conclusion which may be drawn from the present information is that chromatophore material is located at least partly in the internal membranes of some of the photosynthetic bacteria. Also, the constant ratio of chlorophyll-to-proteinfound in highly purified chromatophores points to a stoichiometric relation between this pigment and the internal membrane matrix-at least in Rsp. rubrum (Holt and Marr, 1965b).However, there is also evidence for the presence of chromatophore material in peripheral membrane, though it is not possible t o decide whether the composition of this material differs from that in the internal membrane. Systematic analysis with respect to enzymic and chemical content of the various chromatophore fractions may help in the solution of these problems. The concentration of succinate dehydrogenase and other membrane-bound enzymes may serve, for instance, as an indication of differentiation of the membrane structures. Clearly the primary requirements are for methods of separating and purifying the internal and peripheral membranes, followed by fractionation into their component parts. Rupture of cells by lytic methods may offer the most promising beginning to such studies.

III. Isolation and Composition of Purified Chromatophores A. ISOLATION PROCEDURES Problems of isolating a pure chromatophore fraction begin with the disruption of the cell envelope, and the harsh methods in common use give ample opportunity for the generation of artifacts by damage to delicatemembrane structures. Some possibilities to be considered in evaluating the purity and the relation of isolated chromatophores to the functioning photosynthetic apparatus in the,intact cell are as follows. (1)Components whichinvivo are loosely attached to the structure may be solubilized and lost during the purification ; for instance, ferredoxin, c-type cytochrome and the cytochromoid pigments (Orlando et al., 1961; Horio and Kamen, 1962). (2) Structures which are not associated with the photosynthetic apparatus may be fragmented and may fractionate with the chromatophores during purification. Soluble cytoplasmic

20

JUNE LASCELLES

constituents might also be non-specifically adsorbed to the isolated particles. (3) The chromatophore structure may itself become fragmented into subunits which may or may not represent the state of the structure in vivo. There are no firmly established criteria for assessing the purity of chromatophores. The pigment-to-protein ratio (specific pigment concentration), and homogeneity upon ultracentrifugation or by electron microscopy are useful guides. Another parameter, infrequently used, is photophosphorylation activity expressed in terms of protein and of chlorophyll (Frenkel and Hickman, 1959; Cohen-Bazire and Kunisawa, 1960). Disruption of cells by the French press is the most usual choice for the initial preparation of crude extracts. Perhaps the best method to be exploited so far is disruption by osmotic shock. With Rsp. rubrum this may be achieved by mixing the cells with glycerol followed by rapid dilution into buffer (Holt and Marr, 1965a).Glycerol-lysisis not effective with all photosynthetic bacteria; however, some, including Rps. spheroides, are lysed by treatment with lysozyme and EDTA (Tuttle and Gest, 1959; Lascelles and Szil&gyi,1965; Gibson, 1965a). Chromatophores can be separated from the crude particulate fraction by sucrose-gradient centrifugation which usually results in two pigmented bands, the “light” and “heavy” chromatophores (Frenkel and Hickman, 1959; Cohen-Bazire and Kunisawa, 1960; Holt and Marr, 1965b; Worden and Sistrom, 1964). It is the light fraction which is usually deemed to contain the pure material, on the basis of specific pigment content and homogeneity of particle size ; the position of this fraction from extracts of Rsp. rubrum and Rps. spheroides corresponds to a density of 1.14 g./ml. (Cohen-Bazireand Kunisawa, 1960;Worden and Sistrom, 1964). Centrifugation in a linear gradient of caesium chloride has also been used with Rps. spheroides (Gibson, 1965a). Ribosomes are a likely contaminant of chromatophores purified only by sucrose-gradient centrifugation. Such contamination was essentially abolished from Rps. spheroides preparations by centrifuging in 27% rubidium chloride prior to sucrose-gradient centrifugation (Worden and Sistrom, 1964). The purest preparations appear to be those from Rsp. rubrum which were subjected to sucrose-gradient electrophoresisafter centrifuging in a linear sucrose gradient (Holt and Marr, 1965b).The specific pigment content of these chromatophores was high (Table 3) but their photophosphorylating activity was not reported ; comparison of this activity with less extensively purified material would be interesting. Sedimentation coefficients reported for purified chromatophores include 1535 and 165s for Rps. spheroides (Worden and Sistrom, 1964;

21 Gibson, 1965a), 120s for Chromatiurn (Bergeron, 1959) and 116s for Chlorobium thiosulphatophilucm (Sykes et al., 1965). THE BACTERIAL PHOTOSYNTHETIC APPARATUS

B. COMPOSITION The detailed analyses of Chromatium chromatophores (Newton and Newton, 1957)set a precedent which, regrettably, has not been followed. Attention has been given mainly to the specific pigment content, which in most preparations reflects the pigment content of the cells from which the chromatophores were prepared (Table 3). Since the highly purified preparations from Rsp. rubrurn (Holt and Marr, 1965b) showed a fairly constant specific pigment content, the variations found in other preparations might be attributed to contamination of the pigmented fractions with non-chromatophore material. Except for the photosynthetic pigments the composition of isolated chromatophores (Table 5) is similar to that of membrane fractions prepared from non-photosynthetic organisms (Weibull and Bergstrom, 1958; Kodicek, 1963). The predominant components are protein and lipid; the small amounts of nucleic acid probably represent ribosomal contamination (Worden and Sistrom, 1964). The carbohydrate found in some preparations may arise largely from contamination by cell-wall components, and analyses of chromatophores should include hexosamine as an indication of such material. Galactolipids are associated specifically with the chloroplast in plants (Benson, 1964)but bacteria do not appear to have a unique type of lipid associated with their photosynthetic apparatus. Phosphatidylethanolamine was found by Newton and Newton (1957)to comprise the bulk of the lipid of Chromatiurn chromatophores. Phospholipids are apparently the predominant form of lipid in other photosynthetic bacteria, assuming that the lipid content of the whole cell (upon which most analyses have been made) reflects that of the isolated chromatophores. Most species of photosynthetic bacteria contain several types of phospholipid ; for instance, Rps. spheroides and Chlorobium thiosulphatophilum contain phosphatidylethanolamine, phosphatidylcholine, phosphatidylglycerol and phosphatidic acid (Lascelles and Sziliigyi, 1965; Wood et al., 1965). Athiorhodaceae grown photosynthetically or aerobically in the dark exhibit no difference with respect to the types of phospholipid, their relative proportion or in their fatty acid components (Lascelles and Sziliigyi, 1965;Wood et al., 1965; Scheuerbrandt and Bloch, 1962; Erwin and Bloch, 1964).Photosynthetically grown cells however, do contain a considerably higher content of total phospholipid (Lascellesand Szilhgyi, 1965; Cohen-Bazire and Sistrom, 1966).

TABLE 5. Composition of Isolated Chromatophores Component Protein Lipid

1

Chlorobium thiosulphatophiluma*' 63 25

Chlorophyll yo dry weight Carotenoids Carbohydrate Nucleic acid Total phosphorus pmoles/mg. Lipid phosphorus protein Chlorophyll Carotenoids Haems mpmoles/ Flavin (acid extractable) Nicotinamide nucleotides Non-haem iron

I

7

9 4

98 19 1.5

Chromatium 61 22 (Phospholipid) 4 1.5 7 0 0.83 0.51 40 (155)' 17 (88)g 2.8 (3.7)' 0.5 0.9 12

Sykes et al. (1965). Hulcher and Conti (1960). Newton and Newton (1957). Bergeron (1959). Bull and Lascelles (1963). Gibson et al. (1962). Values in parentheses from Hulcher and Conti (1960); others from Newton and Newton (1957).

Rhodopaeudomonas ~pheroides'*~ 58 25

5 1 4 1 0.62 0.40 112 44 2.9

-

cl

a

z

F

u)

Bu

THE BACTERIAL PHOTOSYNTHETIC APPARATUS

23

1. Catalytic Components

Analysis of purified chromatophores has shown the presence of cytochromes, flavines, and nictotinamide nucleotides (Table 5). Other electron-transfer catalysts can be presumed to be present, at least in preparations which photophosphorylate. Analytical data are not available for the ubiquinone content of chromatophores, though it is known to be present in high concentration in the cells of photosynthetic bacteria, particularly when grown anaerobically in the light (Fuller et al., 1961; Sugimura and Rudney, 1962; Carr and Exell, 1965). Its presence in chromatophores has been indicated by light-induced spectral changes (oxido-reductions) in the region 240-340 mp (Clayton, 1962a). Ferredoxin has now been found in a number of photosynthetic bacteria (Buchanan et aZ., 1965; Yamanaka and Kamen, 1965) and its reduction by illuminated chromatophores from Chlorobium thiosulphutophilum has been shown (Evans and Buchanan, 1965). Extracts of this organism and of Chromutium catalyse a ferredoxin-linkedreduction of NAD in the dark with hydrogen plus hydrogenase as source of reducing power (Weaver et al., 1965). The concentration of ferredoxin in chromatophore preparations has not, to the reviewer's knowledge, been determined but it is presumably present in preparations which show photoreduction of nicotinamide nucleotides. A non-haem iron protein has been found in Chromatium chromatophores, and may contribute to the large amounts of non-haem iron shown in the analyses (Table 5). This protein is quite distinct from ferredoxin and has a potential of +0.35 V ; its function is unknown (Bartsch, 1963). Similar iron proteins have been found in several speciesof Athiorhodaceae (De Klerk and Kamen, 1966). 2. Enzymic Activities

The photophosphorylation activity found by Frenkel and Hickman (1959) with purified chromatophores from Rsp. rubrum was from 110 to 175 pmoles Pi esterified per pmole of chlorophyll per hour. Photoreduction of NAD in the presence of succinate occurred at the rate of 23-26 pmoles NADHzper pmole of chlorophyllper hour. The photophosphorylation values were obtained in the absence of phenazine methosulphate and succinate, which stimulate this activity in purified chromatophores (Geller and Lipmann, 1960 ; Cohen-Bazire and Kunisawa, 1960). Most studies of photophosphorylation have been with chromatophores from Rsp. rubrum partly because this organism provides relatively stable preparations. Purified material from Rps. spheroides exhibits feeble activity (less than 1 pmole Pi esterified per hour per pmole of 3*

24

JUNE LASCELLES

chlorophyll)which is not enhanced by phenazine methosulphate (Gibson, 1965b).Crude extracts of the organism are also far less active in comparison with other organismsbut some improvement occurs when the extracts are made in the presence of ascorbate. Possibly in Rps. spheroides an essential factor(s)is more easily dissociated and inactivated by oxidation. Photophosphorylation activity is usually expressed in terms of chlorophyll, and has been found to vary inversely with the pigment content of chromatophore preparations (Cohen-Bazireand Kunisawa, 1960;Drews, 1964).When expressed on a protein basis, relatively constant values have been obtained with preparations of widely differing pigment content ;the rate is therefore limited by a dark reaction rather than by the primary photochemical event. Factors which may become limiting include some of the oxidation-reduction catalysts which may not be flrmly attached to the structure. Cytochrome c2, for instance, is readily leached from the chromatophores of Rsp. rubrum with consequentloss of photophosphorylation activity; this activity is restored by addition of the cytochrome to the depleted preparations (Horio and Kamen, 1962). Ferredoxin and other iron proteins are also likely to be lost in purification since they are found predominantly in the soluble fraction of cell extracts (Bartsch, 1963; Yamanaka and Kamen, 1965). Purified chromatophores from Rsp. rubrum have succinate dehydrogenase activity which shows essentially no variation in preparations from organisms of very different pigment content (Cohen-Bazire and Kunizawa, 1960). The enzyme might therefore be an integral part of the chromatophore structure, This is not, however, the case with preparations from the green sulphur-bacteria. The enzyme is located in particles which are separable from the bulk of the pigmented material by differential centrifugation (Fuller, 1963; Holt et al., 1966b).

C. FRACTIONATION OF CHROMATOPHORES 1. ChlorophylLProtein Complexes

The object of many fractionation studies with chromatophores has been to isolate a pure pigment-protein complex, but the physiological status of such complexes is uncertain in the absence of test systems to examine their photochemical properties. The green sulphur-bacteria have so far provided the purest preparations (Olson, 1966).Two soluble complexes with red absorption maxima at 770 and 809mp have been isolated from alkaline extracts of Chbrobium thiosuhphtophilum and Chloropseudomoms ethylicum. The material absorbing at 809 mp has been crystallized and consists entirely of protein and pigment. It has a molecular weight of 3.6 x 104, with three moles of

THE BACTERIAL PHOTOSYNTHETIC APPARATUS

25

pigment per mole of complex. The pigment is apparently bacteriochlorophyll a whereas the bulk of the chlorophyllin the organisms is Chlorobium chlorophyll 660 or 650. In vivo experiments suggest that the 809 complex is probably the photosynthetic reaction-centre in the green bacteria (Olson and Romano, 1962; Olson and Sybesma, 1963). Detergent treatment of chromatophores has shown that there are differences between the various pigment-protein complexes in their manner of binding to the chromatophore structure. Light-harvesting forms of chlorophyll in chromatophores from Rps. spheroides, Rsp. rubrum and Chromatiurn are readily solubilized by treatment with deoxycholate or with Triton X- 100,whereasthe reaction-centre form remains attached to the particles (Clayton, 1962b; Bril, 1958,1960). The solubilized pigment is readily destroyed by light unlike the particle-bound material. 2. Multi-Enzyme Complexes

The pigment-protein complexes function in vivo in conjunction with oxidation-reduction catalysts and phosphorylation systems. An understanding of the arrangement of these components in the functional photosynthetic unit requires detailed studies of the individual catalytic and structural units; this means dissection of the chromatophore and, hopefully, re-assembly of the components in some semblance of their natural state. Fractionation studies have not yet reached this degree of sophistication, but have provided leads for future exploitation. Lipase treatment of chromatophores from Rsp. rubrum considerably increased their specific chlorophyll content without altering their behaviour on sucrose-gradientcentrifugation (Cohen-Bazireand Kunisawa, 1960). The treated material did not have photophosphorylation or succinate dehydrogenase activities, presumably due to loss or inactivation of essential components. Treatment of chromatophores of Rps. spheroides with cholate plus deoxycholate has given material which still exhibits photophosphorylation (Gibson, 1965~). The bile salts removed 70% of the lipid (mostly phospholipid) and the sedimentation coefficient dropped from 160s to 1205. The depleted material was unstable and was readily disrupted into two pigmented fractions in the presence of salts. These relatively mild procedures for subfractionation may be usefully extended in the future. Gibson suggests that core particles, containing the photosynthetic pigments and photophosphorylation system, are covered in vivo by a thin layer of lipid. This notion requires confirmation by refined techniques of electron microscopy. The non-ionic detergent, Triton X-100, appears to be the best weapon so far for disruption of chromatophores into subunits with biochemical

26

JUNE LASCELLES

activity. It was first applied by Bril(1958)who showed that it solubilized the 850 mp-form of chlorophyll from chromatophores of Rps. spheroides. The detergent has recently been used to fractionate chromatophores from Chromatium and Rsp. rubrum (Garcia et al., 1966a, b). Chromatium chromatophores were split into two pigmented fragments, the light (L) and heavy (H) fractions, separable by sucrose-gradient centrifugation. The H fraction contained reaction-centre chlorophyll (P-890) which exhibited the typical photo-oxidation response. It also catalysed the anaerobic photo-oxidation of reduced phenazine methosulphate in the presence of ubiquinone-6 and was rich in cytochromes. The L-fraction contained the bulk of the chlorophyll (P-800 and P-850) but was virtually devoid of P-890, cytochromes and of photo-oxidative activity. Electron microscopy showed membrane material in the L fraction whereas the H fraction consisted apparently of subunits. Treatment of chromatophores of Rsp. rubrum with Triton X-100 also gave heavy and light pigmented fractions (Garcia et al., 1966b). Both contained reaction-centre chlorophyll but the H fraction contained most of the total pigment. Only the L fraction catalysed the photoreduction of NAD (reduced dichlorophenolindophenol as electron donor) and had succinate dehydrogenase activity. A third fraction, banding above the L fraction, contained bacteriopheophytin rather than chlorophyll and had succinate dehydrogenase activity and cytochrome c. This material appeared to exist per se in the chromatophore, since such particles were not released by further treatment of H and L fractions with detergent. Electron microscopy showed subunits in the L, and membranous material in the H, fractions corresponding respectively to the H and L particles from Chromatium. The observations support the possibility that light-harvesting chlorophyll is located in the exterior membrane of the chromatophore, and the photochemically reactive form together with associated catalysts is situated in the interior-a logical arrangement.

REACTIVITY D. IMMUNOLOGICAL Not surprisingly, chromatophores are highly antigenic. Antisera have been prepared by injection of rabbits with Chromatium chromatophores ; these antisera contain antibodies which precipitate the pigmented particles (Newton and Levine, 1959). The antigens of the chromatophores apparently included components from the cell surface since the antisera also reacted with intact cells. The immunological reactivity of pigmented subfractions, derived by treatment with disulphide-cleaving reagents, has also been examined (Newton, 1962). Chromatophores from Rsp. rubrum also induce the synthesis of specific

THE BACTERIAL PHOTOSYNTHETIC APPARATUS

27

antibodies which precipitated the photosynthetic pigments from crude cell extracts (Newton, 1960). Extracts prepared from pigmented cells, which had been aerated in the dark for a short period, lost their reactivity with the specific antiserum, suggesting that some dissociation of the reactive material had occurred. The value of immunology as a tool may become more apparent when highly purified chromatophores are used as antigen. Such techniques might, for instance, be applied to members of the Athiorhodaceae to determine whether the chromatophores contain specific proteins which are lacking in particulate fractions from non-pigmented cells.

IV. Formation of the Photosynthetic Apparatus Those Athiorhodaceae capable of either photosynthetic or aerobic growth provide an outstanding example among bacteria of intracellular differentiation in response to the environment. The adaptive phenomena exhibited by these organisms is analogous to the development of plant chloroplasts in response to light. I n each case, synthesis of the photosynthetic pigments is accompanied by major changes in the synthesis of enzymes and cofactors required for photosynthetic metabolism. The plant has the added sophistication of a specialized organelle, the chloroplast, to house the photosynthetic apparatus and this may represent an evolutionary progression from the bacterial chromatophore.

A. DIFFERENCES BETWEEN MEMBERSOF THE ATHIORHODACEAE GROWN PHOTOSYNTHETICALLY AND AEROBICALLY Analysis of members of the Athiorhodaceae grown in the dark with high aeration or anaerobically in the light shows that pigment synthesis is accompanied by many changes in enzyme content and in catalysts associated with photosynthetic electron-transport (Table 6). The cytological changes which accompany pigment synthesis have already been discussed. The high concentration of haemoproteins and of ubiquinone in cells grown anaerobically in the light is consistent with their function in photosynthetic metabolism. There is no evidence for chemical differences between the b- and c-type cytochromes found in photosynthetic and aerobic cells. However, a unique type of haemoprotein (cytochromoids, RHP) has been found in many photosynthetic bacteria grown anaerobically in the light (Kamen, 1963; Bartsch, 1963). Such pigments have two haem c prosthetic groups per molecule, and are characterized by their ability to combine with carbon monoxide. Their formation is apparently associated with the development of the photosynthetic apparatus since

w

OD

TABLE 6. Concentration of Components in Rhodopaeudomonas spheroides Grown Aerobically asd Photosynthetically Concentration in cells grown h

t

Aerobic-dark

Component

1

Bacteriochlorophyll Carotenoids Haemoproteins Total mpmoles/mg. Cytochrome b protein Cytochrome c CO-bindingpigment Ubiquinone (&lo) Lipid phosphorus Ribdose diphosphate carboxylase mpmoles product/ 6-Aminolaevulinic acid synthase 8-Aminolaevulinic acid dehydrase

>

Anaerobic-light*

0.1 0.5

48 22

0.20 0.08 0.06 0.06 3.02 90 60 20 45

0.59 0.17 0.19 0.23 6.64 157 1800 205 105

Reference 1 1 2

*The strainwedwas N.C.I.B. 8253. Cultures were grown in a malate-glutamatemedium (Lascelles,1959)with high aeration or anaerobically

in light of about 250 foot candles intensity.

References: (1) Bull and Lascelles (1963); (2) Porra and Lascelles (1965); (3) Carr and Exell (1965); (4) Lascelles and Szilhgyi (1965); (5) Lascelles (1960b); (6) Lascelles (19608).

THE BACTERIAL PHOTOSYNTHETIC APPARATUS

29

they have not been detected in aerobically grown cells (Taniguchi and Kamen, 1965).However, a function for these peculiar haemoproteins in photosynthetic electron-transfer reactions is yet to be found. The terminal oxidase in aerobically-grown Rsp. rubrum may be a form of cytochrome 0,a carbon monoxide-binding pigment with a protohaem prosthetic group (Taniguchi and Kamen, 1965; Chance et al., 1966). It is not known whether this type of oxidase is also present in photosynthetically grown cells. Such organisms respire vigorously but the respiration could be mediated by a cytochromoidpigment (Horio and Kamen, 1962). An a type cytochrome has been found in a strain of Rps. spheroides when grown aerobically (Motokawa and Kikuchi, 1966).It exhibits the typical binding with carbon monoxide, and appears to function as a terminal oxidase. No evidence for this type of pigment was found in the strains of Rps. spheroides examined by Porra and Lascelles (1965),in which respiration was apparently mediated by an o-type cytochrome. Ribulose diphosphate carboxylase is an enzyme typically associated with photosynthetic metabolism. It is found in high concentration in members of the Athiorhodaceae grown anaerobically in the light, but is low or undetectable in aerobically grown cells (Lascelles, 1960b). Its presence in members of the Athiorhodaceae is anomalous since these organisms grow only on fixed carbon compounds. The carboxylase is present in members of the Thiorhodaceae grown on organic substrates though the concentration of the enzyme is lower than that in cells grown with carbon dioxide as sole carbon source (Hurlbert and Lascelles, 1963).

B. ADAPTIVE SYNTHESIS OF

THE

PHOTOSYNTHETIC APPARATUS

The versatility of some species of Athiorhodaceae makes them useful for study of the synthesis and regulation of the photosynthetic apparatus. The organisms can be grown devoid of the photosynthetic system, and its development can be followed upon subsequent incubation either anaerobically in the light or in the dark with low aeration. I n the anaerobic-light system, the photosynthetic apparatus is developed non-gratuitously since ATP production under anaerobic conditions depends on photosynthetic reactions (Schon and Drews, 1966). The low-aeration system has the advantage of allowing adaptation to proceed gratuitously, and, most important, it can be applied to mutants which cannot grow photosynthetically (Lascelles, 1959;Bull and Lascelles, 1963;Lascelles, 1966a, b)The photosynthetic apparatus formed by organisms grown in the dark with low aeration appears to be fully functional since chromatophores from Rsp. rubrum exhibit photophosphorylation just as actively

30

JUNE LASCELLES

as preparations from photosynthetically grown cells (Cohen-Bazire and Kunisawa, 1960).Also, electron microscopy has shown the same type of internal structure with increasing invagination of the peripheral membrane as the pigment content increases in response to decreased aeration (Cohen-Bazire and Kunisawa, 1963; Biedermann et aZ.,1967). The kinetics of development of individual components of the photosynthetic apparatus have been examined in Rps. spheroides as it adapts from conditions of high to low aeration. Haemoproteins and ubiquinone are formed in parallel with chlorophyll (Porra and Lascelles, 1965; Carr and Exell, 1965)but the appearance of ribulose diphosphate carboxylase is delayed (Lascelles, 1960b). The formation of enzymes of chlorophyll synthesis and of particulate protein is discussed later. 1. Photosynthetic Mutants

Numerous mutant strains of Rps. spheroides have been isolated which are blocked at single stages in the chlorophyll biosynthetic pathway and are therefore unable to grow anaerobically in the light (Lascelles, 1964). I n the low-aeration systems, such mutants accumulate chlorophyll precursors and the usual carotenoids (Griffiths and Stanier, 1956; Griffiths, 1962; Lascelles, 1966a). Carotenoid-less mutants, which make chlorophyll and are therefore capable of photosynthetic growth, have also been isolated (Griffiths and Stanier, 1956; Jensen, 1963). Another class of mutants (“albinos”) form neither carotenoids nor chlorophyll, and are consequently only able to grow aerobically (Griffiths and Stanier, 1956; Lascelles, 1966b; Drews and Schick, 1966). One such albino, strain L-57, has been examined in some detail (Bull and Lascelles, 1963; Lascelles, 1966b).Under low aeration, the organism does not form photosynthetic pigments or ribulose diphosphate carboxylase, whereas the wild type does so under these conditions. Both mutant and wild type, however, show the same increase in contents of b and c type cytochromes and a carbon monoxide-binding pigment; it is not known whether the haemoproteins formed by the mutant are identical with those of the wild type (Porra and Lascelles, 1965).The mutant synthesizes free haem but not magnesium tetrapyrroles from 6-aminolaevulinic acid, and presumably has the iron branch of the tetrapyrrole pathway (Lascelles, 1966b). I n accord with the absence of photosynthetic pigments, the mutant does not form internal membrane when grown under conditions which promote this in the parent strain (Germaine Cohen-Bazire and Rio Kunisawa, personal communication); nor does it show an increase in phospholipid content under these conditions (Lascelles and Szil&gyi, 1965).

THE BACTERIAL PHOTOSYNTHETIC APPARATUS

31

C. CHLOROPHYLL SYNTHESIS There is some justification for airing a special interest in chlorophyll synthesis and its regulation, since the formation of this pigment seems to play a central role in the final elaboration of the chloroplast and of its bacterial counterpart. 1. Biosynthetic Pathway

Haem and chlorophyll synthesis share a common pathway to the stage of protoporphyrin, where divergence into the iron and magnesium branches occurs (Fig. 8). Knowledge of the intermediates of the magnesium branch is fragmentary and is derived largely by intuition from products accumulated by Chlorella and by Rps. spheroides blocked in chlorophyll synthesis either by mutation or by inhibitors of chlorophyll synthesis such as 8-hydroxyquinoline (Bogorad, 1966;Jones, 1963a, b, c, 1964; Lascelles, 1966a).The only step to be demonstrated so far in cellfree extracts is the methyl transferase which catalyses the reaction : Magnesium protoporphyrin +S-adenosylmethionine-t Magnesium protoporphyrin monomethylester + S-adenosylhomocysteine This activity was found in the chromatophore fraction of Rps. spheroides and other Athiorhodaceae (Gibson et al., 1963). 2 . Chlorophyll Synthesis and Chloroplast Development

The development of the chloroplast in plants is closely associated with chlorophyll synthesis; the evidence for this may be summarized as follows. (1) Formation of chlorophyll upon illumination of etiolated leaves or of Euglena is strictly dependent on protein synthesis since pigment formation is prevented by inhibitors of protein and of nucleic acid synthesis (Bogorad, 1966). Such inhibition of chlorophyll synthesis may be only partly attributable to the prevention of the development of enzymes required for the biosynthesis. Addition of actidione to Euglena after greening has begun drastically curtails chlorophyllformation, suggesting the requirement for a protein which functions in stoichiometric rather than in catalytic amounts (Kirk and Allen, 1965). (2) Protochlorophyllide a (magnesium vinylpheoporphyrin a s ) occurs in etiolated plants in combination with protein, the red absorption maximum being 650 mp ;this complex is located in the proplastids (Klein et al., 1964). Conversion of the pigment to chlorophyllide a involves the reduction of ring D of the tetrapyrrole nucleus and occurs upon illumination. Only the bound form of the pigment is reduced; free

8 Glycine pyridoxal PO4

+

___j

-8COa

8 6-aminolaevulinic acid

8 Succinyl-CoA

- 4C09

coproporphyrinogen I11 t-- uroporphyrinogen I11

-2COr

-4H

I coproporphyrin I11 - 6H

protoporphyrinogen ---+

Mg-protoporphyrin

+methyl

+2H

+Fe

protoporphyrin ---+

j+%

4 porphobilinogen

uroporphyrinogen I

uroporphyrin I11

I.

J.

- 4KHs

I

-6H

J. haems

uroporphyrin I

Mg-protoporphyrin

Mgprotoporphyrinmono- --+ --+ methyl ester

+ Ha0

Mg-2-vinylphaeoporphyrina+ ----+ chlorophyllide a ----+ (protochlorophyll a )

Mg-2,4-divinylphaeoporphyrin a5

+2H __f

+2H

2-devinyl-2-hydroxyethylbacteriochlorophyll iphytol chlorophyllide

FIG.8. Outline of the iron-and magnesium branches of the tetrapyrrole biosynthetic pathway.

THE BACTERIAL PHOTOSYNTHETIC APPARATUS

33

protochlorophyllide(absorption maximum at 631 mp), accumulated from exogenous 6-aminolaevulinic acid, is not converted (Granick, 1963 ; Klein and Bogorad, 1964). The protochlorophyllide complex or holochrome has been purified from etiolated bean leaves as an apparently homogeneous protein of molecular weight about 600,000 and sedimentation coefficient of 1 8 s (Boardman, 1966). The complexed pigment is reduced to the chlorophyllide upon illumination, but the hydrogen donor is not known. The status of the protein moiety is far from clear. It is similar in properties to Fraction 1 protein which comprises about 25% of the dry weight of the chloroplast. This fraction may consist largely of ribulose diphosphate carboxylase ; purified preparations of this enzyme (molecular weight 515,000) closely resemble the holochrome protein in many physical properties (Trown, 1965). It is possible that the isolated holochrome may represent the protochlorophyllide combined with a smaller protein which has become associated with the carboxylase during purification. I n vivo association of pigment with the enzyme, a t least in its active form, does not seem to be required for chlorophyll synthesis, since mutants of Chlamydomonas which lack the carboxylase still form chlorophyll (Levine and Togasaki, 1965). (3) The light-induced conversion of the protochlorophyllide holochrome to chlorophyllidea is accompaniedby changesin the fine structure of the proplastids, the tubular elements becoming transformed into vesicles (Virgin et al., 1963). The phenomena appear to be related since the structural changes are induced specifically by light of wavelengths required to effect conversion of the pigment (Klein et al., 1964). There is also evidence that the final lamellar structure of the chloroplast is linked with the esterification of chlorophyllide a with phytol (Klein, 1962; Eilam and Klein, 1962). 3. Chlorophyll Synthesis and Membrane Formation in Photosynthetic

Bacteria I n bacteria there is evidence for an obligatory linkage of chlorophyll and protein syntheses. Pigment synthesis by Rps. spheroides in lowaeration systems is prevented by inhibitors of protein and nucleic acid synthesis or by deprivation of amino acids, purines or pyrimidines in the case of auxotrophic mutants (Lascelles, 1959; Bull and Lascelles, 1963; Higuchi et al., 1965; Biedermann et al., 1967). The dependence of chlorophyll formation on protein synthesis has also been shown with photosynthetic cultures of Rps. spheroides, which form pigment in response to a decrease in light intensity (Sistrom, 1962a, b) and with Rsp. rubrum upon transfer from high aeration to photosynthetic conditions (Drews, 1965).

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Some, at least, of the-enzymes required for chlorophyll synthesis are adaptive (see later) and their formation could account for the obligatory link with protein synthesis. However, pigment synthesis is still sensitive to inhibition of protein formation in cells containing the full complement of biosynthetic enzymes (Sistrom, 1962a, b ; Bull and Lascelles, 1963; Drews, 1965). There is other evidence which points to a specific association of chlorophyll synthesis with the formation of membrane protein. I n low-aeration systems, Rps. spheroides incorporates labelled amino acids into chromatophore protein at a faster rate than into the soluble protein fraction (Bull and Lascelles, 1963). The preferential incorporation into the particulate fraction occurs in parallel with chlorophyll formation, and appears to be specifically connected with it (Bull and Lascelles, 1963; Altshuler and Lascelles, 1967). The phenomenon is not shown when cells are incubated under high aeration, which represses pigment synthesis. Also, albino mutants do not show preferential incorporation when incubated under conditions which promote it in the wild type. It is possible that the preferential incorporation represents the formation of structural protein which accommodates the photosynthetic pigments in the membrane. Fractionation of the membrane proteins is clearly necessary to establish this.* The integration of chlorophyll and membrane formation is also suggested by the increase in phospholipid content in Rps. spheroides which occurs as pigment is formed under low aeration (Lascelles and Szilhgyi, 1965). The albino mutant, L-57, does not show this increase when incubated similarly. There is a distinct possibility that protein-bound intermediates participate in chlorophyll synthesis by the bacteria, and perhaps this protein may become an integral part of the pigmented membrane structure. Evidence for bound intermediates is provided by mutant strains of Rps. spheroides which accumulate chlorophyll precursors ; these are bound to the particulate fraction of cell extracts but are released by treatment with Tween 80 (Lascelles, 1966a).This procedure is too mild to release chlorophyllfrom its complexes;possibly, the phytol residue, which is lacking in the precursors, is needed for the firm integration of pigment molecules into the membrane structure. The less attractive possibility, t$at the bound precursors are artifacts of the fractionation procedures, cannot be dismissed. OF CHLOROPHYLL SYNTHESIS D. REGULATION Mechanisms for the regulation of chlorophyll synthesis are almost completely obscure, a state of affairs which can be partly attributed to ignorance of the enzymic steps. One firm fact is that 6-aminolaevulinic

* See note added in proof on p. 42.

THE BACTERIAL PHOTOSYNTHETIC APPARATUS

35

acid synthase plays a central role in the regulation, being controlled by repression and by feedback inhibition, but the precise mechanisms of this control are unknown. 1. Repression of Biosynthetic Enzymes

The concentrations of some of the enzymes of chlorophyll synthesis rise and fall in response to environmental changes in a manner which is consistent with control of pigment synthesis by enzyme repression. I n Athiorhodaceae, the onset of chlorophyll synthesis in cells transferred from high to low aeration or to photosynthetic conditions is preceded by a 5-10-fold increase in 6-aminolaevulinic acid synthase and other early enzymes of the biosynthetic pathway (Lascelles, 1959, 1960a; Drews, 1965; Drews and Oelze, 1966; Higuchi et al., 1965).I n the converse type of experiment, synthesis of both enzyme and pigment is repressed when pigmented cells are subjected to high aeration (Lascelles, 1960a). There is reason to believe that the enzymes of the magnesium branch are totally repressed by high aeration. Gibson et al. (1963) could not detect the methyl transferase in non-pigmented Rps. spheroides. Also, whole cells of this organism do not form magnesium tetrapyrroles from 6-aminolaevulinic acid when grown with high aeration, though such compounds are formed by pigmented cells (Lascelles, 1966a, b). 2. Role of 6-Aminolaevulinic Acid Synthase

The parallel between the activity of 6-aminolaevulinic acid synthase and the chlorophyllcontent of Athiorhodaceae is consistent with a central function for this enzyme in regulating the amount of pigment formed under various conditions. Repression of this enzyme, however, cannot account for the immediate cessation of chlorophyll synthesis which occurs upon the introduction of oxygen into cultures growing anaerobically in the light and containing the full complement of biosynthetic enzymes (Cohen-Bazire et aZ., 1957; Lascelles, 1960a; Sistrom, 1965). A direct effect on the action of the enzyme is suggested by the fact that there is no accumulation of intermediates in such oxygen-repressed systems. However, there is no evidence from in vitro experiments that the synthase is inhibited by oxygen; rather, it tends to be activated by aeration of crude extracts (Burnham and Lascelles, 1963). One possibility is that oxygen acts indirectly enhancing oxidation of succinate via the tricarboxylic acid cycle, and thereby deprives the synthase of succinyl-CoA (Lascelles, 1964).Knowledge of intracellular pool levels of succinate and succinyl-CoA under various conditions is required to support this. Rapid destruction of 6-aminolaevulinic acid synthase may also be a factor in chlorophyllregulation. The enzyme is highly unstable in extracts

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and partly purified preparations from Rps. spheroides and other bacteria (Burnham and Lascelles, 1963 ;J. Lascelles, unpublished observations). The enzyme is also unstable in wivo, since there is rapid loss in activity when protein synthesis is prevented by inhibitors or by deprivation of amino acids (Bull and Lascelles, 1963). Destruction of the enzyme also occurs when cultures growing anaerobically in the light are transferred to highly aerobic conditions (Lascelles, 1960a). Metabolic instability of the synthase may be of more general significance, since the drug-induced enzyme in liver is rapidly destroyed upon termination of protein synthesis (Marver et aZ., 1966). A control mechanism which involves rapid degradation of a key enzyme is not intellectually satisfying, particularly as one cannot point to many comforting precedents. The present evidence, however, does not allow the dismissal of this possibility. Feedback inhibition of 3-arninolaevulinic acid synthase. I n plants, there is evidence for feedback control of 6-aminolaevulinic acid synthase, exerted by the protochlorophyllide holochrome complex (Bogorad, 1966). The amount of this precursor which is accumulated by etiolated leaves is only a small fraction of the amount of chlorophyll formed from endogenous sources upon illumination. When the light source is removed, the chlorophyllide accumulates again to the original concentration. Free protochlorophyllide does not affect the action of the synthase since its presence in high concentration relative to the complexed form does not prevent chlorophyll synthesis from endogenous substrates (Granick, 1963; Klein and Bogorad, 1964). There is indirect evidence for regulation of the action of the synthase by magnesium tetrapyrroles in bacteria. Rps. spheroides and Rps. mpsalata accumulate coproporphyrin I11 which arises by spontaneous oxidation of the biosynthetic intermediate coproporphyrinogen 111, when chlorophyll synthesis is limited by iron deficiency (Lascelles, 1956 ; Cooper, 1963). Coproporphyrin is also accumulated by Rps. spheroides in the presence of ethionine (Gibson et d.,1962) or under conditions of methionine limitation (Lessie and Sistrom, 1964). The formation of coproporphyrin is accompanied by inhibition of chlorophyll synthesis, and this effect is attributable to a block at the methyl transferase step which requires S-adenosylmethionine, derived from methionine (Gibson et a,?.,1963). &ccumulation of the porphyrin may result from the failure of a feedback control on the 3-aminolaevulinic acid synthase, exerted normally by a component of the biosynthetic pathway beyond the methylation step. The regulator substance cannot be chlorophyll itself since some chlorophyll-deficient mutants form coproporphyrin like the wild type when ethionine is present. A possible candidate for the hypothetical regulator substance is a protochlorophyllide-type of pigment associated with a specific protein, as may be the case in plants.

THE BACTERIU PHOTOSYNTHETIC APPARATUS

37

3. Regulation of the Branched Pathway

Tetrapyrrole biosynthesis by the photosynthetic bacteria provides a good example of a branched pathway stemming from common intermediates. I n addition to the iron and magnesium branches, there is the path leading to vitamin B12,which is present in relatively high concentration in many of these organisms (Cauthen et al., 1967).There must be control mechanisms which ensure the flow of precursors into the appropriate channels to maintain a molar ratio of chlorophyll, haems and vitamins B12of about 1:20: 2000. The magnesium branch is also under some independent control, at least in the Athiorhodaceae, since chlorophyll synthesis can be repressed without preventing the formation of haems and vitamins B12(Lascelles, 1966b; Cauthen et al., 1967). Feedback inhibition of 8-aminolaevulinic acid synthase by haem has been found with partly purified preparations from Rps. spheroides, with supporting evidence from experiments with intact cells for the operation of sucha controlinvivo (BurnhamandLascelles, 1963).Theeffect of haem on the 8-aminolaevulinic acid synthase does not clarify the regulation of the magnesium branch of the biosynthetic pathway. One possibility is that there are two forms of the synthase; one may be specifically concerned with chlorophyllsynthesis under the control of a component of the magnesium pathway, and the other may be associated with haem synthesis. Tenuous evidence for two enzymes (or possibly one enzyme located in different specialized environments) is provided by the albino types of mutant of Rps. spheroides (Lascelles, 1966b; unpublished observations). Such mutants form haems but do not synthesize magnesium tetrapyrroles. These observations alone point to a loss of the magnesium-branch enzymes, possibly under the control of a single gene. However, the albinos also fail to accumulate coproporphyrin under conditions which promote its formation by the wild type or by mutants which are blocked at single steps of the magnesium pathway. The ability to accumulate coproporphyrin may be a manifestation of the unregulated activity of the “magnesium-branch synthase”. Failure of the albino mutants to accumulate the porphyrin could therefore be attributed to the absence of this fork of the synthase. It might be added that there is as yet no direct evidence for two forms of the enzyme or for its compartmentation in the cell.

4. Genetic Control of the Photosynthetic Apparatus There is a complete lack of knowledge of the genetic regulation of the formation of the bacterial photosynthetic apparatus due to failure to find a suitable experimental system, such as recombination or transduction.

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We are therefore forced to resort to speculations based on information about regulation of analogous multi-enzyme systems. Albino mutants, such as Rps. spheroides strain L-57, exhibit many phenotypic changes resulting, apparently, from a single genetic lesion. Examples are known in other complex systems of interactions between individual components which affect their synthesis and organization in the final complex. I n the pyruvate oxidase complex of Escherichia coli, for instance, a mutation affecting the formation of a single enzyme may alter the rate of synthesis of other enzymes of the system and also their capacity to form complexes with each other (Henning, 1966). The closest parallel to the albino mutants is provided by bleached mutants of Euglena and respiratory-deficient strains of yeast and Neurospora. I n these organisms a single genetic lesion results in many changes in the synthesis of enzymes and cofactors of photosynthesis or respiration, coupled with the inability to form normal chloroplasts or mitochondria (Gibor and Granick, 1964; Schiff and Epstein, 1965; Sherman and Slonimski, 1964; Slonimski, 1967). I n Euglena and yeast, both cytoplasmic and nuclear DNA are involved in defining the synthesis of the complete organelle. I n the bacteria, an episomal factor could participate in the control of the photosynthetic apparatus. The discovery of a satellite DNA in some photosynthetic bacteria gives some hope in this direction but there was no evidence that this material was located in the chromatophore fraction (Suyama and Gibson, 1966). It is difficult to envisage how a single mutation can result in such a multitude of phenotypic changes as occurs in photosynthetic and respiratory mutants. Recent work with respiratory-deficient mutants of Neurospora has pointed to a central role for structural proteins in the formation and organization of other mitochondrial proteins (Woodward and Munkres, 1966; Munkres and Woodward, 1966). Structural protein isolated from the mitochondrial fraction of some cytoplasmic mutants differed from that of the wild type by single amino acid replacements. The resulting conformational change affected the capacity of the protein to combine with malate dehydrogenase and other mitochondrial components. The prixtiary effect of the mutation upon the Structural protein had other secondary consequences. The mutants were deficient in cytochromes a and b, but made cytochrome c in excessive amounts ;synthesis of riboflavin and unsaturated fatty acids was also affected, these compounds being present in higher concentration in the mutants than in the wild type. The implications of this work can be applied as a working hypothesis to the case of the development of the bacterial photosynthetic apparatus. One can imagine “foundation molecules”, possibly structural proteins, which have a co-ordinating role in the formation and organization of the

THE BACTERIAL PHOTOSYNTHETIC APPARATUS

39

various components of the photosynthetic apparatus. It might be synthesis of these proteins which is primarily subject to repression or derepression. If chlorophyll formation proceeds via protein-bound intermediates, the protein moiety may be such a “foundation molecule”. For some clarification of the many mysteries we require to examine photosynthetic mutants and revertants in depth. Unfortunately, genetic transfer systems have yet to be discoveredin the photosynthetic bacteria but a diligent search might provide a reward for the brave. REFERENCES Allen, M. B. (1966). In “The Chlorophylls”, (L. P. Vernon and G. R. Seeley, eds.), p. 511. Academic Press, New York. Altshuler, T. and Lascelles, J. (1967). Arch. Mikrobiol. in press. Bartsch, R. G. (1963). I n “Bacterial Photosynthesis”, (H. Gest, A. San Pietro and L. P. Vernon, eds.), p. 315, Antioch Press, Yellow Springs. Benson, A. A. (1964). Annu. Rev. plant Physiol. 15, 1. Bergeron, J. A. (1959). I n “The Photochemical Apparatus, its Structure and Function”, p. 118. Brookhaven Natl. Lab., Upton, New York. Biedemann, M., Drews, G., Marx, R. and Schroeder, J. (1967). Arch. Mikrobiol. 56, 133. Boardman, N. K. (1966). In “The Chlorophylls”, (L. P. Vernon and G. R. Seeley, eds.), p. 437. Academic Press, New York. Boatman, E. S. (1964). J . cell Biol. 20, 297. Bogorad, L. (1966). In “The Chlorophylls”, (L. P. Vernon and G. R. Seeley, eds.), p. 481. Academic Press, New York. Bril,C. (1958). Biochim. Biophys. Acta 29, 458. Bril, C. (1960). Biochim. Biophys. Acta 39, 296. Buchanan, B. B., Evans, M. C. W. and Arnon, D. I. (1965). In “Non-Heme Iron Proteins”, (A. San Pietro, ed.), p. 175. Antioch Press, Yellow Springs. Bull, M. J. and Lascelles, J. (1963). Biochem. J . 87, 15. Burnham, B. F. and Lascelles, J. (1963). Biochem. J . 87, 462. Carr, N. G. and Exell, G. (1965). Biochem. J . 96,688. Cauthen, S. E., Pattison, J. and Lascelles, J. (1967). Biochem. J . 102, 774. Chance, B., Horio, T., Kamen, M. D. and Taniguchi, S. (1966). Biochim. Biophys. Acta 112, 1. Clayton, R. K. (1962a). Biochem. Biophys. res. C m m u n . 9 , 4 9 . Clayton, R. K. (1962b). Photochem. Photobiol. 1, 201. Clayton, R. K. (1963). Biochim. Biophys. Acta 75, 312. Clayton, R. K. (1966). I n “The Chlorophylls”, (L. P. Vernon and G. R. Seeley, eds.), p. 609. Academic Press, New York. Clayton, R. K. and Sistrom, W. R. (1966). Photochem. Photobiol. 5, 661. Cohen-Bazire, G. (1963). I n “Bacterial Photosynthesis”, (H. Gest, A. San Pietro and L. P. Vernon, eds.), p. 89. Antioch Press, Yellow Springs. Cohen-Bazire,G., Sistrom, W. R. and Stanier, R. Y. (1957). J . cell. comp. Physiol. 49, 25. Cohen-Bazire, G. and Kunisawa, R. (1960). Proc. nat. Acad. Sci. Wash. 46, 1543. Cohen-Bazire, G. and Kunisawa, R. (1963). J. cell Biol. 16, 401. Cohen-Bazire, G., Pfennig, N. and Kunisawa, R. (1964). J. cell Biol. 22, 207. Cohen-Bazire,G. and Sistrom, W. R. (1966). In “The Chlorophylls”, (L. P. Vernon and G. R. Seeley, eds.), p. 313. Academic Press, New York.

40

JUNE LASCELLES

Cooper, R. (1963). Biochem. J. 89, 100. De Klerk, H. and Kamen, M. D. (1966). Biochim. Biophys. Acta 112, 175. Drews, G. (1960). Arch. Mikrobiol. 36, 99. Drews, G. (1963). Zentr. Bakt. Parasitenk. Abt. 1. Or@. 190, 508. Drews, G. (1964). Arch. Mikrobiol. 48, 122. Drews, G. (1965). Arch. Mibobiol. 51, 186. Drews, G. and Giesbrecht, P. (1965).Arch. Mikrobiol. 52, 242 Drews, G. and Giesbrecht, P. (1966). Arch. MilcrobioZ. 53, 255. Drews, G. and Oelze, J. (1966). Zentr. Balct. Parasitenk. 11 Abt. 120, 1. Drews, G. and Schick, J. (1966). 2. Naturfmsch. 21b, 1097. Dworkin, M. (1958).J. gen. Physiol. 41, 1099. Eilam, Y. and Klein, S. (1962).J. cell Biol. 14, 169. Eimhjellen, K. E., Aasmundrud, 0. and Jensen, A. (1963). Biochem. Biophys. res. Commun. 10, 232. Erwin, J. and Bloch, K. (1964).Science, 143, 1006. Evans, M. C. W. and Buchanan, B. B. (1965). Proc. nat. Acad. Sci. Wash. 53,1420. Frenkel, A. W. (1956).J. biol. Chem. 222, 823. Frenkel, A. W. (1958).J. Amer. chem. SOC.80,3479. Frenkel, A. W. and Hickman, D. D. (1969).J. biophys. biochem. Cytol. 6, 285. Fuller, R. C. (1963).In “General Physiology of Cell Specialization”, (D. Mazia and A. Tyler, eds.), p. 223. McGraw-Hill, New York. Fuller, R. C., Smillie, R. M., Rigopoulos, N. and Yount, V. (1961).Arch. Biochem. Biophys. 95, 197. Garcia, A., Vernon, L. P. and Mollenhauer, H. (1966a). Biochemistry 5, 2399. Garcia, A., Vernon, L. I?.and MoUenhauer, H. (196613). Biochemistry 5, 2408. Geller, D. M. and Lipmann, F. (1960).J. biol. Chem. 235,2478. Gest, H. (1966). Nature, Lond. 209, 879. Gibbs, S. P., Sistrom, W. R. and Worden, P. B. (1965).J. cell. Biol. 26, 395. Gibor, A. and Granick, S. (1964).Science 145,890. Gibson, K. D. (19654. Biochemistry 4,2027. Gibson, K. D. (1966b). Biochemistry 4, 2042. Gibson, K. D. (19650). Biochemistry 4, 2052. Gibson, K. D. (1966).J. Bmt. 90,1059. Gibson, K. D., Neuberger, A. and Tait, G. H. (1962). Biochem. J . 83, 539. Gibson, K. D., Neuberger, A. and Tait, G. H. (1963). Biochem. J. 88, 325. Giesbrecht, P. and Drews, G. (1962). Arch. MikrobioZ. 43, 152. Giesbrecht, P. and Drews, G. (1966). Arch. Mikrobiol. 54, 297. Granick, S. (1963). In “The Mechanism of Photosynthesis”, p. 176, Symposium No. 6, 6th. International Congress of Biochemistry, Moscow, 1961. Pergamon, Oxford. Griffiths, M. (1962).J. gem. Mkrobiol. 27, 427. Grfiths, M. and Stanier, R. Y. (1966).J. gem. Microbiol. 14, 698. Henning, U.(1966). Angew. Chem. 5,785. Hickman, D. D. and Frenkel, A. W. (1959).J. biophys. biochem. Cytol. 6, 277. Hickman, D. D. and Frenkel, A. W. (19654. J. cell Biol. 25, 261. Hickman, D. D. and Frenkel, A. W. (1965b).J . cell Biol. 25, 279. Higuchi, M., Goto, K., Fujimoto, M., Namiko, 0. and Kikuchi, G. (1965).Biochim. Biophys. Acta 95, 94. Holt, A. S. (1966). In “The Chlorophylk”, (L. P. Vernon and G. R. Seeley, eds.), p. 111. Academic Press, New York. Holt, S. G., Conti, S. F. and Fuller, R. C. (1966a).J. Bud. 91, 311. Holt, 8.C., Conti, S. F. and Fuller, It. C. (196613).J. Bact. 91, 349.

THE BACTERIAL PHOTOSYNTHETIC APPARATUS

41

Holt, S. C. and Marr, A. G. (1965a). J. Bmt. 89, 1402. Holt, S. C. andMarr, A. G. (1965b).J . Buct. 89, 1413. J. Buct. 89, 1421. Holt, S. C. and Marr, A. G. (1965~). Horio, T. and Kamen, M. D. (1962). Biochemistry 1, 1141. Hulcher, F. H. and Conti, S. F. (1960). Biochem. Biophys. res. Commun. 3,497. Hurlbert, R. E. and Lascelles, J. (1963).J. gen. Microbiol. 33, 445. Jensen, S. L. (1963). I n “Bacterial Photosynthesis”, (H. Gest, A. San Pietro and L. P. Vernon, eds.), p. 19. Antioch Press, Yellow Springs. Jensen, A., Aasmundrud, 0. and Eimhjellen, K. E. (1964). Biochim. Biophys. Actu 88, 466. Jones, 0. T. G. (1963a). Biochem. J . 86,429. Jones, 0. T. G. (1963b). Biochem. J. 88, 335. Jones, 0. T. G. (1963~).Biochem. J. 89, 182. Jones, 0. T. G. (1964). Biochem. J. 91,572. Kamen, M. D. (1963).I n “Bacterial Photosynthesis”, (H. Gest, A. San Pietro and L. P. Vernon, eds.), p. 61. Antioch Press, Yellow Springs. Kashket, E. R. and Brodie, A. F. (1963a). Biochim. Biophys. Actu 78, 52. Kashket, E. R. and Brodie, A. F. (196313).J. biol. Chem. 238, 2564. Kirk,J. T. 0. and Allen, R. L. (1965). Biochem. Biophys. res. Commun. 21,523. Klein, S. (1962). Nature, Lond. 196, 992. Klein, S. and Bogorad, L. (1964).J. cell Biol. 22, 443. Klein, S., Bryan, G. and Bogorad, L. (1964).J. cell Biol. 22,433. Kodicek, E. (1963). I n “Recent Progress in Microbiology”, (N. E. Gibbons, ed.), p. 23. Toronto University Press, Toronto. Lascelles, J. (1956). Biochem. J . 62, 78. Lascelles, J. (1959). Biochem. J. 72, 508. Lascelles, J. (1960a).J. gem Microbiol. 23, 487. Lascelles, J. (1960b).J. gen. Microbiol. 23,499. Lascelles, J. (1964). Tetrupyrrole Biosynthesis and its Regulation, p. 106. Benjamin, New Y ork Lascelles, J. (1966a). Biochem. J. 100, 175. Lascelles, J. (196613). Biochem. J . 100, 184. Lascelles, 5. and Szildgyi, J. F. (1965).J. gen. Microbiol. 38, 85. Lessie, T. G. and Sistrom, W. R. (1964). Biochim. Biophys. Actu 86, 250. Levine, R. P. and Togasaki, R. K. (1965). Proc. nat. Acad. Sci., Wash. 53, 987. Marr, A. G. and Cota-Robles, E. H. (1957).J. Buct. 74, 79. Marver, H. S., Collins, A., Tschudy, D. P. and Rechicgl, M. (1966).J.biol. Chem. 241, 4323. Motokawa, Y . and Kikuchi, G. (1966). Biochim. Biophys. Actu 120, 274. Munkres, K. D. and Woodward, D. 0. (1966).Proc. nut. Acud.Sci. Wmh. 55,1217. Newton, J. W. (1960). Biochim. Biophys. Acta 42, 34. Newton, J. W. (1962). Biochim. Biophys. Actu 58,474. Newton, J. W. and Levine, L. (1959). Arch. Biochem. Biophys. 83, 456. Newton, J. W. and Newton, G. A. (1957). Arch. Biochem. Biophys. 71,250. Olson, J. M. (1966).I n “The Chlorophylls”, (L. P. Vernon and G. R. Seeley, ads.), p. 413. Academic Press, New York. Olson, J. M. and Romano, C. A. (1962). Biochim. Biophys. Acta 59, 726. Olson, J. M. and Stanton, E. K. (1966).I n “The Chlorophylls”, (L.P. Vernon and G. R. Seeley, eds.), p. 381. Academic Press, New York. Olson, J. M. and Sybesma, C. (1963). I n “Bacterial Photosynthesis”, (H. Gest. A. San Pietro and L. P. Vernon, eds.), p. 413. Antioch Press, Yellow Springs.

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Orlando, J. A., Levine,L. and Kamen, M. D. (1961). Biochim. Biophy8. Acta 46, 126. Pangborn, J., Marr, A. G. and Robrish, S. A. (1962).J . Bact. 84, 669. Porra, R. J. and Lascelles,J. (1965). Biochem. J . 94,120. Rabinowitch, E. I. (1951). “Photosynthesis”, Vol. 11, part 1, p. 702. Interscience, New York. Schachman, H. K., Pardee, A. B. and Stanier, R. Y. (1952). Arch. Biochem. Biophys. 38, 245. Scheuerbrandt, G. and Bloch, K. (1962). J . biol. Chem. 237, 2064. Schiff, J. A. and Epstein, H. T. (1965). I n “Reproduction: Molecular, Subcellular and Cellular”, (M. Locke, ed.), p. 131. Academic Press, New York. Schmidt, K., Pfennig, N. and Jensen, S. L. (1965). Arch. Milcrobiol. 52, 132-146. Schon, G. and Drews, G. (1966). Arch. Milcrobiol. 54, 199. Sedar, A. W. and Burde, R. M. (1965). J . cell Biol. 27, 53. Sherman, F. and Slonimski, P. P. (1964). Biochim. Biophys. Acta 90, 1. Sistrom, W. R. (1962a). J . gen. Microbiol. 28, 599. Sistrom, W. R. (1962b).J . gen. Microbiol. 28, 607. Sistrom, W. R. (1965). J . B a t . 89,403. Sistrom, W. R. and Clayton, R. K. (1964). Biochim. Biophys. Acta 88, 61. Slonimski, P. P. (1967). Fed. Proc. in press. Stanier, R. Y. (1960). Harvey Lectures, 1958-1959, pp. 219. Stanier, R. Y. (1961). Bact. Rev. 25, 1. Stanier, R. Y. (1963).In “GeneralPhysiology of Cell Specialization”,(D. Mazia and A. Tyler, ede.), p. 242. McGraw-Hill,New York. Sugimura, T. and Rudney, H. (1962). Biochim. Biophys. Acta 62, 167. Suyama, Y. and Gibson, J. (1966). Biochem. Biophys. res. Commun. 24, 549. Sykes, J., Gibbon, J. A. and Hoare, D. S.? 1965). Biochim. Biophys. Acta 109, 409. Taniguchi, S. and Kamen, M. D. (1965). Biochim. Biophys. Acta 96, 395. Trown, P. W. (1965). Biochemistry 4, 908. Tuttle, A. L. and Gest, H. (1959). Proc. nat. Acad. S’ci. Wash. 45, 1261. Van Niel, C. B. (1962). Alznu. Rev. p b n t Physiol. 13, 1. Vatter, A. E. and Wolfe, R. 8. (1958).J . Bact. 75,480. Vatter, A. E., Douglas, H. C. and Wolfe, R. S. (1959).J . Bact. 77, 812. Vernon, L. P. (1964). Annu. Rev. plant Physiol. 15, 73. Vernon, L. P. and Ke, B. (1966). In. “The Chlorophylla”, (L. P. Vernon and G. R. Seeley, eds.), p. 569. Academic Press, New York. Virgin,H. I.,Kahn, H. A. andvonWettstein,D. (1963).Photochem. Photobiol. 2,83. Weaver, P., Tinker, K. andvalentine, R. C. (1965). Biochem. Biophys. res. Commun. 21, 195. Weibull, C. and Bergstrom, L. (1958). Biochim. Biophys. Acta 30, 340. Wood, B. J. B., Nichols, B. W. and James, A. T. (1965). Biochim. Biophys. Acta 106, 261. Woodward, D. 0. and Munkres, K. D. (1966). Proc. nut. Acad. Sci. Wash. 55,872. Worden, P. B. and Sistrom, W. R. (1964). J . cell Biol. 23, 135. Yamanaka, T. and Kamen, M. D. (1965). Biochem. Biophys. res. Commun. 18,611.

Note added in proof. Recently, preferential synthesis of membrane protein has been demonstrated in growing cultures of Rps. Spheroides as they adapt from aeroio-dark to anaerobic-light conditions (Gray, 1967). Pigment synthesis is also accompanied by an increase in the proportion of rapidly labelled RNA which is rapidly degrading in the presence of proflavin (Ferretti and Gray, 1967; Cost and Gray 1967.)

The Plasmids of Staphylococcus aureus and their Relation t o Other Extrachromosomal Elements in Bacteria M. H. RICHMOND Department of Nolecular Biology, University of Edinburgh, West Mains Road, Edinburgh 9, Scotland I. Introduction

. . .

A. Historical B. Curing C. Stability . D. The Plasmid Location of the Penicillinase Genes 11. Types of StaphylococcalPlasmid . . A. Plasmid-BorneMarkers . B. Classification of Plasmids . . C . Distribution and Abundance of Plasmid Types D. Plasmid Nomenclature 111. Transduction of Plasmids . A. Preparation of Transducing Phage . B. Transductants Formed in Negative Recipients C. Transductants Formed in Plasmid-Carrying Recipients . D. Compatibility . E. The Role of Attachment in Recombination and DiploidFormation F. Chromosomal Attachment of Plasmid Markers . G. ThePlasmidMap H. Size of the PenicillinasePlasmids IT.A Comparison of Penicillinase Plasmids with Other Extrachromosomal Elements in Bacteria V. Acknowledgements . References .

.

.

.

.

.

43 43 44 45 48 51 51 55 58 59 60 60 60 61 71 75 78 80 83 84 86 87

I. Introduction A. HISTORICAL It has been known for years that certain hereditary characters are unstable in some staphylococcal strains in the sense that they are lost from cultures at a rate greater than can reasonably be explained by normal spontaneous mutation rates ;this phenomenon implies either an abnormally high mutation rate-a genetic hot spot-in the genes 43

44

M. H. RICHMOND

concerned, or a high rate of loss of the entire gene responsible for the character in question, One of the first characters examined in this way was penicillinase production. Although the phenomenon had been mentioned by Voureka (1948),Barber (1949)was the first to examine this matter quantitatively. She showed that, of 32 hospital isolates examined for the stability of penicillinase production, 17 contained penicillinaseless variants when slopes of the strains had been restored for about 6 months a t 2’. Although these results were hardly obtained under “natural” physiological conditions, the instability of penicillinase production in some strains.of Staphylococcus aureus has been amply confirmed, notably by Fairbrother et al. (1954), Novick (1963), Borowski (19631, May et al. (1964) and Asheshov (1966a). However, although the loss of the ability to synthesize penicillinase occurs widely-and has even been shown to occur in vivo (Hamburger et al., 1958; 1960)-the reverse process had never been shown unequivocally to occur under natural conditions. I n recent years other “unstable” characters have been demonstrated in staphylococci. May et al. (1964)have shown that the genes responsible for tetracycline resistance are lost from staphylococci at high frequency, and this is particularly common among strains of the 52/52A/80/81 complex (Asheshov, 1966a; Asheshov and Winkler, 1966). Similarly, Mitsuhashi and his collaborators (Hashimoto et al., 1964; Mitsuhashi et al., 1965) have shown that erythromycin resistance may be an unstable character in some strains, as may resistance to mercury (Hg2+) (Richmond and John, 1964), cadmium (Cd2+),lead (Pb2+)and arsenate (H2As0r or HAsOf) ions (Novick, 1967). Servin-Massieu (1961) has reported that the type and amount of pigment produced by some staphylococcal strains may also be lost at a relatively high frequency, particularly under rigorous culture conditions. B. CURING Certain growth conditions may markedly accelerate the rate of loss of penicillinase genes from 8.aureus. Fairbrother et. at. (1954) showed that the incidence of penicillinase-less variants in certain strains was greatly increased by growth at 44”, and this result has been confirmed by May et al. (1964) and by Asheshov (1966a). These last two groups of workers also showed that the loss of the tetracycline-resistance genes was accelerated in some strains-particularly those in the 52/52A/80/81 complex (Asheshov and Winkler, 1966)-but that the purging effect of growth at elevated temperature was not universal, either for penicillinase or for the tetracycline-resistance genes.

THE PLASMIDS OF STAPHYLOCOCCUS AUREUS

45

The clear-cut experiments of Hirota (1960) on the curing of the F (the sex, or fertility) factor from Escherichia coli by acridine dyes has led a number of workers to try to cure staphylococci of their penicillinase genes in a similar manner. Hashimoto et al. (1968) reported that overnight growth of staphylococcal strains in the presence of 25 pg. acriflavine/ml. led to the appearance of penicillinase-less variants-from 0.1 to 3.5% of the total population-in 17 out of the 18 strains tested. Similarly, Harmon and Baldwin (1964) reported 6.2% of penicillinsensitive cells'in a staphylococcal culture grown overnight in the presence of 10 mg. [sic] acridine orange/ml. On the other hand, Novick (1963) has been unable to show any significant curing effe& of acridine dyes on staphylococcal penicillinase genes; nor have P. H. A. Sneath, E. Asheshov (personal communications), or the author, in independent experiments. I n view of these conflicting reports, it is important to try to assess which is correct:At the moment it seems probable that acridine dyes do not cure the penicillinase genes, and the most likely explanation for the discrepancy between the reports of different workers is that neither Baldwin nor Mitsuhashi studied a statistically sjgnificant number of clones from untreated cultures to establish an qccurate enough "control" rate against which to compare the rate of loss of the penicillinase genes after treatment. Another procedure which has been reported to cure genes linked to an F factor in E . co2i is to grow a thymine-requiring mutant of the strain in question in the presence of growth-limiting concentrations of thymine (Clowes et al., 1965). However, growth of a thymidine-requiring mutant of a penicillinase-producingstrain of S. aurew in the presence of limiting amounts of thymidine had no effect on the spontaneous rate of loss of the penicillinase genes (M. H. Richmond, unpublished observation). C. STABILITY Although it is clear from the work already described that the penicillinase- and tetracycline-resistance genes may be unstable in aureus, this instability cannot be demonstrated in all strains. The probability of loss of penicillinase genes from a staphylococcal culture can be calculated from the proportion of penicillinase-synthesizingorganisms in the culture at a given density by means of the equation :

s.

r = -(2 -P)" 2"

where r is the proportion of penicillinase-producing cells in the total culture, p is the probability of loss of a penicillinase plasmid, and n is the

46

M. R. RICHMOND

number of generations the culture has completed from the one-cell stage (Richmond, 1966a). Therefore, in a colony which has grown thirty generations and which is found on examination to contain 1 % of negatives, the penicillinase genes are lost at a probability of rather less than 1:1000 cell divisions. And if the proportion of negatives in the clone is lo%, then the probability of loss is about 1: 150.

000 limit

------1/25

I

l

l

1:24 000 1:6000 1:1500 1:375 1:12000 1:3000 1:750 1:187 Frequency of negatives

FIG.1. Stability distribution among 67 independently isolated hospital staphylococci of phage groups I and 111. Data from Richmond (1966a).

I n practice, it is relatively easy to determine the proportion of penicillinase-less variants present in 20,000-30,000 cells-yet an incidence of one negative/30,000 cells corresponds to probability of loss of about 1 :lo4 and this is still considerably greater than the normally accepted value of about 1 :los for the rate of spontaneous mutation. This gap of about lo4 between the spontaneous mutation rate and the rate of loss

THE PLASMIDS OF STAPHYLOCOCCUS AUR.EUS

47

that is readily measured experimentally has made it difficult to decide whether the penicillinase and the tetracyline genes are relatively unstable in all staphylococcal strains-but it seems unlikely. Figure 1 shows the stability distribution among 67 independently isolated hospital staphylococci from phage groups I and I11 (Richmond, 1966a). This experiment was carried out in such a way that 25,000 staphylococcal colonies were screened from each strain to determine the incidence of negatives. I n all, 33 of the 67 strains showed no penicillinase-lesscells in 25,000 colonies; so in these strains the penicillinase genes, if lost at all, disappeared with a probability of less than 1:lo4. Of the remaining 34 strains, three produced as many as 30 negative wlls/25,000 colonies. In this series of experiments there was no apparent correlation between a high degree of instability of the pencillinase genes and phage pattern. Asheshov (1966a) examined 50 strains of staphylococci for loss of penicillin and/or tetracycline resistance. Of the 50 strains, three showed a loss of one type of resistance when grown at 37’ which was not significantly increased by growth at a higher temperature. On the other hand 12 strains showedlossof resistance to penicillin when grown at 43.5” and, of the 12, three showed an independent loss of tetracycline resistance. All of the 12 strains which showed a high rate of loss of resistance markers at high temperature belonged to the complex of staphylococcal strains having ;t.phage pattern 52/528/80/81. I n these experiments, therefore, it is clear that both the penicillinase and tetracycline genes are unstable in some strains, but it is not possible to conclude that they are stable in any. Although penicillinase-less variants can only be demonstrated in a proportion of staphylococcal strains, there is one strain in which there is good evidence that the penicillinase genes are truly “stable”. This strain is PS80 (the propagating strain for typing phage 80) and Asheshov (1966b) has been unable to detect the appearance of penicillinase-less variants from this strain. Although about 100,000 colonies have been examined in a single experiment for the presence of negatives, this evidence would still be insufficient were it not for two other facts. First, despite the inability to show penicillinase-less variants, mercury-sensitive variants do appear at a reasonable frequency; and secondly, the sensitivity to ultraviolet radiation of phage capable of transducing the penicillinase genes from this strain suggests that the penicillinase genes in PS80 are chromosomal. This strain-and the state of the penicillinase genes in it-will be discussed in detail later (see pp. 49 & 78). Recently K. G. H. Dyke (unpublished experiments) has studied the stability of the penicillinase plasmids in the 97 “hospital” staphylococci 4

48

M. H. RICHMOND

studied early by Dyke and Richmond (1967). Here penicillinase-less variants have been obtained from 93 out of 97 strains; but in one of the remaining strains there is preliminary evidence for a chromosomal penicillinaseregion. It appears, therefore, that a site for the penicillinase genes on a plasmid is much more common than a chromosomal location among naturally-occurring staphylococci.

D. THE PLASMID LOCATION OF PENICILLINASE GENES The high rate of loss of the penicillinase genes from S. a w e m led Novick (1963) to suggest that they were not carried as part of the bacterial

chromosome but were present on an extrachromosomal piece of DNA, which he suggested should be called a plasmid. This term has been proposed by Lederberg (1952) for extrachromosomal particles of various types, and Novick felt it to be important to avoid the term episome which has a precise definition involving the ability of the genes to pass relatively freely backwards and forwards between the chromosomal and extrachromosomal states (see p. 84). Even now, when considerably more is known about the behaviour of penicillinase plasmids, free passage of the genes in question in and out of the chromosome has not been shown unequivocally and for this reason the term plasmid is retained-although the penicillinase plasmids of S. auretm have an obvious similarity in certain respects to the episomes of the enterobacteria (see p. 84). Novick (1963) based his claim that the penicillinase genes in S. aweus were usually extrachromosomal on the following points : 1. The high rate of loss from the cell. This property of extrachromo-

soma1 particles in bacteria had been noticed frequently (e.g. Watanabe and Fukusawa, 1961). 2. I n certain strains, other genetic determinants are linked closely to the penicillinase genes and these are nearly always lost at high frequency en bloc with the penicillinase genes. 3. Where it is possible to transduce the penicillinase genes, the linked genetic determinants are cotransduced with high efficiency. Usually there is a greater than 95% cotransduction of all characters at a frequency of about 1:lo5infecting phage. 4. When such transducing phage preparations are inactivated by ultraviolet radiation, the frequency of transduction falls exponentially with the irradiation dose at a slope similar to that of the ultraviolet-radiation inactivation slope for the plaque-forming activity of the phage itself. Of these criteria, only the last is really convincing although the

THE PLASMIDS OF STAPHYLOCOCCUS AUREUS’

49

examination of strain PS80 (see p. 78) has produced circumstantial evidence to support the validity of the first three of the points cited. The conclusion as to whether a gene is chromosomal or extrachromosoma1 on the basis of its sensitivity to ultraviolet radiation when undergoing transduction is based on Arber’s (1960) experiments concerning the transduction of genetic markers between E. coZi strains using phage P1. I n these experiments, one type of donor strain carried a variety of chromosomal markers, while the other carried either the episomal F particle or prophage A. Transduction of the chromosomal markers to a suitable recipient by phage P1 occurred at a frequency of about 1:lo7 and this frequency could be increased about ten-€old by ultraviolet irradiation of the transducing phage. But the ability of phage P1 to transduce the extrachromosomal F or h characters occurred at a frequency of about 1:lo5 and decreased logarithmically with ultraviolet irradiation. The reason for this difference in the behaviour of transducing phage preparations depending on the original genetic location of the transduced genes is not well understood. One suggestion is that ultraviolet irradiation activates repair enzymes carried in the phage particles (or causes them to be formed in the recipient cell) and that this process has the effect of facilitating the integration of transduced chromosomal genes into the chromosome of the recipient, while having no effect on the transfer of extrachromosomal genes. Whatever the reason for the response of the transducing phage to irradiation, Arber’s observations-which were made on E. coZi-appear to hold for S. aureus as well. Korman and Berman (1962) showed that ultraviolet irradiation of transducing phage increased the frequency of transduction of streptomycin resistance-and this gene is believed to be chromosomal in 8.aureus. On the other hand, Novick (1963) showed that there was an exponential decrease in the transduction frequency of the penicillinase genes on irradiation under circumstances in which there was some additional evidence that the genes in question were located on a plasmid (points 1-3 above). Perhaps the most convincing evidence that experiments of the Arber type indicate an extrachromosomal location in X. aureus comes from a study of strain PS80 (Asheshov, 1966b).This strain is resistant to tetracycline and to mercury salts, and synthesizes penicillinase. Both tetracycline-sensitive and mercury-sensitive variants can be obtained a t a relatively high frequency, but the loss of these two characters always appears to be due to independent genetic events. On the other hand, penicillinase-less variants do not appear from this strain-at least at a rate greater than the experimental limit of 1:lo6. This evidence suggests, prima facie, that the mercury and tetracycline genes are carried on

50

M. H. RICHMOND

separate plasmids, while the penicillinase gene is chromosomal. Since a penicillinase-lessvariant cannot be obtained from PSSO, another strain must be used as recipient in transduction experiments, and strain 17855 which was sensitive to penicillin, tetracycline and mercury salts was chosen for this purpose. Propagation of typing phage 80 on PSSO leads to a phage preparation capable of transducing all these genetic markers, although the tetracycline marker is transduced at a frequency of about 1 :5 x l o 5 whereas the penicillinase genes are transferred only at a

0

25

50

7.5

Period of exposure to ultraviolet rodiation (inin)

FIU.2. Transduction frequency of resistance to penicillin (o),to HgCl2 (A) and to tetracycline (A)as a function of the period of exposure to ultraviolet irradiation of the transducing phase. Donor strain: StaphyZococcw aureus PS80. The phage survival curve is also shown (0). Data from Asheshov (1966a and unpublished experiments).

frequency of about 1 :lo8. This difference in rate, in itself, suggests the extrachromosomal location of tetR (Arber, 1960). Ultraviolet irradiation of the transducing phage preparation has the effect shown in Fig. 2. The transduction efficiency of the HgR and tetR markers falls exponentially, whereas transduction of the penicillinase genes, when unlinked to the Bg marker (seep. 52), is increased about ten-fold (Asheshov 1966b). The internal consistency of these results therefore supports the view that a high rate of spontaneous loss and a decrease of transducing efficiencyon irradiation are characteristic of an extrachromosomal gene in S. aureus.

THE PLASMIDS O F STAPHYLOCOCCUS AUREUS

51

11. Types of Staphylococcal Plasmid

A. PLASMID-BORNE MARKERS A number of different types of penicillinase plasmid have been identified on the basis of the genetic determinants they carry. Originally the different types of plasmid were distinguished according to the immunological type of penicillinase they synthesized, whether they carried resistance markers for mercury salts and erythromycin, and the extent to which the penicillinase was extracellular (Richmond, 1965a ; Novick and Richmond, 1965).Although not all the possible combinations of markers were found, in practice it was possible to detect the presence of ten different plasmid types (called 01 to K ) in varying abundance among hospital staphylococci (Dyke and Richmond, 1967). I n addition, tetracycline resistance is plasmid-borne but apparently is always on a plasmid separate from that carrying the penicillinase genes (May et al., 1964). The identification of more genetic determinants on staphylococcal plasmids (Novick, 1967) has further complicated the situation and, a t the moment, the following markers may be present in the plasmid state in S. aureus : 1. The Penicillinase Region

This is a group of genes responsible for the production of penicillinase.

It probably consists of a structural gene p (for penicillinase) and a gene involved in response to inducer, i (for inducibility) and one responsible for “setting the rate” of penicillinase synthesis, pr (for promoter) (Richmond, 1966b). Three immunological variants of the p gene have been identified (Richmond, 1965b).They lead to the synthesis of the A-, B- and C-types of penicillinase, and the genes responbible are called p A ,pBand p c respectively. 2 . The “Extracellukcrity” Region (exo)

This region determines the degree of extracellularity of the penicillinase under standard cultural conditions. I n the early work (Richmond, 1965a; Novick and Richmond, 1965) two characters were recognized-high ( > 25%) and low ( < 25%) extracellularity. However, measurement of the proportion of enzyme in the extracellular state in a number of hospital staphylococci (Fig. 3) shows a biphasic distribution in the quantity of extracellular enzyme (K. G. H. Dyke, unpublished experiments). It seems from these results that the boundary between “high” and “low” liberation should be set at 10 rather than 25%. Bearing in

52

M. H.RICHMOND

mind the difficulties of measuring the true quantity of an extracellular enzyme, and the doubts that must exist as to whether small amounts are truly “extracellular” (Pollock, 1962), it is even possible that the types of extracellularity shown by staphylococcal strains should be classified as all or none (i.e. + or -), rather than setting the boundary at some arbitrary level.

% penicillinase extracellular

FIG.3. The proportion of penicillinase present in the culture medium of 97 staphylococcal strains carrying various pencillinase plasmids.

3. Erythromycin Resistance (eroR)

This marker confers resistance to all macrolide antibiotics, the cells carrying the marker being about 100-1000 times more resistant to the antibiotic than those which do not. The marker is plasmid-borne only in a small minority of erythromycin-resistant staphylococcal strains. The biochemical basis of the resistance is unknown. 4. Resistance to Mercury Salts (HgR) This marker confers a six- to ten-fold increase in resistance to Hg2+ salts and their organic derivatives such as phenylmercuric nitrate. Resistance does not depend on the accumulation of -SH groups within resistant cells-a possible mechanism for resistance because of the avid reaction of -SH groups with Hg2+-but probably reflects a change in membrane properties which excludes the mercury ions from the cell interior (M. H. Richmond, unpublished observations). 5. Resistance to Cadmium Salts (CdR)

This marker is genetically distinct from the HgR marker described above and confers a twenty- to fifty-fold increase in resistance on cells which carry it. The biochemical basis of resistance is unknown. As with the HgR marker, cadmium resistance could involve accumulation of -SH compounds in resistant cells, but this does not seem to occur.

v

a

FIG.4. Staphylococcal colonies (a)before, and (b) after stainmg t o show the presence of penicillinme(darkcolonies). Note how the targot-likecolonies in Fig. 4a are those which do not synthesize penicillinme (Fig. 4b) and therefore do not ca.rrya penicillinme plasmid. Photographs are reproduced by kind permission of Dr. R. P. Novick.

01 0

54

M . H . RICHMOND

Once again, modification of membrane properties is the more likely cause of resistance; but the separate genetic identity of the HgR and CdR marker implies that the biochemical processes conferring resistance are distinct in these two cases. Staphylococci which are resistant t o Cd2+ are also resistant t o Zn2+-but with this ion the difference in the level of resistance is less than with Cd2+(Novick, 1967). 6. Resistance to Arsenate ( h a R )and Arsenite ( A s i R ) I o n s

These are the markers conferring resistance t o arsenate and arsenite ions respectively. Phosphate seems to exert a competitive protection against the inhibitory effect of arsenate but not arsenite, and consequently the phosphate composition of growth media is critical in the detection of arsenate-resistant staphylococci. 7. Other Plasmid-Carried Murkers

A number of other characters may be located on penicillinase plasmids but less is known about their distribution and character. Novick (1967)

FIG.5. The edge of a region of confluent staphylococcal growth on solid medium, stained to show penicillinase production (dark area). The arrows show small, faster growing penicillinase-less sectors at the edge of the growth. The surface of the growth is aIso studded with minute penicillinase-less papillae. Photograph reproduced by kind permission of Dr. R. P. Novick.

THE PLASMIDS OF STAPHYLOCOCCUS AUREUS

55

has detected a marker responsible for resistance to Pb2+which is genetically distinct from the Cd", HgR and Asa" regions, but little is yet known of the properties of this marker. Si&larly, certain plasmids seem to carry a determinant which expresses itself as a dominant sensitivity to Bi3+ions. Again, nothing is known about the biochemical basis of this bizarre observation. The carriage of certain plasmids seems to be associated with changes in colonial morphology. Figure 4a shows a mixture of unstained plasmidpositive and plasmid-negative clones to demonstrate this point. Those parts of the mixture carrying a penicillinase plasmid can be seen as the dark regions in the stained version of the same field (Fig. 4b). Although the colonial morphology appears to be plasmid-linked in these experiments, it is not expressed in every host to which the plasmid is transferred. The colonial morphology marker may, in fact, just be a rather special example of the differences in rate of colonial growth often found where growth of penicillinase-less variants is compared with their plasmid-carrying relatives (see Fig. 5).

B. CLASSIFICATIONOF PLASMIDS The characteristics of ten penicillinase plasmids which had been classified on the basis of the type of penicillinase synthesized, their resistance to erythromycin and mercury salts, and whether more or less than 25% of the penicillinase is excreted into the growth medium, are shown in Table 1. However, the recognition that the division between "low" and "high" extracellularity should be 10% and not 25%, and the discovery of the Cd" and Asa" markers, has meant that this classification has to be re-organized, and this is also shown in Table 1. 1. Plasmid a (A-type enzyme, e m s ; HgR; > 25% exo) is now divided into two different plasmid types: the more fully defined a-plasmid (A-type enzyme; eras; HgR; CdR;h a R ; > 10% exo) and a new h plasmid (A-type; eras; HgR; Cds; Asas; > 10% exo). 2. Plasmid /3 (C-type enzyme; e m s ; HgR; < 25% exo) is now (C-type enzyme ; e m S ; HgR; CdR; AsaR; < 10% exo). 3. Plasmid y (A-type enzyme; em"; Hg"; > 25% e m ) is now (A-type enzyme ; e m R ; HgR; CdR; Asa" ; > 10yoexo). 4. Plasmid 6 (B-type enzyme; e m s ; Hg"; > 25% exo) is now (B-type enzyme; e m s ; Hg"; Cd"; AsaR; > 10% exo). 5. Plasmid E (A-type enzyme; e m s ; Hgs; > 2 5 % e m ) becomes two different types of plasmid: the more fully defined eplasmid (A-type enzyme ;emS;Hgs ; Cds ; Asas ; > 10% exo) and a new p-plasmid (A-type enzyme ; eros ;Hgs ; Cd" ;Asa" ; > 10yoexo).

TABLE1. The “Original” (Richmond, 1965a;Novick and Richmond, 1965)and the “Revised” Plasmid Classifications Plasmid &

I

A-type penicillinase B-type penicillinase C-type penicillinase Reaction to Eg2+ Reaction t o erythromycin Proportion of extracellular penicillinase

A-type penicillinase B-type penicillinase C-type penicillinase Reaction to Hg2+ Reaction t o erythromycin Reaction to Cd2+ Reaction t o HASO& Proportion of extracellular penicillinase a

b

\

a

j

3

y

8

r

[

T

8

+

-

f

-

+

-

-

+

+

+ R S

R R

S S

S S

R S

-

R S

-

+ + - R S

R S

S S

~

K

I

-

+

Earlier version

S S

>25 <25 > 2 5 >25 >25 > 2 5 > 2 5 <25 <25 <25

+ - + - + - -

f

-

-

-

+ + + + - -

R S R R

R S R R

R R R R

S S R R

S S S S

S S S S

R S S S

R S R R

S S S S

> 1 0 <10 > 1 0 > 1 0 >10 > 1 0 > 1 0
+

+ 7

- S s s R

S S

R R

>10 >10

Present version

J

The 5 plasmid is often accompanied in the cell by CdRand AmRmarkers which appear to be chromosomal. In two out of four cases so far, plasmid 6 is accompanied by an HgR marker which appears to be chromosomal.

57

THE PLASMIDS OF STAPHYLOCOCCUS AUREUS

eras;

6. Plasmid 5 (C-type enzyme; Hgs; > 25% exo) is now (C-type HgS; Cds; Asas; > 10% exo). Plasmid 5 is often accomenzyme; panied in the cell by CdR and AsaR markers which appear to be chromosomal (K. G. H. Dyke, unpublished experiments). 7. Plasmid 7 (C-type enzyme; HgR; > 25% exo) now becomes (C-type enzyme ; eroS;EgR;Cds ; Asas ; > 10% exo). 8. Plasmid 0 (A-type enzyme; HgR; < 25% exo) now becomes HgR; CdR; AsaR; < 10% exo). (A-type enzyme; 9. Plasmid L (A-type enzyme; eroS; H f ; ~ 2 5 %exo) now becomes Hgs; Cds; Asas; < 10% exo). (A-type enzyme;

eras;

eras;

eras; eras;

eras;

eras;

The K-plasmid (C-type penicillinase; Hgs; < 25% exo) disappears from the classification as all five strains examined in the survey by Dyke and Richmond (1967) excreted between 15 and 25% of their enzyme into the medium. However, examples of this plasmid do exist within the staphylococcal population. Apart from these plasmids a number of other combinations of markers have recently been detected, although it has been decided not to add them to the classification until they have been investigated further. These plasmids and their characters are shown in Table 2. TABLE2. Genetic Markers Carried by Plasmids Which are Not Yet Included in the General Classification (see Table 1) Strain No. A-type penicillinase B-type penicillinase C-type penicillinase Reaction to Hg2+ Reaction to erythromycin Reaction to Cdz+ Reaction to HAsO42Proportion of extracellular penicillinase

1060

3951

608

3892

-

-

-

+-

S S

R R

S S S R

S S

10

10

10

+

+

+ R

S

-

S S S S

s

According to this classification, therefore, a t least ten distinct penicillinase plasmids have been detected under natural conditions so far. I n addition to these plasmids, however, one other related plasmid is known. It is carried by strainPS80, and, althoughit lacks the penicillinase region, it is clearly related to the penicillinase series of plasmids by possessing CdR, h a R and HgR regions. I n this strain, the penicillinase ( p A )region is probably chromosomal. I n addition to the penicillinase plasmids, staphylococcal strains often carry a plasmid conferring tetracycline resistance. These are found most

38

M. H. RICHMOND

commonly in strains carrying the cc-penicillinase plasmid (the plasmid common in hospital staphylococci) but they also occur with E-, 5-, 8-, L- and A-plasmids.

C. DISTRIBUTION AND ABUNDANCE OF PLASMID TYPES Any attempt to assess the distribution of plasmid types in the natural population of staphylococci is precarious because of the difficulty of ensuring that repeated isolates of the same strain are not included in the survey. However, if strains of distinct phage pattern and wide geographical separation are chosen for the survey, the chance of duplicating strains is largely eliminated. After taking these precautions, K. G. H. Dyke (unpublished experiments) studied the distribution of plasmid types among 97 hospital isolates of phage group I and I11 strains, and his results are shown in Table 3. The CL-, E-, 5-, 9-,7-, L - , A- and p-plasmids were found in various proportions, although the a-plasmid was by far the most common. No correlation between plasmid type and phage pattern emerged. TABLE3. Distribution of Plasmid Types among 97 “Hospital” Staphylococci of Phage Groups I and I11 Plasmid type

No. of strains

cc

43 0 0 0 13 26

P

Y

6. E

5 e

1 1

rl

10 0 2 1

I

x K

P

TOTAL

97

a Apparently confined t o Group I1 strains and therefore absent from this series.

A previous survey has shown that the immunological B-type of penicillinase is always associated with staphylococci from phage group I1 (Richmond, 1965b). I n this case, 12 group I1 strains were tested, but in only one case was there clear-cut evidence that the penicillinase genes were extra-chromosomal ;that is that plasmid 6 was involved.

THE PLASMIDS OF STAPHYLOCOCCVS AW7REVS

59

Two plasmids ( p and y ) listed in Table 1 appear to be extremely uncommon. The p-plasmid has been isolated once only. This was in strain 147-a strain isolated in a food poisoning outbreak in the 1930s (Segalove, 1947).Its rarity nowadays may just reflect that environmental conditions, as far as the staphylococcus is concerned, may have changed somewhat since the 1930s. The y-plasmid is unusual in that it carries genes conferring resistance to erythromycin. This plasmid has been detected a number of times in Japan (Mitsuhashi et al., 1965), but these isolates may well be the same strain. It is interesting that this plasmid may be a recombinant between a penicillinase plasmid ( ? a )and a piece of bacterial chromosome carrying an erythromycin-resistance region. Furthermore, it may represent the first step in the accumulation of resistance markers in a single extrachromosomal element ; a process familiar enough to those concerned with the R-factors of the Enterobacteriaceae (see Datta, 1965, for a review of this topic).

NOMENCLATURE D. PLASMID The nomenclature now used for X.aurew penicillinase plasmids is an extension of that proposed by Novick and Richmond (1965). The first figure in the designation is the strain number, and this is followed by the Greek letter which describes the type of plasmid carried and which refers to the plasmid as a whole. The Greek letter is followed by letters and subscript figures indicating the markers which are carried on the plasmid. The symbols p A ,pBand p c refer to the structural gene for the immunological type of penicillinase while the symbol i, when used without letters and figure subscripts, refers to the penicillinase inducibility locus. This region may be considered t o consist of two parts : il, indicating that part of the i gene in which mutation gives rise to mutants of the magnoconstitutive type; and i t ,the region which givesrise to baso-constitutive mutants. (For details of the behaviour of these mutants, see Richmond. 1967b). Mutations in the p and i genes are numbered in series; e.g. pxiZ indicates the structural mutant No. 2 in the penicillinase A-type gene; and iijs indicates the constitutive mutant No. 6 in the i i region. Resistance to mercury and cadmium salts is designated as HgR and CdR respectively. Similarly resistance to erythromycin is designated eroR, t o arsenate, AsaR, and to arsenite, h i R . Strains which carry no penicillinase plasmid fall into two classes: those that are naturally-occurring plasmid-less strains and those which are known to have lost a plasmid. The former are designated simply by their strain number, e.g. 8325; and the latter have (N) added, e.g. 147 (N).

60

M. H. RICHMOND

I n laboratory strains, in which recombination has occurred betweer different plasmids, the designation indicates which of the markers comet from which plasmid if this is known. Thus 8 3 2 5 1 ~i+@. . HgR...y .eroR: designates strain 8325 carrying a recombinant plasmid, the penicillinasc and mercury resistance regions being contributed by plasmid E and erythromycin resistance by plasmid y. I n plasmid diploids, the respectivc components are separated by an oblique stroke, e.g. : 147(cc.i+p$.H g R . . y .eroR//3. iip;. H@ .eras) .

III. Transduction of Plasmids A. PREPARATION OF TRANSDUCING PHAGE So far the only method available for transferring staphylococcal plasmid-borne markers from cell to cell is transduction: all attempts t c discover a mating system in 8. aureus have failed to date. Broad13 speaking, two main methods of carrying out transduction experimentE have been used. One is to propagate a staphylococcal phage-usually one of the international series of typing phages-in the donor culture by lytic cycle; the other is to induce prophages carried in the donor by irradiating the cells with ultraviolet radiation. As far as the first method is concerned, typing phages 47, 53, 80 and 81 have been used most frequently, but phages 29, 52A and 79 alsa appear to be effective-at least for the transduction of the tetR marker (Pattee and Baldwin, 1961). The efficiency of transferring plasmid markers in this way is variabIe and may range from 1 :lo6 to 1 :lo8. If on the other hand, the transducing phage is obtained by ultraviolet irradiation, the transducing frequency obtained is much more variablebut can sometimes be as high as 1 :5 x lo3 (M. €€.Richmond, unpublished experiments). Although both of these methods of phage production have been used fruitfully, it is important to stress that transduction by a phage preparation propagated by lytic cycle is preferable. On the whole, phage obtained by this means is homogeneous whereas ultraviolet irradiation of a staphylococcal culture frequently induces a number 01 phages, only some of which will be able to transduce. However, for experiments in which it is desirable to transfer plasmids reciprocally between two strains, it may be necessary to rely on the phages which are in the prophage state in the strains concerned.

FORMED IN NEGATIVE RECTPIENTS B. TRANSDUCTANTS Transduction of the penicillinase genes was first described by Morse (1960) and Ritz and Baldwin (1958, 1961), and since that time it has

THE PLASMIDS OF S!l’APHYLOCOCCUS AUREUS

61

been shown that all the genetic markers detectable on penicillinase plasmids can be transduced (Novick, 1963; Richmond and John, 1964; Novick and Richmond, 1965). Similarly the tetracycline-resistance gene(s)can be transferred by appropriate transducing phage preparations (Pattee and Baldwin, 1961 ; Collins and Macdonald, 1962; Asheshov, 1966a), but the penR and tetRregions are never cotransduced. When the recipient in a transduction experiment is a negative or derived negative, the plasmid from the donor is transferred with a frequency of 1 :105-1 :los, and the incoming plasmid is normally held in the plasmid state in the recipient (Novick, 1963). However, cotransfer of all plasmid markers to every transductant does not always occur, and this implies that certain recipients receive an incomplete plasmid. This process, which has been called “plasmid dissociation” (Novick and Richmond, 1965), is normally uncommon but its incidence can be greatly increased by irradiation of transducing phage preparations. Table 10 (p. 74) compares the markers that were transferred to a recipient, with those present on the plasmid in the donor, in one experiment in which the transducing phage was irradiated with ultraviolet radiation (Novick, 1967). The nature of the genetic deletions obtained in this way throws some light on the order of the genetic markers on the plasmid, and this point is discussed later (p. 80).

C. TRANSDUCTANTS FORMED IN PLASMID-CARRYINGRECIPIENTS If the recipient cell already contains a penicillinase plasmid, one of a number of transductant patterns emerges depending on the types of plasmid involved, and on how soon the transductants are selected after mixing the recipient with the phage preparation. These patterns can be classified as follows : 1. Recombination or apparent displacement; 2. Formation of transient diploids; 3. Formgtion of normal stable diploids; 4. Formation of associated stable diploids. Broadly speaking, the first two of these patterns are found in transduction experiments in which the donor and recipient plasmids are of the same (or closely similar) type, whereas the latter two occur when the two plasmids involved are different. 1. Plasmid Recombination and Apparent Displacement

If the two plasmids involved in a transduction experiment are of the same type (e.g. =/a,/3//3, or y / y ) , and selection is delayed for 5-6 hr. after mixing the phage and the recipient cells, the transductants are normally haploid and contain a relatively large number of plasmid

M. R. RICHMOND

62

recombinants. A typical experiment of this type is as follows :

#(a.iipi) -+ 8325(~.i+p2,,) Donor Recipient

(1)

I n this experiment, the transductants were selected with penicillin (which allows survival of the i-p+, i-p- and i+p+genotypes but not i+p-), and the relative abundance of parental and recombinant plasmids is shown in Table 4. The donor parental phenotype and both possible recombinants were found, and the recombination between the i i andpZlz sites occurs with a probability of about 5%. At first sight, crosses of this type open up the possibility of mapping the plasmids by three-point crosses, but there are certain difficulties involved in the interpretation of such experiments and these are discussed in detail later (p. 80). i

TABLE4. Relative Abundance of Parental and Recombinant Transductants Obtained by Selecting the Cross $(a.i - p i ) -+ 8325 (a.i+pG2)with Penicillin

Genotype Parent (donor) (a .$ - p i ) Recombinants (a.i-pz2) (..i+pi)

No. of transductants

Frequency of transductants

% of donor rate

612 37 29

1:2~105 1:1,2~107 1 : 9 x 10s

6 4.8

-

Recombinants are also common when certain plasmids of different types are placed together in a cell by transduction. Pairs that behave in this way are a/y and yla. Even when a/. crosses of this general type are performed with plasmids marked in such a way that it is possible to enrich the transductants with diploids should they occur-for example in the cross

~(a.iipi.CdR.AsaS.eroR) ---f 8325(~.i+pZ,~. CdS.AsaR.eros) (2) selected with either cadmium + arsenate ions or arsenate + erythromycin, transient diploids occur to an extent of less than 2% of all the transductants, as long as selection is delayed about 6 hr. after the phage is mixed with the recipient. Table 4 shows that transductants in which the incoming plasmid ( a .i-pi) appears to have displaced the resident ( a .i+pxI2)plasmid are the most common transductants selected from cross (1). If, as seems likely, the recipient plasmid is held in the cell a t an attachment site (see later), then the apparent displacement of a resident by an incoming plasmid can occur in two ways. Either a recombination between the two plasmids occurs near the point of attachment but outside the range

63

THE PLASMIDS OF STAPHYLOCOCCUS AUREUS

of known genetic markers; or the two plasmids compete for the attachment site in the recipient cell. Which of these two processes is responsible for the apparent displacement found in transduction experiments of this kind is not yet known. 2. Transient Diploids

If transduction ( 2 ) (see above) is carried out and selected either with cadmium + arsenate ions, or with arsenate + erythromycin within 50 min. of mixing the phage and the recipient cells, doubly resistant transductant colonies are obtained at a frequency of about 1 :lo6. If these transductants are now picked into ice-cold liquid medium so that no growth is possible, and plated to give single colonies on a variety of selective media, the characteristics of the colonies that arise show clearly that the transduced cell that was originally plated on the double selective medium was diploid. Furthermore, the composition of the colonies grown from such cells under double selection shows that the diploid state has survived in about 10-15y0 of the cells to the time the original colony was picked and replated. Examination of the remainder of the colony shows that it is made up of doubly resistant recombinants and singly resistant segregants (Table 5). TABLE5. Typical Composition of Transductant Clones from the Cross +(a.i-ph.CdR.AsaS.eroR --t 8325(cc.i+p,l,.CdS.AsaR.eros) when Selected on Medium Containing (a) Cadmium+ Arsenate, and (b) Arsenate + Erythromycin

No. of clones examined

+

(a) Selected on Medium Containing Cadmium Arsenate Donor i-p: .CdR.Asa'.eroR Recipient i+p&. Cd' ,AsaR.ero' Recombinant , i - p z CdR.AsaR.ero' Diploid i-p: .CdR Asas. eroR/i+pz2.Cd' AsaR.e r 8

.

.

61 46

3

.

12

TOTAL

+

(b) Selected on Medium Containing Arsenate Erythromycin Donor i-p: .CdR.Asas.eroR Recipient i+pGa.Cd' AsaR.eroS Recombinant i - p t CdR,AsaR.eroS Diploid i - p i CdR.Asa'. eroR/i+p&.Cd'. AsaR.ero'

. .

122

38 35 1 12

.

TOTAL

96

Single transductant clones were emulsified in ice-cold medium and plated on nonselective medium t o determine the relative abundance of the various possible genotypes in each clone.

64

M. H. RICHMOND

The survival of the singly resistant segregants in these colonies was unsuspected. It appears to be due to the persistence of phenotypic protection against a selective agent for a time after the genetic element conferring resistance to that selective agent has been lost by segregation. The instability of this type of diploid is shown by the fact that a clone containing 10-15% of diploid cells, picked from a double selective plate, contains less than 10% of diploids after 4 hr. growth in a liquid nonselecting medium. By this stage, the clone is almost completely composed of segregants of either parental or recombinant genotype. But the fact that diploids survived even this long in the colony argues that transient diploids are not formed by abortive transduction but can survive a sufficient number of generations to contribute to the composition of a fully grown colony, provided double selection is maintained. On the basis of the results of this and the previous section, therefore, it appears that transduction of an a-plasmid (or an w y recombinant) to a cell already containing an a-plasmid leads t o the formation of transient plasmid diploids. While the diploid state survives, recombination may take place, but segregation to give a haploid cell, containing either a single parental or recombinant plasmid, occurs rapidly. Selection for both plasmid components of the diploid, if applied up to 50 min. after mixing the phage with the recipient cells, may lead to the survival of a predominantly diploid clone for a considerable time; but even this process fails ultimately to preserve a relatively pure clone of diploids for, as time goes on, the colony becomes composed increasingly of doubly resistant recombinants. If the selection is delayed until 6 hr. after transduction, most of the transient diploids have segregated and only cells carrying a single parental or recombinant plasmid survive. 3. Normal Diploids With certain pairs of dissimilar plasmids (e.g. alp, pla, Ply, yip) recombination is uncommon, and relatively stable plasmid diploids are formed more or less regardless of the delay in selecting the transductants. Formation of diploids of this type was fist s h o h in cross (3) selected with penicillin (Richmond, 1964, 1965c):

i-p;) 4 8325(a.i+pAp) (3) Subsequently, however, similar diploids have been constructed in the reverse direction by selecting either with erythromycin (if a y or a . . . y recombinant plasmid is carried in the donor: cross (3) ) or with cadmium or arsenate ions if Cds or Asas alleles are on the recipient r$@.

THE PLASMIDS O F STAPHYLOCOCCUS AURJCUS

65

(crosses (4) and ( 5 )) ;similar methods may be used to construct diploids involving the /3- and y-plasmids (Novick, 1967).

.

8325(u.i-p; . . y.eroR) ---f 147(/3.i+p&.eroS) 8325(a.i-p;. CdR) -+ 147(/3.i+p6.Cds) 8325(a.i-p;. AsaR) --f l47(/3.i+&.AsaS)

(4) (5) (6)

Broadly speaking, all normal a//3 diploids seem to show a number of characteristics in common whichever plasmid is in the recipient cell when the diploid is constructed, and these characters can best be described by referring to the behaviour of the typical diploid 147(a.i+pi . . . y.eroR//3.i-p&.eroS)(Table 6). It is true that the characters are not found in a//3diploids where certain types of regulatory gene mutation in the penicillinase region-notably those involving a micro-inducible or micro-constitutive mutation-are carried on one of the plasmids. But such diploids are described in detail by Richmond (1966b, 1967b) and are not discussed further here. (a) The rate of segregation of the normal diploid to the two possible haploid states-by loss either of the a- or the 13-plasmid-occurs with a probability which reflects the spontaneous rate of loss of a and /3 when present alone in a cell (Richmond, 1965~). With strains 147 and 8325, the rate of segregation of the plasmids is approximately 1 :1000 divisions, whether they are haploid or part of a diploid. Every diploid clone, therefore, tends to be contaminated with approximately equal numbers of haploid segregants. (b) If one plasmid in the diploid carries an intact i+(fully inducible) allele while the other carries i- (fully constitutive), the diploid is inducible. I n the diploid 147(a.i+pi . . . y .eroR//3.i-p;. eras) the uninduced level of enzyme expression in the diploid is about 8 enzyme units/mg. dry wt. organisms as opposed to about 3 units/mg. for the haploid strain 147(a,i+p+. . . y.eroR) (Table 6). Thus the i+ allele represses the expression of i- in the trans position in the diploid, but in this case is not quite as effective at repressing C-type enzyme expression as a similar region would be acting cis (Richmond, 1965~). I n a diploid carrying two if alleles [e.g. 147(a.ifp; . . . y .eroR//3.i+p& .eras) 1, the basal level is about 1 unitlmg. compared with 5 unitslmg. for 147(a.ifp1 . . . y.eroR) and 3 units/mg. for 147(/3.i+&.eroS).The presence of two i+ regions in a single cell therefore causes a somewhat lower level of expression of both structural genes than is found for a single structural gene in the haploid state (K. Smith, unpublished experiments). (c) I n i+/i- or i+/i+diploids, the A- and C-types of penicillinase 5

TABLE6. Phenotypic Characteristics of the Stable Diploid 147(u.i i p z . . .y .eroR/8.i&p;. eras) Penicillinase synthesis (unitslmg. dry wt. bacteria) I

uninduced Strain

Genotype

Immunological typeofenzyme

induced 7

A-type

C-type

'

A-type

C-type

263

-

p1

Inducible donor Constitutive recipient Diploid

147(u.;+p:. ..y.eroR)" 147(8.i&p$. era') 147(~.i+pi.. .y . eroR/p.i.&v$ .eras)

Segregants

147(a(.ifpf.. .y.eroR) 147(8.iG3p;.eras)

A C A+C

-

3

217

-

C

248 128

c-------v---J 86

A

-

147

2.4

-

-

264

293

-

275

291

a The expression of the (a.i + p t . . .y . eroR) plasmid is shown in strain 147 rather than in the normal host, 8325, to allow closer comparison with the expression of this plasmid as a part of a plasmid diploid in strain 147. b About half of this enzyme was immunological A-type and about half C-type.

I

B 3

THE PLASMIDS OF STAPHYLOCOCCUS A V R B V S

67

(characteristic of the a- and /?-plasmidsrespectively) are synthesized in approximately equal amounts in the absence of inducer (Richmond, 1965~).Furthermore, if the penicillinase genes on both the plasmids in the diploid can be induced (as is the case with i+/i- and i+/if diploids) then the A- and C-types of enzyme are also equally represented in induced cultures (Table 6). The maximum quantity of penicillinase that appears to be synthesized by an a//? diploid in either strain 8325 or 147 (with or without induction) is about the same as found in a fully induced or magno-constitutive haploid. This is best demonstrated by comparing the quantity of penicillinase synthesized by the diploid 147(a.iipJ/?.i-p6) with147(a.i-p;) and 147 (/?.i-p&) (Table 7). I n all these cases the maximum quantity of penicillinase synthesized amounts to about 1% of the dry weight of the cell. In a//?or /?/y diploids, both A- and C-type enzyme are liberated into the growth medium t o the extent found in haploid cultures (Table 8). Thus the proportion of A- and C-type enzyme in the growth medium of an a/@ (or @/a)diploid is unequal since about 45% of A-type enzyme is normally liberated from a haploid cell carrying an a-plasmid while only about 5% of the C-type enzyme is extracellular in a culture carrying the /?-plasmid alone. These five characteristics allow a tentative picture of the behaviour of penicillinase plasmid diploids of the normal type to be drawn, particularly with respect to penicillinase production. The fact that the two plasmid components of the diploid co-exist in the cell for relatively long periods without recombining, and the fact that the presence of two plasmids in a cell does not seem to affect the stability of either, suggests that, in this type of diploid at least, the plasmids are held in the cell by two separate attachment sites. According to this model, these sites would have the property of regulating the rate of plasmid multiplication to that of the host cell and also ensuring that each daughter cell received at least one copy of the plasmid at division. The loss of plasmids, to yield derived negatives, might then reflect the failure of either of these two processes. Although this model postulates a separation of the two plasmid components of a diploid so that they do not interact genetically, this need not apply to their products ; indeed the diploid characteristics described above indicate strongly that interaction of plasmid products does occur in a diploid of this type. First, the fact that an i+/i-diploid is fully inducible, despite the presence of the constitutive (i-) genotype, argues that the i-gene product is able to influence the expression of the structural gene in the other plasmid. Whether this trans effect of the 5*

TABLE7. Phenotypic Characteristicsof the Stable Diploid 147(cz.i;pi.. .y.ero*/P.i.&p; .eras) Penicillinase synthesis (unitslmg. dry wt. bacteria)

,

c

uninduced Strain Constitutive A-type strain Constitutive C-type strain Diploid

Genotype

} 147(cr.i;pz.. .y.eroR)” } 147(~.i~,.p;.eros) 147(~.i;pf...y.ero”/P.i&p; .eras)

Immunological , type of enzyme A-type

C-type

A-type

C-type -

A

307

-

320

-

C

-

261

-

242

288

A+C

281

&

161

.. .

induced

127

++

141

140

CI The expression of the (a. i ; p 2 . y ero’) plasmid is shown in strain 147 rather than in the normal, 8325, to allow closer comparison with the behaviour of the same plasmid when part of a plasmid diploid in strain 147.

F

w L-d

l

TABLE8 Liberatian of A- and C-type penicillinase to the Culture Medium by Various Haploid and Diploid Strains

iM

Amount of induced penicillinase (unitslmg. dry wt. bacteria) Strain

Genotype

Immunological typeofenzyme '

A-type

Inducible A-type strain Constitutive C-type strain Constitutive A-type strain i+/i-Diploid

147(a.i+p:. ..y.eroR) 147(j3.iG3p$.era') 147(a.i-p= ...y.eroR) 147(a.i + p i ...y .eroR/p.i&p$ .e r d )

A C A A+C

285

i-/i-Diploid

147(a.iipf.. .y.eroR/j3.i&,p$ .eros)

A+C

177

C-typ;

-

-

% Total enzyme in extracellular state ' A-type

C-type '

45

-

.

w

-

261

-

-

45 38

6 -

%

128

5

E

130

43

6

295

A C A

0

345 167

307

Segregants : Inducible A-type strain 147(a.i+pfI. .y.eroR) Constitutive C-type strain 147(8.iG3p$.ero') Constitutive A-type strain 147(a.i;pi .. y.eroR)

E

294 I

347

261

-

~

a

8 2 B

47

-

-

6

41

-

22

aa W

70

M. R. RICHMOND

i-gene product is mediated at the genetic level, as postulated by Jacob and Monod (1961), or at the ribosomal site of protein synthesis, is not yet clear. Secondly, the fact that haploid and diploid cultures produce the same amount of penicillinase protein suggests that there is a limit imposed by the cell on the level of expression of a plasmid-borne structural gene; and that, in the event of two structural genes being present together in the cell, there is competition between the two gene products for expression at the available sites. Since staphylococcal penicillinase is an extracellular enzyme (Richmond, 1963) and is probably synthesized at the bacterial cytoplasmic membrane, a limit to the number of sites on this structure that could be used for penicillinase synthesis could account for the ceiling in enzyme production that appears to exist both in haploid and diploid cells. But the fact that enzyme can be liberated to achieve either 50% or 5% extracellularity in the haploid state, or even a different degree of extracellularity depending on which plasmid is being expressed in a diploid, argues that the sites must be non-specific as far as their ability to process the products of different penicillinase plasmids is considered. 4. Associated Stable Diploids

When transduction experiments of the type shown in equations

(a),

(5) and (6) (p. 65) are carried out, about 95-98% of the transductants are normal stable diploids of the type just described. I n 245% of cases,

however, interaction of the two plasmids in the diploid occurs to form an “associated” diploid (Richmond, 1967a). This process has been shown to occur, as a rare event, when the a-plasmid is introduced into strain 147 which already carries a P-plasmid and may occur when the a- and #I-plasmids are present together in other strains. Such associated diploids differ from normal diploids in the following properties : (a) Both plasmids may be lost together from an associated diploidto form a derived negative-at about 10,000 times the rate found with a normal stable diploid, where the rate of loss of both plasmids is the product of the rate of independent loss of each component. (b) Propagation of phage on associated diploids leads to a preparation capable of cotransducing both plasmids from the diploid, Phage propagated in this way on normal stable diploids is capable of transducing either plasmid from the diploid but not both. (c) Associated stable diploids may revert to the normal stable type. When this occurs, segregation to the haploid may follow (rather

THE PLASMIDS OB STAPHYLOCOCCUS AUREUS

71

than to the derived negative-see point (a) and the haploid segregants obtained in this way are now found to containrecombinants, unlike segregants from normal diploids. The first two of these characters imply that, in this type of diploid, the a- and p-plasmids are behaving as a single unit capable either of being transduced by a single phage particle or lost together from the cell. The presence of a connexion between the two plasmids, at least at some stage of their existence, is implied by the third character listed above, namely the appearance of recombinants among the segregants after the diploid dissociates. The most likely model to explain associated diploids of this kind would be one in which the plasmids behaved as though they were circles (Fig. 6; Richmond, 1967a).An associated diploid would then be formed by any single crossover between the two component circles of the diploid, and this structure could then behave as a single unit both with respect to loss from the cell or cotransduction. Furthermore, recombinant plasmids would arise by any crossover in the double circle in the opposite sense from the first wherever it occurred in the structure. This model suggests that the a-plasmid should have two different attachment sites in the cell-one in which a is compatible with to form normal stable diploids, and the other which can only be used when a has recombined with p. This second site could well be the normal attachment site for /3 in strain 147.

D. COMPATABILITY The observation that certain pairs of penicillinase plasmids can co-exist in a single staphylococcal cell over considerable periods whereas, with other pairs, recombination and reversion to the haploid state is common, led Novick and Richmond (1965) to postulate the idea of compatibility. Two plasmid types which could co-exist in the same cell were said to be compatible, while those pairs that segregated rapidly, either with or without recombination, were called incompatible. And TABLE9. The Allocation of Various PIasmids to Compatibility Groups Compatibility group

I Plasmids Genetic region involved in compatibility maintenance

a,y,

I1 E,

mc1

5

B

mCn

Data taken from Novick and Richmond (1965) and Richmond (1965a). -**

72

M. R. RICHMOND

on the basis of this behaviour two compatibility groups were compiled (Table 9) and called variously Corn I and I1 (Novick and Richmond, 1965) andPZa I and I1 (Richmond, 1965a). According to this original definition, i-

i+

.-

I

HgR

;+

HgR

i+

FIG.6. A possible mechanism for the formation and separation of associated plasmid diploids in XtaphyZococcus aureus. (a) Normal diploid. (b) First stage of formation of associated diploid; apposition of the two circles. (c) Formation of the associated diploid by a cross-over. (d) Re-formation of two circles by a cross-over in the opposite sense from the first. (e)Final form: note the new position of the eroR marker in relation to p:. Data from Richmond (1967a).

THE PLASMIDS OF STW'HYLOCOCCUS AURBUS

73

therefore, compatibility merely described whether two plasmids could co-exist in a single cell long enough to be detected; the term implied nothing as to the mechanisms involved. Although the discovery of associated diploids (Richmond, 1967a) could be accommodated within the original definition of compatibility, the discovery of transient diploids (see above) has confused the situation since two a-plasmids, which were formerly thought not to be able to co-exist in the cell, are now known to be capable of forming a diploid, albeit transitorily ;the distinction between compatible and incompatible pairs of plasmids now appears as a matter of degree rather than a clearcut difference. It follows therefore that, if the compatibility idea is to retain its usefulness, it must be modified, preferably to reflect the possible mechanisms involved in maintaining plasmids in the staphylococcal cell. Broadly speaking, two types of model could explain compatibility : either two plasmids survive in a single cell because insufficient homology exists between their DNAs to allow rapid recombination; or the plasmids are held in the cell at different attachment sites while incompatible plasmids compete with one another for the same site. By and large, the first of these two possibilities seems unlikely. Apart from the fact that an attachment site may well be necessary to ensure the efficient distribution of plasmids to daughter cells on division, it seems probable that the a- and /3-plasmids must have considerable stretches of DNA base sequence in common since they both carry the genetic regions corresponding to the penicillinase r, and i genes, and the regions responsible for resistance to mercury, cadmium and arsenate ions. Although it is not possible to be dogmatic on this point, the insertion of the tac region into the E . coZi chromosome from the P'lac state occurs at places where the homology is almost certainly a good deal less than would be the case between the a- and /3-plasmids in S. aureus (Beckwith and Signer, 1966). Furthermore, a and /3 have the same number and type of genetic markers in common as do a and y , yet, on the original definition, the first two plasmids are compatible whereas the second two are not. As mentioned previously, an attachment site might be expected to have two main functions: to regulate the rate of multiplication of the plasmid to that of the cell, and to ensure that, after the plasmid had replicated, one copy was transferred to each daughter cell on division. If this model is correct, therefore, the site might not only influence the compatibility relationships of the plasmids, it could also affect stability. One possible site of attachment is the chromosome. And this could clearly explain the behaviour of a few staphylococcal strains (e.g. PSSO; Asheshov, 1966b: and perhaps S26; Poston, 1966) in which the

74

M. H. RICHMOND

penicillinase markers have all the properties of chromosomal genes. If the incoming markers are “attached” by integrating them into the chromosome, the problems of regulating their rate of synthesis and of transferring them correctly to daughter cells at division are largely solved, since both these processes would then be carried out as part of the general process of regulating chromosome replication and partition. But there is good evidence that, in most cases, transduced penicillinase genes-either in the haploid or diploid state-are still plasmid-borne and it seems that their properties exclude the chromosome as an attachment site, at least if attachment occurs by “recombination in” of the transduced markers. An alternative location for the attachment site is some point on the membrane. If this were so, then the plasmid site could be similar in properties to that used to regulate chromosome synthesis and partition in S. aurezcs; in many ways the plasmids could then be regarded as additional chromosomes. In fact the only difference between plasmid and the chromosome, apart from size, would then be that failure to replicate or partition the chromosome would be lethal to one daughter cell while the same lesion affecting plasmid multiplication would just result in a plasmid-less daughter cell. And this difference derives solely from the fact that chromosomal genes are essential for survival of the

.

TABLE 10. Dissociated Plasmids Obtained from ( a .i + p i .AsaR.PbR.CdR.EgR mC.eroR)by Treating Transducing Phage with Ultraviolet Light and Selecting the Transductants on (a) erythromycin agar, (b) cadmium acetate agar, and (c) penicillin agar. Data from Novick (1967).

Transductant genotype

(a) Erythromycin agar penR.AsaR.PbR.CdR.HgR.mC.eroR PbR.CdR.HgR.mC.eroR CdR.HgR.mC.ero’ HgR.mC.eroR mC.eroR

No. of transductants 1500 1

eroRa

1 13 1 17

(b) Cadmium acetate agar penB.AaaR.PbR.CdR.Hg’ .mC .eroR

2000

penR.AsaR.PbR.CdR.HgR.mC

(c) Penicillin agar penR.AaaR.PbR.CdR.HgR.mC .ero’ penR.AaaR.PbR.CdR.HgR.mC a

Selective marker

eroR

CdB

8

6000 2

This class of eroR transductants are integrated into the chromosome.

pefiR

THE PLASMIDS OF STAPHYLOCOCCUS AZ7R.EUS

75

cell whereas plasmid genes frequently are not except under special conditions. If penicillinase plasmids are maintained in staphylococcal cells by this type of mechanism, a genetic region correspondingto the attachment site should be present on the plasmid and this region should express itself both in terms of the compatibility and stability of the plasmid. I n practice, there is some evidence that both “compatibility” and “stability” are determined by genetic regions, and that they lie close together on the plasmid, or may even be different manifestations of the same gene. Novick (1967) has shown by studying the behaviour of deletions covering various regions of the y-plasmid that there is a region lying between H f l and eroR, which is necessary for the retention in the cell of the erythromycin-resistance gene(s)in the plasmidstate (seeTable 10). If this “compatibility maintenance” (mC) region is deleted, the cells can only retain the eroR marker if it becomes integrated into the chromosome. Fragments lacking the mC region are compatible with both a and 6, and Novick’s experiments at least point to a genetic region responsible for maintaining a plasmid in an extrachromosomal state in the cell. I n the a-plasmid there is also evidence for genetic regions involved in stability and compatibility which are closely linked genetically and seem to lie on the same region of the plasmid as Novick’s mC locus (M. H. Richmond, unpublished observations). Under certain circumstances, transduction of the a-plasmid to strain l47(N) gives transductants in which the a-plasmid is particularly unstable, being lost at a rate of about 1:50 divisions (Richmond, 1966a). If this “unstable” a is now transferred to another 147 strain-this time already carrying a 6-plasmid-recombination may occur as a very rare event to give “unstable” p-plasmids. The acquisition of the instability marker is always accompanied by a change in compatibility characteristics of 6. I n this case, the “unstable” ,l3 becomes compatible with a “stable” 6, but incompatible with a. These results suggest therefore that there are genetic regions governing “stability” and “compatibility”, and that these could well be the same or very close to one another. They also have the properties that would be expected of the plasmid-borne region involved in the attachment of the plasmid to the cell.

E. THEROLEOF ATTACHMENT IN RECOMBINATION AND DIPLOID FORMATION If the attachment hypothesis is correct, it becomes possible to suggest the mechanism underlying plasmid recombination and diploid formation, and therefore to give a more precise definition to the idea of compatibility. According to this hypothesis, incompatible plasmids are those using

76

M. € RICHMOND I .

the same attachment site, while a compatible pair use different sites. Thus a normal a/P diploid survives because the two plasmids are attached to different sites and do not normally interact genetically-at least to the extent of forming recombinants. However, as a rare event, one of the plasmids may leave its attachment site and recombine with the other plasmid to form an associated structure. I n the associated diploids that have been examined (Richmond, 1967a) it is the /3-plasmid which seems to leave its site to recombine with a, because the diploid has the stability characteristic of a rather than 8; that is-on the attachment site hypothesis-the associated diploid is attached via the a attachment site. The fact that transduction of the a-plasmid into a cell already carrying usually leads to normal rather than associated diploids presumably reflects a preference on the part of the incoming plasmid to take up an attachment site, if available, rather than to recombine into an existing plasmid. When an a-plasmid is transduced to a cell carrying cc (or a /3-plasmid the relevant attachment site is already filled and it to a cell carrying /I), is reasonable to suppose that the incoming plasmid has no alternative site to which to attach. However, the incoming plasmid does have a high degree of genetic homology with the resident plasmid, and therefore recombination between the two might be expected to occur. If the plasmids are circular, the &st recombination event between the two plasmids establishes an “open-eight” structure; that is an a/a diploid will be formed, and both a-plasmids will be held in the cell at the M. attachment site in the form of an associated diploid (Fig. 7). Once the incoming a is recombined with the resident plasmid in this way, the cell will remain diploid and the stability of the associated structure (i.e. its tendency to form derived negatives) will be determined by the resident a-plasmid. Associated a/a diploids of this kind will probably survive only for a short time since any recombination in the opposite sense to the first (Fig. 7) will establish two separate plasmids, one still attached to the site and the other “free” in the cytoplasm. However, after this process has occurred, the distribution of genetic markers on the attached and free plasmids will vary depending on the precise location of the two recombinational events involved. It is quite possible by this means to achieve an exchange of all of the recipient plasmid markers to the free state or vice versa. This mechanism can explain therefore the origin of the large number of transductants in which the incoming plasmid seems t o have displaced the recipient plasmid entirely and which were found among the progeny of cross (1)(see Table 4,and p. 61). If the mechanism of plasmid recombination does involve an “associated” diploid as an intermediate, the question arises as to whether the

THE PLASMIDS OF STAPHYLOCOCCUS AUREUS

77

penR

mC

S

S

(f)

penR

FIG. 7. A possible mechanism for recombination between similar plasmids in Staphylococcus uureu8. (a)Plasmid haploid with theplasmidattachedto amembrane site S. (b)Diploid immediately after the insertion of the second plasmid by transducing phage. (c) First stage of recombination; apposition of the two circles. (d)Second stage: formation of an attached associated diploid by a single cross-over. (e) Re-formation of two separate plasmids, one still attached and the other free, by a cross-over in the opposite sense to the first. (f) Final stage: separation of the two plasmids. Note the apparent transfer of all the genetic markers from the donor to the recipient and vice versa. Such a mechanism could account for the apparent displacement of a resident by an incoming plasmid.

it5

M. H. XICHMIIUNU

“transient” diploids found to occur in a/acrosses soon after transducing phage and recipient are mixed are these intermediates. Were they so, the two plasmid components of transient diploids should be “associated” at least in some cases. I n an attempt to test this point, the transient a/adiploid 8325(a.4-p:. CdR.Asas. eroR/a.i+pxiz.Cds .AsaR.eras) was constructed, and a clone rich in transient diploids emulsified and picked into 2 ml. saline. This saline suspension was then irradiated and the resulting phages used to infect 8325(N). Transductants were selected for the Cd and AsaR markers separately, to measure the frequency of transduction of haploids from the transient diploid, and also for CdR and AsaR together. The CdR.AsaR transductants occurred at about 1yoof the incidence of AsaR and 50% of CdR transductants. However, when the (CdR.AsaR) transductants were examined further, four out of five were found to be diploid in the sense that they segregated haploids containing various combinations of the Cd, Asa and ero markers carried by the parents, whereas the remaining clone was a haploid recombinant. It follows from these experiments therefore that associated diploids do exist, at least to some extent, among the “transient” diploid class, but they probably do not account for all of the transient diploids present. Another point that is, as yet, not properly explained in this experiment is why the resident plasmid (a.i+pxlz.Cds.AsaR.eras) is transducedmore efficiently to 8325(N) from the transient diploid than the recently introduced plasmid (a.i-pi. CdR.Asas. emR).

F. CHROMOSOMAL ATTACHMENT OF PLASMID MARKERS As mentioned previously, certain genes which are plasmid-borne in the great majority of naturally-occurring staphylococci may be chromosomal in others. Thus the i and p A genes appear to be carried on the chromosome in 8.aureus PSSO (Asheshov, 1966b) while the CdR, Asa” and HgR markers are still present in the extrachromosomal state. Similarly, Dr. K. G. H. Dyke has shown that one class of staphylococcal strains found in hospitals is resistant to cadmium and arsenate ions, produces C-type penicillinase, but is sensitive to mercury salts. Examination of these strains shows that the penicillinase p c and i genes are plasmid-borne but, in many cases, the AsaR and CdR markers are chromosomal. Poston (1966) has also shown that the penicillinase genes behave as though they were chromosomal in some strains of S. aureus. I n this case, transduction of the penicillinase-, streptomycinand tetracycline-resistance genes from strain S26. PR.SR. TR. to strain S26. Ps .Ss .Ts was stimulated by ultraviolet irradiation in a manner similar t o that described by Arber (1960). There was, however, no

THE PUSMIDS OF STAPHYLOCOCCUS AUREUS

79

evidence for cotransduction of the penicillinase, tetracycline or streptomycin genes in these experiments. It would appear from all of these results, therefore, that integration of the plasmid-borne markers into the chromosome of 8. aureus-at least in some strains-is perfectly possible. Although the penicillinase genes are chromosomal in PSSO, it is not clear whether this is so in all cells in a culture, or whether some transition between the chromosomal and the plasmid state is possible. I n an attempt to answer this question, E.H. Asheshov (unpublished experiments) has studied the transduction of the penicillinase and metalresistance genes from PSSO to a strain 17855(N). The parental strain 17855 was resistant to cadmium and arsenate ions, produced C-type penicillinase, but was sensitive to HgC12.I n this strain, the penicillinase genes are plasmid-borne but the Cd" and Asa" markers are chromosomal. Strain 17855(N)is the derived negative with respect to the penicillinase genes; that is, it is sensitive to penicillin and HgCI, but still resistant to cadmium and arsenate ions by virtue of its chromosomal markers. When strain 17855(N)is mixed with phage SO propagated on PSSO, and the transductants are selected with penicillin, A-type penicillinaseproducing transductants are found. But when these are scored to sea whether the HgR marker has been cotransduced, this is found to be so in 20% of the penicillin-resistant transductants, despite the fact that the p i gene is apparently chromosomal in PSSO whereas the HgR is extrachromosomal. When the transductants which had received the i and p genes only from PSSO were compared with those that had received i, p and HgR, it wa8 found that the penicillinase region seemed to be chromosomal in the first class of transductants but was in the plasmid state, and linked to HgR, in the second. This transduction experiment was then repeated but the transductants were selected for the Hg" marker and scored for penicillinase production. Once again about 20% of the HgR clones were penicillinase producers despite the apparently different locations of the penicillinase and Hg" genes in PSSO. Examples of i+pi.H f , i+pt.HgR and HgR transductants were then taken and examined to see whether a transducing lysate obtained from them was stimulated by ultraviolet irradiation in the manner described by Arber (see p. 49). Stimulation was found when the i+pi.Hgs transductant was the donor but not for ifpi. Hg" and Hg". This result confirms therefore the chromosomal location of the penicillinase region in the i+pi.HgS transductants and the plasmid location in the other two classes. The fact that a phage preparation was capable of cotransducing the i, p and Hg regions to the plasmid state (but not vice versa) from a strain in which the penicillinase genes are chromosomal and the mercury gene

80

M. R. RICHMOND

plasmid-borne suggests that, in some cells, the penicillinase region leaves the chromosome to enter the plasmid state. However, if this were so, it should be possible to obtain derived negatives from PSSO, but this has never been shown. Another possibility which must therefore be considered is that some cells in the PS80 culture become transiently diploid with respect to the penicillinase region, one copy of the gene being chromosomal and the other on a plasmid. Alternatively, the HgR region may become integrated in certain cells. Whatever the detailed mechanisms involved in these experiments may be, it seems clear that a 'given set of penicillinase and associated genes (e.g. CdR or AsaR) may be chromosomal in some strains and plasmid-borne in others. Up to now there is no absolutely unequivocal evidence that these genes may move from the chromosomal to the extrachromosomal state in a single strain, but the results are highly suggestive. The possibility of transition from the chromosomal to the plasmid state has a bearing on the relationship between the penicillinase plasmids of X. aureus and the episomes of E. COG, and this point is discussed further later (see p. 84). Further indirect evidence for the possible chromosomal location of the penicillinase genes in S. aureus has been produced by Harmon et al. (1966). They used a penicillin-sensitive, methionine-requiring mutant as recipient, and claimed that transduction of the penicillinase region from a suitable penR.met+ donor leads to the cotransduction of the penicillinase genes and the gene responsible for cystathionine synthesis, a gene involved in the methionine biosynthesis pathway. The authors claim that these experiments show that the penicillinase genes are truly episomal since they appear to be extrachromosomal in the strains in question yet are capable of mobilizing one of the genes of the methionine biosynthetic pathway. However, although the genes of the methionine path are thought to be chromosomal in 8. aureus, Baldwin and his collaborators give no experimental evidence on this point, and their conclusions must therefore remain suspect for this reason. Furthermore, attempts to repeat these experiments with Baldwin's strains have failed (R.P. Novick, personal communication).

G. THEPLASMID MAP The whole question of determining the order of the genetic markers on the penicillinase plasmids depends to a great extent on whether the plasmids are circular linkage groups or not, and to a less extent on their size. Standard methods of determining marker order by three point crosses tend to break down if the linkage groups are circles with the markers scattered around an appreciable portion of the structure

THE PLASMIDS O F STAPHYLOCOCCUS A U R 3 U S

81

(Stahl and Steinberg, 1964; Hopwood, 1965). Thus, although it is possible to order markers unambiguously on the circular E . coZi chromosome, this is only because the markers involved in most crosses lie on a small region of the chromosome compared with the whole structure. The evidence that the penicillinase plasmids of 8. aureus are circular is circumstantial. Circularity might be expected as with other extrachromosomal elements. The build-up and break-down of associated plasmid diploids (see p. 70) suggests a circular nature (Richmqnd, 1967a),and preliminary mapping experiments along the lines advocated by Stahl and Steinberg (1964) tend to confirm this view (M. H. Richmond, unpublished experiments). Campbell (1962)has suggested that the F particle behaves as a circle, and Broda et aZ. (1964)have obtained genetic evidence on the integration of F into the E. coli chromosome which is consistent with this view. Elsewhere, Mitsuhashi (1965) has published a recombinational analysis which shows that the R-factors may be circular. Furthermore, the DNA of some but not all phages-which themselves can be considered as extra-chromosomal elements of a special type-can be inferred to be circular from their behaviour in the ultracentrifuge (Fiers and Sinsheimer, 1962). I n view, therefore, of the possibility of circularity existing among extrachromosomal particles in general, and the penicillinase plasmids in particular, any mapping data obtained by conventional three-point crosses must be regarded with some suspicion. I n view of these difficulties, the best mapping data concerning the penicillinase plasmids comes from Novick’s (1967)experiments in which deletions of various regions of the plasmids are obtained by irradiating transducing phage preparations with ultraviolet radiation (see also p. 75). Starting with the plasmid y, which carried the penicillinase i and p genes, the markers for CdR,h a R ,PbR,HgR, eroR and the compatibility maintenance site (mC,),Novick obtained the deletions listed in Table 10 when the transductants were selected on a variety of different selective media. Although not absolutely unequivocal, these results imply a probable marker order : I

(i,p).AsaR.PbR.CdR.HgR.mC.eroR

A certain amount of additional evidence supports this view. Transduction

of the CL . . . y recombined plasmid to a negative recipient-e.g. 8325(N)-leads to the transfer of dissociated plasmids in a small proportion of cases. Since the parent plasmid carries the markers i , p A , AsaR, PbR, CdR, HgR, mC, eroR, and the most common dissociated plasmid transductant is i . p .AsaR.PbR.CdR.H g R .mC (Richmond, 1966b), these results are consistent with Novick’s (1967) marker order.

82

M, H. RICHMOND

Attempts have been made to determine the linkage relationships of some of the plasmid markers by recombinational analysis (Novick, 1967 ; M. H. Richmond, unpublished experiments). For this purpose transductional crosses were performed between donor and recipient strains carrying an a . . . y recombined plasmid on the one hand and an a-plasmid on the other. A typical experiment carried markers as follows :

+(a.iipA+.Hq'

. . . y.eroR)

-+ 8325(a.i+p,,,. HgR.eros)

Transductants were selected for eroR and scored for the unselected marker. Recombination between ero and p occurred to about 16% of

..y .era') +8325(cr.i+p&. H f .ero"). Dotted lines represent scorable cross-overs. (b), (c) and (d)Diagrammatic representation of the results obtained from the cross shown in (a). Certain recombinants are illustrated by heavy (more common) or dotted (less common) lines. The relative ratios of the two types of recombinant in the pairs shown in (a), (b) and (c) are shown in the right of the figure. Data from Novick (1967). FIU.8. (a)Diagrammatic representation of the cross $(G.i - p z . H#.

the eroR transductants, while recombination between i- and p - occurred at a frequency of about 0.2%. The relative incidence of certain ; / p cross-overs was used to establish the map order. The three pairs of recombinant classes and the routes used to form them are shown in Figure 8 where the more common cross is shown in bold face, and the less common as dashed lines. The relative incidence of recombinants in the relevant pairs is also shown in Figure 8. On the basis of a linear linkage group, the only order consistent with these findings is i .p.H g .ero, with the Hg marker closer to p than to ero. But other orders are possible if a circular linkage group exists. Be that as it may, the order obtained by

T H E PLASMIDS OF STAPEYLOCOCCES A E R E E S

83

recombination analysis is, at worst, compatible with the marker order obtained by Novick's (1967) deletion experiments. Subsequent recombination experiments, using suitably marked strains, have also shown that the Cd marker lies between p and Hglero, but once again the order is only unambiguous if a linear model is assumed. I n summary, therefore, the mapping data suggest that the likely order of markers on the y and a . . . y recombined plasmids is:

i . p .Asa. Pb .Cd .Hg .mC. ero although these positions may have to be revised in the light of further experiments. I n the case of plasmids, such as a and j3, which carry no eron marker, the order is almost certainly the same but for the absence of the ero region. Thus 01 and appear to carry a deletion of the whole ero region when compared with y and a . . . y.

H. SIZEOF

THE

PENICILLINASE PLASMIDS

Since penicillinase plasmids can be transferred from cell to cell by phage particles, an upper limit to their size will be set, to some extent, by the amount of DNA that can normally be accommodated in a phage head. Although no information is available on exactly how much DNA this amounts to in 8. aureus phages, the E. coli phages T, and T4contain DNA of molecular weight about 1.2 x lo8 (Davison et al., 1961 ; Cairns, 1961). This amount of DNA corresponds to about lo6 base pairs. Another indication of the size of the penicillinase plasmids comes from a comparison of recombination frequencies with the distance between two known point mutations in the penicillinase structural gene p A . The two-point mutants used in this work (No: 2 and No. 54)each cause a change in the nature of the penicillinase molecule synthesized, No. 2 causing insertion of isoleucine for threonine, and No. 54 causing an exchange of asparagine for aspartic acid (R. Ambler, unpublished experiments). The two amino acid positions involved in these mutants lie about 75 residues apart in the single penicillinase polypeptide chain (R. Ambler, unpublished experiments) and the mutational events are therefore about 220 base pairs apart in the gene, if colinearity of gene and protein is universal (Yanofsky et al., 1967). Since the recombination frequency between p y and p)54 is about 0.07 %, whereas recombination between p and ero is about 16% and between p z and i i is about 0.2% (M. H. Richmond, unpublished experiments), it can be calculated, assuming uniform recombination along the map, that the total distance between the ii' and ero is about lo6 base pairs. Thus, on this evidence,

84

M. H. RICHMOND

the amount of DNA between the extreme markers detected on the plasmids so far is about the same as found in the average phage particle, and amounts to about 1-2% of the DNA in the staphylococcal cell. It follows, therefore, that it is unlikely that there is much plasmid-borne genetic information outside the known genetic markers.

IV. A Comparison of Penieillinase Plasmids with other Extra-chromosoma1 Elements in Bacteria The plasmids of X.aureus share their extrachromosomal status with the resistance-transfer factors (R) (Watanabe, 1963; Datta, 1965), colicin (col) factors (Fredericq, 1957) and fertility- or sex-factors (F) (Hayes, 1953); and with the FP particles of the pseudomonads (Holloway, 1955, 1956; Holloway and Jennings, 1958; Holloway and Fargie, 1960). I n addition, a great range of other extrachromosomal elements are found in organisms other than bacteria; for example the kinetoplast and kinetosomes of protozoa (Jinks, 1964), the mitochondria of animal cells (Lehninger, 1964), and the chloroplasts and chromophores of plant cells (Kirk and Tilney-Bassett, 1967). However, these non-bacterial structures probably represent a completely different extrachromosomal situation from the bacterial elements, and they will not be considered further here. Ofthe bacterial extrachromosomal elements, the least is known about the pseudomonad PP particles. Broadly speaking, they appear to behave like the F-particle in E . COGin that they can infect FP- cells to the FP+ state, but little further information has appeared since the original publications at the end of the 1950s (Holloway, 1956; Holloway and Fargie, 1960). The main comparison must therefore lie between the staphylococcal plasmids on the one hand and the R-, F- and col-particles of the Enterobacteria on the other. The F-, cob- and R-factors share many properties. One consequence of their extrachromosomal location is that they can be lost spontaneously from a culture at a frequency greater than that expected from spontaneous mutation rates, and loss of the particles is usually associated with the loss of all of the genetic markers carried on them. Thus cultures carrying resistance-transfer factors always contain some drug-sensitive cells (Watanabe and Fukusawa, 1961) and the same is true for coZ+ (Fredericq, 1957) and F+cells (Hayes, 1963). Hirota (1960) showed that F+ cells could be efficiently purged of their F particles by growth in the present of sub-inhibitory concentrations of acridine dyes, and the same is true for cells carrying R-factors (Hirota and Iijima, 1957), although the drug is much less effective in this case. Stocker et al. (1963), however, showed that multiplication and transfer of the col factor is unaffected

THE PLASMIDS OF STAPHYLOCOCCUS AUREUS

85

by acriflavine. Sensitivity to acridine dyes, therefore, is not an obligate consequence of the extrachromosomal state. I n addition to their common extrachromosomal status, the -F, col- and R-factors share two other important properties. The first is the ability to promote their own transfer by conjugation, to cells which lack the equivalent particles, and the other is their ability to mobilize chromosomal markers and transfer them by conjugation. Bacterial conjugation is usually apparent as the unidirectional transfer of genetic information between two strains. This was first reported for the F-factor by Hayes (1952) and by Lederberg et al. (1952), and subsequently for the R-factors by Akiba et aZ. (1960). Similarly transfer of the coZ-factor by conjugation was found by Clowes (1961). I n all these cases, transfer was originally studied between strains of the same species, but it soon appeared that all the factors could be transferred widely among Gram-negative bacteria more or less regardless of species boundaries (Hayes, 1963; Datta, 1965). Normally cells lacking a given factor could accept it by transfer from a donor but the similarity of the COZ-, F- and R-factors is underlined by the fact that, in certain cases, the carriage of one type of factor modifies the ability of that cell to accept another (Clowes, 1963; Meynell and Datta, 1966). The ability of the extrachromosomal particles to mobilize parts (or in some cases all) of the bacterial chromosome implies that, a t least for some part of its existence, the P-, col- and R-factors integrate into the bacterial chromosome. In the case of the F-particle, this integration takes two forms: either a relatively stable interaction in which the factor becomes part of the chromosome and is able to promote the transfer of the whole chromosome‘to an appropriate recipient at high frequency by conjugation-the so-called Hfr state (Hayes, 1953); or a freely reversible integration which allows the F-particle to incorporate limited regions of the chromosome while remaining extrachromosomal for at least a great deal of the time-the so-called 3’’ state (Adelberg and Burns, 1959,1960).Both the col- and R-factors are capable of mobilizing the chromosome in a manner analogous to F-to form HfRT (Sugino and Hirota, 1962)and HfcolT (Stocker et aZ., 1963)strains; but, to date, a situation analogous to the P‘ state has not been found. However, the ability of the extrachromosomal particles to undergo a slowly reversible integration into the chromosome has many analogies to the integration of temperate bacteriophage, and Jacob and Wollman (1958) have coined the term “episome” to describe the genetic elements which behave in this way. The formal definition of this term is that episomes are genetic elements which are capable of undergoing a reversible integration into the bacterial chromosomewithout adding to the genetic complement of the cell (Jacob and Wollman, 1958). Since the col-, Ii- and R-factors

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fulfill these criteria, they, as well as temperate phage, may be regarded as episomes. I n summary, therefore, the COZ-, F- and R-episomes are predominantly extrachromosomal elements which can promote their own transfer by conjugation to suitable recipient cells and which can integrate reversibly into the bacterial chromosome. I n certain respects the penicillinase plasmids are similar to the colF- and R-episomes. They behave as extrachromosomal elements which are lost from the cell at relatively high frequency, and the unpublished experiments of E. H. Asheshov (see p. 78) suggest strongly that plasmidborne genetic markers can enter the chromosomal state in at least some strains of 8.azLreu8. This phenomenon could, therefore, be analogous to the integration of true episomes. The difference between the penicillinase plasmids and the E. COG episomes is most marked in their inability to promote conjugation. Conjugation has never been observed to occur in staphylococci, nor, for that matter, in any Gram-positive bacteria. Although it is possible that the absence of conjugation in staphylococci may reflect a fundamental difference in the structure of Gram-positive cells, rather than an innate difference in the nature of the extrachromosomal genes, this possibility does seem rather unlikely. By and large the penicillinase plasmids seem to form plasmid diploids more readily than do the episomes of E . coZi, but this difference may be more apparent than real. Although diploids of two F-particles do not seem to occur readily, this may be solely because possession by a cell of a single F-particle impedes the uptake of a second or, alternatively, recombination between the two F-particles may be very rapid. A closer analogy to the staphylococcal plasmid situation is provided by strains which carry more than one cob or R-factor. In these cases, the two episomes are probably held in the cell separately, by the use of distinct attachment sites, much as has been postulated for the a//3 plasmid diploids in S. aureus. All in all, therefore, the penicillinase plasmids seem to represent a very similar class of extrachromosomal elements to the COZ-, F- and R-episomes of the Gram-negative bacteria despite their inability to promote conjugation.

V. Acknowledgements I would like to express my thanks to Drs. Elizabeth Asheshov, Richard Ambler and Keith Dyke for telling me about some of their unpublished experiments and allowing me to report them.

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REFERENCES Adelberg, E. A. and Burns, S. N. (1959). Genetics 44, 497. Adelberg, E. A. and Burns, S. N. (1960). J. Bact. 79, 321. Akiba, T., Koyama, K., Ishiki, Y., Kimura, S. and Fukushima, T. (1960). Jap. J. Microbiol. 4, 219. Arber, W. (1960). Virology 11, 273. Asheshov, E. H. (1966a). J . gen. Microbiol. 42, 403. Asheshov, E. H. (1966b). Nature, Lond. 210, 804. Asheshov, E. H. and Winkler, E. (196G). Nature, Lond. 209,638. Barber, M. (1949). J . gen. Microbiol. 3, 274. Beckwith, J. R. and Signer, E. (1966). J. molec. Biol. 19, 254. Borowski, J. (1963). Ann. Inst. Pasteur 104, 535. Broda, P. M. A., Beckwith, J. R. and Scaife, J. (1964). Genet. Res., Camb. 5 , 489. Cairns, J. (1961). J. molec. Biol. 3, 756. Campbell, A. M. (1962). Adwanc. Genet. 11, 101. Clowes, R. C. (1961). Nature, Lond. 190, 986. Clowes, R. C. (1963). Genet. Res., Camb. 4, 162. Clowes, R. C., Moody, E. E. M. and Pritchard, R. H. (1965). Genet. Res., Camb. 6, 147. Collins, A. M. and Macdonald, S. (1962). J. Path. Bact. 83, 399. Datta, N. (1965). Brit. med. Bull. 21, 254. Davison, P. F., Freifelder, D., Hede, R. and Levinthal, C. (1961). Proc. mat. Acad. Sci. Wash. 47, 1123. Dyke, K. G. H. and Richmond, M. H. (1967). J. din. Path. 20, 75. Fairbrother, R. W., Parker, L. and Eaton, R. B. (1954). J. gen. Microbiol. 10, 309. Fiers, W. and Sinsheimer, R. L. (1962). J. molec. Biol. 5 , 424. Fredericq, P. (1957). Annu. Rev. Microbiol. 11, 7. Hamburger, M., Walker, W. F., Clark, K. L. and Carleton, J. (1958).Tram.Amer. Clin. Climat. Assoc. 70, 49. Hamburger, M., Carleton, J., Walker, W. F. and Clark, K. L. (1960). Arch. int. Med. 105,668. Harmon, S . A. and Baldwin, J. N. (1964). J. Bact. 87,593. Harmon, S. A., Baldwin, J. N., Wei-Chen Tien and Critz, D. B. (1966). Canad. J. Microbiol. 12, 973. Hashimoto, M., Kono, K. and Mitsuhashi, S. (1964). J . Bact. 88, 261. Hayes, W. (1952). Nature, Lond. 169, 118. Hayes, W. (1953). Cold Spr. Harb. Symp. p a n t . Biol. 18, 75. Hayes, W. (1963). “The Genetics of Bacteria and their Viruses”, Blackwell, Oxford. Hirota, Y. (1960). Proc. nat. Acad. Sci. Wash. 46, 57. Hirota, Y. and Iijima, F. (1957). Nature, Lond. 180, 665. Holloway, B. W. (1955). J. gen. Microbiol. 13, 572. Holloway, B. W. (1956). J. gen. Microbiol. 15, 221. Holloway, B. W. andFargie, B. (1960). J. Bact. 80, 362. Holloway, B. W. and Jennings, P. A. (1958). Nature, Lo&. 181, 855. Hopwood, D. A. (1965). J. molec. Biol. 12, 514. Jacob, F. and Wollman, E. (1958). C.R. Acad. Sci., Paris, 247,154. Jacob, F. andMonod, J. (1961). ColdSpr. Harb. Symp. qwcnt. Biol. 26, 193. Jinks, J. L. (1964). “Extrachromosomal Inheritance”, Prentice-Hall, New Jersey. Kirk, J. T. 0. and Tilney-Bassett,R. A. E. (1967). “The Plastids”, W. H. Freeman & Co., London & San Francisco. Korman, R. Z. and Berman, D. T. (1962). J. Bact. 84, 228.

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Lederberg, J. (1952). Physiol. Rev. 32, 403. Lederberg, J., Cavalli, L. L. and Lederberg, S. (1952). Genetics 37, 720. Lehninger, A. L. (1964). “The Mitochondrion”, W. A. Benjamin, New York & Amsterdam. May, J. M., Houghton, R. H. and Perret, C. J. (1964). J. gen. Microbiol. 37,157. Meynell, E. and Datta, N. (1966). Genet. Res., Camb. 7 , 141. Mitsuhashi, S. (1965). Gunma J. med. Sci. 14, 169. Mitsuhashi, S., Hashimoto,M., Kono, K. andMorimura, M. (1965). J.Bact. 89,988. Morse, M. L. (1960). Proc. nat. Acad. Sci. Wash. 45, 722. Novick, R. P. (1963). J. gen. Microbiol. 33, 121. Novick, R. P. (1967). Fez. Proc. 26, 29. Novick, R. P. and Richmond, M. H. (1965). J. Bact. 90,467. Pattee, P. A. and Baldwin, J. N. (1961). J. Bact. 82, 875. Pollock, M.R. (1962). I n “The Bacteria”, (I.C. Gunsalus and R. Y .Stanier, eds.), vol. 4, p. 121, Academic Press Inc., New York. Poston, S. M. (1966). Nature, Lond.210, 802. Richmond, M. H. (1963). Biochem. J. 88,452. Richmond, M. H. (1964). Proc. 6th Intern. Congr. Biochem., New York, p. 237. Richmond, M. H. (1965a). Brit. med. BUZZ.21, 260. Richmond, M. H. (1965b). Biochem. J. 94, 584. Richmond, M. H. (1965~).J. Bact. 90, 370. Richmond, M. H. (1966a). Postepy Mikrobiologie 5 , 371. Richmond, M. H. (1966b). J. gen. Microbiol. 45, 51. Richmond, M. H. (1967a). J. gen. Microbiol. 46, 85. Richmond, M. H. (1967b). J. rnolec. Biol. 26, 357. Richmond, M. H. and John, M. (1964). Nature, Lond. 202, 1360. Ritz, H. L. and Baldwin, J. N. (1958). Bact. Proc. 40. Ritz, H. L. and Baldwin, J. N. (1961). Proc. SOC.e q . BioZ. Med. 107,678. Segalove, M. (1947). J. infect. DW. 81, 228. Servin-Massieu, M. (1961). J. Bact. 82, 316. Stahl, F. W. and Steinberg, C. M. (1964). Genetics 50, 531. Stocker, B. A. D., Smith, S. M. and Ozeki, H. (1963). J . gen. Microbiol. 80, 201. Sugino, Y. and Hirota, Y. (1962). J. Bact. 84,902. Voureka, A. (1948). Lancet i, 62. Watanabe, T. (1963). Bmt. Rev. 27, 87. Watanabe, T. and Fukusawa, T. (1961). J. Bact. 81, 669. Yanofsky, C., Drapeau, G. R., Guest, J. R. and Carlton, B. C. (1967). Proc. nut. Acud. Sci. Wash. 57,296.

The “Life Cycle” of Bacterial Ribosomes WILLIAM S. KELLEY AND MOSELIOSCHAECHTER Is

.’

Department of Microbiology, Tufts University School of Medicine, Boston, iWassachusetts, U.S.A. I. Introduction . A. Problems Particular t o the Study of a Ribosomal “Life Cycle” B. Scope of the Review C. Studies of Ribosome Structure and Composition. . D. Cellular Localization of Bacterial Ribosomes. 11. The Role of Ribosomes in the Control of RNA Synthesis . 111. The Production of Ribosomal RNA . IV. The Production of Ribosomal Proteins V. The Assembly of Ribosomal Subunits . A. Studies on Cells in Balanced Growth . B. Studies of Metabolically Inhibited Cells . C. In Vitro Experiments on Ribosome Assembly . D. Conclusions . VI. The Formation of Functional Ribosomes . VII. The Participation of Ribosomes in Protein Synthesis . VIII. The Release of Ribosomes from Messenger RNA . IX. Conclusions . X. Acknowledgements References .

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89 90 91 91 97 99 101 107 112 112 115 124 126 226 131 133 136 137 137

I. Introduction During the last decade, bacterial ribosomes have been the object of intense study by many workers. At the time of this writing only a few generalizations about their function and origin seem possible. Bacterial ribosomes are ribonucleoprotein particles, essential for protein synthesis and perhaps involved in the control of RNA synthesis. I n any sense but the superficial little can be said about their function on a molecular scale. At present no generally acceptable mechanism has been described which accounts for the assembly of ribosomes in the bacterial cell. Obviously a field which is the subject of such intensive study and which contains so many unsolved problems is sure to evolve very rapidly. A 89

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review aimed solely at cataloguing data is likely to be quickly outdated and of limited interest. Therefore we have attempted to focus on a central theme, that ribosomes in growing bacteria undergo a “life cycle”. We will define this “life cycle” as the events of inception, maturation, and repeated functioning of a ribosome, and divide it into a series of biochemical reactions. Formally we shall consider each reaction as a separate event, although in the bacterial cell they are quite probably interrelated in a complex manner. These reactions are :

A. The synthesis of ribosomal (IRNA. -) B. The synthesis of ribosomal proteins. C. The formation of ribonucleoprotein particles that are precursors of complete ribosomes.

D. The assembly of these precursor particles into completed ribosomes.

E. The participation of ribosomes in protein synthesis. F. The release of ribosomes from messenger (m-) RNA into a pool of free particles.

Our review attempts a critical consideration of each step of the “life cycle”, and we shall discuss the steps in terms of the nature of the reactants and the reaction products. Where such information is available we shall also discuss aspects of the timing and regulatory mechanisms. By organizing the available information on this basis, we have created several redundancies. I n many cases some aspects of a set of experiments apply to more than one of our designated reactions. Hence, many experiments have been cited several times, each, we hope, in a different light. We hope that the net effect is not confusion or tedium but rather a story that can be read as a series of discrete but related chapters.

A.

PROBLEMS PARTICULAR TO THE STUDY OFA RIBOSOMAL “LIFECYCLE”

As in any field of scientific study, experiments designed to investigate ribosome assembly and function have problems of methodology peculiar to themselves. In general we would prefer to discuss data which describe the “life cycle” in cells in balanced growth, unimpaired by external inhibitors or by lack of an essential nutrient. Bacterial cells growing at a steady state are generally taken to represent a baseline of microbial ( 6 normalcy” and, ideally, we are aiming at understanding “normal” function. However, few experiments have actually been performed under such circumstances. Various strategies have been used to approach the problem. I n general, work is made difficult because a simple chemical characterization of a, ribosome is impossible. Instead, ribosomes are generally defined on the -’-

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basis of their sedimentation characteristics in preparative and analytical ultracentrifuges. Used alone, these methods specify little of the molecular nature of ribosomes and, in addition, are fairly insensitive for the detection of small numbers of precursor particles and intermediates in ribosome assembly. Therefore it has been necessary for experimenters to detect precursors by their kinetic behaviour in labelling experiments or by perturbing bacteria in such a way that they accumulate precursor or precursor-like particles. Both approaches have specific drawbacks. The mathematical interpretation of kinetic data is complicated by other dynamic cell processes. Perturbing growing cultures by various drugs and metabolic shift conditionshas indeed been a popular and fruitful approach to the study of ribosome assembly, allowing analytical study of the accumulated particles. Obviously, though, cells so inhibited or perturbed are not in balanced growth. Much work has been done in aitro and some of the most interesting recent data are the result of such studies. When these data apply at present to the in vivo process, they will be included in our discussion. B. SCOPEOF

THE

REVIEW

The bacterial ribosome’s “life cycle” as we have defined it involves more than a simple discussion of the individual reactions. Ribosome production and the formation of polysomes (the aggregates of ribosomes and m-RNA active in protein synthesis) are connectedand, sincethesyntheses of r- and m-RNAs are chemically similar processes, an adequate model must relate the regulation of synthesis of one to the other. The kinetics and mechanism of m-RNA synthesis and polysome formation will also be considered, therefore, but principally in light of how they affect the “life cycle” of a ribosome.

C. STUDIESOF RIBOSOME STRUCTURE AND COMPOSITION To introduce our review of the “life cycle” we shall begin with a brief summary of datadescribing ribosome structure on a physical and chemical basis. The first intensive study of the physico-chemical properties of bacterial ribosomes was conducted by Tissibres and Watson (1958) and Tissibres et al. (1959). These workers noted that 80-90% of the RNA of exponentially growing Escherichia coli was in the form of ribonucleoprotein particles. The RNAiprotein ratio of these particles was about 65 :35, and their structural integrity was apparently dependent on magnesium ions. I n alumina-ground extracts, particles with sedimentation coefficients of 30,50,70 and 100s were observed, the relative number of each depending

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WILLIAM S. EELLEY AND MOSELIO SCHAECHTER

on the magnesium ion concentration. These experiments indicated that reversible, magnesium-dependent aggregations took place in a manner summarized by the equation : Increasing Mg2+ -+ 2(305)+2(50S) + 2(70S) + (100s) Using purified 70s ribosomes Britten and McCarthy (1959) confirmed this interpretation by analysing the sedimentation behaviour of the particles at various Mg2+concentrations. These data are reproduced in

I

I

I

I

I l l l l

Magnesium acetate concentration

(M

x lo-’)

FIG.1. Variation in the quantity of the various forms of ribosomes as a function of the concentration of magnesium acetate. Data were obtained from Schlieren diagrams by cutting out and weighing the peaks from enlarged prints. Reprinted from the Carnegie Institution of Washington Yearbook 58,266 (1959), by permission of the authors.

Fig. 1.Note that, at magnesium concentrations of less than M , most of the 70s particles have dissociated to 305 and 50s subunits whereas, at concentrations of lop2 M and greater, the 70s and 100s species are predominant. Tissieres et al. (1960)have shown that in vitro protein synthesis proceeds optimally at M Mg2+.Further, McQuillen et aZ. (1959)and Schlessinger and Gros (1963)have shown that nascent proteins sediment in association with 705 ribosomes. For these reasons the 705 ribosomes are probably the ones which support protein synthesis. Electron micrographs (e.g. Hall and Slayter, 1959; Huxley and Zubay, 1960) of isolated ribosomes show the 305 and 505 particles clearly, and substantiate the physical data that each 70s particle contains one 305 and one 50s subunit; 100s particles appear as dimers of 70s particles joined through the 30s subunits (Hall and Slayter, 1959). An electron

THE

L

‘

CYCLE” ~ OF ~ BACTERIAL ~ ~ RIBOSOMES

93

micrograph of ribosomes arranged on m-RNA in a polysome is shown in Fig. 2 . Although most of the 30s and 50s particles aggregate to form 70s particles at magnesium ion concentrations of M and greater, not all do so. This small number of particles can be separated from the 70s particles by differential centrifugation. First noted by Green and Hall

FIG.2. Electron micrograph of a bacterial polyribosome with membrane particle attached. The preparation was positively stained with uranyl acetate. Magnification x 270,000. Reproduced by courtesy of Dr. Henry Slayter, Children’sHospital, Boston, Massachusetts.

(1961), these particles are called “native” 305 and 50s particles to distinguish them from the “derived” 30s and 50s subunits produced by reversible disaggregation of 70s ribosomes at lower magnesium concentrations. Recent data (Kelley and Schaechter, 1968) indicate that, in lysates of Bacillus megaterium prepared in buffer with a magnesium-ion concentration as great as 4 x lo-’ M, 5-10y0 of the total cellular ribosomes are found as 30s and 505 particles. 6

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WILLIAM S. KELLEY AND MOSELIO SCHAECHTER

In lysates prepared by a gentler method of disruption than the alumina grinding used in the earliest studies, as much as 80% of the total ribosomal material of the cell is found in oligomeric aggregates of ribosomes and m-RNA, the polysomes. Analysis of such lysates shows that the relative number of polysomes,ribosomes and subunits varies with changes in the magnesium concentration of the lysing medium (Kelley and Schaechter, 1968). These results are shown in Pig. 3. Note that, at magnesium ion concentrations below approximately 6-8 x M , most of the 705 ribosomes dissociate into their 30s and 50s subunits, as shown by the earlier work of Britten and McCarthy (1959; see Fig. 1). However, polysomes seem to be considerably more stable at these magnesium-ion

Mg2+concentration ( M x lo-*)

FIG.3. Percentage of total cellular ribosomes of various types in lysates prepared at different magnesium acetate concentrations. Data were obtained by measuring areas of enlarged prints of Schlieren diagrams. Reproduced from Kelley and Schaechter (1968).

concentrations, and dissociate completely only when the magnesium-ion concentration is less than 2 x M . As we shall discuss below, these observations not only indicate a difference between 70s ribosomes and polysomes, but may also be important clues about the nature of the assembly process by which polysomes are formed. When discussing ribosomes and their subunits, it is expedient to regard them as homogeneous and identical to one another as one usually considers a population of enzyme molecules. I n fact, this need not be so since the S value of a particle is not a sensitive measure of all chemical differences.In subsequent sections of this review, the question of whether all ribosomes are identical will play an important role in our discussion. At present, chemical methods of analysis cannot resolve this point. We

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do know from several lines of evidence that the two classes of subunits, 3 0 s and 50s)differ from eachother bothin size and composition.Although their densities and hence their RNAIprotein ratios seem to be identical (Brenner et al., 1961)) the RNAs and proteins of the subunits differ. Kurland (1960) demonstrated that the 3 0 s subunits contain RNA molecules with an S value of 16 whereas 50s subunits contain 2 3 s RNA. This result and many later ones were confused by the fact that the usual methods of RNA extraction almost always produce some 1 6 s RNA from the 50s particles. Midgley (1965) has shown that this phenomenon is dependent upon details of the extraction technique, and that carefully extracted 50s subunits yield 2 3 s and not 1 6 s RNA. Analyses of the nucleotide composition of these RNAs (Bolton, 1959; Spahr and Tissi&res,1959; Midgley, 1962) show that both have virtually identical base composition. However, fingerprints of partial ribonuclease digests (Bolton, 1960; Aronson, 1962) and hybridization experiments (Yankofsky and Spiegelman, 1963) have demonstrated that the sequences of these two types of RNA are different. The 1 6 s RNA has a molecular weight of about 5 x lo5 and the 238, 1.1 x l o 6 (Stanley and Bock, 1965). This is consistent with each particle containing only one RNA molecule. I n addition to 2 3 s and 1 6 s RNA, another class of RNA first reported by Rosset and Monier (1963) has been found to be associated with all bacterial ribosomes. This class sediments at 55 and can be confused with t-RNA in sucrose density-gradients but can be separated from it by acrylamide gel electrophoresis (Loening, 1967). It differs from t-RNA by its base composition, greater chain length, lesser degree of methylation, near absence of rare bases, and lack of acceptor capacity for amino acids. It is associated with the 50s subunit. A t present, the function of this component is unknown. For references on this subject, see recent papers by Comb and Zehavi-Willner (1967) and Brownlee and Sanger (1967). The amino-acid content of the ribosomes has been analysed, and differences in the proteins of 3 0 s and 50s particles have been found. Many of the amino acids are basic and some polyamines are present (Spahr, 1962). End-group analysis of the peptides shows tlfat most of the Nterminal amino acids are methionine and alanine and, on the basis of bhe number of ends, the average molecular weight of a ribosomal protein was estimated as 2.5 x l o 4 (Waller and Harris, 1961). This value has recently been confirmed by studies of Moller and Chrambach (1967)) Moller and Widdowson (1967)) and Traut et al. (1967). Various chromatographic techniques have been used to examine ribosomal proteins (e.g. Spitnik-Elson, 1964) but the most fruitful one has been electrophoresis on polyacrylamide gels (e.g. Leboy et al., 1964; Traut, 1966). This techniques shows that there are thirty or more proteins in the 7 0 s particles, and that different proteins are found in the 30s and 50s subunits (Leboy

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WILLIAM S. KELLEY AND MOSELIO SCHAECHTER

et al., 1964; Traut, 1966). Several such acrylamide gel columns are shown in Fig. 4.The work of Traut et al. (1967) demonstrates conclusively that there are at least nineteen distinctly different proteins of molecular weight approximately 2-5 x lo4 in a preparation of 705 ribosomes, thus eliminating the possibility that the different bands observed in electrophoresis patterns are the result of polymerization of a few simple peptide subunits. The pattern of bands appearing in the acrylamide gels varies among different E. coli strains (Leboy et al., 1964). The patterns change

FIG.4. Polyacrylamide gel electrophoresiscolumns of 50S, 70s and 30s ribosomal proteins. Reprinted from Traub et al. (1966a), by permission of the authors.

apparently as the result of bacterial mutations to ochre suppression (Reid et al., 1965) and amber suppression (Bollen et d., 1965). We conclude that the 30s and 50s particles differ substantially from each other in their protein as well as their RNA content. ' Apparently Mg2+and, to some extent, polyamines, function to hold the peptides and RNA complex in a discrete structure i n vitro. Goldberg (1966) and Choi and Carr (1967) have estimated that the in vitro molar ratio of bound Mg2+to phosphate is 0.5. This Mg2+is apparently not all tightly bound €or Rodgers (1964) showed that 35% of the Mg2+in 705

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97

ribosomes exchanges in 2 min. at 0” in vitro. He concluded that this fraction was responsible for holding the 30s and 50s particles together, the more slowly exchanging material being bound within the subunit. Lowering the Mg2+concentration by treatment with EDTA or dialysis against low-Mg2+buffer apparently allows a loosening of the structure, making the particles unfold and become less compact (Rodgers, 1966; Gesteland, 1966; Gavrilova et al., 1966; Weller and Horowitz, 1964). Polyamines also function to stabilize the RNA-protein structure and will substitute for Mg2+ to some extent in test-tube experiments (e.g. Cohen and Lichtenstein, 1960; Colbourn et al., 1961; Silman et al., 1965). Conversely, high ionic-strength conditions, such as those used in equilibrium density-gradient centrifugation with caesium chloride, can cause dissociation of RNA and protein even at 5 x 10W M Mg2+(Meselson et al., 1964).At lower Mg2+ concentrations, this dissociation can be blocked by the addition of cross-linking agents such as formaldehyde (Spirin et al., 1965). By regulating the Mg2+concentration carefully, it is possible to remove only some of the protein components and produce dense RNArich particles. Under ‘carefully defined conditions, the proteins and stripped ribosomes can be recombined to give particles that resemble the starting material (Lerman et al., 1966; Hosokawa et al., 1966; Staehelin and Meselson, 1966a; Spirin and Belitsina, 1966). Electrophoresis and binding experiments have shown that apparently certain proteins, perhaps surface ones, are removed in this way (Staehelin and Meselson, 1966b; Raskas and Staehelin, 1967; Traub et al., 1966a, b; Nomura and Traub, 1967). For further discussion of the structure of RNA and particularly of ribosomes, the interested reader should consult the monograph of Spirin (1964).

D. CELLULARLOCALIZATION OF BACTERIAL RIBOSOMES For an understanding of the “life cycle” of ribosomes, it would be helpful to know whether at any time ribosomes are localized in any particular portion of the bacterial cell. Such a relationship between ribosomes and other structural elements of the cell might shed light on the mechanisms which control ribosome assembly and function. At present, however, the infordation available is not clear-cut. To examine this question, only two methods are currently available, namely cell fractionation and electron microscopy of ultrathin sections. These methods have been applied to the cells of higher organisms with much success, but bacterial cells are singularly intractable to this sort of approach. Unlike the higher-order cells, bacteria lack well-defined intracellular compartments which aid in isolation of cell fractions. Also, unlike animal cells which are surrounded by a pliable but well-defined membrane, bacteria are encased in comparatively rigid girdles of cell-wall

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WILLIAM S. KELLEY AND MOSELIO SCHAECHTER

FIG.5. Electron micrograph of an ultrathin section of Acinetobacter, a Gramnegative bacterium. Cells were pre-fixed with glutaraldehyde by the method of Glauert and Thornley (1966). Ribosomes are visible as dark bodies scatterednearly a t random throughout the cytoplasm. Reproduced by courtesy of Dr. Audrey Glauert, Strangeways Research Laboratory, Cambridge, England.

THE “LIFE CYCLE” OF BACTERIAL RIBOSOMES

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material which make them unsuitable for the more commonly used preparative methods. Thus, it is little wonder that the techniques applicable to animal cells have yielded little information when applied to bacteria, and that special methods have had to be developed. After differential centrifugation of bacterial lysates, it is common experience to find many ribosomes (upward of 50% of the total) in association with the cytoplasmic membrane (Schlessinger, 1963; McQuillen et al., 1959; Aronson, 1966).This finding has suggested that protein synthesis takes place on membrane-bound ribosomes. This recalls the situation in cells of higher organisms which possess a “rough” (i.e. ribosomerich) endoplasmic reticulum where much protein synthesis takes place. Some authors (Schlessingeret aZ., 1965; Van Iterson, 1966)have reported electron-microscopic evidence for intracytoplasmic filaments associated with ribosomal material. The nature of these filaments is obscure and they have not been isolated from the cells in purified state. I n sections of growing bacteria, there are often so many ribosomes that virtually the whole cytoplasm is packed with particles in no particular orientation. I n such preparations, it is not possible to determine a preferential localization within the cell. Sections of bacteria not replete with ribosomes usually show a fairly random localization of these particles. I n the electron micrograph shown in Fig. 5 , the ribosomes seem scattered about the cytoplasm and few are seen near the envelopes of the cell. Interestingly, ribosomes have never been actually observed within the bacterial nucleus in thin sections. Maaloe and Kjeldgaard (1966) have estimated that, within the bacterial nuclear region, the DNA strands are quite tightly packed so that ribosomes may be excluded on steric grounds.

11. The Role of Ribosomes in the Control of RNA The efficient and regular pattern of bacterial cell growth suggests that physiological processes are interrelated and, on some levels, may serve to regulate each other (e.g. Dean and Hinshelwood, 1966). There are several indications that ribosomes may play a regulatory role of this sort in the synthesis of ribosomal and non-ribosomal RNA. I n vitro ribosomes participate in RNA synthesis catalysed by DNAdependent RNA polymerase. Byrne et al. (1964) have shown that ribosomes attach to nascent RNA strands, that is, to incomplete molecules still attached to their DNA template. Shin and Moldave (1966) demonstrated that the addition of ribosomes to such a system increases the extent but not the initial rate of the reaction. Attachment of ribosomes to nascent RNA chains has also been demonstrated in several in vivo experiments. Using B. megaterium infected with

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phage at multiplicities that result in the inhibition‘of synthesis of host RNA, Schaechter and McQuillen (1966) showed that SO% of the newlymade RNA was attached to ribosomes. Since this RNA was made during a very brief “pulse”, these authors concluded that most of these RNA chains were still being synthesized. This finding implies that the ribosomes attach to the free end of the nascent RNA chains. Analogous results were reported by Das et al. (1967) in cultures of E. coli kept at 0”. If attachment of ribosomes is a necessary step in the completion of nascent RNA chains, there are several possible explanations for this requirement. Stent (1966) has proposed that ribosomes function by traversing the nascent RNA and by directly coupling RNA synthesis to protein synthesis. I n his theoretical model, such movement of the ribosomes would serve to pay out the newly-made portion of the RNA molecules from the DNA template. The absence of such co-ordinate polymerase action and ribosome motion might explain why a molecule of RNA polymerase appears to function only once in in vitro systems (Bremer and Konrad, 1964). Under these artificial conditions, there would be no mechanism for releasing RNA from the DNA template. There are some data to support this theory. Several groups of experimenters have observed that the synthesis of m-RNA is a polarized process beginning at the 5’ and ending at the 3’ end (Shigeura and Boxer, 1964; Bremer et al., 1965; Maitra et al., 1965). It has also been shown that m-RNA is translated in this same 5’ to 3‘ direction (Wahba et al., 1966; Terzaghi et al., 1966). The oldest portion of an RNA molecule is thus read first, meaning that the attachment of ribosomes to m-RNA and the initiation of protein synthesis can actually begin before synthesis of the messenger is completed. I n experiments with certain /3-galactosidase extreme polar mutants of E . coli, it was shown that when enzyme formation was impaired at the level of protein synthesis the corresponding messenger for /3-galactosidasewas not made (Attardi et al., 1963). These experiments suggest that protein synthesis and m-RNA synthesis are linked. This co-ordinate synthesis is not obligatory under all conditions. Bacteria treated with a variety of antibiotics which inhibit protein synthesis (e.g. chloramphenicol, tetracycline and streptomycin) continue to synthesize RNA at a rapid rate while protein synthesis is arrested. Experiments of Naono et al. (1966) using E. coli depleted of ribosomes by magnesium starvation showed that m-RNA production is proportional to the number of ribosomes within the cell. Ribosomes might control RNA production in other ways as well. Thus, the free ends of nascent m-RNA molecules may be sensitive to nuclease attack, and ribosome attachment might protect them. This is supported by the in witro findings of Takanami and Zubay ( 1964) and Takanami et al.

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(1965) that the portion of synthetic polyribonucleotides or phage RNA attached to ribosomes in vitro is resistant to ribonuclease. Another possibility is that the nascent end of the RNA molecule may inhibit RNA polymerase, as suggested by Maitra etal. (1964) and Krakow (1966)who found that in vitro RNA synthesis is stimulated by the addition of ribonuclease. Attachment of ribosomes may function mechanically to clear the RNA from the template. As mentioned above, in ultrathin sections ribosomes are never seen within the nuclear region of bacteria. Perhaps a ribosome attached to a nascent RNA chain serves as a “float”, keeping the RNA molecule from intertwining with the DNA and providing it with a cytological sense of direction. It is well to note that these mechanisms are not mutually exclusive and may actually function in combination. Some indirect evidence has also been presented for an inhibitory rather than a stimulatory role of ribosomes in the synthesis of ribosomal RNA. This suggestion is based on the observation that amino-acid starvation results in both an inhibition of r-RNA synthesis and an increase in the number of free ribosomes as polysomes break down (Morris and DeMoss, 1966; Ron etal., 1966b).

111. The Production of Ribosomal RNA Presumably, r-RNA is produced by the same mechanism as other cellular RNA, by the action of RNA polymerase on ribonucleotide triphosphates directed by a DNA template. The size and location on the genetic map of the genes concerned with r-RNA synthesis have been studied with hybridization techniques. I n principle, an RNA molecule is the complementary copy of theDNAstrand on which it was made. If mixed in vitro with denatured, single stranded DNA, the RNA will make a specific double stranded structure with its homologous region on the DNA. Such studies in E . coli (Yankofsky and Spiegelman, 1962a,b ;Attardi etal., 1965),B. megaterium (Yankofsky and Spiegelman, 1963) and B. subtilis (Oishi and Sueoka, 1965) have shown that the portions of the bacterial chromosome complementary to r-RNA are small. The two types of r-RNA do not compete with each other in hybridization tests in Bacillus species, indicating that they differ in nucleotide sequence (Yankofsky and Spiegelman, 1963; Oishi et al., 1966). I n E. coli some competition has been reported by Attardi et al. (1965). Various estimates of the fraction of the chromosome complementary to the r-RNA have been made on the basis of these studies. Yankofsky and Spiegelman (1962a, b) found that 0.2% of the E. coli chromosome was complementary to 235 r-RNA, corresponding to about ten cistrons; in B. megaterium they found values of O . l S ~ Oand 0.14% complementary ?

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to 235 and 1 6 s respectively, corresponding to about thirty-five and forty-five cistrons, respectively (Yankofsky and Spiegelman, 1963). Similar estimations have been made by Attardi et al. (1965). These estimates all confirm the fact that each chromosome contains few copies of the genetic loci complementary to r-RNA and that together they comprise less than 1% of the genome. The r-RNAs of various bacteria apparently have many nucleotide sequences in common. This is demonstrated by hybridization experiments between r-RNA and DNA from different bacterial species. The high degree of cross hybridization observed indicates that few gross evolutionary changes have taken place in this part of the bacterial chromosome in the organisms tested (Attardi et at,1965; Dubnau et al., 1965b; Doi and Igarashi, 1966; Takahashi et al., 1967). Experiments designed to determine the location of the r-RNA cistrons on the genetic map are difficult. This is due in part to the nature of most genetic mapping experiments. Mapping commonly involves estimations of biochemical changes in the ultimate gene product, a protein, or phenotypic properties resulting from such changes in the recombinant progeny. I n the case of r-RNA, we wish to map changes in the primary gene product, RNA itself. Since, as we shall discuss below, there is no conclusive evidence that, bacterial r-RNA codes for particular proteins, classical genetic tests are inadequate. As a consequence, mapping of the r-RNA cistrons is restricted to techniques which are relatively coarse and do not give map positions with the precision of the usual methods. Mapping experiments have been carried out by synchronizing bacterial cells with regard to the replication cycle of their chromosome. The time when ribosomal cistrons replicate may be assumed to be related to their location on the genetic map. If the experiment is carefully designed, at some point in the replication cycle each chromosomal locus is suddenly doubled and this, it could be assumed, is soon followed by a doubling in the amount of the corresponding RNA. If synchronized cultures are sampled at different times of replication, the r-RNA loci can be determined either directly by quantitative hybridization of the DNA with r-RNA or indirectly by measuring the increase in r-RNA production. Rudner et al. (1964, 1965) assayed the base ratios of RNAs produced at various times in the division cycle of synchronized cultures of E . cobi. They found that, at two times after synchronized growth had begun, the RNA synthesized had a composition like that of r-RNA. Two different strains of E . coli with different chromosomal localization of the sex factor were examined and the results of experiments with each strain were similar. By estimating the rate of chromosomal replication and assuming that it was uniform over the length of the genetic map of the chromosome, they inferred the positions of the two loci correspondingto

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r-RNA. Similar results were obtained by Manor and Haselkorn (1967~) in a study of RNA synthesis in E . coli fractionated according to cell size. An extensive study of the location of r-RNA cistrons was carried out by Cutler and Evans (1967).They synchronized the growth of E . coli and at different times added a “heavy” precursor of DNA (Bbromouracil). This permitted the isolation of specific segments of the genome by equilibrium density-gradient centrifugation. By hybridizing these segments with r-RNA they showed that there are two widely separated r-RNA-specific regions, one of which maps near the streptomycin locus. Synchronous culture experiments can also be performed with B. subtitis. With this brganism, synchronous replication may be achieved by

ade

16s, 2 3 s RNA thr I

I eu I

met I I

FIG.6 . Tentative positions of 16s and 23s RNA loci on the Bacillus subtilis chromosome. The positions of the markers for adenine (ade), threonine (thr),leucine (leu) and methionine (met) are shown. The designation for the RNA locus is not to be taken as an accurate representationof the relative size of this locus. Reprinted from Oishi and Sueoka (1965), by permission of the authors.

inducing germination of spores. Not only can the DNA of these cells be hybridized with RNA, but it can also be used to transform suitable B . subtilis mutants to locate other genetic markers. If the DNAs are labelled with a density marker such as 15Nor bromouracil before replication takes place in a “light” medium, the old “heavy” and new “light” DNA can be separated on the basis of their differences in density. Conversely, “light” spores can be germinated in “heavy” medium. Using this method, Oishi and Sueoka (1965) and Dubnau et at. ( 1965a) separated newly-replicated DNA from unreplicated DNA. The time of appearance of replicated markers was assayed by transformation and, on the assumption that it is related to the position on the chromosome, was used to construct a genetic map. By estimating the hybridization capacity of the newly-synthesized DNA, the authors were able to locate the r-RNA cistrons in the vicinity of the streptomycin locus. The possible significance of this finding is discussed in Section I V (p. 108). A genetic map obtained from these experiments is shown in Pig. 6. The significant result of this work is the demonstration that the regions of the chromosome responsible for r-RNA production have been localized even though their precise location has not been determined. The products of the reaction directed by these templates are 16s and 23s RNA. Early experiments (e.g. McCarthy and Aronson, 1961;

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McCarthy et al., 1962;-Takai et al., 1962) implied that 16s and 235 molecules might be assembled as oligomers of a small polynucleotide with a sedimentation coefficient of about 8S-14S. The subsequent discovery of the existence of the unstable m-RNA in growing bacteria (e.g. Gros et al., 1961; Bolton and McCarthy, 1962) suggested that small RNA molecules are mostly m-RNA or fragments thereof. More recent analytical work of Lindigkeit and Handschack (1965) is consistent with this interpretation. Some evidence has been presented indicating that newly made r-RNA does not have sedimentation properties exactly identical to those of mature r-RNA (e.g. Hayashi et al., 1966). I n the cases reported, sucrose-gradient centrifugation of RNA extracted from a mixture of mature and nascent ribosomes showed that the nascent 16s RNA sediments slightly more rapidly than the mature 1 6 s RNA molecules. Sypherd and Fansler (1967)have concluded that the secondary structures of the nascent and mature r-RNAs differ. Dubin and Gunalp (1967) have found differences in the degree of methylation of nascent and mature r-RNA. There is to date no report of bacterial r-RNA larger than 235. I n the cells of higher organisms the two kinds of r-RNA molecules are synthesized jointly as a 455 chain which is later split (e.g. Scherrer et al., 1963). This phenomenon may reflect the need in higher order cells for a transport system between the nucleus and the cytoplasm. Estimates of the amount of r-RNA produced at any given instant vary. I n B. subtilis, Levinthal et al. (1962) showed that about 20% of the RNA labelled during a short pulse of RNA precursors did not decay vhen the cells were treated with actinomycin D, and that it was largely r-RNA. Bolton and McCarthy (1962) showed that in Proteus wulgaris a larger fraction, about 65% of the newly-made RNA, was r-RNA on the basis of hybridization studies. These differences may reflect the variability of different bacteria since Salser (1966) showed that more unstable RNA is formed in B. subtilis than in E. coli. Whatever the exact proportion of r- to m-RNA synthesized in a short time, a great deal of r-RNA must be produced and this production is apparently directed by less than 1% of the chromosome. The production of 16s and 2 3 s RNA molecules seems to be quite well co-ordinated. Although they are distinctly different molecules they are produced in virtually the same numbers so that the molecular ratio is close to 1 :1 and their weight ratio 1 :2. It is not known how this balance is achieved. How long does a growing bacterium take to synthesize a molecule of r-RNA? While the answer to this question is not available, certain limits can be established from the fact that a bacterial cell in balanced growth must double its ribosome complement every generation. If, in E . coli

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growing with a doubling time of 30 min., there are about 10,000 ribosomes, five or six ribosomes must be made per second if the rate of ribosome synthesis does not vary throughout the cell-division cycle. Since the number of cistrons corresponding to each class of r-RNA has been estimated to be ten or less in E . coli (see above), it is possible to calculate that per cistron one r-RNA molecule is made every few seconds. Note that the process may be intermittent, and therefore intrinsically faster, and that the rate of output is not necessarily the same as the rate of synthesis of individual molecules. The rate of synthesis is given by the time required for one RNA polymerase molecule to transcribe the cistron. If several polymerases are engaged in the transcription of the same cistron, the time of synthesis will be longer than that of output. If, say, ten polymerases are working simultaneously, the time of synthesis would be ten times longer. These calculations have bGen presented in more detail by Kjeldgaard (1967). An experimental attempt to determine the rate of r-RNA synthesis was carried out by Zimmermann and Levinthal (1967). They postulate that the time required for making a 1 6 s RNA molecule is half that for making a 2 3 s molecule since the 16s is half as large. Very short-term labelling experiments indicate that this is the case. If a culture of B. subtilis is pulse labelled with radioactive uracil, the specific activity of the 30s particle containing the 16s RNA is initially higher than that of the 50s particle which contains 2 3 s RNA. This effect was demonstrated by allowing the maturation of ribosomal subunits to proceed while RNA synthesis was inhibited by actinomycin D. Invoking a number of logical assumptions, the authors interpret this to mean that in a short time, less than that required to synthesize a whole 2 3 s molecule, 16s molecules can be labelled and released from their template as completed molecules. After this short lag, uniformly labelled 2 3 s molecules are produced and the 50s and 305 particles achieve the same specific radioactivity. The experimental data indicate that the time required for the synthesis of a 16s molecule is about 18 sec. at 37”. By extension, an RNAmolecule with a molecular weight of 1 x lo6 would be made in about 30 sec. Another rate determination was performed by G. Mangiarotti, D. Apirion, D. Schlessinger and L. Silengo (personal communication) using E. coli grown in a medium containing a high concentration of sodium sulphate. Under these conditions, the organisms grow slowly and are readily lysed by gentle means. By analysing the RNA of newly-synthesized ribosomal precursors after various times of labelling, they determined the rate ofr-RNA synthesis. The time of synthesis for each class of r-RNA was 2 min. at 37”. The discrepancy between these values and those obtained by Zimmermann and Levinthal (vide supra) may be due to differences in the organisms studied and in the experimental plans.

-

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Within this range of times, comparable values have been estimated for the synthesis of m-RNA in a few specific cases (Imamoto et al., 1965; Leive, 1965b). Although all these measurements should be considered tentative they suggest that the rates per nucleotide for synthesis of mand r-RNA may be comparable. If this is so, the relatively small region of the DNA correspondingto the r-RNA cistrons must be transcribed many more times than the cistrons for typical m-RNA molecules. Ribosomal-RNA production seems to be controlled by a specific chromosomal locus called the RC locus (Stent and Brenner, 1961). This control was discovered by examining amino-acid auxotrophic mutants of E. coli. Most mutants are unable to grow or proguce RNA in the absence of their required amino acid. Synthesis of RNA by these mutants is under stringent amino-acid control. However, a certain class of E. coli amino-acid auxotrophs can synthesize appreciable amounts of RNA in the absence of the required amino acid. This RNA is complexed with protein into ribosome-like particles (see Section V, B, p. 115). These relaxed mutants are actually double mutants which have mutated in the RC locus as well as in the amino-acid locus. Many workers have demonstrated that RNA made by relaxed mutants is largely ribosomal (e.g. Dagley et al., 196213; Nakada, 1965a; Sypherd, 1965b). Chloramphenicol has been shown to relieve stringency in stringent strains (Kurland and Maal~e, 1962).I n the presence of high concentrations of chloramphenicol,protein synthesis is blocked and amino acids may become more available for regulation of RNA synthesis. Under these conditions, E. coli produces ribosome-like particles which contain RNA and little protein (e.g. Nomura and Watson, 1959). The ribosome concentration in a bacterial cell is proportional to the cell’s growth rate as shown in 8almonella typhimurium by Ecker and Schaechter (1963). Shifting a bacterial culture from a poor to a rich medium (shift-up) causes a rapid increase in RNA synthesis which takes place before other biosynthetic rates increase (Kjeldgaard et al., 1958; Koch, 1965).Much of the RNA synthesized immediately after the shiftup has the nucleotide composition of r-RNA rather than m-RNA (Mitsui et aZ., 1963).Aronson and Holowczyk (1965) showed that, although this RNA has the same base ratio as r-RNA, nucleotide sequence studies revealed that it is more heterogeneous. These experiments imply that the cell’s capacity for r-RNA production is not at its limit before the shift since its output increases drastically after the shift, before much new DNA carrying additional ribosomal cistrons or much protein such as RNA polymerase can be formed. Apparently the production of new ribosomes takes precedence over all other functions in adapting a bacterial cell to faster growth (Maal~e and K jeldgaard, 1966).

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Production of r-RNA can also be shut off by external perturbations. Infection of the bacterium with a virulent bacteriophage may interrupt host-RNA synthesis (Nomura et al., 1960, 1962; Schaechter and McQuillen, 1966). Certain drugs apparently depress r-RNA synthesis somewhat selectively. This phenomenon has been reported for levorphanol (Simon and Van Praag, 1964; Simon et al., 1966a) and for 2,4-dinitrophenol”(Simon et al., 1966b). In summary, r-RNA synthesis in growing bacterial cells probably proceeds by the transcription of small regions of the bacterial chromosome by an enzyme which we assume to be RNA polymerase. Transcription apparently takes place with high efficiency, for less than 1yoof the genome is transcribed. At any one time this small segment of DNA directs the synthesis of 20-60y0 of the RNA being synthesized. The process is apparently controlled by amino acids and can be interrupted by certain drugs. A plausible explanation of the kinetics of labelling of 16s and 235 RNA is that the r-lkNA cistrons are each being transcribed by several polymerase molecules simultaneously, and that this transcription is coordinated so that neither type of r-RNA molecule is produced in exces8.

IV. The Production of Ribosomal Proteins Ribosomal proteins can be identified only by their integration into ribosomal particles, hence studies of their synthesis have been a part of studies of ribosome assembly. It is generally assumed that they are synthesized by the same sort of mechanism its other cellular protein. There has been no demonstration of the existence of specific polysomes which synthesize ribosomal protein. The principal N-terminal amino acids of E . coli ribosomal proteins are methionine and alanirie, which are the most abundant N-terminal residues of other E . coli proteins (Waller, 1963,1964).This finding implies that N-formylmethionine, hypothesized to be the initiator amino acid in the synthesis of other proteins (Adams and Capecchi, 1966),serves the same function for ribosomal proteins. If these assumptions are true, ribosomes in effect have an autocatalytic role in the cell, synt.hesizing protein for the completion of other ribosomes. If ribosomal proteins are synthesized by the same mechanism as other proteins, some m-RNA must direct the synthesis. Here again we must argue by inference, for no demonstration of the chemical and physical nature of such a m-RNA has ever been made. One hypothesis is that r-RNA may itself serve as this messenger (Ecker, 1965; Nakada, 1965a; Roberts, 1965; Stent, 1966).Such an hypothesis is teleologically attractive for its economy. Several limiting assumptions must be made if it is to be applicable; we shall consider them below.

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If r-RNA acts as the messenger for ribosomal protein, the two molecules must have the same genetic locus on the bacterial chromosome. The experiments described in the preceding section allow tentative assignment for the location of the r-RNA cistrons on the genetic map. While a direct mapping of ribosomal proteins is not yet possible, mapping of properties apparently due to changes in their function can be carried out. Foremost among these changes is the sensitivity of bacteria to aminoglycoside antibiotics such as streptomycin. Indeed, as we noted in Section 111(p. 103), streptomycin sensitivity h a p s near the loci for r-RNA. A number of experiments have shown that the effect of streptomycin both in vitro and in vivo is to derange protein synthesis by interaction with the ribosome (Flaks et al., 1962a, b; Spotts and Stanier, 1961). This effect is apparently due to misreading of codons on the m-RNA (Davies et al., 1964),for the ability of the ribosomes to form polysomes is unimpaired (Cox et al., 1964; Davies, 1964). The site of action of streptomycin was originally designated as the 305 subunit of the 705 ribosome (Cox et al., 1964; Davies, 1964) and more recently has been found to be one of the core components of this subunit (Staehelin and Meselson, 1966b; Traub et al., 1966a).Other experiments (Leboy et al., 1964)have shown that K12 strains of E. coli differ from other strains in the electrophoretic properties of one 30s ribosomal protein. Although recent evidence (Furano, 1966) shows that this protein may not necessarily be associated with streptomycin sensitivity or resistance, transduction experiments have shown its locus to be near or identical to that of streptomycin sensitivity or resistam\ It is largely on the basis of arguments such as these that the r-RNA and protein loci appear to be related genetically. Further mapping data may resolve this question. Any theory which proposes that r-RNA codes for ribosomal protein must consider the number of r-RNA cistrons available for coding and the number of different proteins which are found in ribosomes. Electrophoresis of the proteins extracted from E. coli ribosomes show that there are about 30 protein components of a 705 ribosome (e.g. Leboy et al., 1964; Traut, 1966),at least 19 of which have distinct differences in their peptide sequences (Traut et al., 1967).A simple calculation shows that, if each ribosome contains a 16s and a 235 RNA molecule identical to those of all other ribosomes and one each of the 30 various peptides, then the amount of r-RNA is insufficient for coding for all the proteins. Assuming an average molecular weight of 350 for a nucleotide residue, a coding ratio of three nucleotide residues per amino-acid residue, and an average molecular weight of 120 for an amino-acid residue, the ratio of template RNA to protein product should be 3 x 350: 120 or about 9: 1. The 2 : 1 weight ratio of RNA/protein of ribosomes (Tissikres et al., 1959) is considerably smaller. Clearly then, if r-RNA serves as m-RNA for the

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production of ribosomal protein, not all ribosomes are identical. At the very least their RNAs must vary sufficiently to produce the variety of proteins obsekved. If each r-RNA cistron on the bacterial chromosome differs from the others there can, however, be enough variation in its information content to produce the diversity of peptides observed. This can be seen by further calculations. The estimated average molecular weight of a ribosomal protein is 2.5 x lo4 (Section 11, p. 95). This represents apolypeptide chain of 2.5 x 104/120 or 200 amino acidresidues. An RNA molecule of 600 nucleotide residues (molecular weight of approximately 2 x lo5)would be required to code for such a polypeptide. If 30 such ribosomal proteins exist, then approximately 6 x lo6 daltons of ,RNA would be needed to specify the required information. As we have seen above, the molecular weight of 2 3 s RNA is approximately 1 x lo6 and that of 16s RNA is approximately 5 x lo5, and several copies of each template are found in each genome. If each r-RNA molecule were translated several times and each cistron were different in sequence, then there would be sufficient information contained in the r-RNA cistjons to code for ribosomal proteins. Estimates of the number of times each RNA would need to be translated are of the order of six (Roberts, 1965) to ten (Stent, 1966). Another completely different hypothesis is that each ribosome contains the same RNA as all other ribosomes, but does not have a complete set of the 30 different proteins. Rather, it contains only those proteins coded for by its 1.5 x lo6daltons of RNA and has several copies of each of these. It also seems possible that ribosomes differ in both their RNA and proteins. This latter model has been discussed by Roberts (1965). At present there is no evidence to favour one of the above models to the exclusion of the others. Indeed, there is no compelling evidence for the messenger role of r-RNA. It seems entirely possible that non-ribosomal RNA may serve as messenger for the synthesis of all or some of the ribosomal proteins. Some evidence exists to show that ribosomes appear to be functionally identical. Among the best is an experiment by Nakada (1963)showing that ribosomes existing in the cell before /3-galactosidase induction function in the synthesis of the induced enzyme. This was demonstrated by a, socalled density transfer experiment. Bacteria were grown in amedium containing heavy stable isotopes before being transferred to a “light” medium containing inducer for &galactosidase and radioactive isotopes. 8-Galactosidase was formed under these conditions before any appreciable amount of new, light, radioactive ribosomes could be detected by equilibrium density-gradient centrifugation. Thus, the pre-existing ribosomes were active in synthesis of a protein entirely new to the cell. Similar conclusions can be drawn from the observation that phage v*

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proteins are synthesized by pre-existing ribosomes after bacteriophage infectionwhilehostRNAsynthesisis stopped (Brenner etal., 1961).Therefore, at least some of the ribosomes must be capable of participating in the synthesis of a variety of proteins. Conversely, there is no evidence for the existence of special classes of ribosomes responsible for the synthesis of specific proteins. The ability of r-RNA to serve as a template to direct in vitro amino-acid polymerization has been invegtigated. Purified RNA extracted from \ribosomes functions in amino-acid incorporation only in the presence of neomycin and after its secondary structure has been broken (Holland et al., 1966). There is contradictory information about the ability of rRNA extracted from the incomplete particles synthesized under relaxed conditions (see Section 111,p. 106)to stimulate amino-acid incorporation i n vitro. Otaka et al. (1964),Willson and Gros (1964)and Nakada (1965a) reported that this RNA was more active than mature r-RNA. Manor and Haselkorn (19674 and Sypherd (1967)confirmed these findings but attributed them to contamination by m-RNA. The physical state of ribosomal proteins before their incorporation into completed ribosomes is unknown. According to the schemes discussed above, if r-RNA serves as the sole messenger for ribosomal protein it must be translated several times before being assimilated into a ribosome. During the time between completion of the first ribosomal protein and the assembly of the ribosome, it is possible that the ribosomal proteins enter a pool of soluble proteins from which they are later withdrawn. If non-ribosomal RNA serves as messenger for ribosomal proteins, i t is also likely that such a pool exists. A number of experimental observations support such an hypothesis. Perhaps the most convincing of these is the fact that, under conditions of “relaxed” growth, RNA synthesis can proceed in the absence of protein synthesis and bacteria accumulate ribonucleoprotein particles. The simplest explanation of this phenomenon is that a pool of proteins exists to be complexed with newly synthesized r-RNA (Kurland and Maalrae, 1962). Recent findings of S. Osawa (personal communication) indicate that r-RNA may aggregate nonspecifically with various cellular proteins. This complicates the interpretation of these experiments. Under special conditions of inhibition, bacterial cells lose their ribosomes; studies of recovery from these traumata have suggested that ribosomal proteins are not entirely destroyed under these conditions but survive as a pool from which they can be re-utilized. When Mg2+is removed from the growth medium, bacteria lose virtually all their ribosomes without great loss of viability (Dagley and Sykes, 1957; McCarthy, 1961; Kennel1 and Magasanik, 1962). When Mg2+is restored to the medium, bacteria respond by synthesizing new r-RNA

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(e.g. Suzuki and Hayashi, 1964) and producing new ribosomes. If Mge+is made available but a required amino acid is withheld, the RNA synthesized is complexed with proteins yielding particles similar to those found under conditions of “relaxed” synthesis (Nakada and Marquisee, 1965). Sinde the cells could not have synthesized proteins under these conditions, the protein complexed with the RNA must have pre-existed in the cell. More recent data of Kennell and Kotoulas (1967a, b, c) and Marchesi and Kennell ( 1967) indicate that these interpretations may be oversimplified. These workers report that cell populations are not homogeneous under conditions of Mg2+starvation and that, while some cells lose their ribosomes, others retain 30s and 50s particles. The net content of particles per unit of culture remains constant throughout the starvation period. Although polysomes break down in these conditions, some protein synthesis does take place. These results point t o the complexities arising from starvation experiments. Results similar to those reported with Mg2+starvation have been found for phosphate starvation. Julien et aZ. (1967) found that phosphate starvation causes degradation of the ribosomes of bacteria; following the addition of phosphate, ribonucleoprotein particles can be formed in the presence of chloramphenicol. Also supporting the hypothesis that protein from degraded ribosomes can be complexed with newly made r-RNA are the data of Nomura and Watson (1959).I n their experiments, prolonged treatment of E . coli cultures with high concentrations of chloramphenicol caused eventual breakdown of the existing 705 ribosomes of the cell. The particles which accumulated under these Conditions were thought to result from complexing of newly-made RNA with the proteins from the degraded ribosomes. However, this interpretation may not be correct since S. Osawa (personal communication)recently showed that non-specific aggregation of r-RNA and cellular proteins can occur. Some evidence suggests that ribosomal proteins may be divided into several functional classes and that the pool size of these classes may differ. Nomura and Traub (1967),Traub et al. (1966a,b), Staehelin and Meselson (1966a, b) and Lerman et aZ. (1966) showed that a class of ribosomal proteins can be removed from ribosomes and, under suitable conditions, can be re-associated in vitro. These proteins seem to be surface components of the ribosomes and are necessary for various essential functions in protein synthesis. It is likely that intensive work now carried out on these proteins will shed much light on their function. Julien et d . (1967)have suggested that perhaps these proteins are added to ribosome precursors as the final step in the assembly process. The size of the pool of free ribosomal proteins has been estimated by Schleif (1967)to be small, consisting of less than 5% of the total proteins

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of the cell. This determination was carried out by comparing the rate of incorporation of labelled amino acids into ribosomal and non-ribosomal proteins in balanced-growth cultures. I n summary, ribosomal proteins seem to be produced by the same mechanism as other proteins. No conclusive evidence shows that r-RNA serves as messenger for the produc\ion of these proteins, although the total number of r-RNA cistrons in the bacterial genome is sufficient to code for the various ribosomal proteins. It is possible that ribosomal proteins exist in part as a pool available for later assembly into ribosomes; perhaps a special class of proteins exists which is added to the partially completed particles to give the finished product.

V. The Assembly of Ribosomal Subunits I n this section we will discuss the aggregation of r-RNA with ribosomal protein to produce the elemental ribosome structures, the 305 and 50s subunits. This subject has been investigated extensively but, as we shall see, details of the assembly mechanism are not yet understood. Most investigators assume that the 30s and 505 particles are assembled by similar mechanisms and that no specific enzymes or energy sources are required for the reaction. Experiments designed to study the assembly process have been confined almost entirely to the examination of precursor particles by sucrose-gradient centrifugation. Studies have been carried out both on bacterial cultures in balanced growth and on cultures in which growth is unbalanced by drug inhibition or by mutation. Before considering the assembly process, it is well to note that very little r-RNA exists free and uncomplexed in the bacterial cell. This suggests that, as soon as it is completed or while it is being made, it associates with proteins from a pre-existing pool. Alternatively, if r-RNA serves a messenger fudction, it may be complexed with ribosomes either during its synthesis or immediately after completion.

GROWTH A. STUDIESON CELLS IN BALANCED When cultures of various bacteria are “pulsed” with radioactive uridine or uracil, about two-thirds of the incorporated label is found as a broadly sedimenting component lighter than the 70s ribosomes. Figure 7 shows the appearance of such material in a lysate of B. megaterium centrifuged through a sucrose gradient (Schaechter et aZ., 1965). The remainder of the incorporated label is in the form of m-RNA bound to 70s ribosomes and sedimenting with the polysomes. The light material contains ribosomal precursors and some m-RNA as shown by hybridiza-

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tion experiments using homologous DNA. It is heterogeneous and appears as an unresolved band containing peaks of about 455 and 25s when fractionated further by sucrose density-gradient centrifugation (Fig. 8). These rapidly labelled precursors are sensitive to amounts of ribonuclease which do not affect mature ribosomes, as shown in Fig. 8. If labelling is extended for longer periods of time, it is seen that the label fist enters the 30s and 505 regions and only later the 705 ribosomes. This has been shown in E. coZi by McCarthy et aZ. (1962)and Nakada and Kaji

"

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FIG.7. Gradient centrifugation of a Iysate from pulse-labelledBacillus meguterium. A culture was labelled for two doubling times by addition of 1.5 pC/ml. 32PO:-. The culture was pulse-labelled for 30 sec. by addition of tritiated uridine. The lysate was layered on a 29 ml. 15-40% sucrose density-gradient and centrifuged for 130 min. at 25,000 rev./min. in an SW25 rotor (Beckman). The ordinate indicates counts/min. The top of the gradient is to the right. Radioactivity of 32P, -o-e-; 3H, ---0--0---. Reprinted from Sehaechter et al. (1965).

(1967) and in B. megaterium by ourselves. I n the case of B. megaterium, label does not enter the 70s material until after a delay of about 5 min. when the culture is growing at 37" with a 30 min. doubling time (W. S. Kelley and M. Schaechter, unpublished observations). The only extensive study of the kinetics of synthesis of ribosome precursors and their assembly into ribosome subunits is the pioneering work of several investigators at the Department of Terrestrial Magnetism of the Carnegie Institution of Washington. These investigators studied the assembly process in E. coZi and used DEAE column chromatography and

114

WILLIAM S. EELLEY AND MOSELIO SCHAECHTER

sucrose-gradient centrifugation at M Mg2+to resolve the precursor components. They interpreted their data on the basis of a mathematical model for the incorporation of radioactive uracil into the various cell

4000 2000

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a

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0 Fraction number Bottom

Meniscus

FIG.8. Gradient centrifugationof a lysate from pulse-labelledBacillus megaterium. A culture was labelled for two doubling times by addition of 0.4 pC/ml. s2PO,8-. The culture was pulse-labelledfor 30 sec. by addition of 200 pC 3H-uridine.A lysate was prepared by the method of Schaechter et al. (1965) and divided into two portions. One was layered directly on a 10-35% convex exponential sucrose densitygradient; the other was treated with 1 pg. pancreatic ribonuclease /ml. in the cold and then layered on another identical gradient. Gradients were centrifuged 34 hr. at 5' b a n SW39L rotor (Beckman).The top of the gradient is to the right. The ordinate indicates counts/min. a2P0,8-, ---o---o-; 3H, ---0-0-. The top gradient is the sample treated wi6h ribonuclease.

fractions. I n addition to the original publications (Britten and McCarthy, 1962; Britten et al., 1962; McCarthy et al., 1962), two reviews of their study are available, to which the interested reader is referred (Britten, 1963; Roberts et aZ.,1963). Their analysis makes it possible to compare

THE “LIFE CYCLE” OF BACTERIAL RIBOSOMES

115

data obtained from different experiments by normalizing both the expressions for the growth rate and specific radioactivity. Assuming a model in which the uracil label flows through two sequential precursors called “eosome” and “neosome”, a plot of experimental values for specific activities of each precursor at various times is well fitted by the theoretical curves. Since these studies were carried out before the existence of labile m-RNA was demonstrated, the mathematical model was constructed on the assumption that there is no significantturnover of RNA. Recent work by Nierlich (1967), Salser (1966) and Koch (1967), to which the reader is referred for details, has shown that the turnover of m-RNA is a major contributor to the kinetics of labelling of RNA. I n brief, the reason is the following. When a labelled precursor enters a cell it is mixed with the unlabelled constituents of the low-molecular-weight pool and is withdrawn from this fraction to be incorporated into RNA. This pool is replenished with unlabelled precursors arising from the decay of the pre-existing m-RNA. The net effect is to mask the kinetics of labelling of RNA which would take place if the pool components were solely of external origin. These considerations make it apparent in the light of newer knowledge that a detailed kinetic analysis constitutes a formidable task. However, the overall conclusion of the Carnegie group that ribosomes are assembled in a step-wise fashion, has been confirmed by other means. Recent re-examination of the precursor particles in E . coli has revealed there are at least two precursors to the 5 0 s subunit and at least one to the 30s subunit (S. Osawa, personal communication ; G. Mangiarotti, D. Apirion, D. Schlessinger and L. Silengo, personal communication).

B. STUDIES OF METABOLICALLY INHIBITED CELLS As indicated above, alarge number of studies have followed an alternative approach to examine the assembly of ribosomes. These studies have focused on the accumulation of RNA-protein particles in bacteria which have been metabolically inhibited. I n what follows we propose to present a sampling of the results that seem most pertinent. The earlier literature of this field has been thoroughlyreviewed by Osawa (1965). Our summary will deal with each type of particle separately. 1. RCR“ Particles A large number of studies have used the so-called “relaxed” mutants, which do not have the usual “stringent” requirements for amino acids in RNA synthesis (see Section 111, p. 106). The mutation in the RC locus does not influence the assembly of ribosomes in the bacterium under normal conditions. However, when the mutation is allowed to express itself, that is, when a required amino acid is withheld, ribonucleoprotein

116

WILLIAM S . KELLEY AND MOSELIO SCRaECHTER

particles accumulate (Dagleyetal., 1962a,b). Neidhardt and Eidlic (1963) showed that the accumulated particles contain 16s and 23s RNA. If the “relaxed” mutant requires methionine, the RNA of the particles produced by “relaxed” synthesis in the absence of methionine is undermethylated (Mandel and Borek, 1963; Dubin and Giinalp, 1967). However, if the bacterium requires a different amino acid such as histidine, methylation is apparently complete (Manor and Haselkorn, 19674. The

I

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3

0

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0 ._ 7J

2

Fraction number

FIG.9. Sucrosedensity-gradientanalysis of “relaxed” particles in a cell extract prepared from Escherichia coli RCRe’.cells after 60 min. methionine starvation in a glycerol-containingmedium. 1%-Uracilwas present during 10-50min. of methionine starvation. The sedimentationcoefficients of the major fractions are indicated in thegraph. -0-o-indicates extinctionat 260mp; -~-o-,radioactivityof material precipitated with trichloroacetic acid. Reproduced from Nakada et al. (1964), with permission of the authors..

particles seem to be fragile in conditions which do not affect normal ribosomes. They are broken by sonication (Dagley et al., 1963),shear forces generated by the French pressure cell (Sypherd, 1965a) and low concentrations of pancreatic ribonuclease (Dagley et al., 1963;Sypherd, 1965a; Manor and Haselkorn, 1967b). Various sedimentation coefficients have been reported, including 18s and 25s (Nakada et al., 1964; Manor and Haselkorn, 1967a); ZOS, 30s and 455 (Sypherd, 1965a); 205 and 235 (Dagley et al., 1963).Chemical analyses indicate that these particles are

THE ‘‘IJXE CYCLE” OB BACTERIAL RIBOSOMES

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poorer in proteins than are the normal ribosomes ;their RNA/proteinratio is about 3 :1. Figure 9 shows a sucrose density-gradient profile of “relaxed” and naturally occurring particles. Whether these particles have any in vivo function is unknown. Nakada (1965a)reports that, in vitro, these particles can incorporate amino acids in the absence of 705 ribosomes and that the product is a heavier particle that resembles a completed ribosome. Muto et aZ. (1966)have been unable to confirm this report. Sypherd (1967) has observed that addition of “relaxed” particles to an amino acid-incorporating system in the presence of 705 ribosomes stimulates the production of acid-insoluble peptides. However, this stimulation may be due to the presence of extraneous m-RNA in the preparation of “relaxed” particles. The RNA of the “relaxed” particles seems to be slightly different in structure from that of mature ribosomes. The undermethylated particles can be methylated in vitro (Gordon and Boman, 1964) or the RNA extracted from them can be methylated (Manor and Haselkorn, 1967a). Methylation of this RNA in vitro does not affect its capacity to serve as a hemplate for in vitro amino-acid incorporation ; also, undermethylated “relaxed” RNA seems to have about the same in vitro template activity as “relaxed” RNA which is normally methylated, but neither seems to function better than RNA from mature ribosomes (Manor and Haselkorn, 1967a). Sypherd and Fansler (1967) have observed that “relaxed” RNA differs slightly from the RNA of mature ribosomes in sedimentation and when separated chromatographically on a methylated albuminkieselguhr column. If further RNA synthesis is inhibited by a chemical such as 2,4-dinitrophenol, the RNA of these “relaxed” particles apparently is digested by the cell but more slowly than m-RNA (Nakada et al., 1964). This also seems to occur if E . coli are infected with virulent phage T4 (Turnock, 1966). This latter result implies that the “relaxed” particles need the synthesis of non-ribosomal RNA for stabilization. When protein synthesis is re-initiated after restoration of the required amino acid, the amount of material in “relaxed” particles decreases and the number of ribosomes increases (e.g. Dagley et al., 1962a; Turnock and Wild, 1964; Nakada, 1965a; Sypherd, 1965b). After restoration of methionine to a starved methionine RCR” auxotroph, the RNA of the particles becomes fully methylated (Nofal and Srinivasan, 1966), and is incorporated into mature ribosomes (Nakada et aZ., 1964). During this process, the RNA “?natures” in its configuration and the unique properties of the “relaxed” RNA in sucrose-gradient centrifugation and methylated albumin-kieselguhr chromatography are lost (Sypherd and Fansler, 1967). This process takes place without degradation of the “relaxed” particles, and apparently is due to preferential

WILLIAM 8. KELLEY AND MOSEUO SUHAEUHTER 118 synthesis of ribosomal proteins which are added to the particles allowing them to mature into complete ribosomes (Ennis and Lubin, 1965b). Among the most interesting experiments performed with “relaxed” particles are those done by Nakada and Unowsky (1966). They demonstrated tha%,when these particles are mixed with ribosomal protein, ribosomal subunits are formed. This reaction requires no biosynthetic activity and no energy source. Particles produced in this way function as ribosomes in in vitro protein-synthesizing systems, incorporating phenylalanine under the direction of polyuridylic acid. This finding strongly suggests that “relaxed” particles are identical or very similar to normal ribosomal precursors.

2. Mutants Defective in Ribosome Assembly

Two mutants which accumulate ribonucleoprotein particles smaller than 705 ribosomes during normal growth conditions have been studied. The first of these is a derivative of E. coli 15reported by MacDonald et al. (1967). It grows slowly in ordinary media and has unusually large RNA/ protein or RNA/DNA ratios. Much of this excess RNA is found as a particle which sediments at approximately 435. The second mutant is derived from E. coli K12. During exponential growth, this organism also accumulated 435 particles (Lewandowskiand Brownstein, 1966). The mutant of MacDonald et al. (1967)is streptomycin-resistant.Aswe have noted i n the section on ribosomal protein (p. 108), streptomycin sensitivity seems to have a suggestivebut uncertain nexus with ribosomal structure. Since the streptomycin-resistance locus maps near the regions of the ribosomal RNA cistrons, the two may be related or even i d e n t i 4 It is possible that some structural differencesin the ribosome resulting in streptomycin resistance might result in formation of incomplete rib@ somes of this sort. 3. Particles Resulting from the Addition of Drugs that

Inhibit Protein Synthesis

Inhibition of protein synthesis by various drugs, such as chloramphenicol, puromycin and others, can result in the formation of protein-poor subribosomal particles which are similar to the “relaxed” particles in several ways. The most extensively studied are the particles formed in the presence of chloramphenicol,usually referred to as “chloramphenicol particles”. (a) Chloramphenicol Particles. Chloramphenicol is a potent inhibitor of protein synthesis in bacterial cells. When added to cells growing in

THE “LIFE CYULE” OF BACTERUL RIBOSOMES

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minimal media, it initially causes an acceleration of RNA synthesis (Gale and Folkes, 1953; Wisseman et aZ.,1954). This has been interpreted as being the result of increased availability of amino acids when protein synthesis is blocked (Kurland and Maalere, 1962). Hence, the effect of chloramphenicol is analogous to the RCR“ mutation allowing RNA accumulation to take place in the absence of protein synthesis. The accumulated RNA is in the form of ribonucleoprotein particles of sedimentation coefficient about 245 and 315 (Nomura and Watson, 1959), containing 75% RNA and 25% protein, and appearing as two species of 14s and 18s in some experiments (Dagleyand Sykes, 1959).As mentioned before, some of this protein may be spuriously attached (S. Osawa, personal communication). Seemingly, these particles are more fragile than ribosomes for they break down easily during sonication (Dagley and Sykes, 1960 ; Aronson and Spiegelman, 1961), ribonuclease treatment, and at low Mg2+concentrations (Nomura and Watson, 1959). The ultracentrifuge studies of Kurland et al. (1962) indicated that the particles were loosely coiled polyelectrolytes, sensitive to high salt concentrations, and sedimenting at 18s and 255 in low ionic-strength buffer. The smaller particle contained 16s RNA and the larger, 23s RNA. More recent studies have shown thqt this RNA is undermethylated (Gordon et al., 1964) like the RNA of RCR“particles, and apparently differs in minor nucleotide composition from that of mature ribosomes (Dubin and Gunalp, 1967).

During the extended period of chloramphenicol treatment necessary to produce large amounts of this material, some of the 705 ribosomes break down (Nomura and Watson, 1959). Approximately one-fourth of the RNA of chloramphenicol particles produced by this treatment is derived from degraded 70s ribosomes. The source of the protein in these particles is unclear, but it too may be derived from pre-existing particles, from pre-existing pools of nascent ribosomal protein, or from the adventitious attachment of non-ribosomal proteins. With these complicated factors in operation, it is difficult to determine if the aggregation of “chloramphenicol RNA” with pre-existing protein represents a normal mode of ribosome assembly. Figure 10 shows a tracing of Schlieren patterns obtained in ultracentrifuge studies of these particles. The short-term effect of chloramphenicol is bacteriostatic, and E . coli can survive in the presence of high concentrations of this drug. When the inhibitor is washed away, the cells recover permitting study of the fate of this so-called “chloramphenicol RNA”. It has been shown that at least some of the RNA synthesized in the presence of chloramphenicol is converted to mature ribosomes without degradation when the inhibitor is removed (Dagley et al. 1962b; Nomura and Hosokowa, $965). Lerman et aZ. (1967) showed that

W J L L U S. E E L L E Y AND MOSELIO SCHAECHTER 120 chloramphenicol particles can combine with ribosomal proteins to produce particles that are physically indistinguishable from 705 ribosomes. Thus, it appears that the particles produced during chloramphenicol inhibition might be some sort of precursor to mature ribosomes. However, this need not mean that they represent a natural precursor particle since they may be shunted off the normal biosynthetic pathway. (b) Pwomycin Particles. Puromycin is another inhibitor of protein synthesis. Its chemical structure resembles that of an aminoacyl t-RNA and

FIG.10. Ultracentrifuge pattern of an extract of chloramphenicol-inhibited

Escherichia coli prepared using buffer containing M Mg2+. 1 indicatesa soluble component ( 5 s ) ; 2,505 particles; 3,70S particles; 4 and 5, “chlorampheniool particles” (24Sand31s).ReprintedfromNomuraand Watson( 1959), bypermission of the authors.

it blocks completion of polypeptides by replacing -aminoacyl t-RNA, resulting in the formation of a peptide bond u h h puromycin as the carboxy terminal group (Traut and Munro, 1964). When bacteria are inhibited by puromycin, they accumulate ribonucleoprotein particles of smaller sedimentation coefficient than 305 ribosomes. Reported sedimentation coefficients for these particles vary from 15-185 and 18-258 (Nakada, 1965b)to 205 and 25s (Hosokawa and Nomura, 1965).At least a two-fold net increase in bacterial RNA content can be observed in the absence o f m y increasein protein synthesis (Dagleyetal., 1962a,b). When puromycin-inhibited cells are allowed to recover in the absence of the

THE “LIFE CYCLE” OF BACTERIAL RIBOSOMES

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drug, the RNA of these particles is incorporated into mature ribosomes. During this recovery phase there seems to be a preferential synthesis of ribosomal protein (Sells, 1964; Hosokawa and Nomura, 1965; Nakada, 1965b). (c) Particlesformed in the. Presence of Streptomycin and Chlortetracycline. Similar accumulation of ribonucleoprotein particles resulting from inhibiting protein synthesis has been observed with streptomycin (Dubin, 1964; Dubin and Giinalp, 1967)and with chlortetracycline (Holmes and Wild, 1965a, b). These effects have not been studied as extensively as those mentioned above. 4. Particles Resulting from the Synthesis of R N A Containing Unnatural Bases Other metabolic inhibitors can cause accumulation of unnatural ribonucleoprotein particles by altering cellular RNA. Two of these inhibitors are 5-flumouracil and 8-azaguanine which are assimilated into the RNA as analoguesfor naturally occurring bases. I n the experiments of Aronson (1961), 5-fluorouracil was incorporated into particles which sedimented with the 305 and 505 ribosomal subunits and were incorporated into ribosomes when the analogue was removed. Although a report by Kono and Osawa (1964)implied that the RNA of such particles was incorporated into ribosomes unchanged, more recent reports have shown that it is unstable after the fluorouracilis removed (Andoh and Chargaff, 1965; Hills and Horowitz, 1966). Similarly the “relaxed” particles of an RCRez mutant produced in the presence of 5-fluorouracil are not incorporated into ribosomes after the inhibitor is removed although other “relaxed” particles formed in the absence of fluorouracil are (Nakada, 19658). The normal nucleotides of 5-fluorouracil-RNA can be re-utilized for synthesis of ribosomes afte5 an excess of uracil is added to the bacterial culture, whereas 5-fluorouracil is excluded (Andoh and Chargaff, 1965;Iwabuchi et al., 1965). A series of reports on the incorporation of 8-azaguanine are found in the literature, e.g. Dagley et al. (1962a, b), Otaka (1960),Otaka et ab. (1962), and Chantrenne.and Devreux (1960). Very little is known about the nature of the accumulated material except that it is unstable when the analogue is removed. 5 . Effects of Deprivation of Certain Ions on Ribosomal Assembly

I n some instances, upsetting the ionic balance of the growth medium of a bacterial culture can cause the accumulation of particles which resemble those described in the inhibition studies mentioned above. For

122 W I L W 9. KELLEY AND MOSELIO SCHAECHTER example, an E . coli mutant that requires high concentrations of K+, when starved of K+, accumulates unstable particles resembling the chloramphenicol particles (Ennis and Lubin, 1965s). If the K+ level is restored to that of a normal growth medium, these particles are rapidly .converted to ribosomes in much the same fashion as “relaxed” or chloramphenicol particles (Ennisand Lubin, 1965b). If bacteria are deprived of Mg2+they lose practically all their ribosomes and resynthesize them when the cation is restored (see Section IV, p. 110). Suzuki and Rayashi (1964) reported that, during recovery from Mg2+ starvation, bacteria accumulate new ribonucleoprotein particles similar to those formed in the presence of chloramphenicol. This effect is more manifest if an amino acid is withheld after the Mg2+is restored, thus preventing protein synthesis (Nakada and Marquisee, 1965). Phosphate starvation produces similar effects. After extensive deprivation, cellular RNA breaks down to soluble oligonucleotides (Maruyama and Mizuno, 1965, 1966). When phosphate is restored to the medium, synthesis of r-RNA takes place and large amounts of precursor particles accumulate which are gradually converted to mature ribosomes (Julien et al., 1967). This conversion seems to occur even more rapidly in the presence of chloramphenicol, showing that under these conditions ribosome synthesis apparently can take place from pre-existing proteins and nascent RNA.

./& &bP

6. Metabolic Shifts Designed to Promote Selective Synthesis of Ribosome Precursors As we described in Section-I11 (p. 106), bacterial cultures may be “shifted up” fro‘m a poor to a rich growth medium by nutritional supplementation. When this shift takes place, the rate of RNA synthesis exceeds that of synthesis of other macromolecules. Several reports indicate that, under these conditions, the principal RNA species formed is r-RNA, as shown by its nucleotide content (Mitsui et al., 1963; Aronson and Holowczyk, 1965). Kono et al. (1964) and Iwabuchi et al. (1965) used sucrose-gradient centrifugation to examine extracts of cells harvested soon after shift-up and found evidence for the formation of a series of ribonucleoprotein particles which they concludedwere precursors of mature ribosomes. The RNA made at various times after the shift-up was mostly 16s and 235 RNA and was active in stimulating amino-acid incorporation (Otaka et al., 1964).Growth in the presence of chloramphenicolor 5-fluorouracil added after the shift-up showed that particles aimilar to the chloramphenicol and fluorouracilparticles described above could be found (Kono

THE ‘‘LIFE UYULE” OF BACTERIAL RIBOSOMES

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FIU.11. Extinction at 260 mp (0-0) andradioactivity (0---0; counts/min.) of ribosome precursors synthesized in Escherichia ooli during a “shift-up”. Sedimentation analyses of crude extracts in pulse-labelling experiments are shown for 15 sec. (a)and 1 min. (b). Starved cells were transferred to 40 ml. of “shift-up” medium. [SH] Adenosine (100 pC) was added 6 min. after the transfer, and incubation continued for 15 sec. and 1 min. Extracts were treated with DNase ( 5 pg.) and centrifuged at 25,000 rev./min. for 9 hr. Reprinted from Kono and Osawa (1964), by permission of the authors.

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WILLIAM S. KELLEY AND MOSELIO SCHAECHTER

and Osawa, 1964; Iwabuchi et al., 1965; Hayashi et al., 1966). These authors concluded that there were at least two stages in the assembly of each ribosome subunit. I n one case (Kono and Osawa, 1964) newlysynthesized ribonucleoprotein particles sedimented as a rather broad and heterogeneous band in sucrose gradients at S values between 1 5 s and 305 (Fig. 11). Osawa (1965) interpreted these data as suggestingthe existence of several ribonucleoprotein intermediates in the synthesis of 305 and 505 subunits. It seems difficult to determine the actual number of intermediates since there could be a broad continuum of incomplete particles differing from one another by a few proteins. Until analytical techniques which would separate such intermediates from each other more efficiently are developed, it will not be clear how many intermediate steps do exist. For further discussion of this work, the reader is referred to Osawa (1965). 7. A General View of Ribosome Assembly Based on "Relaxed" Syn-

thesis, Drug Inhibition, Recoveryfrom Ion Deprivation and Shift . Experiments The one underlying theme in all of the studies cited above (pp. 115-124) is (hat under certain conditions bacterial protein synthesis can be severely limited or-stopped entirely while RNA synthesis can continue. I n the apparent abeence of protein synthesis, ribonucleoprotein particles appear. I n our discussion of ribosomal protein production we considered the possibility that a pool of protein could exist within the cell and be combined with r-RNA synthesized at a later time, and we described experiments which support that idea. I n the section to follow, we will discuss some in witro experiments with ribosomes that also support the idea that ribosome assembly can take place when a pool of ribosomal protein and a partially completed ribosomal particle are allowed to combine.

C. I n vitro EXPERIMENTS ON RIBOSOME ASSEMBLY

A number of experiments reported recently have indicated that it is possible to remove certain proteins from ribosomes, and that these proteins can be replaced to generate active ribosomes. Several groups of workers have performed similar experiments based on the observation of Meselson et al. (1964) that partial degradation of ribosomes can be accomplished by centrifugation in 5 M caesium chloride solution (Fig. 12). By this treatment, it is possible to produce particles of 4 2 s and 235 from the 605 and 305 particles respectively (Staehelin and Meselson, 1966a; hosokawa et al., 1966). These particles have lost some of their proteins which can be recovered at the meniscus of the caesium chloride gradients. These particles are inactive in in vitro amino acid-incorporating systems.

THE “LIFE CYCLE” OF BACTERIBL RIBOSOMES I

‘ 5 x 10-3MI

125

I

Fraction number

FIU.12. Effect of Mg2+concentration on the distribution of ribosomesfrom Escherichia coli in caesium chloride density-gradients. The Mg2+ concentration and peak densitiesare indicated for each distribution. Reprinted from Meselson et al.(1964), by permission of the authors.

When the 425 and 23s particles are recombined with their respective “split protein” fractions, they reconstitute ribosomes which are active in vitro. The proteins separated by the caesium chloride treatment differ electrophoretically from those remaining in the ribosomal “core” (Traub et al., 1966b; Gesteland and Staehelin, 1967). When particles are reconstituted from 235 “cores” of ribosomes from bacteria resistant to streptomycin, and the “split proteins” of ribosomes from a sensitive strain, in,

126

W I L L I A M 8. EELLEY AND MOSELIO SUHAEUHTER

vitro amino-acid incorporation using these particles is unaffected by streptomycin. If the cmverse reconstitution experiment is performed, the particles produced are sensitive to streptomycin, thus indicating that streptomycin sensitivity is a function of the “core” of the 305 subunit (Staehelin and Meselson, 1966b; Traub et aZ., 1966a).By similar studies, it has been shown that the “cores” themselves do not bind m-RNA (Raskas and Staehelin, 1967) and that distinct components from the “split proteins” from 305 and 505 particles are responsible for t-RNA binding, m-RNA binding and in vitro amino-acid incorporation (Nomura and Traub, 1967).Similar dissociationexperiments have been carried out by Lerman et al. (1966)which demonstrate step-wise dissociation and reaggregation under slightly different conditions. These experiments strongly suggest that ribosomes can be assembled by step-wise addition of specific proteins to an RNA core.

D. CONCLUSIONS I n conclusion, we might say that the assembly of ribosomal subunits is the most studied and yet the least understood of those processes we have discussed so far. The reaction apparently proceeds via a number of precursor particle stages, and can be arrested at intermediate degrees of completion. The precursors which accumulate seem to be the result of the complexing of an r-RNA chain with various ribosomal proteins. It seems likely that the time for completing a ribosome is sufficientlylong that, at any time, a substantial portion of the cellular RNA exists as a pool of precursor particles.

VI. The Formation of Functional Ribosomes I n the preceding section we discussed the reactions by which 305 and 505 ribosomal subunits are formed from r-RNA and ribosomal proteins. The purpose of this section is to consider the mechanisms by which these subunits combine to give functional ribosomes. Of all sections of our review this is the most speculative since, until recently, little work has been done on this important step. As we described in Section V (p. 113), in labelling experiments radioactivity accumulated in 305 and 50s particles before appearing in mature ribosomes (McCarthy et aZ., 1962; Nakada and Kaji, 1967). In these experiments, polysomes were broken by shear forces and the functionally mature ribosomes were found as 705 particles. These data indicate that free 305 and 50s particles are intermediates on the pathway of ribosome assembly.

THE “LIFE CYCLE” 033 BACTDRLAL RIBOSOMES

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Particles identified as sedimenting in the 305 and 505 regions of sucrose gradients are actually a mixture of two kinds, “old” and “new”. “New” particles arise by ribosomal-neosynthesis while “old” ones result from the cyclic functioning of ribosomes in protein synthesia. The direct participation of the 30s and 505 subunits in the ribosomal cycle was demonstrated by Kaempfer et al. (1967). Their work was based on the previous observation of Meselson et ab. (1964) that ribosomal particles are preserved for at least three generations of cell growth. Kaempfer et al. (1967) showed that the 50s and 305 subunits rather than the 705 particles are conserved, and that the 705 particles dissociate into 505 and 305 components which re-aggregate at random during cell growth. This was demonstrated by growing bacteria in a medium containing heavy isotopes and transferring them to light medium. Zonal centrifugation in sucrose gradients and equilibrium centrifugation in caesium chloride revealed that the heavy ribosomal subunita formed before transfer, and the light ribosomal subunits formed after transfer, were distinctly s e p e rate entities whereas the 705 ribosomes found after the transfer were actually combinations of heavy and light subunits. All four possible combinations were found; heavy 50 and heavy 30, heavy 50 and light 30, light 50 and heavy 30, light 50 and light 30. So-called “chase” experiments add supporting evidence to this scheme. I n the chase experiment, uracil- or uridine-labelled bacteria are washed free of excess labelled base or nucleoside or are presented with a large excess of non-labelled molecules to dilute the soluble labelled precursor. During subsequent growth, RNA synthesized from this precursor is unlabelled or of low specific activity. If a particle containing RNA represents a rapidly synthesized and utilized intermediate in the biosynthesis of ribosomes, its specific radioactivity will decrease drastically during a chase. On the other hand, if a particle is a component of a cyclic metabolic pathway, during a chase its specific radioactivity will tend towards the same value as that of the other components of the cycle. This value will decrease for all components at the same rate as new material is synthesized. The specific radioactivity of the 305,505 and 705 particles decreased at approximately the same rate during a chase experiment. This has been shown for E . coli by Aronson et ab. (1960) and by G. Turnock (personal communication) and for B . megatehm by ourselves. There is no defkitive information about the properties of the two kinds of particles sedimenting in the 305 and 505 regions in sucrose gradients, the “old” and the “new”. They seem to be physically inseparable although the data of G. Turnock and R. E. MacDonald (personal communication) indicate that, in E . coli, the “new” particles have slightly smaller sedimentation coefficients. Chemical differences between the “new” and

128

WILLIAM S: KELLEY AND MOSELIO SCHAECHTER

“old” subunit particles have not been studied. Both particles seem to belong to the class of 305 and 505 subunits termed “native” by Green and Hall (1961). The average flux of 305 and 505 particles through this intermediate stage must be quite large for a cell in balanced growth. It can be estimated as the sum of the newly-synthesized particles and those recycled. In Section 111 (p. l04),we calculated that a culture of E . coZi growing with a doubling time of 30 min. at 3 7 O , and containing about 104ribosomes,must manufacture about five new ribosomes per second. At the same time, the existing ribosomes are synthesizing protein. The average time required for a cell to synthesize a protein has been estimated to be about 20 sec. (Maaloe and Kjeldgaard, 1966).If we assume that all or nearly all of the cell’s ribosomes are functioning in protein synthesis and working a t this average rate, about one-twentieth of them should complete the synthesis of a protein during a 1-sec. interval. This means that approximately 5% or 500 of the cell’s ribosomes are in the process of detaching from messenger molecules in a given second. This estimate may be incorrect by a considerable factor, but it implies that the ratio of “old” to “new” 305 and 505 particles is quite large and would explain the data of the chase experiments quite nicely. With these observations and calculations in mind, let us consider several possible schemes for the assembly of functional ribosomes. Basing our schemes only on the observations we have cited, that 305 and 505 particles are precursors of polysome-bound 705 ribosomes and that they are a mixture of newly made and recycled particles, we can draw at least four possible cycles. Let us consider each of these four hypothetical models in terms of other available data to see which is most closely compatible with the experimental results. _____j.

30S+50S

-

polysomes

!

SCHEME A

Scheme A This model postulates that the 305 and 505 subunits react with other components of the cell to form polysomes from which the 305 and 50s particles are released after functioning in protein synthesis. I n this cycle, free 705 ribosomes are not found except as artifacts of preparation or as non-functional metabolip end-products.

129

THE “LIFE CYCLE” OF BACTERIALRIBOSOMES

SCHEME B

Scheme B This model states that 305 and 50s subunits are joined to form free 705 particles which in turn attach to m-RNA. The particles are released from the polysomes as 30s and 50s subunits. 30S+50S

____f

polysomes

SCHEME C

Scheme C This model has free 70s particles as the component released from polysomes after protein synthesis. I n this scheme, the m-RNA orients the 30s and 505 subunits in the formation of 70s particles. 305+50S

t-70s

-705

4L

polysomes

LJ’

I

I SCHEME D

Scheme D I n this model, the free 70s particle is the entity which attaches to and is released from the m-RNA. I n this case, the free 705 pasticles are essentially in chemical equilibrium with their subunits, the joining process being independent of m-RNA and polysome formation. The choice between these schemes depends on whether or not “free” 705 ribosomes exist, and if they do, on whether they are formed during the assembly of polysomes, during the degradation of polysomes, or during both processes. The few experiments pertinent to these questions will be discussed below. Scheme A is essentially that proposed by Mangiarotti and Schlessinger (1966) based on their finding that lysates of E . coli prepared by special methods of cell disruption do not contain free 705 ribosomes. Kelley and

WILLTAM 8. KELLEY AND MOSELIO SUHA.EUHTER 130 Schaechter (1967)have shown that, in B. rnegaterizcrn lysates, “free” 705 ribosomes disaggregate at low Mg2+concentrations at which polysomebound 705 ribosomes are preserved. This implies that a special class of “free” 705 particles may exist. However, the condition of ribosomes within the growing cells cannot be determined with certainty from the examination of cell lysates alone. The joining of subunits could take place during the process of polysome formation (asin schemes A and C).,Schlessinger et al. (1967)showed that, in an in vitro amino acid-incorporating system, 705 ribosomes are formed only in the presence of m-RNA, t-RNA, K+, and Mg2+.A mechanism for the formation of polysomes from ribosomal subunits has been presented on the basis of recent studies of the initiation of protein synthesis. It is currently thought that the initial amino acid of peptide chains is N-formylmethionine (Adams and Capecchi, 1966) and that protein synthesis begins with the binding of N-formylmethionine-t-RNAto ribosomes. Nomura and Lowry (1967)reported that initiation of protein synthesis in vitro is a function of the 305 subunit and not the 705 ribosome. They found that, in the presence of natural m-RNA (f2 phage RNA), N-formylmethionine-t-RNA binds only to the 305 subunit, Mixtures of 305 and 505 subunits or 705 particles could not bind the aminoacyl-tRNA, and if 505 Rubunits were added after binding had taken place the reaction was not reversed. This implies that polysome assembly is sequential, the 305 subunit binding to m-RNA before the 505 subunit. Under some conditions, it appears that ribosomes are released from polysomes as 705 particles, supporting schemes C or D. When the release of ribosomes is allowed to proceed but resynthesis of m-RNA is inhibited, polysomes break down and free ribosomes accumulate. These were found to be 705, and not 305 and 505 particles, after actinomycin D treatment of B. megaterium (Schaechter et al., 1965)and E . coli (R. E. Kohler, E. Z. Ron and B. D. Davis, personal communication) and starvation of E . coZi for iDs earbon source (Dresden and Hoagland, 1967). On the other hand, Zimmermann and Levinthal(l967) have shown that, if actinomycin D treatment of B. subtilis is carried out in a trislbuffered medium, the accumulated particles are 305 and 505 .while 705 particles accumulate if a phosphate-buffered medium is used/In vitro treatment of cell extracts with puromycin effects the release of’ribosomesfrom polysomes. Schlessinger et al. (1967) have reported that the released particles are 305 and 505 subunits, whereas R. E. Kohler, E. Z. Ron and B. D. Davis (persona1 communication) found that they are 705 ribosomes. Even without such contradicting results, we could not be sure of all the relevant effects of these perturbations and cannot choose between various models on the basis of this information alone.

131 I n summary, we have discussed the possible modes by which newly made 305 and 50s ribosome subunits might enter a metabolic cycle and participate in polysome formation. The results of density-transfer and I&bellhgand chase experiments imply that such a ribosomal metabolic cycle actually exists. THE “LIFE CYULE” OF BACTERIBZ RIBOSOMES

VII. The Participation of Ribosomes in Protein Synthesis The subject of protein synthesis is very extensive and we will limit ourselves to those aspects that seem immediately relevant to the “life cycle” of ribosomes. In growing bacterial cells, protein synthesis is carried out on polysomes, aggregates of ribosomes attached to m-RNA. I n lysates made from cells labelled in vivo, newly-made protein is fleetingly associated with these structures (Schaechter, 1963).Such a transient association has also been found for newly-made /3-galactosidase by enzyme assay (Kiho and Rich, 1964). Since polysomes are sensitive to shear forces, a variety of methods for gentle lysis of bacteria have been developed to prevent erstensivebreakage (e.g. Schaechter, 1963;Dresden and Hoagland, 1965; Ron et al., 1966%;Mangiarotti and Schlessinger, 1966; Godson, 1967; Currdliife, L967a). I n general, these methods use detergents to lyse bacterial protoplasts or backria the cell walls of which have been weakened. Bacterial polysomes have been characterized by several criteria. (a) They are readily and quantitatively degraded to 705 particles by ribonuclease in concentrations which have no demonstrable effect on the sedimentation behaviour of ribosomes (Schlessinger, 1963 1963).(b) They sediment as highly heterogeneous material, 705 ribosomes. The sedimentation characteristics (which reflect the frequency distribution of individual polysome classes)vary with the method of preparation but the sedimentation coefficient usually falls between 100s and several hundred S. (c)‘They contain most of the cellular m-RNA.-This was demonstrated by hybridization techniques, sedimentation characteristics, and experiments which measure its rapid turnover (Schaechter et aZ.,‘1965; Schaechter and McQuillen, 1966). (d) As mentioned above, they contain nascent proteins. It is not clear whether polysomes can be formed under conditions where protein synthesis is blocked. Perhaps under such conditions ribosomes attach to m-RNA and make “sterile” passages without synthesizing proteins. Indeed, genetic experiments of Imamoto et aZ. (1966) and Malamy (1966)suggest that this may happenin certain bacterial mutants. It is difficult to establish a sensible criterion for the purity of ribosomal and polysomal preparations. Hence, it is difficult to determine analytically which components are required in vivo for polysome formation and

132

WILLIAM 9. KELLEY AND MOSELIO SCRAECHTER

function. Ribosomes bind substances such as antibiotics (Flaks et al., 1962b; Vazquez, 1966) a n d cellular enzymes (Neu and Heppel, 1964), apparently in an adventitious manng. Therefore, preparations may contain factors unrelated to in vivo function. Knowledge of the minimum requirements for the formation of polysomes comes from in vitro experiments with such preparations, and their relevance to in vivo circumstances cannot be assessed at present. In vitro, mixtures of ribosomes with certain synthetic polyribonucleotides result in formation of polysomelike aggregates which incorporate amino acids. Aggregation occurs most readily with polyribonucleotideswhich do not have a complex secondary structure (Takanami and Okamoto, 1963). This binding apparently does not depend on the interaction between bases (Szer and Nowak, 1967). The synthetic messengers se6m to bind to the 305 component of the ribosome (Takanami and Okamoto, 1963). Other constituents of the amino acid-incorporating system also bind to ribosomes, e.g. t-RNA, earlier thought to attach to the 505 particle (Cannon et al., 1963) and more recently to the 305 (Kaji et al., 1966). Amino-acid incorporation requires the presence of both ribosomal subunits. Ribosomal proteins are responsible for t-RNA and m-RNA binding. Raskas and Staehelin (1967) and Nomura and Traub (1966) have shown that centrifugation in 5 M caesium chloride strips proteins from ribosomes which are responsible for these binding capacities (see Section V, p. 124). This ability can be restoredby carefulre-aggregationof the stripped protein with the RNA-rich “cores”. Messenger-RNA apparently contains special sites to which ribosomes attach. Dahlberg and Haselkorn (1967) have shown that turnip yellow mosaic virus RNA contains five sites of this sort, indicating that attachment need not take plaae on13 at the end of an RNA molecule. A codon specific for N-formylmethionine initiates protein synthesis (e.g. Adams and Capecchi, 1966). However, it is entirely possible that ribosome attachment may take place elsewhere and translation begin only when the N-formylmethionine codon is reached. Ribosomes are believed to move along the m-RNA molecule, the genetic information of which is translated into the amino-acid sequence of the nascent protein. The direction of travel is from tkie 5’ to the 3‘ end of the m-RNA molecules. This conclusion is based on in vitro studies of peptide synthesis directed by polyribonucleotides of known composition, where it has been shown that the N-terminal amino acid of this peptide could only have been specified by reading in the 5’ to 3‘ direction (e.g. Wahba et al., 1966). This has also been concluded independently by Terzaghi et aZ. (1966) who showed that “frame-shift” mutants in the lysozyme cistron of phage T4 produced changes in amino acid sequence consistent only with 5’ to 3‘ reading.

THE “LIFE CYCLE” OF BACTERIAL RIBOSOMES

133

It is interesting to note that RNA synthesis also seems to proceed in this 5‘ to 3’ direction (Shigeura and Boxer, 1964; Maitra et al., 1965; 1965). Thus, the oldest portion of the m-RNA molecule Bremer &Z., would be read first and it is possible that ribosomes attach and traverse it while the m-RNA molecule is being synthesized (see Section 11, p. 100). The loading of a m-RNA molecule with ribosomes to give a complete polysome should require at least as much time as the synthesis of the peptide or peptides coded for by the m-RNA. This time of translation has been estimated to be 10-20 sec. at 37” for an average polypeptide (McQuillen et al., 1959; Maalere and Kjeldgaard, 1966) and would be correspondingly longer for translation of a polycistronic message. However, the time required to complete a polysome by attachment of ribosomes to nascent m-RNA chains might be greater than that for translation, If ri6osomes move along the RNA chain as it is paid out from its template, the time of polysome formation will be that required for synthesis of the m-RNA. This time has been estimated for only a few specific cases and has been reported to be several minutes at 37” for the synthesis of the large polycistronic m-RNA of the tryptophan operon (Imamoto et aZ., 1965)and about 2 min. at 30” for &galactosidase messenger (Leive, 1965a).Kinetics of labelling with radioactive precursors have shown that the average time required for formation of “mature” polysomes in phageinfected B. megaterium is between 30 and 120 sec. (Schaechter and McQuillen, 1966).This is the time needed to reach a steady-state value of the ratio of pulse-labelled RNA to ribosomes. The relationship of the timing of these three processes-translation, m-RNA synthesis, and loading of polysomes-cannot be directly interpreted at present. We have briefly discussed the formation of polysomes from the points of view of the interaction of the various components, the position of attachqent and direction of movement of ribosomes along m-RNA, and the timing of the processes. For lack of factual information we have avoided considering the fundamental question, why ribosomes are required for the synthesis of proteins.

VIII. The Release of Ribosomes from Messenger RNA After participating in protein synthesis, ribosomes are released from the m-RNA of the polysomes to become part of a pool of free particles. The signal for this release is not known at present. Perhaps, as with ribosome attachment to m-RNA, the release mechanism depends on recognition of certain codons or nucleotide triplets in the m-RNA. Indeed, there are special codons that terminate the synthesis of proteins. This is borne out by experiments with certain bacteriophage mutants called 8

134

WILLIAM 9. EELLEY AND MOSEUO SCHAEUHTER

“amber” and “ochre” in which codsns normally sigulfymg certain amino acids have changed to the chain-terminating codons, UAG and UAA, respectively. Consequently, when ribosomes come in contact with these codons,they prematurely terminate the peptides being synthesized. Thus, infection of a normal bacterium with a mutant phage of this type results in the incomplete translation of the cistron containing the mutation and in the formation of a peptide that is correspondingly smaller than normal. Although it has not been demonstrated that such terminating triplets exist at the end of a normal cistron, it seems possible that they play a physiological role in protein synthesis. It is not known, however, if termination of the peptide chain causes the ribosomes to be released from m-RNA. Ribosome detachment from m-RNA can be influenced by certain antibiotics. Puromycin, an aminoecyl-t-RNA analogue, induces breakdown of polysomes and release of ribosomes, probably by premature detachment of the ribosomes from the m-RNA. A similar breakdown of polysomes is also seen when the drug actinomycin D is added. I n this case, however, the breakdown seems to be $he result of a detachment of ribosomes from the end of the messenger molecules. Thus, actinomycin D, unlike puromycin, allows the completion of molecules that are in the process of synthesis at the time of addition of the drug. The addition of chloramphenicol or chlortetracycline, which also inhibit protein synthesis, does not result in the destruction of polysomes over a short period of time (Weber and DeMoss, 1966; Cundliffe, 196713). It is likely that these drugs stop protein synthesis by blocking the movement of ribosomes along the m-RNA. I n accordance with this prediction, the addition of chloramphenicol to cultures that have been treated with actinomycin D results in the preservation of the polysomes in experiments of short duration. Evidently, if movement of ribosomes along the messenger is prevented, the effect of actinomycin D on polysomes is not manifest (M. Schaechter and P. Graze, unpublished data). Experiments with these inhibitors also show that m-RNA seems to be stabilized by the attachment of ribosomes. Thus, the addition of actinomycin D or puromycin leads to the breakdown of m-RNA (Levinthal et al., 1963).It was found that addition of chloramphenicolor chlortetracycline blocks this breakdown. As discussed in Section VI (p. 130), it is not clear at present whether ribosomes are released from the messenger as 70s particles or as pairs of 305 and 505 subunits. I n any case, they, unlike the m-RNA, are quite stable in growing cells and are not degraded. Meselson et al. (1964)showed this by carrying out “density shift” experiments with E . COG.Organisms grown in “heavy” [15N, 2H]-containing medium were transferred to “light” [14N,’HI medium. After three generations of bacterial growth in

THE “LIFE CYCLE” OF BACTERIAL RIBOSOMES

135

‘light” medium, the ribosomes originally present still retained a “heavy” abel as shown by their density in caesium chloride gradient centrifuga;ion. At equilibrium, only particles of “heavy” and “light” buoyant iensity were found. The absence of particles of intermediate densities

Fraction number

FIG. 13. Density distributions produced by purified 505 ribosomes 0, 1 and 3 generationsafter transfer of bacteria growingin [%HI 15N,SZP] medium to light nonradioactive medium. Unlabelled 50s ribosomes provide a density reference. -o-o---, indicates extinction at 260 mp; ---*---e---, radioactivity of 32 Pin particles. Reprinted from Meselson et al. (1964), by permission of the authors.

136

WIJAIAM S. KELLEY AND MOSELIO SCEAECHTER

shows that ribosomes or their subunits were conserved and not broken down into smaller components. The results for the 50s particles are shown inFig. 13. I n summary, ribosomes are released from m-RNA molecules, perhaps at a signal which corresponds to the “amber” or “ochre” codons. After their release, ribosomes are stable and can be reutilized for many generations in making new polysomes. The release of ribosomes from m-RNA can be influenced by various antibiotics which either preserve the polysomes or cause their breakdown. Whether ribosomes are normally released as 30s and 505 particles or as 705 ribosomes is not known at present.

IX. Conclusions We have organized our discussion around the theme that ribosomes have a “life cycle”. Perhaps three general conclusions may be derived about this “life cycle”. First, the assembly of 30s and 505 ribosome subunits proceeds in steps, by a series of poorly characterized intermediates containing r- RNA and various amounts of ribosomal proteins. Second, the process of aggregation of these particles into completed ribosomes seems to be related to the process of polysome formation. And, third, after polysomes have completed their function in the synthesis of proteins, the ribosomes and m-RNA part company, the ribosomes to be reutilized for the formation of other polysomes and the messenger to be degraded and metabolized by the cell. We have summarized,this “life cycle” in Fig. 14. Within this general framework, many details are not yet clear, especially those dealing with timing, co-ordination and biochemical mechanisms. It is likely that these details will be clarified in the near future because the subject is being investigated with vigour and enthusiasm. Amino -Ribosomal acids proteins “iree“ ri bosornes

Messenger RNA

Ribo- -Ribosomal nucleotides RNA

Polysornes

FIG.14. The “life cycle” of the bacterial ribosome.

THE “LIFE CYCLE” OF BACTERIAL RIBOSOMES

137

X. Acknowledgements The investigations reported from the authors’ laboratory have been supported by grants 3R01 A105103 from the National Institutes of Health, U.S. Public Health Service, and GB-5849 from the National Science Foundation. One of us (M.S.) holds a Career Development Award from the National Institutes of Health, U.S. Public Health Service. We wish to thank the following persons for their helpful comments and suggestions :J. E. Davies, B. D. Davis, H. Engelberg, E. P. Geiduschek, D. Gillespie, E. B. Goldberg, R. 0. R. Kaempfer, N. 0. Kjeldgaard, 0. Maalrae, M. H. Malamy, R. MacDonald, D. R. D. Shaw, G. Turnock, D. G. Wild and R. A. Zimmermann. The literature survey for this article was completed on 1st July 1967. REFERENCES Adams, J. M. and Capecchi,M. R. (1966). Proc. nut. Acad. Sci. Wash. 55, 147. Andoh, T. and Chargaff,E. (1965). Proc. nut. A m d . Sci. Wash. 54,1181. Aronson, A. I. (1961). Biochim. Biophys. Acta 49, 98. Aronson, A. I. (1962). J . molec. Biol. 5, 453. Aronson, A. I. (1966). J . molec. Biol. 15, 505. Aronson, A.I., Bolton,E.T., Britten,R. J.,Cowie,D. B., Duerksen, J.D., McCarthy, B. J., McQuillen, K. and Roberts, R. B. (1960).Carnegie Inst. of Wash. Yearbook 59, 257. Aronson, A. I. and Holowczyk, M. A. (1965). Biochim. Biophys. Acta 95, 217. Aronson, A. I. and Spiegelman,S. (1961). Biochim. Biophys. Acta 53,84. Attardi, G.,Huang,P.C. andKabat, S. (1965). Proc.nat.Acad.Sci. Wash.53,1490. Attardi, G., Naono, S., Rouviere, J., Jacob, F. and Gros, F. (1963). Cold Spring H a r b . S y w . quant. Biol. 28,363. Bolton, E. T. (1959). Carnegie In&. of Wash. Yearbook 58, 274. Bolton, E. T. (1960). Carnegie Inst. of Wash. Yearbook 59, 260. Bolton, E. T. and McCarthy, B. J. (1962). Proc. nut. Acad. Sci. Wash. 48,1390. Bollen, A., Herzog, A. and Thomas, R. (1965). Arch. Internat. Physiol. Biochem. 73, 557. Bremer, H. and Konrad, M. W. (1964). Proc. nut. Acad. Sci. Wash. 51,801. Bremer, H., Konrad, M. W., Gaines, K. and Stent, G. S. (1965).J . molec. Biol. 13, 540. Brenner, S., Jacob, F. and Meselson, M. (1961).Nature, Lo&. 190,576. Britten, R . J. (1963).Ann. N . Y . Acad.Sci. 108,273. Britten, R. J. and McCarthy, B. J. (1959). Carnegie In&. Wash. Yearbook 58, 266. Britten, R. J. and McCarthy, B. J. (1962). Biophy8.X. 2,49. Britten, R. J., McCarthy, B. J. and Roberts, R. B. (1962). Biophys. J . 2,83. Brownlee, G. C. and Sanger, F. (1967).J . molec. Biol. 23, 337. Byme, R., Levin, J., Bladen, H. A. and Nirenberg, M. W. (1964). Proc. nut. Acad. Sci. Wash. 52, 140. Cannon, M., Krug, R. and Gilbert, W. (1963).J . molec. Biol. 7,360. Chantrenne, H. and Devreux, S. (1960). Biochim. Biophys. Acta 39, 486. Choi, Y .S. and Cam, C. W. (1967).J . molec. Biol. 25,331. Cohen, S. S. andLichtenstein, J. (1960).J.bioLChem. 235,2112.

138

WILLIAM S . KELLEY AND

MOSELIO SCHAECHTER

Colbpum, J. L., Witherspoon, B. H. and Herbst, E. J. (1961). Biochim. Biophys. A& 49, 422. Comb, D. G. and Zehavi-Fllner, T. (1967).J.mokc. Bwl. 23,441. Cox, E. C., White, J. R. and Flaks, J. G. (1964). Proc. m t . Acad. Sci. Wash. 51,703. Cundliffe, E. (1967a), J.gen. Microbiol. 46, viii. h d l i f f e , E. (196713). Molec. Pharmacol (in Press). Outler, R. G. and Evans, J. E. (1967).J. molec. Biol. 26,91. Dagley, S. and Sykes, J. (1957). Nature, Lond. 179,1249. Dagley, S. and Sykes, J. (1959). Nature, Lond. 183,1608. Dagley, S. and Sykes, J. (1960). Biochem. J. 7 4 , l l P . Dagley, S., Turnock, G. and Wild, D. 0. (1963). Biochem. J. 88,555. Dagley, S., White, A. E., Wild, D. G. and Sykw, J. (19624. J.gem. Microbiol. 29,59, Dagley, S., White, A. E., Wild, D. G. and Sykes, J. (1962b). Nature, Lond. 194,25. Dahlberg, J. E. andHaselkorn, R. (1967).J. m o k . Bwl. 24, 83. Das, H. K., Goldstein, A. and Lowney, L. I. (1967).J.molec. Biol. 24,231. Davies, J. E. (1964). Proc. m t . Acad.Sci. Wash. 51,659. Davies, J. E., Gilbert, W. and Gorini, L. (1964). Proc. nat. A d . Sci. Wash.51,883. Dean, A. C. R. and Hinshelwood, C. (1966). “Growth, Function and Regulation in Bacterial Cells”, Oxford University Press, London. Doi, R. H. and Igarashi, R. T. (1966).J. Bact. 92,88. Dresden, M. and Hoagland, M. (1965). Science, 149,647. Dresden, M. and Hoagland, M. (1967). J. biol. Chem. 242, 1065. Dubin, D. T. (1964). J. molec. Biol. 8, 749. Dubin, D. T. and Giinalp, A. (1967). Biochim. Biophys. Acta 134, 106. Dubnau, D., Smith, I. and Mamur, J. (1965a). Proc. mt.Acad. Sci. Wash. 54,724. Dubnau, D., Smith, I., Morell, P. and Marmur, J. (1965b). Proc. nat. Acad. Sci. Wash. 54, 491. Ecker, R. E. (1965). Proc. nat. Acad. Sci. Wash. 54, 1465. Ecker, R. E. and Schaechter, M. (1963). Biochim. Biophys. Acta 76,275. Ennis, H. L. and Lubin, M. (1965a). Biochim. Biophy8. Acta 95,605. Ennis, H. L. and Lubin, M. (1965b). Biochim. Biophys. Acta 95,624. Flaks, J. G., Cox, E. C. and White, J. R. (1962a). Biochem. Biophys. res. Commun 7, 385. Flaks, J. G., Cox,E. C., Witting, M. L. andWhite, J. R. (196213).Biochem. Biophys res. Cornmum. 7, 390. h a n o , A. V. (1966). J. biol. Chem. 241,2237. Gale, E. F. and Folkes, J. P. (1953). Biochem. J. 53,493. Gavrilova, L. P., Ivanov, D. A. and Spirin, A. S. (1966).J.molec. Biol. 16,473. Gesteland, R. F. (1966). J. mokc. Biol. 18, 356. Gesteland, R. F. and Staehelin, T. (1967).J. molec. Biol. 24.149. Glauert, A. M. and Thornley, M. J. (1966).J. R. micr. SOC. 85,449. Godson, G. N. (1967). Methods in Enzymology (in Press). Goldberg, A. (1966). J. molec. Biol. 15, 663. Gordon, J. and Boman, H. G. (1964). J. molec. Biol. 9, 638. Gordon, J., Boman,’H. G. and Isaksson, L. A. (1964). J. molec. Biol. 9,831. Green, M. H. and Hall, B. D. (1961). BiOphY8. J. 1, 517. Gros, F., Gilbert, W., Hiatt, H. H., Attardi, G., Spahr, P. F. and Watson, J. D (1961). Cold Spring Hcurb. Symp. qwnt. Biol. 26, 111. Hall, C. E. and Slayter, H. S. (1959). J. mokc. Biol. 1, 329. Hayashi, Y., Osawa, S. and Miura, K. (1966). Biochim. Biophys. Acta 129, 519. Hills, D. C. and Horowitz, J. (1966). Biochemistry 5,1625.

THE “LIFE CYCLE” OF BACTERIAL RIBOSOMES

I39

Holland, J. J., Buck, C. A. and McCarthy, B. J. (1966). Biochemistry 5,358. Holmes, I. A. and Wild, D. G. (1965a). Biochem. J. 94, 8P. Holmes, I. A. and Wild, D. G. (1965b). Biochem.J. 97,277. Hosokawa, K., Fujimura, R. K. and Nomura, M. (1966). Proc. wt.A d . Sci. Wash. 55, 198. Hosokawa, K. and Nomura, M. (1965). J. wwlec. Biol. 12,225. Huxley, H. E. and Zubay, G. (1960). J. molec. Biol. 2, 10. Imamoto, F., Morikawa, N. and Sato, K. (1965). J. molec. Biol. 13, 169. Imamoto, F., Ito, J. and Yanofsky, C. (1966). ColcESpringHarb. Symp. quant. Biol. 31, 235. Iwabuchi, M., Kono, M., Oumi, T. and Osawa, S. (1965). Biochim. Biophys. Acta 108, 211. Iwabuchi, M., Otaka, E., Kono, M. and Osawa, S. (1966). Biochim. Biophys. Acta 114, 83. Julien, J., Rosset, R. and Monier, R. (1967). Bull. SOC.chim. Biol. 49, 233. Kaempfer, R. 0. R., Meselson, M. and Raskas, H. J. (1967). J. moZec. Biol. (in Press). Kaji, H., Suzuka, I. and Kaji, A. (1966). J. biol. Chem. 241, 1251. Kelley, W. S. and Schaechter, M. (1967). I n preparation. Kennell, D. and Kotoulas, A. (196%). J. Bact. 93, 334. Kennell, D. and Kotoulas, A. (1967b). J. Bact. 93, 345. Kennell, D. and Kotoulas, A. (1967~). J. Bact. 93, 367. Kennell, D. and Magasanik, B. (1962). Biochim. Biophys. Acta 55, 139. Kiho, Y. and Rich, A. (1964). Proc. nat. Acad. Sci. Wash. 51, 111. Kjeldgaard, N. O., Maalse, 0. and Schaechter, M. (1958).J.gen. Microbiol. 19,607. Kjeldgaard, N. 0. (1967). Advanc. microbial Physiol. 1, 39. Koch, A. L. (1965). Nature, Lond. 205, 800. Koch, A. L. (1967).J. theoret. Biol. (in Press.) Kohler, R. E., Ron, E. Z. and Davis, B. D. (1967). In preparation. Kono, M. and Osawa, S. (1964). Biochim. Biophys. Acta 87, 326. Kono, M., Otaka, E. and Osawa, S. (1964). Biochim. Biophys. Acta 91,612. Krakow, J. S. (1966). J. biol. Chem. 241, 1830. Kurland, C. G. (1960). J. molec. Biol. 2, 83. Kurland, C. G. and Maalse, 0. (5962). J. molec. Biol. 4, 193. Kurland, C. G., Nomura, M. and Watson, J. D. (1962). J. wwlec. Biol. 4,388. Leboy, P. S., Cox, E. C. and Flaks, J. G. (1964).Proc. nat. Acad.Sci. Wash.52,1367. Leive, L. (1965a). Biochem. Biophys. res. Commun. 20, 321. Leive, L. (1965b). J. molec. Biol. 13, 862. Lerman, M. I.,Spirin, A. S., Gavrilova, L. P. and Golov, V. F. (1966).J. molec. Biol. 15, 268. Lerman, M. I., Zimmermann, R. A., Gavrilova, L. P. and Spirin A. S. (1967). Moleculcvr Biology.(USSR)1, 12. Levinthal,C., Keynan, A. and Higa, A. (1962). Proc. mt.Acad. Sci. Wash.48,1631. Levinthal, C., Fan, D. P., Higa, A. and Zimmermann, R. A. (1963). Cold Spring Harb. S y q . qmnt. Bwl. 28, 183. Lewandowski, L. J. and Brownstein, B. L. (1966). Biochem. Biophys. Tea. Commun. 25, 554. Lindigkeit,R. and Handschack, W. (1965). Biochim.Biophys. Acta 103,241. Loening, U. E. (1967). Biochem. J. 102,251. McCarthy, B. J. (1961). Biochim. Biophys. Acta 55, 880. McCarthy, B. J. and Aronson, A. I. (1961). Biophys. J. 1, 227.

140

WILLIAM S. KELLEY AND MOSELIO SCHAECHTER

McCarthy, B. J., Britteri, R. J. and Roberts, R. B. (1962). Biophys. J. 2, 57. McQuillen, K., Roberts, R. B. and Britten, R. J. (1959).Proc. nat. Acad.Sci. Wash. 45, 1437. MacDonald, R. E., Turnock, 0.and Forchhammer, J. (1967). Proc. mt. Acad. Sci. Wash. 57, 141. Maalse, 0. and Kjeldgaard, N. 0. (1966). “Control of Macromolecular Synthesis”, W. A. Benjamin, New York. Maitra, U., Tal, M. and Hurwitz, J. (1964). Abstr. S i ~ h Imtermt. Congr. Biochem. I-lZ!, p. 72. Maitra, U., Novogrodsky, A., Baltimore, D. and Hurwitz, J. (1965). Biochem. Biophys. res. Commun. 18, 801. Malamy, M. (1966).Cold Spring Hurb. S y m p . quunt. Biol. 31,189. Mandel, L. R. and Borek, E. (1963). Biochemistry 2, 560. Mangiarotti, G. and Schlessinger, D. (1966).J.molec. Biol. 20,123. Manor, H. and Haselkorn, R. (1967a).J. molec. Biol. 24, 269. Manor, H. and Haselkorn, R. (1967b).J. molec. Biol. 24, 323. Manor, H. and Haselkorn, R. (19674. Nature, Lo&. 214, 983. Marchesi, S. L. and Kennell, D. (1967).J. Bact. 93, 357. Maruyama, H. and Mizuno, D. (1965). Biochim. Biophys. Acta 108, 593. Maruyama, H. and Mizuno, D. (1966). Biochim. Biophys. Acta 123, 510. Meselson, M., Nomura, M., Brenner, S., Davern, C. and Schlessinger, D. (1964). J. molec. Biol. 9, 696. Midgley, J. E. (1962). Biochim. Biophys. Acta 61, 513. Midgley, J. E. (1965). Biochim. Biophys. Acta 95, 232. Mitsui, H., Ishihama, A. and Osawa, S. (1963). Biochim. Biophys. Acta 76, 401. Moller, W. and Chrambach, A. (1967).3. molec. Biol. 23, 377. Moller, W. and Widdowson, J. (1967).J. molec. Biol. 24, 367. Morris, D. W. and DeMoss, J. A. (1966).Proc. nut. Acad. Sci. Wmh. 56,263. Muto, A., Otaka, E. and Osawa, S. (1966).J. molec. Biol. 19,60. Nakada, D. (1963). Biochim. Biophys. Actu 72, 432. Nakada, D. (1965a).J. molec. Biol. 12, 695. Nakada, D. (1965b). Biochim. Biophys. Acta 103,455. Nakada, D., Anderson, I. A. C. and Magasanik, B. (1964).J. molec. Biol. 9,472. Nakada, D. and Kaji, A. (1967). Proc. mt. Acad. Sci. Wash. 57, 128. Nakada, D. and Marqukee, M. J. (1965).J. molec. Biol. 13,351. Nakada, D. and Unowsky, J. (1966).Proc. mt. A d . Sci. Wmh. 56,659. Naono, S., Rouviere, J. and Gros, F. (1966). Biochim. Biophys. Acta 129,271. Neidhardt, F. C. and Eidlic, L. (1963). Biochim. Biophys. Actu 68,380. Neu, H. C. andHeppel, L. A. (1964).Proc. nut. Acad.Sci. Wash. 51,1267. Nierlich, D. 0. (1967).Sciewe 158, 1186. Nofal, S. and Srinivasan, P. R. (1966).J. molec. B w l . 17,548. Nomura, M., Hall, B. D. and Spiegelman S. (1960).J. molec. Biol. 2, 306. Nomura, M. and Hosokawa, K. (1965).J.molec. Biol. 12,242. Nomura, M. and Lowry, C. V. (1967). Proc. nat. Acud. Sci. Wash. 58, 946. Nomura, M., Okamoto, K. and Asano, K. (1962).J. molec. B w l . 4, 376. Nomura, M. and Traub, P. (1967).1% “Organizational Biosynthesis” (H. J. Vogel, ed.), (in Press). Nomura, M. and Watson, J. D. (1959).J. molec. Biol. 1,204. Oishi, M., Oishi A., and Sueoka, N. (1966).Proc. mt.Acad. Sc(. Wmh. 55,1095. Oishi, M. and Sueoka, N. (1965).Proc. nat. Acad. Sci. Wash. 54,483.

THE “LIFE CYCLE” OF BACTERIAL RIBOSOMES

141

Osawa, S. (1965). Progr. nucleic acid Res. 4, 161. Otaka, E. (1960).Exp. Cell Res. 21,229. Otaka, E., Osawa, S., Oota, Y., Ishihama, A. and Mitsui, H. (1962). Biochim. Biophys. Acta 55, 310. Otaka, E., Osawa, S. and Sibatani, A. (1964). Biochem. Biophys. res. Commun. 15, 568. Raskas, H. and Staehelin, T. (1967).J. molec. Biol. 23, 89. Reid, P. J., Orias, E. and Gartner, T. K . (1965). Biochem. Biophys. res. Commun. 21, 66. Roberts, R. B. (1965).J. theoret. Biol. 8, 49. Roberts, R. B., Britten, R. J. andMcCarthy, B. J. (1963).I n “Molecular Genetics”, (J.H. Taylor, ed.), Part 1, p. 291, Academic Press, New York. Rodgers, A. (1964). Biochem. J. 90, 548. Rodgers, A. (1966). Biochem. J. 100, 102. Ron, E. Z., Kohler, R. E. andDavis, B. D. (1966a).Science 153,1119. Ron, E. Z., Kohler, R. E. and Davis, B. D. (196633).Proc. mt,Acad. Sci. Wash. 56, 471. Rosset, R. and Monier, R. (1963).Biochirn. Biophys. Acta. 68, 653. Rudner, R., Prokop-Schneider, B. and Chargaff, E. (1964). Nature, Lond. 203, 479. Rudner, R., Rejman, E. and Chargaff, E. (1965). Proc. nat. Acad. Sci. Wash. 54, 904. Salser, W. (1966).Ph.D. thesis: M.I.T., Cambridge, Mass. Schaechter, M. (1963).J. molec. Biol. 7, 561. Schaechter, M. and McQuillen, K. (1966).J. rnolec. Biol. 22, 223. Schaechter, M., Previc, E. P. andGillespie, M. E. (1965).J.molec. Biol. 12,1119. Schemer, K., Latham, H. and Darnell, J. E. (1963).Proc. nut. Acad. Sci. Wash. 49, 240. Schleif, R. (1967).J. molec. Biol. 27, 41. Schless+ger, D. (1963).J. molec. Biol. 7, 569. Schlessinger, D. and Gros, F. (1963).J.molec. Biol. 7,350. Schlessinger, D., Mangiarotti, G. and Apirion, D. (1967). Proc. nat. Acad. Sci. Wash. 58, 1782. Schlessinger, D., Marchesi, V. T. and Kwan, B. C. K. (1965).J. Bact. 90, 456. Sells, B. H. (1964).Biochirn. Biophys. Acta 80, 230. Shigeura, H. T. and Boxer, G. E. (1964). Biockem. Biophys. res. Gommun. 17,758. Shin, D. H. and Moldave, K. (1966).Biochern. Biophys. res. Commun. 22, 232. Silman, N., Artman, M. and Engelberg, H. (1965). Biochim. Biophys. Acta 103, 231. Simon, E. J. and Van Praag, D. (1964).Proc. mt. Acad. Sci. Wash. 51, 1151. Simon, E. J., Van Praag, D. and Aronson, A. I. (1966a).Molec. Pharmacol. 2,43. Simon, E. J., Cohen, S. S. and Raina, A. (196613).Biochem. Biophys. res. Commun. 24, 482. Spahr, P. F. and TissiBres, A. (1959).J. rnolec. Biol. 1,237. Spahr, P. F. (1962). J. rnolec. Biol. 4, 395. Spirin, A. S. (1964). “Macromolecular Structure of-Ribonucleic Acids”, Reinhold, New York. \i Spirin, A. S. and Belitsina, N. V. (1966).J. molec. Biol. 15, 282. Spirin, A. S., Belitsina, N. V. and Lerman, M. I. (1965).J. rnolec. Biol. 14, 611. Spitnik-Elson, P. (1964).Biochim. Biophys. Acta 80,594. Spotts, C. R. and Stanier, R. Y. (1961).Nature, Lond. 192, 633. 9

142

WILLIAM 9.KE L L E Y AND MOSELIO SCHAECHTER

Staehelin, T. and Meselson, M. (1966a).J. molec. Biol. 16, 245. Staehelin, T. and Meselson, M. (196613).J. molec. Biol. 19, 207. Stanley, W. J. Jr. and Bock, R. M. (1965). Biochemistry 4, 1302. Stent, 0. S. (1966). Proc. Roy. SOC.B, 164, 181. Stent, G. S. and Brenner, S. (1961). Proc. nat. Acad. Sci. Wash. 47, 2005. Suzuki, H. and Hayashi, Y. (1964). Biochim. Biophys. Acta 87, 610. Sypherd, P. S. (1965a).J. Bact. 90,403. Sypherd, P. S. (196513).J. Bact. 90, 411. Sypherd, P. S. (1967). J. molec. Biol. 24, 329. Sypherd, P. S. and Fansler, B. S. (1967). J. Bact. 93, 920. Szer, W. and Nowak, L. (1967).J. molec. Biol. 24, 333. Takai, M., Kondo, N. and Osawa, S. (1962). Biochim. Biophys. Acta 55, 416. Takahashi, H., Saito, H. and Ikeda, Y. (1967). Biochim. Biophys. Acta 134, 124. Takanami, M. and Okamoto, T. (1963). J. molec. Biol. 7, 323. Takanami, M. and Zubay, G. (1964). Proc. a&. Acad. Sci. Wash. 51,834. Takanami, M., Yan, Y. and Jukes, T. H. (1965).J. molec. Biol. 12,761. Terzaghi, E., Okada, Y., Streisinger, G., Emrich, J., Inovye, M. and Tsugita, A. (1966). Proc. mat. Acad. Sci. Wash. 56,500. Tissibres, A., Schlessinger, D. and Gros, F. (1960). Proc. nat. Acad. Sci. Wash. 46, 1450. TissiAres, A. and Watson, J. D. (1958).Nature, Lond. 182, 778. TissiBres, A., Watson, J. D., Schlessinger, D. and Hollingworth, B. R. (1959). J . molec. Biol. 1, 221. Traub, P., Hosakawa, K. and Nomura, M. (1966a).J.molec. Biol. 19,211. Traub, P., Nomura, M. and Tu, L. (1966b).J . molec. Biol. 19,215. Traut, R. R. (1966). J. molec. Biol.21,571. Traut, R. R., Moore, P. B., Delius, H., Noller, H. and Tissibres A. (1967).Proc. mt. Acad. Sci. Wash. 57, 1294. Traut, R. R. and Munro, R. (1964).J. molec. Biol. 10, 63. Turnock, G. (1966).J. molec. Biol. 17, 289. Turnock, G. and Wild, D. G. (1964). Biochem. J. 90, 32P. Van Iterson, W. (1966).J. cell Biol. 28, 563. Vazquez, D. (1966). Biochemical Studies of Antimicrobial Drugs S y m p . Soc. gen. Microbiol. 16, 169. Wahba, A. J., Salas, M. and Stanley, W. M. (1966). Cold Spring Harb. S y m p . quant. Biol. 31, 103. Waller, J.-P. (1963). J. molec. Bib. 7, 483. Waller, J.-P. (1964). J. molec. Biol. 10, 319. Waller, J.-P.andHarris,J.I. (1961).Proc. mat. Acad.Sci. Wash.47,18. Weber, M. J. and DeMoss, J. A. (1966). Proc. mt. Acad. Sci. Wash. 55, 1224. Weller, D. L. and Horowitz, J. (1964). Biochim. Biophys. Acta 87, 361. Willson, C. and Gros, F. (1964). Biochim. Biophys. Acta 80,478. Wisseman, C. L., Smadel, J. E., Hahn, F. E. and Hopps, H. E. (1954). J. Bact. 67, 662. ' Yankofsky, S. A. and Spiegelman, S. (1962a). Proc. mat. Acad. Sci. Wash. 48,1069. Yankofsky, S. A. and Spiegelman, S. (1962b). Proc. nat. Acad. Sci. Wash. 48, 1466. Yankofsky, S. A. and Spiegelman, S. (1963). Proc. mat. Acad. Sci. Wash. 49, 538. Zimmermann, R. A. and Levinthal, C. (1967). J. molec. Biol. (in Press).

Budding of Yeast Cells, Their Scars a n d Ageing K. BERAN Department of Technical Microbiology, Institute of Microbiology, Czchoslocuk Academy of Xciences, Prugue, Czechoslocakia I. Introduction . 11. Composition and Structure of the Cell Wall of Yeast

.

A. Macromolecular Wall Components . B. Structure of the Wall 111. Mechanism of the Primulin-lnduced Fluorescence . IV. Budding of Yeast Cells and Its Mechanism A. Budding of Yeast Cells as Observed in the Optical and Electron Microscopes B. Examination of Buds and Scars Using the Fluorescence Microscope . C. Mechanism of Budding. D. Some Further Perspectives . V. Ageing of Cells and Age Distribution in a Population A. Maximum Reproductive Capacity of an Individual Cell B. Age Distribution in a Population References

.

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143 145 145 147 148 148 148 149 154 161 162 162 164 169

I. Introduction Recent years have witnessed considerable activity concerning the composition, structure and function of the yeast cell wall, a structure which appears to be the site of a number of processes which reflect the functional state and relationship between the proliferating yeast cell and its environment. Our present knowledge of the molecular components of the cell wall and of their structural arrangement is too limited to permit a final conclusion to be drawn. The present review is an attempt to relate results of direct observations on some structures of the cell surface of intact budding cells in a fluorescence microscope with the results of other investigations, and to point out some other applications of this simple method. The fluorochromeprimulin had been used previously for distinguishing between viable and dead yeast cells (Meisselet nl., 1961). It was found in 143

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FIG.1. Photomicrographs of yeasts showing different types of reproduction. (a) Saccharomyces cerevisiae showing a birth scar (indicated by an arrow) and bud scars ; magnification about x2000; (b)Saccharomycodes ludwigii showing multiple scars; magnification about ~ 2 0 0 0 ; (c) Schizosaccharomyces pombe showing division scars; magnification about x 2000; (d) Endomyees rnugnusii; magnification about x 2000.

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this laboratory that, at higher concentrations (1:lOOO), primulin makes it possible to observe directly the scars in all types of vegetative reproduction of yeast cells (Streiblovh and Beran, 1963a, b). By following the formation of structures by induced fluorescence during the course of reproduction, together with their submicroscopic structure in an electron microscope, it was possible to define three new types of yeast cell walls (Streiblovh et al., 1964; Beran et al., 1964; Streiblovh and Beran, 1965; Streiblovh et al., 1966; Streiblovh, 1966a, b). Figure 1 shows examples of scars on yeast cells reproducing in four different ways, namely multipolar budding (Xaccharomyces cerevisiue), bipolar budding (Xaccharomycodes ludwigii), division (Schizosacchuromyces pombe) and terminal growth (Endomyces magnusii).

11. Composition and Structure of the Cell Wall of Yeast A. MACROMOLECULAR WALLCOMPONENTS I n order to give proper scope to this discussion, we must mention the composition and organization of the cell-wall components of yeasts. As may be seen from a number of publications devoted to this problem, the yeast cell wall is mostly composed of polysaccharides accompanied by some proteins, lipids and glucosamine. The ratio of the various types of components varies from species to species. The main differences are observed in the amounts (or even absence) of mannans, lipids and glucosamine, particularly in the form of chitin (cf. the review by Phaff, 1963). The present discussion will be limited to representatives of the group of the multipolar-budding yeast, Xaccharomyces cerevisiae. The cell wall of this species is relatively well studied, even if our information on the macromolecular form of cell-wall composition is incomplete. It is known, however, that mannan represents the most readily extractable component, that glucan is present in two forms, one being soluble in weak alkaline solutions, the other being insoluble, and thbt polysaccharide and protein molecules are present in complexes. These complexes, together with the other components of the cell wall, account for its integrity. The most frequently isolated complex is a mannan-protein (Northcote and Horne, 1952; Palcone and Nickerson, 1956; Eddy, 1958; Eddy and Rudin, 1958; Korn and Northcote, 1960) which has identical properties in all cases (Nickerson, 1963). The extraction of glucomannan-protein complexes (Eddy, 1958; Kessler and Nickerson, 1959; Korn and Northcote, 1960)serves as further evidence for the existence of bonding between the polysaccharide and the protein component of the cell wall. Although we do not know much about the mutual role of glucose and aannose in

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the molecules of these complexes, it is assumed that we are dealing with a copolymer of glucan and -mannan bound into a complex by the protein (Korn and Northcote, 1960 ; Nickerson, 1963). The alkali-insoluble component of the cell wall is, according to Kessler and Nickerson (1959), a glucan-protein with traces of mannose. However, even glucan isolated by different workers (Belland Northcote, 1950) contains some amino acids and a considerable quantity of chitin (Korn and Northcote, 1960). Its insolubility depends on factors other than those which contribute to the insolubility of cellulose (Northcote, 1953). The growth rate of the yeast X.cerevisiae affects only slightly the ratio of glucan to mannan in the cell wall but, in the extractable fractions, the ratio of these two components changed systematically in favour of the more readily extractable mannan and glucan as the growth rate decreases (McMurroughand Rose, 1967). The protein in the cell wall contains relatively large amounts of sulphur (Falcone and Nickerson, 1956.; Kessler and Nickerson, 1959). According form. to the above authors, most of the sulphur exists in the -S-SThe protein is acid in character because of a high content of aspartic and glutamic acids, and both Kessler and Nickerson (1959) and Korn and Northcote (1960) deduce from their results that the protein in the individual fractions is the same. The amount of protein in the cell wall is also little influenced by the growth rate but is substantially decreased when the yeast is grown under conditions of NH4+limitation (McMurroughand Rose, 1967). The role of proteins in cell wall integrity is evident from the reported lysis of the wall by powerful proteolytic enzymes (Nickerson, 1964).

Glucosamine was found in all the isolated fractions (Eddy, 1958; Korn and Northcote, 1960; Kessler and Nickerson, 1959). Korn and Northcote (1960) conclude on the basis of the solubilities of the individual fractions that only 9% of the total amount of glucosamine in the wall is present in the form of chitin, the presence of which had been demonstrated chemically (Roelofsen and Hoete, 1951)) by X-ray analysis (Houwink and Kreger, 1953) and by the detection of acetylglucosaminein the wall lysate following treatment with enzymes (Eddy, 1958). The existing work on lipids has been concerned solely with their extraction, and there is no clear evidence on the form in which they exist in the cell wall. Little is known about the bonding between soluble complexes and between these and the insoluble glucan component. The presence of highly acid proteins in all three of the fractions prepared by Kessler and Nickerson (1959) led to the view that these complexes may contain protein carboxyl groups esterified by polysaccharide hydroxyl groups. This assumption was borne out by the formation of hydroxamates with cellwall preparations in the presence of excess alkali and by the presence of

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acidic amino acids in polysaccharide fragments after treatment with proteolytic enzymes (Nickerson, 1964). The function of glucosamine is less clear. The compound cannot derive from chitin as it is found in the soluble fractions. For this reason, Eddy (1958) and Korn and Northcote (1960) assume that glucosamine can serve as a link between polysaccharides and proteins. The function of disulphide bridges in the cell-wall protein and its relation to the wall properties will be mentioned later. OF THE WALL B. STRUCTURE

It is beyond doubt that the yeast cell-wall is a highly organized structure, but it is not yet fully understood how the various macromolecular components are embedded in it. The soluble fractions have a globular character (Kessler and Nickerson, 1959) and the insoluble glucan, which maintains the shape of the cells even after extraction of soluble components, apparently accounts for the shape and the rigidity of the cell wall. It occurs at least partly in the form of fibrils which have been seen on the internal face of the intact cell wall in electron micrographs (Houwink and Kreger, 1953; Nickemon et al., 1961) and in thin sections of X-contrasted specimens (Hagedorn, 1964). The glucan aggregates into heavy fibrils on treatment with hot HC1 (Houwink and Kreger, 1953). These fibrils are probably of glucan-protein character and form the ridges of scars visible in an electron microscope after treatment with acids (Houwink and Kreger, 1953) or alkalis (Northcote and Horne, 1952). The hypothesis of a stratiform character for the cell wall continually receives fresh support. The first reports, which were based on electronmicroscopic studies, mention at least two layers (Northcote and Horne, 1952; Agar and Douglas, 1955; Bartholomew and Levin, 1955). This view of the stratiform character of the yeast cell wall and of the specific localization of mannan on the cell surface is further supported by the cytochemical studies of Mundkur (1960, 1963) who removed the mannan layer from the yeast S. cerevisiae and investigated the double membrane, by studies on the basis of yeast flocculation (Eddy and Rudin, 1958) and in the work of Marquardt (1962), Hagedorn (1964) and Sentandreu and Villanueva ( 1965)’.The last named authors observed three electron-dense layers formed by material of similar electron opacity in sections of heatdestroyed cells of Candida utilis. E. StreiblovA (1967, personal communication), using the freeze-etching method, also discovered a layer on the inner face of the wall. Evidence on the localization of chitin at the site of mother scars was supplied by Houwink and Kreger (1953) and recently by Bacon et aZ. (1966).

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111. Mechanism of the Primulin-InducedFluorescence Recognizing the participation of fibrils in the structural arrangement of the yeast cell wall, and the properties of colloid particles of primulin formed from its dipolar molecules in an aqueous solution (Ziegenspeck, 1949; Frey-Wyssling, 1959)) one can understand the induced fluorescence of yeast cells as being due to the colloid particles penetrating the interfibrillar spaces of the wall and being adsorbed on the fibril surfaces. The fluorescenceintensity then depends on the spatial arrangement of the fibrillar components, and on the size and orientation of interfibrillar spaces, and thus it is possible to obtain a view of the wall texture. Three types of fluorescence have been described in yeast (StreiblovA, 1966c): (a) diffuse, green-yellow fluorescence of the wall, probably corresponding t o disperse textures; (b) maximum green-yellow fluorescence of scar and septa1 ridges, corresponding to maximum accumulation of microfibrils; (c) minimum green-yellow fluorescence of scar plugs and bands, probably corresponding to the parallel arrangement of these structures.

IV. Budding of Yeast Cells and Its Mechanism A. BUDDINGOF YEASTCELLS

AS OBSERVEDIN ELECTRON MICEOSCOPES

THE

OPTICAL AND

The course of budding by yeast cells has been described by Lindegren (1945)and by Lindegren and Haddad (1953)on the basis of direct observztions, and by Agar and Douglas (1955) using electron microscopy of ultra-thin sections. It appears from these studies that budding is initiated by a small swelling of the mother-cell wall which grows continually into a bud. At a certain size, a plug is formed which severs the connection between the mother and daughter cells. The base of the bud is constricted even before the daughter cell is broken off, the constriction proceeding from the mother cell. Barton (1950)studied the budding process of S. wrevisiae var. ellipsoideus using viable, plasmolysed and stained cells, and described microscopic structures which were apparent after separation of daughter cells. The birth scars were found at places where the daughter cell was connected with the mother cell, and bud scars a t the place on the mother-cell surface where the bud was formed. These structures are permanent throughout the life span of the cell and no new cells can be formed at the place of an older scar. Barton (1950)also showed that the bud scars form a wedge with a central thickening, surrounded by a slightly raised circular ridge of cell-wall material. The general character of the scars is concave in bud scars and convex in birth scars.

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Studies of the cell-wall structure and of scars, using electron microscopy of whole cells (Bartholomew and Mittwer, 1953),of isolated walls (Northcote and Horne, 1952),of thin sections (Agar and Douglas, 1955; Bartholomew and Levin, 1955)and of carbon replicas (Bradley, 1957)all basically support the findings of Barton (1950). OF BUDSAND SCARSUSING THE FLUORESCENCE B. EXAMINATION MICROSCOPE

Fluorescence microscopy after primulin treatment shows that both the mother-cell wall and the bud wall display a slight fluorescence, but that a structure strongly fluoresces at the bud base, the structure remaiiiing on the surface of the mother cell in the form of a ring (Fig. 2 ) even

FIG.2 . A budding cluster of Saccharomyces cerevisiae. Magnification about x 1500.

after separation of the daughter cell. Two types of scars may be distinguished in the fluorescence microscope. Birth scars (Fig. la) where primulin is not accumulated at the edge; these scars have a diameter greater than that of a bud scar. The birth scar plug is convex and it fluorescences weakly. Bud scars have a morphology as described previously. During the formation of'bud scars, the material of the wall forms a circular thickening which is filled by a convex plug. The edges of the scars display a strong fluorescence while the plug fluoresces only weakly. One may assume, on the basis of the mechanism of action of primulin, that the cellwall microfibrils possess a diffuse texture as postulated by Nickerson (1963, 1964), the scar edges being composed from accumulated microfibrils, probabiy of glucan, which are circularly orientated (Houwink and Kreger, 1953; Northcote and Horne, 1952).

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The main difference between these two types of scars is in the structure of their edges. A weak fluorescence of the scar edges indicates that they might contain material different from the remaining part of the cell wall. With the bud scars, this is an assumption based on cytochemical studies of wall polysaccharides (Mundkur, 1960)and of studies of chitin localization (Houwink and Kreger, 1953; Bacon et al., 1966).Since the bud-scar plug probably does not represent an integral part of the original wall (Agar and Douglas, 1955, 1957; Vitols et al., 1961; Marquardt, 1962; Hagedorn, 1964) the cell-wall type in yeasts reproducing by multipolar budding can be schematically represented as in Pig. 3 (Streiblovh, 1966a, b). A fibrillar structure was found even during regeneration of protoplasts of X. c e r h k i a e (Eddy and Williamson 1959; NeEas, 1965a) when, in a

FIG.3. A schematic drawing of the cell-wall type in multipolar-budding yeast, Light areas indicate original wall of mother cell; striped areas, bud scars; dark area. birth scar.

liquid medium, a fine fibrillar meshwork is synthesized (Fig. 4);but this does not correspond to the native fibrils of an intact cell wall. Complete regeneration of protoplasts is achieved only in a gel medium of gelatin or agar (NeBas, 1961,1962; Svoboda, 1966),a behaviour whichis foundwith protoplasts of all yeasts which reproduce by budding (Svoboda and NeBas, 1966; Svoboda, 1967). The regeneration process passes through several stages (NeBas, 1956, 1965a). At first, the protoplasts undergo hypertrophic growth ; the irregular shapes divide and grow further and their cytological structure does not differ from that of protoplasts either qualitatively or quantitatively (NeBas and Svoboda, 1967). The bodies then become denser, they attain a more regular shape and an atypical wall appears which can undergo budding ;this process gives rise to bodies from which morphologically normal cells can be formed.

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The course of the regeneration process followed by primulin-induced fluorescence (StreiblovA et al., 1967) indicates that, even during the first stages of protoplast growth, some fluorescenee is observed (Fig. 5 ) although originally the protoplast showed no fluorescence whatsoever.

FIG.4.Electron micrograph showing the fibrillar mesh of a regenerating protoplast ofSaccharomyces cerevisiae. After 24 hr. growth on the surface of an agar-containing medium, the protoplasts were exposed to osmotic shock and the regenerated surface was isolated by centrifugation in capillaries. The specimen was chromium-plated at an angle of 45". Magnification about x 10,000. From NeEas (1965a).

This fluorescence corresponds to the fine fibrillar mesh of irregularly orientated fibrils observed in the electron micrographs. At this stage of growth, the fibrils have a tendency to group into flat bundles which are identical with the fluorescent granules apparent in the midst of the slight fluorescence of the remaining mesh (Fig. 6). The f i s t generation formed

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FIG.5. Diffuse fluorescence of growing protoplasts of Saccharomyces cerevisiae. For details of the preparation of protoplasts of S. cerevkiae, laboratory strain 7, see StreiblovA et al. (1967). The protoplasts were prepared by means of digestive juice of Helix pomatia and cultivated in synthetic medium (medium N1) containing gelatin prepared according to NeEas (1962). The growing protoplasts were transferred into centrifugation test tubes using 0.6 M-KC1, incubated for 10 min. a t 36" and centrifuged. The sediment was centrifuged once more in capillaries and transferred onto slides using an isotonic solution of primulin ( 1 :lo00 in 20% glucose solution or 0.6 M-KC1).Magnification about x 2000.

FIG. 6. Fluorescence of granules in regenerating protoplasts of Xaccharomyces cerevisiae. Details of the preparation of protoplasts, their cultivation, and the preparation of microscopical specimens are given in the caption to Fig. 5. Magnification about x 2000.

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FIGS. 7a and b. The first generation formed by budding of regenerated protoplasts of Saccharomyces cerevisiae (Fig. 7a),and a regenerating protoplast with a n atypical scar (shown by the arrow; Fig. 7b). Preparation of the protoplasts, conditions for regeneration, and preparation of microscopical specimens are given in the caption for Fig. 5 . Magnification of each micrograph is about x 2000.

by budding from regenerating bodies (Figs. 7a, b) does not resemble normal cells. The bodies are usually larger and the buds do not possess an intensely fluorescent edge. Subsequent generations of cells are normal. From the point of view of the structures discussed on the basis of observations of secondary fluorescence of primulin, it would seem that fluorescent edges of mother scars are formed only if the wall fibrils have a

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certain arrangement but, morphologically, the budding is similar even if the cell wall is atypical in its structure. It can also be assumed that this indicates an important role of the amorphous matrix of the wall for the structural arrangement of the native microfibrils. OP BUDDING C. MECHANISM

The sequence of biochemical and physical events that take place during yeast budding was formulated by Nickerson and his coworkers (Falcone and Nickerson, 1958; Nickerson and Falcone, 1959; Nickerson et al., 1961; Nickerson, 1963, 1964). This elegant theory represents an attempt at compiling existing knowledge of cell-wall components and the arrangement of these polymers in the wall and relating these data to views on the function and enzymic cleavage of covalent disulphide bonds in the cell-wall protein. Although this theory has not yet been fully proved, it represents a t present the only fertile base from which the mechanism of budding can be further treated. The first enzymic step, which initiates budding by cleavage of covalent bonds between the macromolecules of the wall, has been deciphered by observations of Nickerson and his coworkers (cf. VCTard, 1958) on dimorphism in Candida albicans and in particular on differences between the normal strain and the division-less mutant, especially with respect to an electron-transferring system and to the activity of protein disuIphide raductase. As it had been established that this enzyme splits the -8-Sbonds in isolated wall fractions, this was taken as evidence for a connection between the -SH groups of the wall and the budding process. Differences in the activities of protein disulphide reductase, an enzyme bridges in the wall glycothat might control the formation of -S-Sproteins which are associated with shape changes, were also observed by Brown and Hough (1966). Cells of 8. cerewisiae cultivated continuously under conditions of NH,+ limitation were elongated as compared with cells grown under conditions of glucose limitation, and they possessed a decreased activity of disulphide reductase in the mitochondria1 fraction. I n this connection, it should be mentioned that McMurrough and Rose (1967) found the cell walls of S. cerevisice grown continuously under conditions of NH,+ limitation to contain only about half the amount of protein compared with walls from glucose-limited cells. Also the proportion of soluble mannanincreasedinwallsof yeast grownunderNH,+limitation, and this was accompanied by pronounced changes in the fine structure of cell wall as seen in thin sections. A number of direct as well as indirect indications can be cited to show or -SH groups in the yeast cell-wall during the importance of -S-Sgrowth, and in contributing to the properties of the wall. Direct proof was

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supplied by Robson and Stokly (1962) who applied tritiated phenyl mercuric chloride to demonstrate a striking reaction of the -SH groups in the walls of C. albicans and the yeast-like organism Eremothecium ashbyii. The authors consider this finding to be evidence in support of the views of Nickerson (1963). Some further information can be gathered from publications dealing with the problem of protoplast formation and with lysis of the yeast cell-wallby various enzyme preparations, especially those of microbial origin. Since the first application of the snail-gut juice to yeast protoplast formation (Eddy and Williamson, 1957),the number of available wall-digesting preparations has greatly increased (Phaff, 1963);it appears that they all contain a glucan-splitting system of similar properties, but differ in the content of other enzymes and hence in their ability to attack intact cells or in the extent of lysis of isolated walls. It was found that the snail-juice preparation, which contains a number of enzymes including mannanase, glucanase, chitinase, lipase but few proteolytic enzymes (Holden and Tracey, 1950), affects different yeast strains with different vigour ;in general, cells in the logarithmic phase of growth will form protoplasts more readily than stationary-phase cells (Sutton and Lampen, 1962; Holter and Ottolenghi, 1960). Millbank and MacRae (1964)fractionated the snail-gut enzymes and showed that, for protoplasts to be formed, the wall mannan must be attacked; glucanase activity alone is not sufficient. Further sensitization of the cell wall towards snail enzymes can be achieved by adding 2-mercaptoethanol or other thiol reagents (Davies and Elvin, 1964; Nurminen et ctl., 1965). Davies and Elvin (1964) observed that cells of Xaccharomyces fragilis, even from the stationary phase of growth, formed protoplasts readily when pre-incubated with 2-mercaptoethanol although without this compound no protoplasts were formed. Thiol reagents affect positively the activity of enzymes prepared from, or contained in, the spent media of various micro-organisms; these media alone generally possess little activity toward intact cells or isolated walls (see e.g. Bender, 1963; Bacon et al., 1965). Bacon and his coworkers (1965)reported that preincubation of the S. cerevisiae cell-wall with 2-mercaptoethanol brought about a practically complete lysis using the cultivation medium of Cytophaga johnsonii, the mannans released forming the predominant component. Intact cells treated in the same manner displayed no microscopic changes but the thiol itself, at pH 7 - 5 ,released the entire invertase content within 24 hr. which is in agreement with the work of Davies and Elvin (1964) on 8.fragilis. It thus appears that, from the point of view of the assumptions of Nickerson and Falcone ( 1956a, b) on the mannan-protein complexes connected by disulphide bridges and on the participation of these bridges in the mechanical properties of cell walls (Nickerson, 1963,1964)and the

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localization of mannan predominantly on the external face of the wall (Mundkur, 1960; Eddy and Rudin, 1958) of S. cerevisiae, the abovementioned effects possess a common basis. The disulphide bonds participate in the structural integrity of the cell wall, and their reduction makes it easier for enzymes to lyse some cell-wall components, e.g. /3-glucosides. I n view of the results of Robson and Stokly (1962)it can be assumed that this is in agreement with the sensitivity of cells from the logarithmic phase of growth toward the snail-gut enzyme. I n connection with the effect of thiol reagents on the yeast cell-wall, it should be mentioned that, in the presence of cysteine, yeast cells increase their volume (A. Kleinzeller, 1966, personal communication). This effect

FIG.8. Sequence of scars on the pole of aSaccharornyces cerevisiae cell. Magnification about x 2000.

can be taken as further evidence for Nickerson’s view that the physical properties of the wall are determined by the cross linkage of cell-wall bonds participate in polymers. According to these views, the -S-Sthe elasticity of the cell wall and, at the point of their reduction by protein disulphide reductase, a change in plasticity ensues resulting in a decrease of stability; the increased internal pressure causes the cell wall t o break and the native protoplast to be blown out explosively. The protoplast then becomes rapidly enveloped in a cell wall but, due to the explosive emergence of the protoplast, the wall fibrils are re-orientated, group together, and form a circular scar edge. This physical stage of budding does not take place at a random site of the cell surface but rather at the point of maximum curvature. This view is supported by the observation of Barton (1950)on the sequence of bud

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formation on the surface of the mother cell. The observation was confirmed by Freifelder (1960) but the position of the first bud is usually dependent on the degree of ploidy. The results of fluorescence microscopy are also in agreement with this finding (Fig. 8). The first mother scar is formed at the pole opposite to the birth scar; subsequent scars are formed in the vicinity of this pole and the birth scar, and the latest ones appear in the quatorial plane where the sequence appears to be more or less random. However, the change of size with scar number is of such character that the originally oval cell has a tendency t o become spherical (Beran et a$., 1966a). Deviations from this most common sequence have also been observed (see Fig. 1). I n the triangular forms of Trigonopsis variabilis (Fig. 9), the scars were located near the apexes of the cell pyramid. However, another sequence was observed in some cells of X. cerevisiae var.

FIG. 9. Localization of scars in the triangular foim of Trigonopsis variabilis. Magnification about x 3000.

ellipsoideus. I n this yeast, the first bud is formed near the pole opposite the birth scar, but other buds appear successively until a regular ring of scars is formed; this continues along a spiral toward the opposite pole (Figs. 10a and b). Hence it follows that budding proceeds gradually through all the loci of curvature, from the maximal to the minimal ones in the quatorial plane, and then again to the greater curvatures. These observations support the view that the budding sequence is not random but that the bud need not always be formed at the point of maximum curvature of the wall. The integrity of the cell wall is probably r i o t destroyed durillg tho bud growth. McClary and Bowers (1965) published a critical study in which they demonstrate, in agreement with the work of Agar and Douglas (1955)and Hagedorn (1964) on sections through budding yeast cells, that the wall of the budding cell maintains its integrity and fundamentally also

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its constant thickness (Fig. 11). These observations indicate that the cell wall grows by intussusception of newly synthesized material up to the finished structure a t a rate which is in agreement with the expanding

FIGS.10a, b. Sequence of scars in Saccharomyccs cerevisiae var. ellipoideus. In Fig. 10a, note the formation of rings from scars according to the position of buds and the beginning of formation of spiral from scars. I n Fig. lob, note the spiral from scars on the third cell a t the bottom. Magnification about x 2000.

volume of the bud. This assumption is in agreement with the results of studies on the cell-wall synthesis and bud formation in 8. cerevisiae by labelling with fluorescent antibody (Chung et al., 1965). By following the cell budding after treatment with the antibody, the authors found that

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FIG.11. An electron micrograph of a section through a budding cell ofSaccharomyces cemvisiae. Reproduced from McClary and Bowers (1965).

FIG.12. An electron micrograph of a metal-shadowed isolated wall of a budding cell of Saccharomyces cerevisiae. The washed cells were disintegrated by shaking with Ballotini beads (grade 12), and the walls isolated by repeated centrifugation in 0.9% NaCl and distilled water until the required purity was reached. The cell walls were shadowedwith chromium at an angle of 30"on theFormvar-coated grids. Magnification about x 10,000.

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the mother-cell wall does not grow during budding. Budding is initiated on a small area of newly synthesized wall which increases in size to a bud and grows further in parallel with the bud. The new cell-wall is synthesized in a circular band produced in the vicinity of the old-wall material and pushes the previously formed wall away from the mother cell. The electron microscope reveals an electron-dense structure a t the bud base on isolated walls (Fig. 12); this structure usually shows bright fluorescence. M. Hayashibe ( 1967, personal communication), working with a synchronous culture of S. cerevisiae and using fluorescence colour brighteners, found such fluorescence even in very small buds. If this indicates that this structure is formed during budding, the seemingly contradictory findings can be mutually accommodated. It is difficult to draw final conclusions a t present, but certain assumptions can be made that can serve as a working hypothesis. The place on the mother-cell surface where the bud is to be formed is predetermined. The cell wall is composed of complexes of macromolecules with a certain structural arrangement that is determined by its function, the complexes being mutually linked by various types of bonds. and -SH groups of proteins certainly are important The -S-Sfor the cell-wall integrity, and participate in the budding process. The predetermined site at the cell surface becomes the site of new cell-wall synthesis, growth of the bud proceeding basipetally near the mother-cell wall. This site thus becomes, in contrast with the remaining mother-cell wall, the active site of a number of processes. It is questionable whether, in view of its simultaneous function, this site represents a full structural analogy to the rather rigid remainder of the cell wall. If it is concluded that this cannot be so, one can assume that budding is initiated by enzyme activity, whether of protein disulphide reductase alone or of a group of enzymes or of a set of wall-synthesizing enzymes. The consequence of this activity is a change in the structure of this wall locus. The re-orientation of fibrils apparent in the fluorescence microscope follows as a consequence of the above change either immediately or more often after the first bud is formed; the wall of the mother cell becomes extended, thereby ensuring a connection between the mother and the daughter cell. This assumption is in agreement with the view that the organizational principle producing orientation of fibrils is present in the amorphous wall matrix and determines the type of texture with respect to its future function. Passive orientation of the fibrils occurs only by a secondary extension of the cell (Streiblod, 1966~). A more detailed study using the fluorescence microscope, in connection with other methods, should either support or refute these assumptions.

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D. SOMEFURTHER PERSPECTIVES By studying wall function on the basis of its macromolecular composition and arrangement, it is possible to draw conclusions regarding its function in maintaining cell shape (Nickerson, 1963). This maintenance of shape has a more profound morphogenetic character, as can be seen from analyses based on regeneration of protoplasts into normal cells (NeEas, 1965b).The normal cell-wall ensures mutual correlations between cytological cell structures and hence protects the biological functions of the cell in proper proportions. It follows,therefore, that factors governing cell-wall synthesis both biochemically and structurally are of considerable importance. During the biosynthesis of the cell wall, the regulating mechanisms are genetically fixed, together with conditions for their realization. It should be left to specialists in the field to form views on the character of the mechanisms playing a role in the transcription of the genetic information in this connection. We know, however, that they must contain timing systems which regulate the sequence of individual processes in relation to the general development of the cell system. The presence of such timing systems in the growth cycle of the yeast cell is indicated by the findings of Gorman et al. (1964), Tauro and Halvorson (1966) and by the fact that these systems can be eliminated with the result that cell growth is separated from division. There is increasing evidence that, in addition to the protein disulphide reductase, designated as the division enzyme (Nickerson and Falcone, 1956a), micro-organisms contain proteins or other substances t h a t are linked with cell division (Hase et al., 1959; Watanabe and Ikeda, 1965a, b). I n the case of yeast, such a substance was isolated from the cold-trichloroacetic acid extract of whole cells (VranQand Fencl, 1964). This substance possessed the same properties as a substance isolated from Chlorelb pyrenoidosa, and the effects of the two substances were reciprocal. The alga contains the substance before cell division and, without it, the cells would not divide (VranB and Fencl, 1964). The substance contains sulphur, suppresses uptake of sulphur by the alga and, when added to an algal suspension or to a yeast culture, it accelerates division but not growth of cell mass. I n yeast, it raises the manometric quotients and the Pasteur ratio. It requires a source of energy to become active ; it is inactive under anaerobic conditions but, in a medium without a nitrogen source, it brings about duplication of the cell number, the overall dry weight being maintained (VranQand Fencl, 1966,1967). Hence it follows that the substance is capable of separating the processes of growth and cell division in that it accelerates budding while the biomass increase is constant. I n this manner, it apparently interferes with the timing

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processes participating in the sequence of processes associated with cell reproduction.

V. Ageing of Cells and Age Distribution in a Population A. MAXIMUM REPRODUCTIVE CAPACITY OF AN INDIVIDUAL CELL Barton (1950), Bartholomew and Mittwer (1953) and Streiblov&and Beran (1963b, using the fluorescence method) established t,hat the scar structure is permanent and that a new bud can never be formed at the site of an old scar. Hence it follows that every mother cell can produce only a limited number of generations. The maximum number of generations can be defined by the surface area of the cell. Calculations of this iheoretical maximum using data obtained by fluorescence microscopy (Beran et al., 1966a) generally agree with the figure of Bartholomew and Mittwer (1953) who assumed that the cell surface might be saturated by 100 scars. Factors limiting the maximum production capacity are not known but it appears that the cell surface area is not one of them. According to Chung and his coworkers (1965), the wall of the mother cell does not grow during budding, which is in agreement with the calculation of the free surface of cells bearing different numbers of scars. According to this calculation, the original surface area of a mature scar-free cell is maintained throughout the life span of the cell, and the total surface area increases by the scar area which is incorporated into the original cell wall of the mother cell. Barton (1950), Bartholomew and Mittwer (1953) and Mortimer and Johnston (1959) found that the size of the mature cell increases during every budding cycle. An alternative role for the cell wall in limiting the maximum productivity might lie in the assumption that a greater wall area of the mother cell takes part in budding than is in the subsequent area of the scar. This would also explain the patterns sometimes observed under the electron microscope in chemically treated walls (Fig. 13). The fluorescence-microscope method has so far revealed a maximum number of thirty scars per cell but, in actual fermentations, the number of generations is limited by cultivation conditions. Mortimer and Johnston (1959) and Johnston (cited in Cook, 1962) separated the forming daughter cells from the mother cell with a micromanipulator and found as many as fifty buds. According to Johnston (1966)the maximum reproductive capacity can represent a property of the strain which might be accounted for by genetic factors. Without drawing any definite conclusions, it appears that the maximum reproductive capacity of an individual cell of S. cerevisiae is limited and that the population consists of individuals that can be grouped according to the number of scars. The 8. cerevisiae cell thus represents a model of ageing of a microbial cell which satisfies the concept of ageing by DNA replication (Beran,

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1967).Cells may be designated accordingly as being of a different “divisional age” (Maruyama and Hayashi, 1966).The cell culture as a whole is then a population model composed of cell groups of various ages, the distribution of which in populations grown under various conditions can be estimated by the fluorescence method. Also, in combination with a method involving fractionating the yeast-cell population in a dextran gradient (Lieblovh et aZ.,1964), which makes it possible to isolate cells

FIG. 13. Electron micrograph of a metal-shadowed surface of a chemically treqted cell wall of Saceharomyees cerevisiae. The cell-walI preparation was the same as described in the caption for Fig. 12. The isolated cells were extracted by a short boiling in dilute solutions of NaOH and HC1. Shadowing was with carbon and chromium, the latter a t an angle of 30”. Magnification about x 20,000.

that have not yet produced a daughter generation and to accumulate cells of higher age, the fluorescence method facilitates the confirmation of physiological differences existing between groups of cells of various age. This type of study was stimulated by the finding that cells differ from one another not only in the number of their scars and in their size but also in the ratio of the principal cell components (LieblovB and Beran, 1965). Since, in this population, cells are compared on the basis of their number of scars, the age thus defined was designated as the “relative cell age” and

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a group of cells of the same relative age was described as an “age category’’ (Beran et al., 1966b).

B. AGE DISTRIBUTION IN A POPULATION The problem of age distribution in a yeast population was touched upon by Hough (1961) and Cook (1962) who suggested the possibility oj following microscopically the number of scars on isolated yeast-cel: walls. In batch fermentations using excess nutrients, and under glucosc limitation in a continuous cultivation using synthetic media, aerated and I D E A L SCHEME FOR POPULATION FORMATION Time interval

0 __

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0

1

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3

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500002500012500 6250 3125

5

6

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9

10

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1563 0781 0391 0195 0098 0 0 4 9 0 0 2 4

< t--

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stirred, we have applied the fluorescence method using 8.cerevisiae (Beran et al., 1966a, b; Beran, 1967). From an examination of an ideal population growth (Pig. 14), when it is assumed that all cells have the same generation time, one can obtain a view of the gradual attainment of the relative cell age on passing from one age category to another, which is accompanied by the production of cells of zero category, and finally an ideal age distribution in such a population. This distribution forms a geometric progression where one half of cells are in the zero category, and the frequency of every subsequent category is one-half of the preceding one. From such a formulation of population growth, it follows that we are dealing with a relatively simple process for which equation (1) has been presented, the equation describing the structure of a population when the frequency distribution of the individual categories in a population is in a steady state.

=- 9n-1

Pa-I

a +g n

In this equation, g is the generation time, p the frequency of the age categories, and a is a constant when the frequencies are in a steady state. The indexes refer to the relative cell age. The equation shows at the same time that, on the basis of frequencies, one may study the growth kinetics of cells of the individual age categories. I n connection with the fact that, during cell growth in the presence of excess nutrients, all cells reproduce at an approximately equal rate, there is some interest in the finding that the time dependence of frequencies of the age categories shows a damped oscillation (Fig. 15).This might mean that cells of different categories respond by a different alteration of their growth rate in response to the cultivation conditions. During growth limited by glucose in a continuous cultivation, one can clearly separate cells of the zero category, the frequency of which rises in proportion with decreasing dilution rate. Their value for the specific growth constant is thus always less than the correspondingvalue of the dilution rate ;but the specific rate of their formation is always higher (Fig. 16). It follows from an interpolation of the curves that this category should cease to reproduce at a certain low dilution rate. Similar results were obtained when investigating changes in age distribution in the course of a transition state passing from a lower to a higher dilution rate (Beran et al., 1966b). Several conclusions and assumptions can be drawn from the growth kinetics of cells of different age categories concerning their significance 10

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FIG.15. Distribution of relative ages in a batch fermentation of Sccccharomycea cerevisiae with the exponential phase of growth prolonged. Curves 0-4 (left-hand ordinate scale) indicate the frequencies of cells of categories 0-4. Curve a (righthand ordinate scale) indicates the total cell count,

for the population (Beran et al., 1966a) and for continuous cultivation (Beran, 1967). (1) If the physiological state of the culture as a whole under given cultivation conditions represents a statistical set of physiological manifestations of individual cells, then from the point of view of age

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distribution it is defined predominantly by the physiologicalstate of those age categories which predominate in the population. The frequency distribution curve of the age categoriesis generally J-shaped (Fig. 17) aswas

t

0.55

-

0.50

-

0.45

-

040-

u

al

e

e

0.35-

f

3

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-

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025-

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00501

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I

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I

I

I

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0.15

0.20

0.25

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I

0.40 0.45

Dilution rate (D)

FIG.16. Relationship between p (curve 1)or the specific formation rate (curve 2) of cells of zero category and the dilution rate, D. The scale for p = K; K indicates the specific formation rate of cells of zero category.

also found for bacteria by Powell (1956). This indicates that young cells predominate in the population. The fact that the mother cell can produce several tens of generations and then stop its reproduction justifies the above views on ageing; but, at the same time, the very low frequencies of

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the higher age categories indicate that, if there is a physiological difference between the old-andthe young cells, its effect on the overall population is negligible. Thus cells of the fifth age category which, in an ideal distribution should represent 1.56% of the population, will affect the overall distribution of cell age by less than 1%. (2) The physiological state of the culture is thus determined by cells of

3 Number of scars

FIG.17. Frequency curves of the relative age distribution. Full circles are data for cells grown in continuous cultivation (D=0.15); open circles, cells grown in a medium containing excess nutrients.

low age categories, and above all by categories 0 and 1 which represent about SOY0 of the population. These ceIls possess the property that the maintenance of the whole population in permanent proliferation depends on their reproduction ; they were therefore named “population carrier categories” (Beran et aZ., 1966a). (3) The view that young cells are decisive for the physiology of the whole population is in contradiction with the finding of a rising frequency

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of cells of the zero category with increasing glucose limitation in a continuous cultivation. It is known that the activity of continuous cultures is generally decreasedunder such conditions and that the opposite would be expected. This leads us to the following conclusions: (a) Cells of this category differ from other cells in the degree of their development. Since these cells are smallest and, from this point of view, rather heterogeneous, they must first conclude their development before they can give rise to a new generation. I n view of the fact that, with excess substrate, they reproduce at the same rate as cells of other categories, they differ from them probably in the higher synthetic activity of their cell components. Some partial results support this view (Lieblovft and Beran, 1966). However, under conditions of substrate limitation, they cannot put their activity to any use, and this is reflected in the lower value of their specific growth constant. (b) It can be assumed that the distribution of the relative yeast-cell age in a continuous cultivation has no fundamental influence on the physiological state of the culture and that other factors are probably in play. The cumulative expression of these factors, under otherwise identical cultivation conditions, is probably a different growth rate. The fact that more than one-half of the cells limited by glucose slow down the rate of reproduction suggests, in connection with the view advanced under (a), that the energy substrate limit probably interferes with their development and hence can affect the physiological state of the whole population. REFERENCES Agar, H. D. and Douglas, H. C. (1955).J . Bact. 70, 427. Agar, H. D. and Douglas, H. C. (1957).J . Bact. 73, 365. Bacon, J. S. D., Milne, B. D., Taylor, I. F. and Webley, D. M. (1965). Biochem. J . 95, 28C. Bacon, J. S. D., Davidson, E. D., Taylor, D. J. and Taylor, I.F. (1966). Biochem. J . 101, 36C. Bartholomew, J. W. and Mittwer, T. (1953). J . Bact. 65, 272. Bartholomew, J. W. and Levin, R. (1955). J . gen. Microbiol. 12,473. Barton, A.A. (1950). J . gem. Microbiol. 4, 84. Bell, D. J. and Northcote, D. H. (1950).J . chem. SOC.1944. Bender, H. (1963). Archiv.f. Mikrobiol. 45, 407. Beran, K. (1967). Mitt. versuchst. Gav.21, 101. Beran, K.,StreiblovB,E. and Pokornf, V. (1964). FoZia Microbiol. 9,358. Beran, K., StreiblovB, E. and LieblovB, J. (1966a). I n “Recent Progress in Yeast Physiology, Biochemistry, Immunology and Pathogenicity”, (A.KockovB, ed.), Publishing House of the Slovak Academy of Sciences, Bratislava. Beran, K., MBlek, I.,StreiblovB,E. and LieblovB, J. (196613).In “Symposium on Microbial Physiology and Continuous Culture”, (E. 0. Powell, ed.), p. 57, H.M.S.O., London.

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Bradley, D. ( 1957).Inc‘ElectronMicroscopy”, (F.S. Sjostrand and J. Rhodin, eds.), p. 268, Almqvist and Wiksell, Stockholm. Brown, C. M. and Hough, J. S. (1966). Nature, Lond. 211, 201. Chung, K. L., Hawirko, P. Z. and Issac, P. K. (1965). Camd. J. Microbiol. 11,953. Cook, A. H. (1962). Mitt. Versuchst. Gar. 12, 169. Davies, R. and Elvin, P. A. (1964). Biochem. J. 93, 8P. Eddy, A. A. and Williamson, D. H. (1957). Nature, Lond. 179, 1252. Eddy, A. A. (1958). Proc. Roy. SOC. B, 149, 425. Eddy, A. A. and Rudin, A. D. (1958). J. Inst. Brew. 64,19. Eddy, A. A. and Williamson, D. H. (1959). Nature, Lond. 183, 1101. Falcone, G. and Nickerson, W. J. (1956). Science 124, 272. Falcone, G. and Nickerson, W. J. (1958). In “Biochemistry of Morphogenesis”, (W. J. Nickerson, ed.), Vol. 8, p. 65, Proc. I V t h . Int. Congressof Biochem., Vienna. symp. VI. Freifelder, D. (1960).J . Bact. 80, 567. Frey-Wyssling, A. (1959). “Die pflanzliche Zellwand”, Springer-Verlag, Berlin. Gorman, J., Taruo, P., La Berge, M. and Halvorson, M. (1964). Biochem. Biophys. res. Commun. 15, 43. Hagedorn, K. (1964). Protoplasm 58, 274. Hase, E., Mihara, S. and Otsuka, H. (1959).J.gen. appl. Microbiol. 5,43. Holden, M. and Tracey, M. V. (1950). Biochem. J . 47, 407. Holter, H. and Ottolenghi, P. (1960). Compt. rend. Lab. Carlsberg 31,409. Hough, J. S. (1961). J. Inst. Brew. 67, 494. Houwink, A. L. and Kreger, D. R. (1953). Antonie wan Leeuwenhoek 1 9 , l . Johnston, J. R. (1966). Antonie wan Leeuwenhoek 32,94. Kessler, G. and Nickerson, W. J. (1959).J. biol. Chem. 234,2281. Korn, E. D. and Northcote, D. H. (1960). Biochem. J. 75,12. LieblovB, J. and Beran, K. (1966). I n “Recent Progress in Yeast Physiology, Biochemistry, Immunology and Pathogenicity”, (A. KockovA, ed.), Publishing House of the Slovak Academy of Sciences, Bratislava. LieblovB, J.,Beran, K. and StreiblovB,E. (1964). F o l k Microbiol. 9,205. LieblovB, J. and Beran, K. (1965). Fed. Europ. Biochem. SOC.,2nd. Meeting. Abstracts A 269, 183. Lindegren, C. C. (1945). Mycologia 37, 767. Lindegren, C. C. and Haddad, S. A. (1953). Ezp. cell Res. 5,549. Marquardt von, H. (1962). 2eitsch.f. Naturforschung 10, 689. Maruyama, Y. and Hayashi, K. (1966). J.ferment. Technol. (Japan) 44,227. McClary, D. 0. and Bowers, W. D. (1965). C a d . J. Microbiol. 11,447. McMurrough, I. and Rose, A. H. (1967). Biochem. J. 105, 189. Meissel, M. N., Medvedeva, G. A. and Alexeva, V. M. (1961). Mikrobiologiya 30,855. Millbank, J. W. and MacRae, R. M. (1964). Nature, Lond. 201, 1347. Mortimer, R. K. and Johnston, J. R. (1959). Nature, Lond. 183, 1751. Mundkur, B. (1960). E z p . cell Res. 20, 28. Mundkur, B. (1963).Naturforschung 18, 1073. NeEas, 0. (1956). Nature, Lond. 177, 898. NeEas, 0. (1961). Nature, Lond. 192, 580. NeEas, 0. (1962). Polia Biol. (Praha) 8, 256. Nedas, 0. (1965a). Folia Biol. (Praha) 11, 97. NeEas, 0. (1965b).I n “The Cell Regeneration”, (0.NeEas, ed.), Vol. 14, p. 155, Acta Facultatis Medicae Universitatis Brunensis, Brno. NeEas, 0. and Svoboda, A. (1967). Folia Biol. ( P r a h ) ,13, 379.

BUDDING OF YEAST CELLS, THEIR SCARS AND AaEING

171

Nickerson, W. J. and Falcone, G. (1956a).Science 124, 722. Nickerson, W. J. and Falcone, G. (1956b). Science 124, 318. Nickerson, W. J. and Falcone, G. (1959). I n “Sulfur in Proteins” (R. Benesh, ed.) p. 409, Academic Press, New York. Nickerson, W. J., Falcone, 0. and Kessler, G. (1961). In “Macromolecular Com plexes” (M. V. Edds, ed.), p. 205, The Ronald Press Comp., New York. Nickerson, W. 5.(1963). Bact. Rev. 27, 305. Nickerson, W. J. (1964). In “Cellular Membranes in Development” (M. Locke, ed.) p. 281, Academic Press, New York. Northcote, D. H. and Horne, R. W. (1952). Biochem. J . 51,232. Northcote, D. H. (1953). Biochim. Biophys. Acta 11,471. Nurminen, I., Oura, E. and Suomalainen, H. (1965).SuomenKemistilehti3 38,282 Phaff, H. J. (1963). Annu. Rev. Microbiol. 17, 15. Powell, E. 0. (1956).J . gen. Microbiol. 15, 492. Roelofsen, P. A. and Hoete, I. (1951). Antonie wan Leeuwenhoek 17, 297. Robson, E. I. and StocMey, M. H. (1962).J . gen. Microbiol. 28,57. Sentandreu, R. andVillanueva, J. R. (1965).Archiv.f.Mikrobiol. 50,103. StreiblovB, E. and Beran, K. (1963s). Exp. cell Res. 30,603. Streiblovi, E. and Beran, K. (1963b). Folia Microbiol. 8,221. StreiblovB, E., Beran, K. and Pokorny, V. (1964). J . Bact. 88,1104. StreiblovB, E. and Beran, K. (1965). Polia Microbiol. 10, 352. StreiblovB, E., Milek, I. and Beran, K. (1966).J . Bact. 91,428. Streiblovit, E. (1966a). In “Recent Progress in Yeast Taxonomy, Cytology Ecology and Genetics” (A. KockovB, ed.), Publishing House of the Slovak Academy of Sciences, Bratislava. StreiblovB, E. (1966b). IXth. Int. Congress of Microbiol., Moscow. Abstract oj Papers, p. 73. StreiblovB, E. ( 1 9 6 6 ~ ) .Thesis: Inst. of Microbiol., Czechoslovac Academy oj Sciences, Prague. StreiblovB, E., Svoboda, A. and NeEas, 0. (1967). In “Symposium on Yeast Proto, plast” (R. Miiller, ed.), p. 91, Akademie Verlag, Berlin. Sutton, D. D. and Lampen, J. 0. (1962). Biochim. Biophys. Acta 56, 303. Svoboda, A. (1966). Exp. cell Res. 44, 640. Svoboda, A. and Nedas, 0. (1966). Natzlre, Lond. 210,895. Svoboda, A. (1967). I n “Symposium on Yeast Protoplast” (R. Miiller, ed.), p. 81 Akademie Verlag, Berlin. Tauro, P. and Halvorson, H. 0. (1966).J . Bact. 92,652. Vitols, E. R., North, J. and Linnane, A. W. (1961). J . biochem. biophys. Cytol. 9 689. Vrani, D. andFencl, Z. (1964).Polia Microbiol. 9,156. Vrani, D. and Fencl, Z. (1966). In “Recent Progress in Yeast Physiology, Bio chemistry, Immunology and Pathogenicity” (A. KockovB, ed.), Publishing House of the Slovak Academy of Sciences, Bratislava. VranB, D. and Fencl, Z. (1967). Folia Microbiol. 12,432. Ward, J. M. (1958). I n “Biochemistry of Morphogenesis” (W. J. Nickerson, ed.) Vol. 8, p. 33, Proc. IVth. Int. Congress of Biochem., Vienna. Symp. M. Watanabe, Y. and Ikeda, M. (1965a). Exp. cell Res. 39,443. Watanabe, Y. and Ikeda, M. (196510).Exp. cell Res. 39,464. Ziegenspeck, H. ( 1949). In “Beitrage zu Fluoreszenzmikroskopie”, G. F r o m e Co., Wien.

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The Repair of Damaged DNA in Irradiated Bacteria B. E. B. MOSELEY Department of General Microbiology, University of Edinburgh, Scotland

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I. Introduction . . 11. Evidence that DNA is an Important Target in Radiation Inactivation 111. Radiation-Induced Chemical and Physical Changes in DNA . . A. Changes in DNA Caused by Ultraviolet Irradiation . , B. Changes in DNA Caused by Ionizing Irradiation . . IV. The Repair of Damaged DNA . . A. Photoreactivation . . B. DarkRepair . . V. Summary . . References . ,

173 174 176 176 181 183 183 187 191 192

I. Introduction Vegetative bacteria show enormous differences in their response to the lethal action of ultraviolet (u.v.) and ionizing radiation (Fig. 1). For example, the dose of U.V. radiation required to inactivate 90% of a suspension of a mutant of Escherichia coli is less than 1 erg/mm.2 (Hill, 1958)whilst a dose of 7000 ergs/mm.2is needed to achieve the same effect with Micrococcus radiodurans (Setlow and Duggan, 1964). A similar situation exists with ionizing radiation. Thus the 90 %-inactivating dose for various Pseudomonas spp. is less than 2 Krad (Kaplan and Zavarine, 1962),whilst for M. radioduransitis 750 Krad (Moseley and Schein, 1964). Even quite recently, attempts were made to explain this variation in resistance to ionizing radiation in terms of cellular targets which had to be irreversibly damaged by the radiation. Calculations were made of the sizes of such targets and the number of “hits” which were required to cause such damage (Oliver and Shepstone, 1964).No consideration was given to the possibility of repair. However, in the last few years the concept of repair of radiation damage in bacterial DNA has emerged. This explains, t o a large extent, why some bacteria are resistant to ionizing and U.V. radiation, and incidentally to other agents which 173

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damage DNA, e.g. nitrogen mustards, mitomycin C, and N-methy1-N’nitro-N-nitrosoguanidine.

00-0

0

Ultraviolet dose (ergs rnrn-’l

(4

500

1000

1500

2000

Ionizing rodiotion dose lkrads)

(b)

FIU.1. Survival curves to illustrate the extremes in the response of vegetative bacteria to irradiation. (a)Ultraviolet-radiation survival curves : (i) Escherichia coli B,-l (Hill, 1958), (ii) Micrococcus radiodurans (Setlow and Duggan, 1964). (b) Ionizing-radiation survival curves : (i) Pseudomonas aeruginosa (Kaplan and Zavarine, 1962), (ii)N . radiodurans (Moseley and Schein, 1964). Note the different scales on the figures.

This article will describe the evidence for DNA being considered the most important target involved in the radiation inactivation of bacteria, the types of damage in DNA caused by radiation which are biologically relevant, and the different mechanisms possessed by bacteria to repair such damage.

11. Evidence that DNA is an Important Target in Radiation Inactivation Evidence from a wide range of experiments implicates DNA as the principal target in the bacterial cell. When bacterial or mammalian cells are grown under appropriate conditions in the presence of certain pyrimidine or purine analogues, they incorporate into their DNA varying amounts of these analogues instead of their corresponding natural bases (Dunn and Smith, 1954; Zamenhof and Griboff, 1954). Such cells, containing appreciable amounts of analogues, are abnormally sensitive to both U.V. and ionizing radiation (Kaplan and Tomlin, 1960; Kaplan et al., 1961; Opara-Kubinska et al., 1961). Using halogenated

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thymidine analogues, Opara-Kubinska et aZ. (1961) were able to show that both Bacillus subtilis cells and transforming factor (DNA) isolated from them were sensitized to the same degree, and they concluded from this that DNA is the principal and indispensable target for radiation effects in bacteria. A different approach to this problem was used by Kaplan and Zavarine (1962) who examined the relationship between the base composition of DNA in eight species of bacteria and their resistance to X-radiation. On plotting the D,, values (the dose required to cause 90 yoinactivation) from the exponential part of the survival curve against the guanine +cytosine (GC) content, a linear relationship was obtained in which resistance and GC content were inversely related, that is, resistance increased with lowering of the GC content. This was shown to apply to bacteria with GC contents varying between 34 and 67 yoof the total bases. DNA base composition is also correlated with bacterial sensitivity to U.V.radiation but in an inverse fashion, i.e. resistance of bacterial species increases with lowering of the adenine +thymine (AT) content (Haynes, 1964). This is due to the main component of lethal damage being the formation of a thymine dimer between adjacent thymine bases on the same DNA strand. The chances of such dimers being formed is, of course, dependent on the AT content of the DNA (see section on pyrimidine dimers, page 179). From the above it would follow that, if the base ratio of DNA were the sole determinant of radiation sensitivity (or resistance) in bacteria, then extreme resistance to both ionizing and U.V. radiations should be incompatible in the same bacterium (Moseley and Schein, 1964). This is not the case, and the most notable exception is Micrococcus radiodurans, originally isolated by Anderson et aZ. (1956), which is the most resistant vegetative bacterium known to both ionizing and U.V. radiation. Its DNA contains 67 yoGC, which should make it one of the most sensitive of bacteria to ionizing radiation. Nevertheless, from an investigation of the response of this bacterium to mixed doses of ionizing and U.V. radiation, it was concluded that the main target for damage was the DNA (Moseley and Laser, 1965) and that it was the possession of a very efficient enzymic repair mechanism for both ionizing and U.V. radiation damage which made it an exception to the rule. I n some respects, Kaplan and Zavarine (1962) were lucky in the choice of their organisms since many mutants have been obtained which do not fit their scheme. All have repair mechanisms which modify the initial radiation damage. Further evidence for considering DNA as the site of radiation damage are the action spectra for U.V. killing (that is plots of the efficiency of u.v. ~ i aand other bacteria. These killing against wavelength) of ~ s c ~ e r i ccoZi are similartonucleic acidabsorptionspectra(Gates,1930; Wyckoff, 1932).

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The experiments described provide mainly indirect evidence for the involvement of DNA in the lethal effect of radiation but the remarkable success of many experiments based on this view enhances its credibility.

III. Radiation-Induced Chemical and Physical Changes in DNA Investigations to identify the chemical changes in the DNA of bacteria, irradiated at dose levels yielding 10-100% survival of cells, are made difficult by the large size of the DNA molecule and the small number of nucleotides involved in lethal damage. Por example, in the DNA of a typical bacterium one lethal hit would produce perhaps only one damaged nucleotide in 3 x l o 6 and, since DNA cannot be handled conveniently in solution at concentrations greater than 2-5 mg./ml., it would be necessary to detect an unknown product at a concentration of pg./ml. in the presence of an enormous background of chemically similar compounds (Freifelder, 1966).It has been common practice therefore to use very high doses of radiation, well beyond the biologically relevant dose range, in order to raise the concentration of damaged molecules to measurable levels, with the resulting problem of assessing the relative biological importance of the observed product. A different approach is to detect chemical changes in DNA by their physical consequences. For example, one chain-break in a long polymer is detectable in that it results in a large change in molecular weight. However, a problem arises with DNA in that, during its isolation from irradiated cells, a substantial number of breaks are caused by hydrodynamic shear forces with the result that the sample contains many more breaks than are caused by the radiation. Nevertheless methods are being developed which considerably decrease the magnitude of these errors and give valuable information (Freifelder, 1966; McGrath and Williams, 1966).

IRRADIATION A. CHANGESIN DNA CAUSEDBY ULTRAVIOLET 1. Chain Breaks

To detect u.v.-induced single-chain breaks, the DNA has to be denatured to separate the strands. The molecular weights of the samples can then be estimated from their sedimentation coefficients. The dose of u .v. required to decrease the molecular weight of Diplococcus pneumonia DNA by 50 "/,is about 2 x lo5ergs/mm.2,while that required to decrease the transforming activity of the streptomycin marker in this DNA t o the same extent is 100-fold less (Marmur, et al., 1961). At the dose required to kill 99 yoof a population of phage T7, no chain breaks were

THE REPAIR O F DAMAGED DNA IN IRRADIATED BACTERIA

177 detected (Freifelder and Davison, 1963a). This evidence would suggest that chain breaks do not contribute to the biological effects of U.V. radiation. 2. DNA Cross-Links DNA strands can be separated by heating or treating with formamide. When DNA in solution is irradiated, strand separation is prevented, apparently because of cross-linking caused by the action of the U.V. radiation (Marmur and Grossman, 1961).More cross-links are produced if the DNA is heated during irradiation up to a temperature at which denaturation is 20 yocomplete but, with further heating, the amount of cross-linking decreases (Glisin and Doty, 1962). Thus a disturbance in the local structural organization of DNA favours cross-linking but, when strand separation begins, no further links are formed. The chemical nature of the cross-links remains unknown although for a given dose of U.V. the extent of cross-linking is proportional to the AT content of several DNAs. Thus it was suggested that some type of thymine dimer might be responsible (Marmur et al., 1961). However Opara-Kubinska et al. (1963) found more cross-links with increasing substitution of 5-bromouracil for thymine in DNA, indicating that thymine dimers cannot be the only cause of cross-linking. The possible biological role of DNA cross-linkingwas suggested by the results of Marmur and Grossman (1961). They found that cross-links could be detected in the DNA isolated from Bacillus subtilis irradiated with doses which inactivate transforming factor. However, this DNA is very resistant to radiation, 0.05 % of the original biological activity remaining even after a dose of lo6 ergslmrn.' at 260 mp (Haug and Goes, 1963).It is possible that some of the biological effect on this DNA at such doses could be due to the formation of cross-links,but on the other hand transforming DNA is not exceptionally resistant to cross-links produced by mitomycin (Iyer and Szybalski, 1963). Cross-links do not appear to be involved in the radiation-inactivation of phage. At doses which inactivate 99 yoof phage T7, no cross-links could be detected and there was approximately one cross-link per molecule only when the survival was down to lo-* (Freifelder and Davison, 1963a). The doses required to produce intrastrand cross-linksare large (about l o 6 ergs/mm.2 causing an average of one cross-link per phage particle; Glisin and Doty, 1962) whereas most bacteria are inactivated at much lower doses, some as low as 1 erg/mm.2 (Hill, 1958). This would exclude cross-linking as a contributing factor to the biological effects of U.V. inactivation in these cases. The exception might possibly be in the case of u.v.-resistant transforming factor.

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3. Nucleic Acid-Protein Cross-Links

There is a progressive decrease in the amount of DNA that can be extracted with detergent from bacteria following increasing doses of U.V. (Smith 1962). This loss of extractability occurs with doses of U.V. that are biologically significant. Thus, at the 99% killing dose for Escherichia coli B/r (1800 ergslmm.’), 11 %of the DNA is not extractable. This amount of DNA can be quantitatively accounted for in the precipitate of denatured proteins and can be recovered after treatment of the precipitate with trypsin. These data suggest that the DNA cross-links to protein. Additional evidence for the cross-linking of DNA and protein is provided by an in, vivo model, in which mixtures of uracil and cysteine, when irradiated, JPield a mixed photoproduct (Smith and Aplin, 1966). The photochemical addition of [35S]cysteineto poly-U, poly-C, and DNA has also been demonstrated (Smith, 1966). However, in spite of the apparent cross-linking of DNA to protein at biologically significant doses of u.v. radiation, the action spectra for U.V. killing of 3.coli and other bacteria (Gates, 1930; Wyckoff, 1932) suggest that nucleic acid and not protein is the absorbing material, whereas protein is the more important target in DNA-protein cross-linking (Smith, 1964). Micrococcus radiodurans may be an exception. The action spectrum for the inactivation of (and for the inhibition of DNA synthesis in) this organism indicates that u.v., at a wavelength of 280 mp, is as efficient as that at 260 mp suggesting that killing in this organism may be due as much to protein damage as,to DNA damage. It is suggested that the repair of DNA damage (e.g. elimination of thymine dimers; see Section 5, p. 179) is so efficient that eventually inactivation of the bacterium is due to some form of protein damage (Setlow and Boling, 1965). Thus the role of DNA-protein cross-linking in U.V. inactivation is not clear, but it would seem to be unimportant except in the case of extremely resistant bacteria such as M . radiodurans. 4. Hydration of Cytosine and Uracil

When uracil is irradiated, it undergoes a chemical alteration accompanied by a loss of U.V. extinction which is reversed by heat or acid treatment (Sinsheimer and Hastings, 1949). Similar results have been obtained with uridine and cytidylic acid. It was suggested that the photoproduct of uracil was formed by hydration of the double bond between C-5 and C-6 (Sinsheimer, 1954) and the synthesis of 6-hydroxy5-hydrouracil with identical properties proved this (Moore, 1958). By

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analogy, the reversible photoproducts of the other compounds are believed to be hydrates at the same position (Shugar, 1962). When cytosine compounds are irradiated, their characteristic extinction peak a t 270 mp disappears and a new one appears at 236 mp. This peak is destroyed by heat or acid treatment of the compound with the reappearance of the original peak (Sinsheimer, 1957). It was this new peak which enabled Setlow and Carrier (1963) to demonstrate the existence of heat-reversible cytosine hydrate in irradiated denatured DNA. However, irradiated native DNA showed no such change. Similarly, although evidence for reversible hydrate formation was obtained in irradiated polyribocytidylic acid (poly-rC), none was found for its formation in the homocopolymer composed of polydeoxyinosinic acid hydrogen bonded to polydeoxycytidylic acid (poly d I :dC; Wierzchowski and Shugar, 1962; Setlow et al., 1965a). This suggests that pyrimidine hydrates a.re not formed in irradiated double-stranded DNA but are formed in single-stranded DNA. Since the formation of pyrimidine hydrates by irradiation is reversible by heat it might be expected that, if the formation of these compounds contributed towards the biological effect of u.v. inactivation, then heating cells after irradiation should decrease inactivation. Such a clear-cut effect would be partially concealed by the effect of heat on other aspects of cell physiology, for example, repair enzymesand DNA synthesis ;indeed cells have been found to be reactivated (Anderson, 1949), unaffected (Anderson, 1949), and sensitized (Setlow, 1959) by post-irradiation heat. Noncellular systems, such as transforming factor from Haemophilus inJuenzae irradiated at 85" (Setlow and Setlow, 1961) and pneumococcal DNA irradiated at 58" (Lerman and Tolmach, 1959), are inactivated at the same rate or only slighly faster than at room temperature. Some irradiated phages may be reactivated by heat but only after adsorption to host bacteria, indicating a cellular mechanism for the effect (Bresch, 1950; Shugar, 1962). These results, and the fact that pyrimidine hydrates are not formed in double-stranded DNA, suggest that pyrimidine hydrates are not an important contributor to U.V. inactivation in these systems.

5. Pyrimidine Dimers On U.V. irradiation, frozen solutions of thymine were found to lose their characteristic U.V. extinction (Beukers et al., 1958) and a thymine photoproduct was isolated by extraction with ethanol and recrystallization from water (Beukers and Berends, 1960). It was identified as a dimer from information obtained by elementary analysis, molecular weight determinations, crystallographic analysis and infrared spectra, and indicated that the two thymine molecules were linked to each other

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by carbon-to-carbon bonds between their respective C-5 and C-6 atoms, forming a cyclobutane ring between the two thymines (Fig. 2 ) .

I \N/ H ' H I

I1

I11

FIG.2. Formation of a thymine dimer. I: thymine; 11: thymine dimer; 111: conformation of dimer in DNA.

There is a wavelength dependency for the formation of, and the monomerization of, thymine dimers, so that an equilibrium is reached between the monomer and dimer which is characteristic for each wavelength, At long U.V. wavelengths (about 280mp) formation of the dimer is favoured while, at shorter wavelengths (around 240 mp), monomer formation is favoured (Setlow, 1961; Wang, 1961). This feature of thymine dimers has been used to implicate them in the biological effect of U.V. irradiation. If DNA is irradiated with 280 mp (more than l o 4 ergs/mm.2) so that the number of dimers is considerably greater than would be formed at 240 mp, and this is followed by irradiation at 240 mp, the number of dimers may be decreased without apparently decreasing the number of other photoproducts (Setlow and Setlow, 1962). Unfortunately most bacterial cultures are completely inactivated by U.V. doses of l o 4ergs/mm.2,but transforming DNA from H.injuenzae is relatively resistant and has measurable biological activity at doses of 280 mp radiation well above l o 4 ergs/mm.z. When this initial treatment is followed by 240 mp radiation, which decreases the numbers of dimers in DNA, some biological activity is restored. The kinetics of biological reactivation and the elimination of dimers are quantitatively similar, and provide evidence that dimers, formed on 280 mp irradiation, are responsible for a large part of the U.V. inactivation of transforming DNA (Setlow and Setlow, 1963).

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Attempts to demonstrate short-wavelength reversal of thymine dimer damage in bacteria have not been successful. It is necessary, because of the large initial dose of 280 mpradiation, to use a very radiation-resistant bacterium such as M . radiodurans. However, short-wavelength reversal cannot be demonstrated in this bacterium (Setlow and Duggan, 1964) possibly because the repair mechanism for thymine dimer damage is so efficient that the initial level of dimer formation is irrelevant for survival (Setlow and Boling, 1965). Other evidence for the role of pyrimidine dimers in U.V. inactivation comes from in vitro DNA synthesis experiments. DNA, u.v.-irradiated at 280 mp, fails to act as the primer in the enzymic synthesis of DNA in vitro. The loss of priming ability can be restored by 240 mp radiation, indicating that thymine dimers act as a block to DNA synthesis (Bollum andsetlow, 1963). In addition to the formation of thymine dimers, evidence for four other types of pyrimidine dimer has been obtained. They are dimers of cytosine (Wacker, 1963; Setlow et al., 1965a), uracil (Wang, 1961; Smietanowska and Shugar, 1961;Smith, 1963),uracil-thymine (Beukers and Berends, 1960; Wacker et al., 1961; Smith, 1963) and cytosinethymine (Setlow et aZ. 1965b). I n most of the investigations to be described, thymine dimers have been isolated by chromatographic procedures which do not distinguish between thymine dimers and uracilthymine dimers (Setlow et al. 1965b).Thus, what is assayed is a mixture of thymine dimers and uracil-thymine dimers, the latter having been formed from cytosine-thymine dimers during the isolation procedure. The role of these mixed dimers and cytosine-cytosine dimers in biological inactivation is not well defined. They are all capable of being monomerized by photoreactivating enzyme, and it is known that cytosinethymine dimers are excised from the DNA of radiation-resistant bacteria (Setlow et al., 1965b). I n these respects they occupy the same role as thymine-thymine dimers. B. CHANGES IN DNA CAUSED BY IONIZING IRRADIATION 1. Chain Breaks

As with U.V. radiation, early work on the effect of ionizing radiation on DNA involved using high doses and extrapolation of the results to biological levels. A repeated observation was that irradiation of DNA with hard X-rays and 1 MeV electrons resulted in a decrease in viscosity (Sparrow and Rosenfeld, 1946; Taylor et aZ., 1948; Butler, 1949; Butler and Smith, 1950)and sedimentation coefficient (Shooter, 1957)suggesting that DNA was depolymerized by the radiation. I n spite of the difficulty of interpreting the results because of the heterogeneity of the molecular

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weight of the DNA which was used, the data made it seem probable that double-strand breakage is a major structural change produced by ionizing radiation. This would certainly contribute to the lethal effects of this type of radiation. The finding that single DNA molecules could be obtained from phage T2 by avoiding hydrodynamic shear (Davison et al., 1961)led Freifelder to an analysis of radiation chain-breakage in phage systems. Using the ultracentrifuge, the resolution was such that, in an initially homogeneous DNA, degradation of 2 yoof the molecules could be detected and hence the method could be used to measure double-strand breakage in the dose-range yielding one break per molecule. Single-strand breakage was measured by heat denaturation of the DNA in the presence of formic acid or in solutions of pH 13 or greater. Under these conditions, the single polynucleotide strands separated and broken and unbroken strands could be resolved (Freifelder and Davison, 1963b; Davison et al., 1964). I n the phage T7 system, double-strand breakage could be correlated with loss of biological viability (Freifelder, 1965). When the phage was irradiated in phosphate buffer, each dead phage particle contained a double-strand break. I n lob3 M-histidine, which simulates broth in which T7 is more resistant to X-rays, the rate of production of double strand-breaks was only 30-40% that for loss of viability. The singlestrand break analysis showed that in histidine, single-strand breaks accumulated about 15-20 times as rapidly as lethal hits, and in buffer each phage particle contained three single-strand breaks at the 95 % survival level. It was concluded that single-strand breaks are not lethal. This agrees with the early 32Pexperiments in which about one in ten disintegrations were lethal in double stranded phage (Stent and Fuerst, 1960). The production of a double-strand break is therefore a lethal advent in phage. An elegant technique devised by McGrath and Williams (1966) has shown this to be true also for E . coli. 2. Base Damage

Strand breakage cannot explain all the lethal effects of ionizing radiation. For example, in the case of T7 phage irradiated in Nhistidine, only a fraction of the loss of viability can be explained in terms of double-strand breakage. The substitution of base analogues such as 5-bromouracil for thymine referred to earlier, which does not increase the rate of strand breakage, sensitizes bacterial DNA (Kaplan and Tolmin, 1960; Kaplan et aZ., 1962; Opara-Kubinska et al., 1961), phage DNA (Stahl et al., 1961) and transforming DNA (Szybalski and Lorkiewicz, 1962)to ionizing radiation. X-Ray sensitivityis also dependent

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upon the base ratio. Finally, the inactivation rate of cells by ionizing, though not by u.v., radiation is oxygen-dependent although there is no oxygen effect on strand breakage in DNA irradiated in vitro (Freifelder, 1966). Although there must be some other form of biological damage to DNA other than that caused by strand breakage, no radiation product in DNA has been characterized. R. B. Setlow and Carrier have found lesions induced in DNA by ionizing radiation which are excised by an extract s in Setlow, 19661, but as yet they remain of M . l y ~ ~ d e i k t i c u(reported unidentified.

IV. The Repair of Damaged DNA A. PHOTOREACTIVATION Kelner (1949a, b) found that, when the U.V. irradiation of Streptomyces griseus conidia, Escherichia coli, Penicillium notatum or yeast cells was followed by exposure to visible light, the organisms had an enhanced ability to survive and form colonies. This phenomenon was called photoreactivation (Fig. 3). It applies only to U.V. radiation damage, there being no correspondingprocess for ionizing radiation damage. An understanding of the molecular processes involved in photoreactivation began when Goodgal et al. (1957) demonstrated that transforming DNA which had been inactivated by U.V. radiation could recover some of its activity when illuminated in the presence of an extract of E. coli. However, because of the presence of nucleases in extracts of E. coli which degrade transforming factor (in competition with the photoreactivation), most of the in vitro studies on photoreactivation have been done with extracts of yeast (Rupert, 1960). Rupert, using yeast extracts, showed that the active agent in photoreactivation is an enzyme which obeys MichaelisMenten kinetics. The enzyme forms a complex with irradiated, but not with unirradiated, DNA in the dark, and the enzyme in this state is protected against inactivation by heat and heavy metals. The complex dissociates in the light but not in the dark and, if the illumination is continued for a sufficient time, the DNA loses its ability to complex with the enzyme (Rupert, 1960, 1961, 1962a, b). These facts are compatible with the enzyme binding to substrate, namely U.V. damage in the DNA, with the subsequent removal of the damage assisted by visible light. The reaction is temperature-dependent in that the rate of photoreactivation of transforming DNA is considerably higher at 37" than at 15", similar to the temperature effect on whole cells (Kelner, 1950). Only one photon is required for the reaction since the amount of reactivation increases linearly with the total number of photons and

- B. E. B. MOSELEY

184

-

U.V.radiation

4

Photoreactivoting enzyme

+ light

...

----h

-A-T

-5

C--G

P.

P Repolyrnerizotion

A

-t--

A C A

.

FIG.3. Alternativemethodsof eliminationof thymine dimersfromDNA. (a)Photoreactivation in which the dimer is monomerized in sih. (b) Dark repair in which the dimer and some adjacent bases are excised and new bases inserted. A indicates an adenine residue; C, cytosine; G, guanine; T, thymine.

is independent of the light intensity. This is true for in wiwo photoreactivation in E. coli (Jagger andLatarjet, 1956)andin witrophotoreactivation of Haernophilus inJEuenzaetransforming factor (Setlow and Boling, 1963). The study of photoreactivation in whole cells is complicatedby another type of photoreactivation described as “indirect” (Jagger and Stafford, 1966). This process can occur in strains of E. COGwhich are incapable of normal photoreactivation (Harm and Hillebrandt, 1962).The two types of photoreactivation, direct and indirect, can be distinguished by the wavelength, the dose and dose-rate of light required t o reverse the biological action of u.v. radiation and also by their different temperature dependencies (Jagger and Stafford, 1965). The wavelength optimum for indirect photoreactivation induces a delay in cell division which is postulated to aid recovery by allowing time for other cellular processes

to operate.

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1. Photoreactivable Damage

Rupert (1962a) established that the photoreactivating system showed Michaelis-Menten kinetics but was not able to specify the nature of the substrate. Wacker (1961) and Wulff and Rupert (1962) found that thymine dimers were removed from DNA by treatment with crude yeast extracts in the light. Final proof of the involvement of thymine-dimers in photoreactivation was provided by Setlow and Setlow (1963) who found that there was an overlap in the biological effect of short-wavelength reversal and photoreactivation. Irradiated transforming DNA, which had been photoreactivated, was not further reactivated by exposure to short-wavelength U.V. radiation, and its photoreactivability was decreased by previous short-wavelength irradiation. They concluded that thymine-dimers are involved in the biological effect of photoreactivation. The nature of substrates for the photoreactivating enzyme has been investigated by Rupert (1961, 1964), using a competition experiment in which irradiated polynucleotides compete with irradiated transforming factor for photoreactivating enzyme in that they decrease the rate of the reactivation of transforming ability. If radiation damage in the synthetic polymers is removed by the photoreactivating enzyme during illumination, their capacity to compete is removed and the damage is considered to be “photoreactivable”. Using this method, Rupert showed that the irradiated synthetic polymer, polydeoxy GC, contained a substrate for the photoreactivating enzyme. Since no thymine dimers could be formed, i t can be inferred that there is a photoreactivable lesion in addition to the thymine dimers. I n confirmation of this observation, E. coli DNA irradiated with increasing doses of short-wavelength U.V. radiation shows a constant content of thymine dimers but an increasing ability to compete for enzyme (Setlow, 1964) indicating that there is a photoproduct other than the thymine dimer available for photoreactivation. Further experiments on competition by a variety of irradiated synthetic polydeoxynucleotides have shown that only those which contain adjacent pyrimidine bases are photoreactivable (Setlow, J. K. et al., 1965). The thymine dimer itself (TpT) and the irradiated (pT)8 do not compete for enzyme (Rupert, 1964). This suggests that the thymine dimer is the substrate for the photoreactivating enzyme only if it is part of a particular structure, possibly in order for the enzyme to bind. Neither irradiated RNA nor synthetic polyribonucleotides are competitors for the enzyme. The kinetics of U.V. induction of competing ability in synthetic polymers agrees with the kinetics of dimer formation (Setlow,J. K. et al., 1965). A constant content of dimers and a constant level of competing ability is produced in poly dA:dT after a short-wavelength irradiation a t

186

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a dose of about 3 x lo4ergs/mm.2The rate of short-wavelength reversal of dimers in poly dI :dC is slower if the irradiated polymer has been heated first, converting the cytosine dimers to uracil dimers. The competing ability of the irradiated polymer is also removed more slowly by shortwavelength reversal. The reason is that the dose for short-wavelength monomerization of uracil dimers is larger than for cytosine dimers. Conversely, both the dimers in, and the competing ability of, the heated polymer are removed more rapidly by photoreactivating enzyme because uracil dimers are more rapidly monomerized by the enzyme than are cytosine dimers (Setlow et al., 1965a). Of the pyrimidine dimers which have been demonstrated or postulated to cause biological damage, thymine dirners are eliminated more rapidly than the other four types, and the mixed dimers cytosine-thymine and uracil-thymine more rapidly than cytosine-cytosine and uracil-uracil dimers (Setlow,J . K. et al., 1965). It is probable that the monomerization of pyrimidine dimers in DNA accounts for the biological effects of direct photoreactivation. 2. Non-Photoreactivable Damage

Photoreactivation has never been observed to restore irradiated cells or DNA to full biological activity. There is always some residual biological damage after photoreactivation has reached a constant level (Jagger, 1958; Rupert, 1960). This occurs even when all thymine dimers and cytosine-thymine dimers have been monomerized. The fraction of damage which is photoreactivable is called the photoreactivable sector (Jagger, 1960). I n E.coli, the photoreactivable sector varies between 0.5 (Jagger, 1960) and 0.8 (Castellani et al., 1964). In H . injluenxae transforming DNA, it is as high as 0.9 (Setlow, 1963). The nature of the non-photoreactivable damage remains unknown. The action spectrum for non-photoreactivable damage in H . injluenxae transforming DNA appears to implicate cytosine (Setlow, 1963) but, since dimers involving cytosine are photoreactivable and cytosine hydrates are unlikely to be involved for reasons given earlier, the problem remains. 3. Occurrence

Following the initial observations (Kelner, 1949a, b) that photoreactivation occurs in griseus, E. COG,P. notatum and Saccharomyces cerevisea,e, it has been found to occur in a wide range of microbes. For example, the photoreactivation of lethal damage occurs in the bacteria Aerobacter aerogenes, Chromobacterium violaceum, Micrococcus pyogenes,

s.

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187

Erwinia carotovora, Proteus spp., Azotobacter chroococcum, Axotobacter “Q” and Xerratia rnarcescens; in the fungi Ustilago maydis, Penicillium chrysogenum and haploid, diploid, triploid and tetraploid strains of Saccharomyces; and in the protozoan Chlamydomonas moewussi (for review see Jagger, 1958). B. DARKREPAIR

It has been known for a long time that the survival levels of irradiated bacteria can be appreciably influenced by post-irradiation conditions that do not involve photoreactivation since they occur in the dark (Hollaender and Claus, 1937). This modification of initial radiation damage has been described in different ways, e.g. reactivation, recovery, restoration and rescue. However i t was the work of R. B. Setlow and his colleagueswhich provided the first experimental evidence for a molecular mechanism for the dark repair of damagedDNA in bacteria (Setlow et al., 1963; Bollum and Setlow, 1963; Setlow and Carrier, 1964). They showed that thymine dimers in U.V. irradiated E. coli B and the radiationresistant mutant E. coli B/r, but not in the extremely radiation-sensitive E. coli. Bs-l, become less susceptible to monomerization by photoreactivation after incubation in the dark even though the dimers are still in the cells. Most of the dimers, however, were no longer in the trichloroacetic acid-insoluble (DNA) fraction of the cell but now appeared in the trichloroacetic acid-soluble fraction as parts of tri- or tetranucleotides and were no longer available to the photoreactivating enzyme. By the time that DNA synthesis resumed in the resistant strains, more than 90% of the dimers had appeared in the trichloroacetic acid-soluble material. In the sensitive strain E. coli, Bs--l, the dimers remained in the trichloroacetic acid-insolublefraction and DNA synthesis did not resume. It was proposed that strains B and B/r were resistant to U.V. radiation because they could “excise” thymine dimers from their DNA, and following some additional steps, e.g. the reclosing of the gaps caused by the excision, DNA synthesis could begin. E. coli Bsp1failed to survive because it could not remove dimers which acted as blocks to DNA synthesis. Similar results were obtained by Boyce and Howard-Flanders (1964a) using U.V. radiation-sensitive and -resistant strains of E. coli K12. The fact that E. coli E/r is more resistant to U.V. radiation than E. coli B, even though it excises dimers more slowly, is probably due to differences in their ability to repair the damage caused by excision of bases (Setlow and Carrier, 1984). Some evidence of repair of the gaps in DNA caused by excision has been provided by Pettijohn and Hanawalt (1964). They irradiated E . coli and incubated the cells in a medium containing 5-bromouracil which,

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when incorporated into the DNA in place of thymine, acts as a density label. They analysed the density distribution by sonication of the DNA extracted at various times, and showed that the first newly synthesized material appeared at random in the DNA in short single-stranded segments of about 20 nucleotides. This is consistent with the picture of the thymine dimers being excised together with a few bases, and the first synthesis being that of small pieces of DNA to repair the gaps, the correct bases being inserted using the bases on the complementary DNA strand as a pattern. This process was called repair replication to distinguish it from normal semiconservative DNA replication. This repair replication did not occur in E. coli Bs--l,or in photoreactivated E. coli, since inneither case was there excision of dimers. Finally, it was shown that, after the repair replication, normal semiconservative DNA synthesis resumed. This is a very attractive hypothesis, the one experimental disadvantage being that these observations were not made at a U.V.dose level at which 100 %of the irradiated cells survived, and it not known what contribution to the overall picture was made by those cells which were dying. Obviously in such a complicated repair process, several steps are necessary, viz. the recognition of the radiation lesion, an incision made in one strand of the DNA near the lesion, excision of the damage and replacement of the excised bases, and finally the rejoining of strands. 1. Recognition

Any process involving repair of damaged regions of DNA must begin with recognition of the damaged region. It has already been shown that the photoreactivating enzyme recognizespyrimidine dimers but, whereas photoreactivation operates mainly on dimer-type damage, the darkrepair process recognizes other types of DNA damage. Repair replication follows treatment of E. coli TAU-bar with the bifunctional alkylating agent, nitrogen mustard, which primarily attacks the N-7 position of guanine (Hanawalt and Haynes, 1965).It has also been observed following treatment of bacteria with N-methyl-N’-nitro-N-nitrosoguanidine (Cerda-Olmedo and Hanawalt, 1967). The fact that u.v.-resistant and -sensitive strains of E. coli are resistant and sensitive respectively to mitomycin C, and show similar DNA breakdown patterns when U.V. irradiated or treated with mitomycin C, has been taken as evidence that mitomycin C damage is repairable (Boyce and Howard-Flanders, 1964b). This would suggest that it is not the precise nature of the base damage which is recognized but some associated secondary structural alteration in the phosphodiester backbone of the DNA (Hanawalt and Haynes, 1965).

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2. Incision

Rorsch and his colleagues (Rorsch et al., 1966) have shown with cell-free extracts of Micrococcus lysodeikticus that an incision step follows recognition of the damage. I n this test system, the replicative form of phage 4 x 1 7 4 was U.V. irradiated, preincubated with an enzyme preparation from M . lysodeikticus, and assayed in spheroplasts of an E. coli strain which lacked the complete repair system and so was incapable of repairing the u.v.-damaged phage. Rorsch et al. (1966) demonstrated that the incubation step resulted in increased survival of phage, and yet no thymine dimers were released. They concluded that the enzyme extract of M . lysodeikticus performed an incision step near the damage, and that the spheroplasts were able to complete the excision and repair the DNA. The repair of damage involving single-strand breaks, such as that produced by ionizing radiation, 32Pdisintegration or methylmethanesulphonate, does not require either a recognition step or an incision to be made, and repair could begin with excision. This would account for those mutants which are sensitive to U.V. radiation but resistant to ionizing radiation. They have presumably lost either the recognition or incising enzyme which would be unnecessary for the repair of ionizing radiation-type damage (Searashiand Strauss, 1964 ;Bridges and Munson, 1966).

3. Excision and Replacement

The evidence for the excision of pyrimidine dimers has already been described. Whether this excision precedes the repair replication or occurs concurrently with it is not clear. Certainly two models have been proposed (Fig. 4). The first scheme postulates that an enzyme excises a short, single-strand segment of the damaged DNA. The resulting gap is enlarged by further enzyme attack, and then the missing bases are replaced by repair replication in the genetically correct sequence, according to the normal base-pairing concept. The second scheme proposes that an initial incision is made near the defective bases. Repair replication begins immediately at this point and is accompanied by a peeling back of the defective strand as the ne'w bases are inserted. This has the advantage for the cell that long, vulnerable regions are not introducedinto the DNA during repair. Present experimental data do not favour one model over the other. Some interesting results have been obtained by Setlow et al. (1967) concerning the number of single-stranded regions in DNA existing at any one time during the repair of u.v. damage. Using a method developed by McGrath and Williams (1966), in which high molecular-weight single11"

190

B. E. B. MOSELEY

stranded DNA of bacteria is isolated directly on an alkaline sucrose gradient, they showed that, in E. cobi B/r which had been U.V. irradiated to cause the formation of 500 dimers per chromosome strand, about 15 breaks are open at any time during the time that 30 dimers per minute are being excised. After 45 minutes of incubation, most of the dimers are excised and the DNA of irradiated cells has normal sedimentation values. This indicates that the breaks are repaired almost as rapidly as the dimers are excised.

1 Repolyrner ization

!

-- - - - - - -

Incision

J

Excision

-- - - - - - -

FIG.4. Alternative models proposed for the dark repair of DNA following an incision near the lesion. (a)Excision of the lesion and some adjacent bases followed by replacement of the bases. (b) Repolymerization accompanied by a peeling back of the defective strand as new bases are inserted; excision in this case follows repolymerization.

Excision repair is inhibited by chemical agents such as caffeine and acriflavine and by the more general metabolic inhibitor, cyanide (Setlow, 1964). Starvation is also an excellent inhibitor of excision. However, excision is not inhibited in E. cobi by chloramphenicol or thymine starvation in thymine-requiring strains (Shuster and Boyce, 1964). 4. Rejoining of fltrands

The decay of 32Pincorporated into DNA can cause single- and doublestrand breaks. The single-strand breaks are not lethal events except in single-stranded phages (Stent and Fuerst, 1960). It is usually assumed that double-strand breaks in DNA are lethal events because of the improbability of the two free ends finding one another and rejoining with no loss of genetic continuity. Dean et ab. (1966) reported that X-irradiated M . radiodurans could repair double-strand breaks, but the method they

THE REPAIR O F DAMAGED DNA I N IRRADIATED BACTERIA

191

used is open to the objection that the double-strand breaks were caused by extraction procedures (Setlow, 1967). The double-strand breaks probably arose from single-strand breaks caused by the irradiation, and the joining of strands was that of single strand breaks. The rejoining of single-strand breaks has also been demonstrated by McGrath and Williams (1966) following X-irradiation of E . coli. They found that equal numbers of breaks were produced in E . coli B;-l and B/r but, in Bse1, there is further DNA breakdown while in B/r the singlestrands breaks disappear with time. It takes about 60 minutes t o repair 20 single-strand breaks during which time there is almost no DNA synthesis. 5 . Extent of the Dark-Repair Mechanism in Bacteria

The excision mechanism has not been extensively studied in bacteria. Besides its presence in E . coli, it has been found in M . radiodurans (Boling and Setlow, 1966), Bacillus megaterium (Donnellan et al., 1966) and B. subtilis (Strauss et al., 1966; Shuster, 1967). M . radiodurans has merited particular interest because, to date, it is the most resistant vegetative bacterium to both ionizing and u.v. radiation. This remarkable resistance to radiation is matched by its resistance to the decay of 32P incorporated in its DNA, full viability being retained after 50,000 disintegrations (and therefore single-strand breaks) per nucleus (M. Swann, personal communication). It is also very resistant to N-methyl-”-nitroN-nitrosoguanidine but sensitive to mitomycin C (Moseley, 1967). I n addition to its ability to repair single-strand breaks (Dean et al., 1966), it has a very efficient mechanism for the excision of thymine dimers (Boling and Setlow, 1966)differing in one respect from E . coli in that the excised dimers do not remain in the trichloroacetic acid-soluble fraction of the cell, but appear in the growth medium. The only other bacterium which has been shown to have a dark-repair mechanism is N . lysodeikticus. It has been shown that cell extracts can reactivate u.v.-irradiated transforming factor (Elder and Beers, 1964), u.v.-irradiated phage (Rorsch et al., 1966) and can excise dimers from DNA (Carrier and Setlow, 1966).

V. Summary Ultraviolet and ionizing radiations are predominantly lethal for bacteria because of the damage they cause in the bacterial DNA. The types of damage they cause are quite different. The main lesion formed 0nu.v. irradiationis the pyrimidine dimer, while single-anddouble-strand breaks are caused by ionizing radiation.

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Two types of enzymic repair mechanisms have been described in bacteria. Firstly, a photoreactivating mechanism, active only for U.V. damage in which pyrimidine dimers are monomerized in situ by the photoreactivating enzyme in the presence of light. Secondly, a darkrepair mechanism in which pyrimidine dimers are excised from the DNA and the resulting gaps repaired by the insertion of new bases. This repair system operates for damage other than that caused by U.V. irradiation, for example that caused by ionizing radiation, mitomycin C, nitrogen mustards, and N-methyl-N’-nitro-N-nitrosoguanidine. REFERENCES Anderson, A. W., Nordan, H. C., Gain, R. F., Parrish, G. and Duggan, D. (1956). Food Technol. 10, 575. Anderson, E. H. (1949). Amer. J . Bot. 36, 807. Beukers, R. and Berends, W. (1960). Biochim. biophys. Acta 41, 550. Beukers, R., Ylstra, J. andBerends, W. (1958). Rec. Trav. Chim. Pays-Bas 77,729. Boling, M. E. and Setlow, J. K. (1966). Biochim. biophys. Acta 123,26. Bolluna, F. J. and Setlow, R. B. (1963). Biochim. biophys. Acta 68, 599. Boyce, R. P. andKOward-Flanders,P. (1964a). Proc. nat. Acad. Sci.Wash. 51,293. Boyce, R. P and Howard-Flanders,P. (1964b). 2.Vererbungsl. 95, 345. Bresch, C. (1950). 2. Naturforsch. 5b, 420. Bridges, B. A. and Munson, R. J. (1966). Biochem. Biophys. res. Commun. 22, 268. Butler, G. C. (1949). Canad. J . Res. 27B, 972. Butler, J. A. V. and Smith, K. A. (1950). J . chem. SOD. 3411. Carrier, W. L. and Setlow, R. B. (1966). Biochim. biophys. Acta 129,318. Castellani, A., Jagger, J. and Setlow, R. B. (1964). Science 143, 1170. Cerda-Olmedo, E. and Hanawalt, P. C. (1967). Mutation Res. 4,369. Davison, P. F., Freifelder, D. and Holloway, B. W. (1964). J . molec. Biol. 8, 1 Davison, P. F., Freifelder, D., Hede, R. and Levinthal, C. (1961). Proc. nat. Acad. Sci. Wash. 47 1123. Dean, C. J., Feldschreiber, P. and Lett, J. T. (1966). Nature, Lond. 209,49. Donnellan, J. E., Stafford, R. S. and Setlow, R. B. (1966). Biophys. Soc. Abstracts, p. 112. Dunn, D. B. and Smith, J. D. (1954). Nature, Lond. 174, 305. Elder, R. and Beers, R. F. (1964). Fed. Proc. 23, 373. Freifelder, D. (1965). PTOC. nat. Acad. Xci. Wash. 54, 128. Freifelder, D. (1966). Radiation Res. Supplement 6, 80. Freifelder, D. and Davison, P. F. (1963a). Biophys. J . 3, 97. Freifelder, D. and Davison, P. F. (19633). Biophys. J . 3 , 4 9 . Gates, F. L. (1930). J . gen. Physiol. 14, 31. Glisin, V. R. and Doty, P. (1962). Biochim. biophys. Acta 61, 458. Goodgal, S. H., Rupert, C. S. and Herriot, R. M. (1957). In “The Chemical Basis of Heredity”, (W. D. McElroy and B. Glass, eds.), p. 341. Johns Hopkins Press, Baltimore. Hanawalt, P. C. andHaynes, R. H. (1965).Biochem. Biophys. res. Commun. 19,462. Harm, W. and Hillebrandt, B. (1962). Photochem. Photobiol. 1, 271. Haug, A. and Goes, E. (1963). Int. J . radiat. Biol. 7,447. Haynes, R. H. (1964). Photochem. Photobiol. 3, 429. Hill, a.F. (1958). Biochim. biophys. Acta 30, 636.

THE REP-

OF DAMAGED DNA I N IRRADIATED BACTERIA

193

Hollaender, A. and Claus, W. D. (1937). Bull. nat. Res. Council, U.S. 100, 75. Iyer, V. N. and Szybalski, W. (1963). Proc. nat. Acad. Sci. Wash. 48, 1409. Jagger, J. (1958). Bact. Rev. 22, 99. Jagger, J. (1960). In “Radiation Protection and Recovery”, (A. Hollaender, ed.), p. 352. Pergamon, Oxford. Jagger, J. and Latarjet, R. (1956). Ann. Inst. Pasteur 91, 858. Jagger, J. and Stafford, R. S. (1965). Biophys. J . 5, 75. Kaplan, H. S. and Tomlin, P. A. (1960). Radiation Res. 12, 447. Kaplan, H. S. and Zavarine, R. (1962). Biochem. Biophys. res. Commun. 8,432. Kaplan, N. S., Smith, K. C: and Tomlin, P. A. (1961). Nature, Lond., 190, 794. Kaplan, H. S., Smith, K. C. and Tomlin, P. A. (1962). Radiation Res. 16, 98. Kelner, A. (1949a).Proc. nat. Acad. Sci. Wash. 35, 73. Kelner, A. (19493). J . B m t . 58, 511. Kelner, A. (1950). Bull. N.Y. Acad. Med. 26, Ser. 2, 189. Lennan, L. S. and Tolmach, L. J. (1959). Biochim. biophys. Acta 33, 371. Marmur, J. and Grossman, L. (1961). Proc. nat. Acad. Sci. Wash. 47, 778. Marmur, J., Anderson, W. F., Matthews, L.,Berns, K., Gajewska, E., Lane, D. and Doty, P. (1961). J . cell. comp. Physiol. 58 (Suppl. l), 33. McGrath, R. A. and Williams, R. W. (1966). Nature, Lond. 212, 534. Moore, A.M. (1958). C d . J . Chem. 36, 381. Moseley, B. E. B. (1967). J . gen. Microbiol. In the press. Moseley, B. E. B. and Laser, H. (1965). Nature, Lond. 206, 373. Moseley, B. E. B. and Schein, A. H. (1964). Nature, Lond. 203, 1298. Oliver, R. and Shepstone, B. J. (1964). Phys. Med. Biol. 9, 167. Opara-Kubinska, Z., Lorkiewicz, Z. and Szybalski, W. (1961). Biochem. Biophys. yes. Commun. 4,288. Opara-Kubinska, Z., Kurylo-Borowska, Z. and Szybalski, W. (1963). Biochim. biophys. Acta 72, 298. Pettijohn, D. and Hanawalt, P. (1964). J . molec. Biol. 9, 395. Rorsch, A., van Sluis, C. A. and van de Putte, P. (1966). Radiation Res. Suppl. 6, 217 Rupert, C. S. (1960). J . gen. Physiot. 43, 573. Rupert, C. S. (1961). J . cell. comp. Physiol. 58 Suppl. 1, 57. Rupert, C. S. (1962a). J . gen. Physiol. 45, 703. Rupert, C. S. (1962b). J . gen. Physiol. 45, 725. Rupert, C. S. (1964). Photochem. Photobiol. 3, 399. Searashi, T. and Strauss, B. (1964). Biochem. Biophys. res. Commun. 20, 680. Setlow, J. K. (1959). Ph.D. Thesis: Yale University. Setlow, J. K. (1963). Photochem. Photobiol. 2, 393. Setlow, J. K. (1964). Photochem. Photobiol. 3, 405. Setlow, J. K. (1966). I n “Current Topics in Radiation Research”, Vol. I1 (M. Ebert and A. Howard, eds.), North Holland Publishing Co., Amsterdam. Setlow, J. K. and Boling, M. E. (1963). Photochem. Photobiol. 2, 471. Setlow, J. K. and Boling, M. E. (1965). Biochim. biophys. Acta 108, 259. Setlow, J. K. and Duggan, D. E. (1964). Biochim. biophys. Acta 87, 664. Setlow, J. K. and Setlow, R. B. (1963). Nature, Lond. 197, 560. Setlow, J. K. and Setlow, R. B. (1961). Proc. nat. Acad. Sci. Wash. 47, 1619. Setlow, J. K., Boling, M. E. and Bollurn, F. J. (1965).Proc. rzat. Acad. Sci. Wash. 53, 1430. Setlow, R.B. (1961). Biochim. biophys. Acta 49, 237. Setlow, R. B. (1964). J . cell. comp. Physiol. 64, Suppl. 1, 51.

194

B. E. B. MOSELEY

Setlow, R. B. (1967). “Radiation Research”. North Holland Publishing Co., Amsterdam. Setlow, R. B. and Carrier, W. L. (1963).Photochem. Photobiol. 2, 49. Setlow, R. B. and Carrier, W. L. (1964).Proc. nat. Acad. Sci. Wash. 51, 226. Setlow, R. B. and Setlow, J. K. (1962).Proc. nat. Acad. Sci. Wash. 48, 1250. Setlow, R. B., Carrier, W. L. and Bollum, F. J. (1965a). Proc. nat. Acad. Sci. Wash. 53, 1111. Setlow, R. B., Carrier, W. L. and Bollum, F. J. (1965b). Biophys. SOC.Abstracts p. 111. Setlow, R. B., Carrier, W. L. and Williams, R. W. (1967).Biophys. SOC.Abstracts, p. 83. Setlow, R. B., Swenson, P. A. and Carrier, W. L. (1963).Science 142, 1464. Shooter, K. V. (1957).Progr. Biophys. biophys. Chem. 8, 310. Shugar, D. (1962). Abh. Deut. Akad. Wissensch. Berlin 1, 72. Shuster, R. C. (1967).J. Bact. 93, 811. Shuster, R. C. and Boyce, R. P. (1964).Biochem. Biophys. res. Commun. 16,489. Sinsheimer, R. L. (1954). Radiation Res. 1, 505. Sinsheimer, R. L. (1957). Radiation Res. 6 , 121. Sinsheimer, R. L. and Hastings, R. (1949).Science 110, 525. Smietanowska, A. and Shugar, D. (1961). Bull. Acad. Polon. Sci. CL, I1 9, 375. Smith, K. C. (1962). Biochem. Biophys. res. Commun. 8, 157. Smith, K. C. (1963).Photochem. Photobiol. 2, 503. Smith, K. C. (1964). In “Photophysiology”, Vol. 2 (A. C. Giese, ed.). Academic Press, New York. Smith, K. C. (1966). Radiation Res. Suppl. 6, 54. Smith, K. C. and Aplin, R. T. (1966).Biochemistry 5,2125. Sparrow, A. H. and Rosenfeld, R. M. (1946).Science 104,245. Stahl, F. W., Crasemann, J. M., Okun, L., Fox, E. and Laird, C. (1961). Virology 13, 98. Stent, G. and Fuerst, C. (1960). Advanc. Biol. med. Phys. 7, 1. Strauss, B., Searashi, T. and Robbins, M. (1966).Proc. nut. Acad. Sci. Wash. 56, 932. Szybalski, W. and Lorkiewicz, Z. (1962). Abhandl. Dewtsch. Akad. Wiss. Berlin, K1. Med. No. 1, 61. Taylor, B., Greenstein, J. P. and Hollaender, A. (1948). Arch. Biochem. Biophys. 16, 19. Wacker, A. (1961).J. Chim.Phy8.58, 1041. Wacker, A. (1963).Progr. nucleic acid Res. 1, 269. Wacker, A., Weinblum, D., Trager, L. and Monstafa, Z:H. (1961).J. molec. Biol. 3, 790. Wang, S. Y. (1961). Nature, Lond. 190, 690. Wierzchowski, K. L. and Shugar, D. (1962).Photochem. Photobiol. 1, 21. Wulff, D. L. and Rupert, C. S. (1962). Biochem. Biophyiys. res. Commun. 7, 237. Wyckoff, R. W. G. (1932).J . gen. Physiol. 15, 351. Zamenhof, S. and Griboff, G. (1954).Nature, Lond. 174, 306.

AUTHOR INDEX Numbers in italics refer to the pages on which references are listed at the end of each article. Bergstrom, L., 21, 42 Berman, D. T., 49, 87 Aasmundrud, O., 2, 4, 5, 40, 41 Berns, K., 176, 177,193 Adams, J. M., 107,130,132,237 Beukers, R., 179, 181, 192 Adelberg, E. A., 85, 87 Biedermann, M., 30, 39 Agar, H. D., 147,148, 149,187,169 Bladen, H. A., 99,137 Akiba, T., 85,87 Bloch, K., 21, 40, 42 Alexeva, V. M., 143,170 Boatman, E. S., 14, 39 Allen, M. B., 4,39 Bock, R. M., 95,142 Allen, R. L. 31,41 Bogorad, L., 31, 33, 36, 39, 41 Alpin, R. T., 178,194 Boling, M. E., 178, 181, 184, 185, 186, 191, Altshuler, T., 34, 39 192,193 Anderson, A. W., 175, 192 Bollen, A., 96, 137 Anderson, E. H., 179,192 Bollum, F. J., 181, 185, 186, 187, 192, 193, Anderson, I. A. C., 116, 117, 240 194 Anderson, W. F., 176, 177,193 Bolton, E. T., 95, 104, 127, 137 Andoh, T., 121,137 Boman, H. G., 117, 119,138 Apirion, D., 130,141 Borek, E., 116, 140 Arber, W., 49, 50, 78, 87 Borowski, J., 44, 87 Arnon, D. I., 23, 39 Bowers, W. D., 157, 159, 170 Aronson, A. I., 95, 99, 103, 106, 107, 119, Boxer, G. E., 100, 133,141 121, 122,127,137,139,141 Boyce, It. P., 187, 188,190,192,194 Artman, M., 97,141 Bradley, D., 149,170 Asano, K., 107,140 Bremer, H., 100, 133, 137 Asheshov, E.H.,44,47,49,50,61,73,78,87 Brenner, S., 95, 97, 106, 110, 124, 125, 127, Attardi, G., 100, 101, 102, 104, 137, 138 134, 135,137,140,142 Bresch, C., 179, 192 Bridges, B. A., 189, 192 Bra, C., 25,26, 39 Britten,R. J., 92, 94, 99, 104, 113,114,126, Bacon, J. 8. D., 147,150, 155,169 127, 134,137,140, 141 Baldwin, J. N., 45, 60, 61, 80, 87, 88 Broadman, N. K., 33, 39 Baltimore, D., 100, 133, 140 Broda, P. M. A., 81, 87 Barber, M., 44, 87 Barthomolomew, J. W., 147,149, 162,169 Brodie, A. F., 18, 41 Brown, C. M., 154,170 Barton, A. A., 148, 149, 156,162,169 Brownlee, G. C., 95,137 Bartsch, R. G., 23, 24, 27, 39 Brownstein, B. L., 118,139 Beckwith, J. R., 72, 81, 87 Bryan, G., 31, 33, 41 Beers, R. F., 191, 192 Bryne, R., 99,137 Belitsina, N. V., 97, 141 Buchanan, B. B., 23, 39, 40 Bell, D. J., 146, 169 Buck, C. A., 110,139 Bender, H., 155,169 Bull, M. J., 14, 22,28,29,30, 33, 34, 36, 39 Benson, A. A., 21, 39 Beran, K., 145,157,162,163, 164,165, 166, Burde, R. M., 18, 42 Burnham, B. F., 35, 36, 37, 39 168, 169,169,170,171 Burns, S. N., 85, 87 Berends, W., 179, 181,192 Butler, G. C., 181, 192 Bergeron, J. A., 21, 22, 39 195

A

B

196

AUTHOR INDEX

C Cain, R. F., 175,192 Cairns, J., 83, 87 Campbell, A. M., 81, 87 Cannon, M., 132,137 Capecchi, M. R., 107,130,132,137 Carleton, J., 44, 87 Carlton, B. C., 83, 88 Carr, C. W., 96,137 Carr, N. G., 23,28,30,39 CIXTier, w. L-9 179,181,186, 187, 189, 191, 192,194 Castellani, A., 186, 192 Cauthen, S. E., 37,39 Cavalli, L. L., 85,88 Cerda-Olmedo, E., 188,292 Chance, B., 29, 39 Chantrenne, H., 121, 137 Chargaff, E., 102, 121,137,141 Choi, Y. S., 96, 137 Chrambach, A., 95,140 C h u g , K. L., 158, 162,170 Clark, K. L., 44, 87 Claus, W. D., 187,193 Clayton, R. K., 6,23,25, 39,42 Clowes, R. C., 45, 85, 87 Cohen, S. S., 97, 107,137,141 Cohen-Bazire, G., 6, 8, 9, 10, 12,13,14, 15, 16, 18, 20, 21, 23, 24, 25, 30, 35, 39 Colboum, J. L., 97,138 Collims, A., 36,41 Collins, A. M., 61,87 Comb, D. G., 95,138 Conti, S. F., 8, 9, 12, 15, 18, 22, 24, 40, 41 Cook, A. H., 164,170 Cooper, R., 36, 39 Cota-Robles, E. H., 12, 41 Cowie, D. B., 127,137 Cox, E. C., 95, 96, 108, 132, 138,139 Crasemann, J. M., 182,194 Critz, D. B., 80, 87 Cundliffe, E., 131,134,138 Cutler, R. E., 103, 138

Davies, R., 155,170 Davis, B. D., 101, 131,139,141 Davison, P. F., 83, 87, 177, 182, 192 Dean, A. C. R., 99,138 Dean, C. J., 190, 191,192 De Klerk, H., 23, 40 Delius, H., 95, 96, 108, 142 DeMoss, J. A., 101, 134, 140, 142 Devreux, S., 121,137 Doi, R. H., 102, 138 Donnellan, J. E., 191,192 Doby, pa,176, 177, 192 Douglas, H. C., 9, 42, 147, 148, 149, 150, 157,169 Drapeau, G. R., 83, 88 Dresden, M., 130, 131, 138 Drews, G., 2, 5, 8, 9, 12, 13, 14, 15, 24, 29, 30, 33, 34, 35, 39, 40, 42 Dubin,D.T., 104, 116, 119,121,138 Dubnau, D., 102,103,138 Duerksen, J. D., 127, 137 Duggan, D., 173,174,175,181,192,193 Dunn, D. B., 174,192 Dworkin, M., 4,40 Dyke, K. G. H., 47,51,57,87

E Eaton, R. B., 44, 87 Eoker, R. E., 106,107,138 Eddy, A. A., 145, 146, 147, 155, 156,170 Eidlic, L., 116, 140 Eilam, Y., 33, 40 Eimhjellen, K. E., 2, 4, 5, 40, 41 Elder, R., 191, 192 Elvin, P. A., 155, I70 Emrioh, J., 100, 132, 142 Engelberg, H., 97, 141 Ennis, H. L., 118,122,138 Epstein, H. T., 38, 42 Erwin, J., 21, 40 Evans, J. E., 103,138 Evans, M. W. C., 23, 39, 40 Exell, G., 23, 28, 30, 39

D Dagley, S., 106, 110, 116, 117, 119, 120, 121,138 Dahlberg, J. E., 132,138 Darnell, J. E., 104,141 Das, H. K., 100,138 Datta, N., 59, 84, 85, 87, 88 Davern, C., 97, 124, 125, 127, 134, 135, 140 Davidson, E. D., 147,150,169 Davies, J. E., 108,138

F Fairbrother, R. W., 44, 87 Faloone, G., 145, 146, 154, 155, 161, 170, 171 Fan, D. P., 134,139 Fansler, B. S., 104, 117, 142 Fargie, B., 84, 87 Feldschreiber, P., 190, 191, 192

197

AUTHOR INDEX Fend, Z.,161, 171 Fiers, W.,82, 87 Flab, J. G., 95,96,108, 132,138,139 Folkes, J. P.,119,138 Forohhammer, J., 118,140 Fox, E.,182,194 Fredericq, P.,84, 87 Freifelder, D.,83, 87, 157, 170, 176, 177, 182,183,192 Frenkel, A. W., 8, 9, 11, 12, 13, 16, 20, 23, 40 Frey-Wyssling, A., 148,170 Fuerst, C., 182, 190,194 Fujimoto, M., 33, 35, 40 Fujimura, R.K.,97,124,139 Fukusawa, T.,48, 84, 88 Fukushima, T.,85, 87 Fuller, R. C., 8, 9, 12, 15, 18, 22, 24, 40 Furano, A.V., 108,138

Griboff, G., 174,194 Griffiths, M., 30,40 Gros, F., 92, 100, 104, 110, 137, 138, 140, 141,142 Grossman, L.,177,193 Giinalp,A., 104,116, 119, 121,138 Guest, J. R.,83, 88

H

Haddad, S. A,, 148,170 Hagedorn, K.,147, 150, 157,170 Hahn, F.E.,119,142 Hall, B.D.,93,107,128,138,140 Hall, C. E.,92,138 Halvorson, M.,161,170,171 Hamburger, M., 44, 87 Hanawalt, P.C., 187, 188. 192 Handschack, W.,104,139 Harm, W., 184,192 Harmon, S.A., 45, SO, 87 Harris, J.I.,95, 142 Hase, E.,161,170 Gaines, K., 100, 133, 137 Haselkorn,R., 103,110,116, 117, 132,138, Gajewska, E.,176, 177,193 140 Gale, E.F., 119,138 Hashimoto, M., 44,45, 59, 87 Garcia, A., 26, 40 Hastings, R., 178,194 Gartner, T.K.,96,141 Haug, A,, 177,192 Gates, F. L.,175, 178,192 Gavrilova,L.P.,97,111,119,126,138,140 Hawirko, P.Z.,158, 162,170 Hayashi, K.,163,170 Geller, D.M., 23, 40 Hayashi, Y.,104, 111, 122, 124,138,142 Gest, H., 1, 3, 12,20,40,42 Hayes, W.,84, 85, 87 GesteIand, R. F.,97, 125,138 Haynes, R. H., 175,188,192 Gibbon, J. A.,21, 22, 42 Hede, R., 83,87,182,192 Gibbs, S.P.,8, 9, 14, 40 Henning, U.,38,40 Gibor, A., 38, 40 Eteppel, L.A., 132,140 Gibson, J., 38,42 Herbst, E.J., 97,138 Gibson, K.D.,12,16,20,21,22,24,25,31, Herriot, R.M.,183, 192 35, 36, 40 Herzog, A.,96, 137 Giesbrecht, P.,2, 5, 8,9, 12, 13, 14,40 Hiatt, H. H., 104, 138 Gilbert, W.,104, 108, 132, 137,138 Gillespie,M.E., 112,113,114,130,131,141 Hickman, D.D.,9, 11, 12, 13, 15, 20, 23, 40 Glauert, A.M.,98,138 Higa, A., 104, 134,139 Glisin, V. R.,177,192 Higuchi, M.,33, 35, 40 Godson, G. N.,131,138 Hill, R.F., 177,192 Goes, E.,177,192 Hiebrandt, B.,184,192 Goldberg, A., 96,138 Hills, D.C.,121,138 Goldstein, A., 100, 138 Hinshelwood, C., 99,138 Golov, V. F., 97, 111, 126,139 Hirota. Y..45, 84, 85, 87,88 Goodgal, S. H., 183,192 Hoagland,-M.,. 130, 131, i38 Gordon, J., 117, 119,138 Hoare, D.S., 21, 22, 42 Gorini, L.,108,138 Hoete, I., 146,171 Gorman, J., 161,170 Holden, M., 165,170 Goto, K.,33, 35, 40 Hollaender, A., 181,187,193,194 Granick, S.,33, 36, 38, 40 Holland, J. J., 110,139 Green, M.H., 93, 128,138 Hollingworth, B.R.,92,108,142 Greenstein, J. P.,181,194

G

198

AUTHOR INDEX

Holloway, B. W., 84,87, 182,192 Holmes, I. A., 121,139 Holowczyk, M. A., 106, 122,137 Holt, A. S., 4, 40 Holt, S.C., 8,9,12,13,14,15,16,17,18,19, 20, 21, 24, 40, 41 Holter, H., 155,170 Hops, H. E., 119,142 Hopwood, D. A., 81,87 Horio, T., 19, 24, 29, 39, 41 Horne, R. W., 145, 147, 149,171 Horowitz, J., 97, 121, 138, 142 Hosokawa, K., 96, 97, 108, 111, 119, 120, 121,124,126,139,140,142 Hough, J. S., 154, 164, 170 Houghton, R. H., 44,88 ~ o ~ w i nA. k ,L., 146,147,149,150, iro Howard-Flanders, R. P., 187, 188, 29: Hueng, P.0..101, 102,137

Hulcher, F. H., 22, 41 Hurlbert, R. E., 29,41 Hurwitz, J., 100, 101, 133, 140 Huxley, H. E., 92,139

I Igarashi, R. T., 102,138 Iijima, F., 84, 87 Ikeda, M., 161,171 Ikeda, Y., 102,142 Imamoto, F., 106,131, 133,139 Inovye, M., 100, 132,142 Isaksson, L. A., 119,138 Ishihama, A., 106,121,122,140,141 Ishiki, Y., 85, 87 Issac, P. K., 158, 162, I70 Ito, J., 131, 139 Ivanov, D. A., 97,138 Iwabuchi, M., 121,122, 124,139 Iyer, V. N., 177,193

J Jacob,F., 70,85,87,95, 100,110,137 Jagger, J., 184,186, 187,192 James, A. T., 21,42 Jennings, P. A., 84,87 Jensen, A., 2, 4, 5, 40, 41 Jensen, S. L., 4,30, 41, 42 Jinks, J. L., 84, 87 John, M., 44,61,88 Johnston, J. R., 162, 170 Jones, 0. T. G., 31, 41 Jukes, T. H., 100,142 Julien, J., 111, 122, 139

K Kabat, S., 101, 102, 137 Kaempfer, R. 0. R., 127,139 Kahn, H. A., 33, 42 Kaji, A., 113, 126, 132, 139, 140 Kaji, H., 132, 139 Kamen, M. D., 19,23,24, 27,29, 39, 40, 41, 42 Kaplan, H. S., 173, 174, 175, 182,193 Kashket, E. R., 18, 41 Ke, B., 3, 42 Kelley, W. S., 93, 94,129, 139 Kelner, A., 183, 186, 193 Kennell, D., 110, 111,139,140 Kessler, G., 145, 146, 147, 154, 170, 1 7 1 Keynan, A., 104,139 Kiho, Y., 131, 239 Kikuohi, G., 29, 33, 35, 40, 41 Kirnura, S., 85, 87 Kirk, J. T. O., 31, 41, 84, 87 Kjeldgaard, N. O., 99, 105, 106, 128, 133, 139,140 Klein, S., 31,33,36,40,41 Koch, A. L., 106, 115,139 Kodicek, E., 21, 41 Kohler, R. E., 101, 131, 139, 141 Kondo, N., 104,142 Kono, K., 44, 45, 59, 87, 88 Kono, M., 121, 122, 123, 124,139 Konrad, M. W., 100, 133,137 Koman, R. Z., 49, 87 Korn, E. D., 145, 146, 147,170 Kotoulas, A., 111,139 Koyama, K., 85, 87 Krakow, J. S., 101,139 Kreger, D. R., 146, 147, 149, 150,170 Krug, R., 132,137 Kunisawa, R., 8, 9, 10, 13, 14, 16, 18, 20, 23, 24, 25, 30, 39 Kurland, C. G., 95, 106,110, 119,139 Kurylo-Borowska, Z., 177, 193 Kwan, B. C. K., 99,141

L La Berge, M., 161,170 Laird, C., 182,194 Lampen, J. O., 155,171 Lane, D., 176, 177, 193 Lascelles, J., 8,14,15,20,21,22,28,29,30, 31, 33, 34, 35, 36, 37, 39, 41, 42 Laser, H., 175, 193 Latarjet, R., 184,193 Latham, H., 104,141 Leboy, P. S., 95, 96, 108, 139 Lederberg, J., 48, 85, 88

199

AUTHOR INDEX

Lederberg, S., 85, 88 Lehninger, A. L., 84,88 Leive, L., 106, 133, 139 Lerman, L. S., 179,193 Lerman, M. I., 97, 111, 119, 126, 139, 141 Lessie, T. G., 36, 41 Lett, J. T., 190, 191, 192 Levin, J., 99,137 Levin, R., 147,149,169 Levine, L., 19, 26, 41, 42 Levine, R. P., 33, 41 Levinthal, C., 83, 87, 104, 105, 130, 134, 139,142,182,192 Lewandowski, L. J., 118,139 Lichtenstein, J., 97, 137 Lieblovai, J., 157, 162, 163, 164, 165, 166, 168, 169,169 Lindegren, C. C., 148,170 Lindigkeit, R., 104, 139 Linnane, A. W., 150,171 Lipmann, F., 23,40 Loening, U. E., 95,139 Lorkiewicz, Z., 174,175,182,193,194 Lowney, L. I., 100,138 Lowry, C.V., 130,140 Lubin, M., 118,122,138

M Maaloe, O., 99,106, 110, 119,128, 133,139, 140 McCarthy, B. J., 92,94,103,104,110, 113, 114, 126, 127,137,139,140,141 McClary, D. O., 157,158,170 MacDonald, R . E., 118,140 Macdonald, S., 61,87 McGrath, R.A., 176, 182, 189, 191,193 McMurrough, I., 146,154,170 McQuillen, K., 92, 99, 100, 107, 127, 131, 133,137,140,141 MacRae, R. M., 155,170 Magasanik,B., 110, 116,117,139,140 Maitra, U., 100, 101, 133, 140 Malamy, M., 131, 140 M&k, I., 165,169,171 Mandel, L. R., 116,140 Mangiarotti, G., 129, 130, 131, 140, 141 Manor, H., 103, 110, 116,117,140 Marchesi, S. L., 111, I 4 0 Marchesi, V. T., 99, 141 M a m u r , J., 102,103,138, 176, 177,193 Marquardt, von, H., 147, 150,170 Marquisee, M. J., 111, 122,140 Marr,A. G., 8, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 41, 42 Maruyama, H., 122,140 Maruyctma, Y.,163,170

Marver, H. S., 36, 41 Ma=, R., 30, 39 Matthews, L., 176, 177, 193 May, J. M., 44, 88 Medvedeva, G. A., 143,170 Meissel, M. N., 143,170 Meselson,M., 95,97,108,110,111,124, 125, 126, 127, 134, 135,137,139,140, 142 Meynell, E., 85,88 Midgley, J. E., 95, 140 Mihara, S., 161, 170 Millbank, J. W., 155, 170 Milne, B. D., 155,169 Mitsuhashi, S., 44, 45, 59, 81, 87, 88 Mitsui, H., 106, 121, 122, 140, 141 Mittwer, T., 149, 162, 169 Miura, K., 104,124,138 Mizuno, D., 122, 140 Moldave, K., 99,141 MBller, W., 95, 140 Mollenhauer, H., 26,40 Monier, R., 95, 111, 122, 139, 141 Monod, J., 70, 87 Monstafa, Z. H., 181, 194 Moody, E. E. M., 45, 87 Moore, A. M., 178,193 Moore, P. B., 95,96,108,142 Morell, P., 102,138 Morikawa, N., 106,133,139 Morimura, M., 44, 59,88 Morris, D. W., 101, 140 Morse, M. L., 60, 88 Mortimer, R. K.,162, 170 Moseley, B. E. B., 173, 174, 175, 191, 193 Motokawa, Y., 29, 41 Mundkur, B., 147,150, 156,170 Munkres, K. D., 38, 41, 42 M m o , R., 120,142 Munson, R. J., 189,192 Muto, A., 117,140

N Nakada, D., 106, 107, 109, 110, 111, 113, 116,117,118,120, 121, 122,126,140 Namiko, O., 33, 35, 40 Naono, S., 100,137,140 NeEas, O., 150,151,152,161,170,171 Neidhardt, F. C., 116,140 Neu, H. C., 132,140 Neuberger, A., 16,22, 31, 35, 36, 40 Newton, G. A., 12,21,22, 41 Newton, J. W., 12, 21, 22, 26, 27, 41 Nichols, B. W., 21, 42 Nickerson, W. J., 145, 146, 147, 149, 154, 155,161,170,171

200

AUTFIOR INDEX

Nierlich, D. O., 115, 140 Nirenberg, M. W., 99,137 Nofal, S., 117,140 Noller, H., 95, 96, 108, 142 Nomura,M., 96,97,106,107,108,111, 119, 120, 121, 124, 125, 126, 130, 132, 134, 135,139,140,142 Nordan, H. C., 175,192 North, J., 150,171 Northcote, D. H., 145, 146, 147, 149, 169, 170,171 Novick, R. P., 44,45,48,49,54,56,59, 61, 65, 71, 72, 74, 75, 81, 82, 83, 88 Novogrodsky, A., 100, 133,140 Nowak, L., 132,142 Nurminen, I., 155,170,171

Oelze, J., 35, 40 Oishi, A., 101,140 Oishi, M., 101,103,140 Okada, Y., 100, 132,142 Okamoto, K., 107,140 Okamoto, T., 132,142 Okun, L., 182,194 Oliver, R., 173, 193 Olson, J. M., 6,24,25,41 Oota, Y., 121,141 Opara-Kubinska, Z., 174, 175, 177, 182, 193 Orias, E., 96,141 Orlando, J. A., 19, 42 Osawa, S., 104,106,110,115,117,121,122, 123,124,138,140,141,142 Otttka, E., 110, 117, 121, 122, 139, 140, 141 OtSUka, H., 161, 170 Ottlenghi, P., 155, 170 Oumi, T., 121,122,124,139 Oura, E., 155,171 Ozeki, H., 84, 85, 88

P Pangborn, J., 18, 42 Pardee, A. B., 8,42 Pmker, L., 44, 87 Parrish, C., 175,192 Pattee, P. A., 60, 61, 88 Pattison, J., 37, 39 Perret, C. J., 44, 88 Pettijohn, D., 187, 193 Pfennig, N., 4, 9, 10, 18, 39, 42

Phaff, H. J., 145, 155,171 Pokornf, V., 145,169,171 Pollock, M. R., 52,88 Porra, R. J., 28, 29, 30, 42 Poston, S. M., 44, 73, 78, 88 Powell, E. O., 167,171 Previc, E. I?., 112, 113, 114, 130, 131, 141 Pritchard, R. H., 45, 87 Prokop-Schneider, B., 102,141

R Rabinowitch, E. I., 5, 42 Raha, A., 107,141 Raskas, H. J., 97, 126, 127, 132, 139, 141 Rechicgl, M., 36, 41 Reid, P. J., 96, 141 Rejman, E., 102,141 Rich, A., 131, 139 Richmond, M. I€.,44,46,47, 51,56,57,58, 59,61, 64, 65, 67, 70, 71, 72, 73, 75, 76, 81, 87, 88 Rigopoulos, N., 23,40 Ritz, H. L., 60, 88 Robbins, M., 191,194 Roberts, R. B., 92, 99, 104, 107, 109, 113, 114,126,127,133,137,140,141 Robrish, S. A., 18, 42 Robson, E. I., 155, 156,171 Rodgers, A., 96, 97, 141 Roelofsen, P. A,, 146,171 Rtksch, A., 189, 191, 193 Romano, C. A., 6,25, 41 Ron, E. Z., 101, 131,139, 141 Rose, A. H., 146, 154,170 Rosenfeld, R. M., 181,194 Rosset, R., 95, 111, 122, 139, 141 Rouviere, J., 100,137,140 Rudin, A. D., 145,147,156,170 Rudner, R., 102,141 Rudney, H., 23,42 Rupert, C. S., 183, 185, 186, 192, 193

S Saito, H., 102,142 Salas, M., 100,132,142 Salser, W., 104, 115, 141 Sanger, F., 95,137 Sato, K., 106, 133, 139 Scaife, J., 81, 87 Schachman, H. K., 8,42 Schaechter, M., 93, 94, 100, 106, 107, 112, 113, 114, 130, 131, 133, 138, 139, 141

201

AUTHOR INDEX

Schein,A.H., 173,174,175,193 Schemer, K., 104,141 Scheuerbrandt, G., 21, 42 Schick, J., 30, 40 Schiff, J. A., 38,42 Schleif, R., 111,141 Schlessinger, D.,91, 92, 97, 99, 108, 124, 125, 127, 129, 130, 131, 134, 135, 140, 141,142 Schmidt, K., 4,42 Schon, G., 29,42 Schroeder, J., 30, 39 Searashi, T., 189,191,193,194 Sedar, A. W., 18,42 Segalove, M., 59, 88 Sells, B. H., 121,141 Sentandreu, R., 147,171 Servin, Massieu, M., 44, 88 Setlow, J. K., 173, 174, 178, 179, 180, 181, 183,184,185,186,192,193,194 Setlow, R. B., 179, 180, 181, 185, 186, 187, 189, 190,191,192,193,194 Shepstone, B. J., 173,193 Sherman, F., 38,42 Shigeura, H. T., 100,133,141 Shin, D. H., 99,141 Shooter, K. V., 181,194 Shugar, D., 179,181,194 Shuster, R. C., 190,194 Sibatmi, A., 110, 122, 141 Signer, E., 73, 87 Silman, N., 97,141 Simon,E. J., 107,141 Sinsheimer, R. L., 81,87, 178,179, 194 Sistrom, W.R., 6,8,9,12,14,15,16,17,20, 21, 33, 35, 36, 39, 40, 41, 42 Slayter, H. S., 92,138 Slonimski, P., 38, 42 Slonimski, P. P., 38,42 Smadel, J. E., 119, 142 Smietanowska, A., 181,194 Smillie, R. M., 23, 40 Smith, I., 102, 103, 138 Smith, J. D., 174,192 Smith, K. A., 181,192 Smith, K. C., 174, 178, 181, 182, 193, 194 Smith, S. M., 84,85,88 Spahr, P. F., 95,104,138,141 Sparrow, A. H., 181,194 Spiegehnan, S., 95, 101, 102, 107, 119,137, 140,142 Spirin, A. S., 97, 111, 119, 126, 138,139, 141 Spitnik-Elson, P., 95,141 Spotts, C. R., 108, 141 Srinivasan, P. R., 117,140

Staehelin,T., 97, 108, 111, 124, 125, 126, 132,138, 141,142 Stafford, R. S., 184,191,192,193 Stahl, F. W., 81, 88, 182,194 Stanier, R. Y., 1,4,8,12,30,35,39,40,42, 108,141 Stanley, W. J., Jr., 95,142 Stanley, W. M., 100, 132, 142 Stanton, E. K., 6, 41 Steinberg, C. M., 81, 88 Stent, G., 182,190,194,100, 106, 107, 109, 133,137,142 Stocker, B. A. D., 84,85, 88 Stockley, M. H., 155, 156,171 Strauss, B., 189,191,193,194 StreiblovB, E., 145,148, 150, 151, 152, 157, 160, 162, 163, 164, 165, 166, 168, 169, 170, 171 Streisinger, D., 100, 132,142 Sueoka, N., 101,103,140 Sugimura, T., 23,42 Sugino, Y., 85,88 Suomalahen, H., 155, 171 Sutton, D. D., 155,171 Suyama, Y., 38,42 Suzuka, I., 132,139 Suzuki, H., 111,122,142 Svoboda, A., 150,151,152,170,171 Swenson, P. A., 187,194 Sybesma, C., 25, 41 Sykes, J.,21,22,42,106,110,116,117,119, 120, 121,138 Sypherd, P. S., 104, 106, 110, 116, 117, 142 Szer, W., 132,142 SzilBgyi, J. F., 15, 20, 21, 28, 30, 34, 41 Szybalski, W., 174, 175, 177, 182, 193, 194

T Tait, G. H., 16, 22, 31, 35, 36, 40 Takahashi, H., 102,142 Takai, M., 104, 142 Takanami, M., 100,132,142 Tal, M., 101, 140 Taniguchi, S., 29, 39, 42 Taruo, P., 161,170,171 Taylor, B., 181, 194 Taylor, D. J., 147, 150, 169 Taylor, I. F., 147, 150, 169 Terzaghi, E., 100, 132,142 Thomas, R., 96,137 Thornley, M. J., 98,138 They-Bassett, R. A. E., 84, 87 Tinker, K., 23, 42 Tissihres, A., 91, 92, 95, 96, 108, 141, 142

202

AUTHOR INDEX

Togasaki, R. K., 33,41 Tolmach, L. J., 179,193 Tomlin, P. A., 174,182,193 Tracey, M. V., 155,170 Trager, L., 151,194 Traub, P., 96, 97, 108, 111, 125, 126, 132, 140,142 Traut, R. R., 95,96, 108, 120,142 Trown, P. W., 33,42 Tschudy, D. P., 36,41 Tsugita, A., 100, 132, 142 Tu, L., 97, 111, 125, 142 Turnock, G., 116, 117, 118,138,140, 142 Tuttle, A. L., 12, 20, 42

U Unowsky, J., 118,140

V Valentine, R. C.,23, 42 Van de Putte, P., 189, 191,193 Van Iterson, W., 99, 142 Van Niel, C. B., 1, 42 Van Praag, D., 107,141 Van Sluis, C. A., 189, 191,193 Vatter, A. E., 7, 8, 9, 42 Vazquez, D., 132,142 Vernon, L. P., 1, 3, 26, 40, 42 Villanueva, J. R., 147,171 Virgin, H. I., 33,42 Vitols, E. R., 150, 171 Von Wettstein, D., 33, 42 Voureka, A., 44, 88 VranB, D., 161, 171

Weaver, P., 23, 42 Weber, M. J., 134,142 Webby, D. M., 155, 169 Weibull, C., 21, 42 Wei-ChenTien, 80, 87 Weinblum,D., 181,194 Weirzchowski, K. L., 179,194 Weller, D. L., 97, 142 White, A. E., 106, 116, 117, 119, 120, 121, 138 White, J. R., 108, 132, 138 Widdowson, J., 95, 140 Wild, D. G., 106, 116, 117, 119, 120, 121, 138,139,142 Winkler, E., 44, 87 Williams, R. W., 176, 181, 182, 189, 191, 193,194 Williamson, D. H., 150, 155,170 Willson, C., 110,142 Wisseman, C. L., 119,142 Witherspoon, B. H., 97,138 Witting, M. L., 105, 132, 138 Wolfe, R. S., 7, 8, 9, 42 Wollman, E., 85,87 Wood, B. J. B., 21,42 Woodward, D. O., 38,41,42 Worden, P. B., 8, 9, 14, 15, 16, 17, 20, 21, 40,42 WulfT, D. L., 185,194 Wyckoff, R. W. G., 175, 178,194

Y Yamanaka, T., 23,24, 42 Yan, Y., 100,142 Yankofsky, S. A., 95,101,102,142 Yanofsky, C., 83,88, 131,139 Ylstra, J., 179,192 Yount, V., 23, 40

W Wacker, A., 181,185,194 Wahba, A. J., 100,132,142 Walker, W. F., 44,87 W d e r , J.-P., 95, 107,142 Wang, D. Y., 180,181,194 Ward, J. M., 154,171 Watanabe, T., 45, 84,88 Watanabe, Y., 161,171 Watson, J. D., 91, 194, 106, 108, 111, 119, 120,138,139,140,142

Z Zamenhof, S., 174,194 Zavarine, R., 173,174,175,193 Zehavi-Willner,T., 95, 138 Ziegenspeck, H., 148,171 Zimmermann, R. A., 105, 119, 130, 134, 139,142 Zubay, G., 92, 100,139,142

SUBJECT INDEX A

Azotobacter agilis, differentiation of membranes in, 18 Azotobacter chroococcum, photoreactivation in, 187

Acinetobacter sp., electron micrograph of thin section of, 98 Acridine dyes, use in curing experiment, 45 B Acrylamide gels, use of in separating Bacillus megaterium, disaggregation of ribosomal proteins, 96 Actinomycin D, effect of on bacterial 7 0 s ribosomes in lysates of, 130 effect of actinomycin on ribosomes in, protein synthesis, 134 Actinomycin, effect of on ribosomes in 130 ribosomal precursor particles from, B. megaterium, 130 Adaptive synthesis of photosynthetic 113 ribosomal subunits in, 93 apparatus in bacteria, 29 Adenosine triphosphate, light -inducedsyn- Bacillus subtilis, effect of actinomycin on ribosomes in, 130 thesis of, 3 Aeration, effect of on composition of formation of cross links in DNA from, Rps. spheroides, 28 177 inactivation of DNA in by radiation, Aerobacter aerogenes, photoreactivation in, 186 175 location of succinate dehydrogenase in Age category of yeasts, 164-169 membranes of, 18 Age distribution in yeast populations, Bacteria, repair of damaged DNA in, 164-169 Ageing of yeasts, 162-169 173 Alanine as an N-terminal amino acid in response of to ionizing radiation, 173 response of t o ultraviolet radiation, ribosomal protein, 95 173 Albino mutants of photosynthetic bacteria, Bacterial photosynthesis, scheme for, 3 30 Amber mutations, 134 Bacterial photosynthetic apparatus, 1 formation of, 27 Amino acids, role of in control of RNA Bacterial photosynthetic pigments, chemsynthesis, 106 istry of, 3 8-Aminolaevulinic acid synthase, possible isoenzymes of in Rps. spheroides, 37 Bacterial ribosomes, life cycle of, 89 role of in regulation of chlorophyll Bacteriochlorophyll,structural formula of, 4 synthesis in bacteria, 34, 35 Bacteriochlorophylla from green sulphurstability of, 35, 36 Anaerobic growth of photosynthetic bacteria, 25 Bacteriochlorophylls,2 bacteria, 1 differences in structure, 5 Antibody, fluorescent, use of, 158 Bacteriophage, effect of on RNA synthesis Antigens in chromatophores, 26 Arsenate, resistance of 8.uureus to, 44,54, in bacteria, 107 Bacteriopheophytin from chromatophores 57, 62, 63, 64, 73, 74, 78-80 Arsenite, resistance of S. aureus to, 54 of Rsp. r u h m , 26 Balanced growth, studies on cells in, 112 Assembly of ribosomal subunits, 112 Assembly of ribosomes, 107 Base composition of DNA in relation to resistance to X-radiation, 175 Associated stable diploids, 70-71, 72, 73, Base damage in DNA caused by ionizing 76,78 radiation, 182 8-Azaguanine, incorporation into ribosomal particles, 121 Bismuth, resistance of S. uureus to, 55 203

204

SUBJECT INDEX

Branched pathway in chlorophyll biosynthesis, regulation of, 37 Budding of yeast, 143-169 mechanism of, 1 5 6 1 6 0

C Cadmium, resistance of S. aureus to, 44,52, 54, 57, 62, 63, 64, 74, 78-80 Candida albicans, mechanism of budding of, 154,155 C a d i d a utilis, structure of cell wall, 147 Carotenoid-less mutants of bacteria, 30 Carotenoids, accumulation of by photosynthetic mutants of bacteria, 30 effect df oxygen on synthesis of in bacteria, 8 in photosynthetic bacteria, 3 Catalytic components of chromatophores,

23 Cell fractionation, in study of ribosome localizationin bacteria, 97 Cell membrane and chromatophore material, 12 Cell wall of yeast, 143,145 composition of, 145-147 structure of, 147 Chain breaks in DNA caused by ionizing radiation, 181 Chain breaks in DNA caused by ultraviolet radiation, 176 Changes in DNA caused by ionizing radiation, 181 Chemical changes in DNA induced by radiation, 176 Chitinase of snail juice, 155 Chitin of yeast cell wall, 145,146,147,150, 165 Chlamydornonas moewusii, photoreactiva. tion in, 187 Chloramphenicol, effect of on protein synthesis in bacteria, 100 effect of on RNA synthesis by bacteria, 106 Chloramphenicolparticles, conversion into ribosomes, 119 nature of, 118 Chloramphenicol treatment of bacteria, effect of on ribosomal content, 111 Chlorella pyrenoidosa, division enzyme of, 161 Chlorobium chlorophylls, 2 occurrence of, 4 Chlorobiurn ZimiCola, appearance of photosvnthetic structure in, 9 Chlorobiurn spp., photosynthesis by, 2

Chlorobium thiosubpktophilurn, appearance of Photosynthetic structure m, 9 composition of chromatophoresfrom, 22 electron micrograph of thin section of, 10 location of enzymes and pigments in membranes in, 18 Chlorophyll content of photosynthetic bacteria, relation to membrane content, 14 Chlorophyll contents of bacterial chromatophores, 15 Chlorophyll precursors, accumulation of by photosynthetic mutants of bacteria, 30 Chlorophyll-protein complexes in chromatophores, 24 Chlorophyll synthesis in bacteria, 31 regulation of, 34 Chlorophyll synthesis, synthesis of enzymes catalysing, 30 Chlorophylls, complexes of in bacteria, 6 in photosynthetic bacteria, 2 regulation of synthesis of, 6 spectra of, 5 Chloroplast development in plants, 31 Chloropseudornonas ethylicum, appearance of photosynthetic structure in, 9 chlorophyll content of, 15 location of enzymes and pigments in membranes in, 18 photosynthesis by, 2 Chlortetracyclineparticles, nature of, 121 Chromatiurn chromatophores, analysis of, 21 Chromatiurn D, composition of chromatophores from, 22 Chromtiurn okenii, appearance of photosynthetic structure in, 9 Chromatiurn sp., release of pigment particles from, 12 Chromatiurn spp., photosynthesis by, 2 Chromatophore, definition of, 3 Chromatophore material, and cell membrane, 12 location and structure o f in bacteria, 8 Chrornatophore protein, content of in Rsp. rnol$schianurn, 14 C h r o ~ o b a c t e ~ i u ~ e photoreactivaceu~, tion in, 186 Cistrons for r-RNA synthesis, genetic mapping of, 102 Codons for termination of protein synthesis, 133 Colicin factors, 83-86 Compatability of plasmids, 71-75 Composition of purified chromatophores, 19,21

205

SUBJECT INDEX

Composition of ribosomes, 90 Constitutive mutants for penicillinase, 59, 65-69 Coproporphyrin, accumulation of by Rps. spheroides, 36 Coupling of RNA and protein syntheses, 100 Criteria for purity of chromatophores, 20 Curing of antibiotic resistance, 44-47 Cytochrome a in photosynthetic bacteria, 29 Cytochrome b in bacterial photosynthesis, 3 Cytochrome c in bacterial photosynthesis, 3 Cytochrome c2, leaching of from Rsp. rubrum, 24 Cytochrome contents of chromatophores, 22,23 Cytochrome o in photosynthetic bacteria, ' 29 Cytochromoid in bacterial photosynthesis, 3 Cytochromoidsin photosynthetic bacteria, 27 Cytophaga johnsonii, effect on yeast cell walls, 155 Cytoplasmic membrane, association of bacterial ribosomes with, 99 Cytosine, hydration of following irradiation of bacteria, 178 Cytosine dimers, 181 Cytosine-thyminedimers, 181

Diploids, associated stable, 70-71, 72, 73, 76, 78 normal in plasmids, 64-71 transient in plasmids, 63, 64, 73, 78 Disulphide bonds in yeast cell-wall, 154, 155, 156,160 Division enzyme, 161 Divisional age of yeast cells, 163-167 DNA, as a target in radiation inactivation in bacteria, 174 changes in caused by ionizing radiation, 181 cross-links in, 177 damaged, repair of in bacteria, 173, 183 Drugs, effect of on synthesis of RNAcontaining particles in bacteria, 118

E

Effect of light on phospholipid content of bacteria, 21 Electron microscopy of photosynthetic bacteria, 8 Electrophoretic mobilities of ribosomal proteins, 108 Endomyces magnusii, photomicrographs of, 144 Environmental effects on pigment synthesis by photosynthetic bacteria, 6 Enzyme activities of chromatophores, 23 Enzymic repair of damaged DNA in bacteria, 175 Eosomes, formation of in bacteria, 115 Eremothcium ashbyii, budding of, 155 D E k n i a carotovora, photoreactivation in, 187 Damaged DNA in bacteria, repair of, 173, Erythromycin resistance of 8.aureus, 44, 183 52, 55, 56, 57, 59, 63 Dark repair mechanisms in bacteria, extent Escherichia coli, absence of 70s ribosomes of, 191 from lysates of, 129 Dark repair of radiation damage in dark repair of radiation damage in, 187 bacteria, 187 effect of deprivation of potassium ions Deoxycholate, effect of on chromatophores, on accumulation of ribosomal particles 25 by 122 Depolymerization of DNA caused by effect of magnesium ion concentration ionizing radiation, 181 on distribution of ribosomes from in Detergent treatment of chromatophores, density gradients, 125 25 effect of radiation on, 173 Differences between Athiorhodaceae grown formation of nucleic acid-protein crossphotosynthetically and aerobically, links in DNA of, 178 27 indirect photoreactivation in, 184 Differentiationof chromatophorestructure location of NADHz oxidase in, 18 in green bacteria, 18 sedimentation analysis of ribosome 2,4-Dinitrophenol, effect of on RNA synprecursors in, 123 thesis in bacteria, 107 separation of particles from a relaxed D~plococcu8 pneumoniae, effect of ultrastrain of, 116 violet radiation on DNA of, 176

206

SUBJECT INDEX

E8cherichia coli, synthesis of ribosomalprecursor particles in, 114 ultraviolet irradiation of, 183 E&&k, seanrmla~on of coproporphyrin by Rps. spheroides in presence of, 36 Euglena, chloroplast development in, 31 Excision in dark repair, 189 Exogenous reductant, need for in bacterial photosynthesis, 1 Extent of dark repair mechanisms in bacteria, 191 Extrachromosal elements in bacteria, 83-86

F Farnesol, esterification with in bacteriochlorophylls, 5 Feedback inhibition of 6-aminolaevulinic acid synthase activity in photosynthetic bacteria, 36 Ferredoxin contents of photosynthetic bacteria, 23 Ferredoxin in bacterial photosynthesis, 3 Fertility factors, 83-86 Flavine contents of chromatophores,22,23 Elavoprotein in bacterial photosynthesis, 3 Fluorescent antibody, use of, 158 Fluorescent dyes, use of, 143, 145, 148, 149,150,151,152,153,157,160,162 5-Fluorouracil, incorporation into ribosomal particles, 121 Formation of bacterial photosynthetic apparatus, 27 Fractionation of chomatophores, 24 Function, possible, of relaxed particles, 117 Functional ribosomes, formation of, 126

G Galactolipidsin plant chloroplasts, 21 Genes for r-RNA synthesis, location of on bacterial genome, 101 Genetic control of photosynthetic apparatus in bacteria, 37 Genetic mapping of r-RNA cistrons, 102 Glucan of yeast cell-wall, 145, 146, 147, 149, 155 Glucanase of snail juice, 155 Glucomannan of yeast cell-wall, 145, 146, 147 Gradient centrifugation of bacterial chlorophylls, 17 Green bacteria, vesicles of, 18

Green plant photosynthesis, 1 Green sulphur-bacteria, chlorophyll-protein complexes from, 24 chlorophylls in, 4 photosynthesis by, 2

H Haem, feedback inhibition of S-aminolaevulinic acid synthase activity by, 37 synthesis in bacteria, 31 Haemophilw inJEzenzae, resistance of transforming DNA from to ultraviolet radiation, 180 transforming factor, photoreactivation of, 183 Haemoproteinsin photosynthetic bacteria, 27 Heat, effect of on irradiated bacteria, 179 Heavy chromatophores in bacteria, 16 Hybridization tests with bacterial DNA and RNA, 101 Hydration of cytosine following irradiation of bacteria, 178 Hydration of uracil following irradiation of bacteria, 178 Hydrogen, reduction of by photosynthetic bacteria, 1 8-Hydroxyquinoline as an inhibitor of chlorophyll synthesis in bacteria, 31

I Immunological reactivity of chromatophores, 26 Inception of bacterial ribosomes, 90 Incision of damaged DNA in dark repair, 189 Indirect photoreactivation, 184 Internal membrane as site for pigment in bacteria, 16 Ionic balance of medium, effect of on formation of ribosomal particles in bacteria, 121 Ionizing radiation, changes caused in DNA by, 181 response of bacteria to, 173 Ions, effect of deprivation of on ribosomal assembly, 121 Iron branch of tetrapyrrole biosynthetic pathway, 32 Irradiated bacteria, repair of damaged DNA in, 173 Isolation of purified chromatophores, procedures for, 19

207

SUBJECT INDEX

K Kinetics of synthesis of components of bacterial photosynthetic apparatus, 30

L Lamellar structure of chromatophores in Rps. vil-idis, 12 Lead, resistance of S. aureus to, 55 Levorphanol, effect of on RNA synthesis in bacteria, 107 Life-cycle of bacterial ribosomes, 89 Light, visible, effect of radiation damage, 183 Light chromatophoresin bacteria, 16 Lipase of snail juice, 155 Lipase treatment of chromatophores, 25 Lipid phosphorus, content of in membranes of photosynthetic bacteria, 14 Lipids of yeast cell-wall, 145, 146 Localization of ribosomes in bacteria, 97 Location of chromatophore material in bacteria, 8 Lysates, bacterial, differential centrifugation of, 99 Lysates of E . coli, absence of 705 ribosomes from, 129 Lysis of Rps. spheroides to prepare chromatophores, 20

M Magnesium branch of tetrapyrrole biosynthetic pathway, 32 Magnesium deprivation, effect of on ribosomal content of bacteria, 110 Magnesium ion concentration, effect of on distribution of ribosomes in density gradients, 125 effect of on ribosomes, 92 Magnesium ions, effect of deprivation of on assembly of ribosomal particles in bacteria, 122 Magnesium-vinylpheoporphyrin, structural formula of, 4 Mannan of yeast cell-wall, 145, 146, 147, 154, 155,156 Mannanraso of snail juice, 155 Map of plasmids, 80-84 Maturation of bacterial ribosomes, 90 Membrane, as site for pigment in bacteria, 16 volume of in Rsp. rnolischianurn, 14 Membrane content of photosynthetic bacteria, relation to chlorophyll content, 14

Membrane formation, relation of to chlorophyll synthesis in bacteria, 33 Membrane protein, synthesis of during chromatophore development, 34 2-Mercaptoethanol,effect on cell-walllysis, 155 Mercury resistance in S. aureus, 44,47, 49, 50, 52, 55, 57, 62, 78-80 Message for synthesis of ribosomal proteins, 107 Messenger RNA, release of ribosomes from, 133 turnover of in bacteria, 115 Metabolic shifts designed to promote selective sythesis of ribosome precursors, 122 Metabolically inhibited cells, studies on ribosome assembly in, 115 Methionine, as an N-terminal amino acid in ribosomal protein, 95 Micrococcus lysodeikticus, incision of damaged DNA in dark repair in, 189 Micrococcus pyogenes, photoreactivation in, 186 M i ~ ~ ~ c ~ ~ c u s r a d i O d Ueffect T a n sof , radiation on, 173, 174,175 effect of ultraviolet radiation on DNA of, 178 Mitomycin C, repair of DNA damaged by in bacteria, 174 Mitomycin C damage to bacterial DNA, repairable nature of, 188 Multi-enzyme complexes in chromatophores, 25 Mutants of bacteria, defective in ribosome assembly, 118 Mutants of photosynthetic bacteria, 6, 30

N NADHz oxidase in membranes of A . agilis, 18 Neosomes, formation of in bacteria, 115 New 30s ribosomal particles, 127 Nicotinamide nucleotide contents of chromatophores, 22, 23 Nicotinamide nucleotides, light-dependent formation of in photosynthetic bacteria, 3 Nitrogen mustards, repair of DNA damaged by in bacteria, 174 N-Methyl-N’-nitro-N-nitrosoguanidine, repair of DNA damaged by in bacteria, 174 Non-haem iron contents of chromatophores, 22, 23

208

SUBJECT INDEX

Non-photoreactivable damage in bacteria, 186 Nucleases, action on m-RNA, 100 Nucleic acid-protein cross-linksin bacterial DNA, 178 Nucleoids, bacterial, absence of ribosomes from, 99

0 Occurrence of photoreactivation in bacteria, 186 Ochre mutations, 134 Ochre suppression in bacteria, effect of on ribosomal proteins, 96 Old 30s ribosomal particles, 127 Organic substrates, reduction of by photosynthetic bacteria, 1 Osmotic shock of Rsp. rubrurn to prepare chromatophores, 20 Oxygen, effect of in repressing synthesis of enzymes of chlorophyll synthesis in bacteria, 35 evolution of by photosynthetic organisms, 1 repression of pigment synthesis by in photosynthetic bacteria, 6

P Pathway for chlorophyll synthesis in bacteria, 31 Penicillinase genes, 45-83 Penicillinase of S. aureu~,44-86 PenicilEiurn chrysogenurn, photoreactivation in, 187 Penioillium notatum, ultraviolet irradiation of, 183 Peptide sequences in bacterial ribosomal proteins, 108 Peripheral membrane as site for pigment in bacteria, 16 Pigment content of bacteria, relation t o number of photosynthetic structures, 9 Pigment production in S. aureus, 44 Pigment synthesis by photosynthetic bacteria, environmental effects on, 6 Pigment synthesis in Athiorhodaceae, 27 Pigments, photosynthetic, in bacteria, 3 Phosphate starvation, effect of on assembly of ribosomal particles in bacteria, 122 effect of on ribosomal content of bacteria, 111 Phosphatidylcholine in chromatophores, 21

Phosphatidylethanolaminein Chromatiurn chromatophores, 21 Phosphatidylglycerol in chromatophores, 21 Phospholipids in photosynthetic bacteria in relation to chlorophyll content, 15 Photodynamic oxidation reactions, protection from in photosynthetic bacteria, 4 Photophosphorylating activity of chromatophores, 23 Photophosphorylation activity, of ghost fractions from bacteria, 12 of isolated chromatophores, 20 Photophosphorylation by bacterial chromatophores, 8 Photoreactivable damage in bacteria, 185 Photoreactivable sector, 186 Photoreactivation of DNA damage in bacteria, 183 Photoreduction, by bacterial chromatophores, 8 of NAD by fractions from chromatophores, 26 Photosynthesis, bacterial, scheme for, 3 Photosynthetic apparatus, bacterial, 1 Photosynthetic bacteria, growth of, 1 Photosynthetic mutants of bacteria, 30 Physical changes in DNA induced by radiation, 176 Plasmid compatability, 7 1-75 Plasmid dissociation, 61 Plasmid map, 80-84 Plasmid recombination, 61-63, 71, 73, 75, 76,77,81-83 Plasmid, attachment of, 71-80 diploid forms of, 63-71,72-80 Plasmids of S . aureus, 43-86 classification of, 55, 56, 57 distribution of, 58, 59 erythromycin resistance, 52, 59, 63, 64, 74,75, 81-83 map of, 80-84 nomenclature of, 59,60 resistance to arsenate and arsenite, 54, 62, 63,64,73,74,78-80,81-83 resistance to cadmium salts, 52, 54, 59, 62, 63, 64, 73, 74, 78, 79, 80, 81-83 resistance to mercury salts, 52, 59, 60, 62, 73, 74, 78, 79, 80, 81-83 size of, 83, 84 tetracycline resistance, 57, 61, 78, 81-83 the extracellularity region, 51 the penicillinase region, 51-84 transduction of, 60-83 Polyamines, role of in ribosomes, 96

SUBJECT INDEX

Polyribosome, bacterial, electron micrograph of, 93 Polysomes, bacterial, effect of ribonuclease on, 131 stability of as affected by magnesiumion concentration, 94 Population carrier categories of yeasts, 168, 169 Potassium ions, effect of deprivation of on accumulation of ribosomal particles in bacteria, 122 Primulin, action and use of, 143, 145, 148, 149, 150,151,153, 157,160,162 Production of ribosomal proteins, 107 Production of r-RNA, 101 Proplastids in plants, 31 Protein, separation of from ribosomal particles, 97 Protein-chlorophyll complexes in chromatophores, 24 Protein disulphide reductase, 154, 156, 160,161 Protein of yeast cell-wall, 145, 146, 147, 154 Proteins, removal of from ribosomes, 124 surface, in ribosomes, 97 Protein synthesis, dependence of chlorophyll synthesis on, 31 inhibition of and formation of particles in bacteria, 118 participation of ribosomes in, 131 Proteus spp., photoreactivation in, 187 Protoplasts of yeast, 150, 151, 152, 153, 155,161 Protoporphyrin, structural formula of, 4 Pseudomoms aerzcginosa,survival curve of, 174 Pseudomoms spp., effect of radiation on, 173 Puromycin, effect of on bacterial protein synthesis, 134 Puromycin particles, nature of, 120 Pyrimidine dimers, formation of in DNA, 179 rates of photoreactivation of, 186 Pyrimidine hydrates, formation of in DNA, 179

R Radiation-induced chemical changes in DNA, 176 RC locus, in bacteria, 115 nature of, 106 RC-’ particles, see Relaxed particles Reaction-centre chlorophyll, in photo. synthetic bacteria, 6

209

Reaction-centre chlorophyll, isolation of from Chomatium chromatophores, 26 Recognition of radiation lesion in dark repair, 188 Recombination of plasmid genes, 61-71, 73,75,76, 77, 81-83 Regeneration of yeast protoplasts, 150, 151,152,153, 161 Rejoining of strands in dark repair, 190 Relative cell age of yeasts, 163 Relaxed particles, composition of, 116 fragility of, 116 in bacteria, 110, 115 possible function of, 117 RNA of, 117 sedimentation coefficients of, 116 Relaxed strains of bacteria, 106 Release of ribosomes from m-RNA, 133 Repair of damaged DNA in bacteria, 183 Repair replication, nature of, 188 Replacement of DNA in dark repair, 189 Repressionof enzymes catalysing synthesis of chlorophylls in bacteria, 36 Resistance of 8. aureus to antimicrobial agents, 44-86 Resistance-transfer factors, 83-86 Rhodomicrobium uannielii, appearance of photosynthetic structures in, 9 Rhodopseudomoms pah~stria,appearance of photosynthetic structures in,9 Rhodopseudomoms spheroides, appearance of photosynthetic structures in, 9 chlorophyll content of chromatophores in, 16 composition of chromatophores from, 22 electron micrograph of thin section of, 13 function of chlorophyllsin, 6 invagination of membrane in, 13 regulation of pigment synthesis in, 6 spectrum of bacteriochlorophyllin, 5 structure of chromatophores in, 8 Rhodopseudomoms spp., photosynthesis by, 2 Rhodopseudomoms uiridis, appearance of photosynthetic structures in, 9 bacteriochlorophyllb in, 6 Rhodospil.illum fulvum, appearance of photosynthetic structures in, 9 Rhodospirillum molischianum, appearance of photosynthetic structures in, 9 chlorophyll content of, 15 electron micrograph of thin section of, 11 invagination of peripheral membrane in, 13

210

SUBJECT INDEX

Rhodospirillurn photometricurn, appearance of photosynthetic structures in, 9 Rhodospirillurn rubwrn, appearance of photosynthetic structures in, 9 chlorophyll content of chromatophores in, 15 chromatophores in, 7 , s gradient centrifugation of chlorophylls from, 17 invagination of membrane in, 13 regulation of pigment synthesis in, 6 relation between chlorophyll and phospholipid contents, 15 scheme for photosynthesis in, 3 structure of chromatophores in, 8 Rhodospirillum spp., photosynthesis by, 2 Ribonuclease, action of on ribosomal precursor particles, 113 effect of on bacterial polysomes, 131 Ribonucleic acid, see RNA Ribonucleoprotein particles, in protein synthesis, 89 RNA/protein ratio of, 91 Ribosomal precursor particles, from B. megaterium, 113 kinetics of synthesis of, 113 Ribosomal proteins, existence of pool of in bacteria, 110 functional classes of, 111 incorporation into relaxed particles, 118 molecular weight of, 95 N-terminal amino acids in, 95 number of in bacterial ribosomes, 108 physical state of before incorporation into proteins, 110 production of, 107 separation of, 95 size of pool of, 111 surface location of, 111 Ribosomal-RNA, aggregation of with proteins, 110 as a possible message for synthesis of r-RNA, 107, 108 control of synthesis of, 106 estimates of amounts produced, 104 possible inhibitory effect of ribosomes on synthesis of, 101 possible role as a messenger in protein synthesis, 110 production of, 101 rates of synthesis of, 104 virtual absence of free form from bacteria, 112 Ribosomal subunits, 92 assembly of, 112 Ribosome assembly, effect of deprivation of ions on, 121

Ribosome assembly, in metabolically inhibited cells, 115 in vitro experiments on, 124 mutants defective in, 118 Ribosome composition, 91 Ribosome concentration in bacteria, 106 Ribosome precursors, metabolic shifts designed to promote selective synthesis of, 122 Ribosome structure, 91 Ribosomes, amino-acid composition of, 95 appearance of in electron micrographs, 92 as contaminants of chromatophores, 20 autocatalytic role of, 107 bacterial, indentical functioning of, 109 life cycle of, 89 cellular localization of, 97 characterization of, 91 dissociation of, 92 participation of in protein synthesis, 131 release of from m-RNA, 133 role of in control of RNA synthesis, 99 sedimentation coefficients of, 91 705, formation of in an in vitro system, 130 stabilization of on attachment t o m-RNA, 134 Ribulose diphosphate carboxylase in photosynthetic bacteria, 29 RNA, in relaxed particles, 117 nucleotide composition of ribosomal, 95 role of ribosomes in control of synthesis of, 99 55,95 16S, 95 235,95 m-RNA, direction of translation of, 100 polarized synthesis of, 100 RNA chains, attachment ofribosomesto, 99 RNA containing unnatural bases, in particles, 121 RNA polymerase, 99, 101 possible inhibition of action of by RNA, 101 RNA synthesis by relaxed strains of bacteria, 106

s Saccharmyces cerevisiae, budding of, 148-169 photomicrographsof, 144, 149, 151, 152, 153,156,158,159,163 photoreactivation of radiation damage in, 183, 187 protoplasts of, 150-153

21 1

SUBJECT INDEX

~ a ~ r o m y c ecereviske, s structure and composition of cell wall, 145-147 synchronous culture of, 160 var. ellipsoideus, budding of, 157, 158 Saccharomyces fragilis, protoplasts of, 155 Saccharomycodes ludwigii, photomicrographs of, 144 Scars of yeasts, 143-169 Schemes for assembly of functional ribosomes, 128-129 Schizosaccharomyces pombe, photomicrographs of, 144 Sedimentation characteristics of ribosomes, 90 Sedimentation coefficients of chromatophores, 20 Selective synthesis of ribosome precursors, 122 Serratia marcescens, photoreactivation in, 187 Specific pigment content of chromatophores as a criterion for purity, 20 Spectra, in vivo, of bacterial chlorophylls, 6

Staphylococcus aureus, antibiotic resistance of, 43-86 pigment production in, 44 plasmids of, 43-86 Streptomyces grkeus conidia, ultraviolet irradiation of, 183 Streptomycin, effect of on protein synthesis in bacteria, 100 interaction of with ribosomes, 108 particles, nature of, 121 sensitivity, relation to ribosome assembly, 118 site of action of, 108 Stringent strains of bacteria, 106 Structural protein in bacterial membranes, 34 Structure of chromatophore material in bacteria, 8 Structure of ribosomes, 91 Subunits, ribosomal, combination of t o give functional ribosomes, 126 Succinate dehydrogenase activity of chromatophores, 24 Succinate dehydrogenase, location of on membranes of B. secbtilis, 18 Sulphur bacteria, green, photosynthesis by, 2 Sulphur compounds, inorganic, reduction by photosynthetic bacteria, 1 Survival curves of bacteria exposed to radiation, 174 Synchronous culture of S. cerevhiae, 160

T Target, DNA as a in radiation inactivation in bacteria, 174 Terminal oxidases in photosynthetic bacteria, 29 Tetracycline, effect of on protein synthesis in bacteria, 100 Tetracycline resistance of S. aureus, 44,47, 49, 50, 57, 61, 79 Tetrapyrrole biosynthetic pathway, 32 Thiocapsa sp., appearance of photosynthetic structures in, 9 Thiopedia sp., appearance of photosynthetic structure in, 9 Thiospirillum jenseni, appearance of photosynthetic structure in, 9 Thymine dimers, and photoreactivation, 185 conformation of in DNA, 180 formation of, 177, 180 Transduction, of pencillinase genes, 48,49, 60,60-83 of plasmids of S. aweus, 60-83 of streptomycin resistance, 49, 78, 79 Transient diploids in plasmids, 63, 64, 73, 78 Translation of m-RNA, time for, 133 Trigonopsis oariabilis, budding of, 157 Triton X-100, effect of on chromatophores, 25

U Ubiquinone contents of chromatophores, 23 Ubiquinone in bacterial photosynthesis, 3 UItraviolet radiation, changes in DNA caused by, 176 effect of on bacteria in relation to base composition of DNA, 175 effect on plasmids, 48,49 response of bacteria to, 173 Undermethylation in RNA of chloramphenicol particles, 119 Undermethylation of ribosomal RNA in bacteria, 116 Unnatural bases, effect of on formation of particles in bacteria, 121 Uracil, hydration of following irradiation of bacteria, 178 Uracil dimers, 181 Uracil-thymine dimers, 181 Ustilago maydis, photoreactivation in, 187-

212

SUBJECT INDEX

v Vesicles of green bacteria, 18 Vitamin BIZ synthesis in photosynthetic bacteria, 37

W Wavelength dependency for formation of thymine dimers, 180

X X-radiation,effect of on bacteriainrelation to base composition of DNA, 175

Y Yeast, ageing of, 162-169 scars of, 143-169 Yeast budding, mechanism of, 154-160 Yeast cell-wall, 143, 145-169 composition of, 146-147 structure of, 147 Yeasts, budding of, 143-169

Z Zinc, resistance of 8.aureus to, 64