MODERNMICROBIALGENETICS

Second Edition

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MODERNMICROBIALGENETICS

Second Edition

E D I T E D B Y

Uldis N. StreipsDepartment of Microbiology and Immunology

School of MedicineUniversity of Louisville

Louisville, Kentucky

Ronald E.YasbinProgram in Molecular BiologyUniversity of Texas at Dallas

Richardson, Texas

A JOHN WILEY & SONS, INC., PUBLICATION

This book is printed on acid-free paper. ;oc>)

Copyright © 2002 by Wiley-Liss, Inc., New York. All rights reserved.

Published simultaneously in Canada.

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Library of Congress Cataloging-in-Publication Data

Modern microbial genetics / edited by Uldis N. Streips, Ronald E. Yasbin.—2nd ed.p. ; cm.

Includes bibliographical references and index.ISBN 0-471-38665-0 (cloth : alk. paper)

1. Microbial genetics. I. Streips, Uldis N., 1942-II. Yasbin, Ronald E.[DBLM: 1. Genetics, Microbial. QW 51 M689 2002]

QH434 .M634 2002579'.135—dc21

2001045534

Printed in the United States of America.

1 0 9 8 7 6 5 4 3

Contents

Preface vii

Preface to the First Edition ix

Introduction xi

Contributors xiii

Section 1: DNA METABOLISM , 1

CHAPTER 1. Prokaryotic DNA ReplicationWilliam Firshein 3

CHAPTER 2. DNA Repair Mechanisms and MutagenesisRonald E. Yasbin 27

CHAPTER 3. Gene Expression and Its RegulationJohn D. Helmann 47

CHAPTER 4. Bacteriophage GeneticsBurton S. Guttman and Elizabeth M. Kutter 85

CHAPTER 5. Bacteriophage X and Its RelativesRoger W. Hendrix 127

CHAPTER 6. Single-Stranded DNA PhagesJ. Eugene LeClerc 145

CHAPTER 7. Restriction-Modification SystemsRobert M. Blumenthal and Xiaodong Cheng 177

CHAPTER 8. RecombinationStephen D. Levene and Kenneth E. Huffman 227

CHAPTER 9. Molecular ApplicationsThomas Geoghegan 243

Section 2: GENETIC RESPONSE 259

CHAPTER 10. Genetics of Quorum Sensing Circuitry in Pseudomonas aeruginosa:Implications for Control of Pathogenesis, Biofilm Formation,and Antibiotic/Biocide Resistance

Daniel J. Hassett, Urs A. Ochsner, Teresa de Kievit, Barbara H. Iglewski,Luciano Passador, Thomas S. Livinghouse, Timothy R. McDermott,John J. Rowe, and Jeffrey A. Whitsett 261

v

vi CONTENTS

CHAPTER 11. Endospore Formation in Bacillus subtilis: An Example of CellDifferentiation by a Bacterium

Charles P. Moran Jr 273

CHAPTER 12. Stress ShockUldis N. Streips 281

CHAPTER 13. Genetic Tools for Dissecting Motility and Development ofMyxococcus xanthus

Patricia L. Hartzell 289

CHAPTER 14. Agrobacterium GeneticsWalt Ream 323

CHAPTER 15. Two-Component RegulationKenneth W. Bayles and David F. Fujimoto 349

CHAPTER 16. Molecular Mechanisms of Quorum SensingClay Fuqua and Matthew R. Parsek 361

Section 3: GENETIC EXCHANGE 385

CHAPTER 17. Bacterial Transposons—An Increasingly Diverse Group of ElementsGabrielle Whittle and Abigail A. Salyers 387

CHAPTER 18. TransformationUldis N. Streips 429

CHAPTER 19. ConjugationRonald D. Porter 463

CHAPTER 20. The Subcellular Entities a.k.a. PlasmidsMichael H. Perlin 507

CHAPTER 21. Transduction in Gram-Negative BacteriaGeorge M. Weinstock 561

CHAPTER 22. Genetic Approaches in Bacteria with No Natural Genetic SystemsCarolyn A. Haller and Thomas J. DiChristina 581

Index 603

Preface

The impetus for this updated edition ofModern Microbial Genetics came from manydiscussions among the authors and editorswith the leadership and participants at thelovely Wind River Conference on Prokary-otic Biology held in Estes Park, Coloradoevery June. The first edition, though compre-hensive, had become outdated and the needfor an up-to-date, advanced textbook formicrobial genetics was palpable. With theable encouragement and cooperation of oureditor Luna Han, at John Wiley & Sons, Inc.,the agreement was reached to publish thistext. So, we welcome you to Modern Micro-bial Genetics II.

We have maintained the same model forchapter authorship. Even though in someways it would be optimal to have a singleauthor for the entire textbook, we felt thatthis in-depth material could be handled farbetter by enlisting experts in their fields toput together chapters of their own respectiveinsights. Moreover, we chose authors who arealso excellent teachers so that the textbookcould be easily adapted to classrooms in ad-vanced undergraduate and graduate courses.

A quick comparison of the two editionsshould point out a universal truth about sci-entific publications: namely, a publishedbook may advance information a step, or atmost a few steps, ahead of other existing

books, but the moment it is published, thebook is miles behind where the informationwill ultimately lead. Because of this, inModern Microbial Genetics II the chaptersare extensively revised and updated, someare removed, and others added. This happensto be the most complete and relevant infor-mation at this point in time from our per-spective. Publication on the Web will furtherallow for more facile updating and diminishthe inevitable dissipation of current informa-tion.

As we stated in the first edition, this bookpresents a vibrant field of knowledge withmany areas anxiously awaiting new investi-gators. After going through this text, one oranother of the chapters may beguile you, thereader, enough to willingly immerse yourselfin the wonderful discipline of microbial gen-etics. Again we say—Welcome!

We wish you success in adding the exten-sive knowledge presented in this textbook toyour previous experience in microbial genet-ics and applying it to your own future goalsand objectives. We look forward to many ofyou joining us in generating information, andperhaps even chapters, for future editionsand updates to this textbook.

Uldis N. StreipsRonald E. Yashin

vii

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Preface to the First Edition

The information presented in this book rep-resents the best efforts by a select group ofauthors, who are not only productive in re-search but who are also excellent teachers, todelineate the limits of knowledge in the vari-ous areas of microbial genetics. We feel theuse of multiple authors provides not only fordepth of material, but also enriches the per-spectives of this textbook. The limits ofknowledge need to be stretched continuouslyfor science to remain exciting and meaning-ful. It should be obvious that this then leavesa vast field for future work, where some ofyou readers will find a lifetime of productiveresearch. Moreover, it should also be obviousthat many of the areas discussed in this bookstill contain pathways and byways whichsometimes have never been explored, andsometimes have side roads waiting for eagerminds to map and meld within the pool ofknowledge which we call modern microbialgenetics. We expect that you will have had

some previous exposure to microbial geneticsand will use this text to build on that experi-ence. As you probe in depth the thoughtprocesses and experiments which were usedto formulate the fundamental concepts inmodern microbial genetics, one or anotherof the included chapters may spark the inter-est in your mind to become a traveler withinthis vast and exciting discipline. If that is thecase—Welcome!

We wish you success in adding the know-ledge presented in this textbook to your pre-vious experience in microbial genetics andapplying it to your future goals and object-ives. We thank the many reviewers whohelped to enhance the accuracy and presen-tation of this material. In this regard, MartiKimmey was most helpful in correlating thevarious chapters.

Uldis N. StreipsRonald E. Yasbin

IX

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Introduction

ULDIS N. STREIPS AND RONALD E. YASBIN

The initial studies, which presaged the emer-gence of the capabilities for the completesequencing of genomes and the study ofwhole organism proteomics in addition tovarious aspects of molecular biology, arenow almost 90 years old. The early reportson bacteriophage by Twort (1915), d'Herelle(1917), and Ellis and Delbruck (1939) and theinitial description of the pneumococcal type"transformation" by Griffith (1928) presha-dowed this explosion of information bylaying down a solid foundation on which tobuild layer upon layer of new ideas and facts.Even though these early workers had nobasis for concluding more than their time inthe flow of events allowed them to conjec-ture, we can envision that an unbreakablethread was formulated by their work. Thescientists in the many subsequent decadeshave woven this initially thin thread into anextensive and mutlicolored tapestry in whichare embedded the stories of the research thatis described in Modern Microbial Genetics II.It is fascinating that for the first yearsthe major debate was on the existence andfunction of DNA. Entering the New Millen-ium, not only can we reproducibly obtainDNA, deliver it to any cell we choose,but we can also unlock every secret in thatmolecule.

In the 1940s and 1950s two major researchthrusts permanently changed the perspectiveson microbial genetics and provided the basisfor the explosion of information in the fieldof molecular biology. These were, first, thedocumentation of DNA as the carrier of anorganism's genetic information by Avery andcoworkers (1944) and the subsequent de-

ciphering of the chemical structure of thismolecule by Watson and Crick (1953), andsecond, the discovery of mobile genetic elem-ents by McClintock (1956).

The seminal work on proving that DNA isthe stuff of heredity, can be manipulated, andindeed is self-manipulating, rapidly led to thedescription in 1950s and 1960s of genetic ex-change in bacteria and in subsequent years tomodern microbial genetics. In this textbookthere are detailed descriptions of three majorareas. The first is DNA Metabolism: howDNA replicates (Firshein), how DNA isrepaired (Yasbin), how DNA is transcribedand the transcription regulated (Helmann)and how DNA recombines (Levene and Huff-man). This section also includes the geneticsof bacteriophage including the T-even phages(Guttman and Kutter), the lambdoid phages(Hendrix), the phages with nucleic acids otherthan double stranded DNA (Leclerc), andhow restriction and modification directs mi-crobial existence (Blumenthal and Cheng).A chapter (Geoghegan) on DNA manipula-tion techniques and application to molecularbiology completes the DNA Metabolismsection.

The second section is on Genetic Responseand includes several chapters on how micro-organisms interact with the environment. Therole and mechanism of bacteria in establish-ing disease states is discussed by Hassett andcoauthors. How cells react to environmentalstress is shown in the chapters by Moran onsporulation and Streips on stress shock. Twoenvironmental organisms that depend on gen-etic versatility are discussed in the chapters onMyxococcus by Hartzell and Agrobacterium

xi

xii INTRODUCTION

by Ream. The ability of microorganisms toconstantly sense their environment is revealedin chapters on two-component sensing byBayles and Fujimoto and quorum sensing byParsek and Fuqua.

The last section on Genetic Exchange in-cludes the latest information on the classicexchange mechanisms (see the Chapters byStreips on transformation, Porter on conjuga-tion, and Weinstock on transduction). Perlindiscusses the genetics of plasmids that do notbelong to the F family. In addition this sectionalso includes recent information about trans-posons and their ability to move from cell tocell (Whittle and Salyers). Finally, the mo-lecular study of bacteria which have no stand-ard genetic systems is described by Haller andDiChristina and concludes this book.

The elucidation of global regulatory sys-tems, which control everything from DNAuptake to emergency responses and overallmicrobial development, are widely discussedin various chapters in this book and they helpto bring the study of molecular biology fullcircle. As described by Helmann, Streips, andMoran, there are genes and operons inbacteria which are coordinately regulatedand defined as regulons. So, from the initialconsideration about the existence and natureof DNA, now assumptions are made abouthow genes network and cooperate in multi-gene regulons to suit the needs of the bacter-ial cell.

McClintock's early work showed thatDNA was not merely a static chemical mol-

ecule, but rather a dynamic structure whichcan be amplified to a myriad of genetic pos-sibilities. So it is once the fundamentalaspects of bacterial genes and their exchangewere elucidated, it became apparent that bac-teria, bacteriophage, and also eukaryotes,through mutation, evolution, and genetic ex-change have arranged and rearranged theirgenetic material to take an optimal advan-tage of their niche in the environment. Thistheme is the constant thread that connectsthe various sections and subject areas ofModern Microbial Genetics II.

This textbook is our approach to link thepioneering work of the past to the moderntechnology available today and to startanswering some of the major questions aboutthe molecular mechanisms operating in mi-crobial cells.

REFERENCESAvery OT, MacLeod CM, McCarty M (1944): Studies

on the chemical nature of the substance inducingtransformation of pneumococcal types. Induction oftransformation by a desoxyribonucleic acid fractionisolated from pneumococcus type III. J Exp Med79:137-158.

D'Herelle F (1917): Sur un microbe invisible antagonistedes bacilles dysenteriques. CR Acad Sci 165:373.

Griffith F (1928): The significance of pneumococcaltypes. JHyg 27:113-159.

McClintock B (1956): Controlling elements in the gene.Cold Spring Harbor Symp Quant Biol 21:197-216.

Twort FW (1915): An investigation on the nature of theultramicroscopic viruses. Lancet 11:1241.

Watson JD, Crick FHC (1953): Molecular structure ofnucleic acids. Nature 171:737-738.

Contributors

Kenneth W. Bayles, Department of Micro-biology, Molecular, and Biochemistry, TheCollege of Agriculture, University of Idaho,Moscow, ID 83844-3052

Robert M. Blumenthal, Department of Micro-biology and Immunology, Medical Collegeof Ohio, Toledo, OH 43614-5806

Xiaodong Cheng, Biochemistry Department,Emory University, Atlanta, GA 30322^1218

Thomas J. DiChristina, School of Biology,Georgia Institute of Technology, Atlanta,GA 30332

William Firshein, Department of MolecularBiology and Biochemistry, Wesleyan Univer-sity, Middletown, CT 06459

David F. Fujimoto, Biology Department LS-416, San Diego State University, San Diego,CA 92182

Clay Fuqua, Department of Biology, IndianaUniversity, Bloomington, IN 47405

Thomas Geoghegan, Department of Bio-chemistry and Molecular Biology, Universityof Louisville School of Medicine, Louisville,KY 40292

Burton S. Guttman, The Evergreen State Col-lege, Olympia, WA 98505

Carolyn A. Haller, School of Biology, Geor-gia Institute of Technology, Atlanta, GA30332

Patricia L. Hartzell, Department of Micro-biology, Molecular Biology, and Biochemis-try, University of Idaho, Moscow, ID 83844-3052

Daniel J. Hassett, Department of MolecularGenetics, Biochemistry, and Microbiology,University of Cincinnati, College of Medi-cine, Cincinnati, OH 45267-0524

John D. Hehnann, Department of Microbiol-ogy, Cornell University, Ithaca, New York14853-8101

Roger W. Hendrix, Pittsburgh BacteriophageInstitute, Department of Biological Sciences,University of Pittsburgh, Pittsburgh, PA15260

Kenneth E. Huffman, Department of Molecu-lar and Cell Biology, University of Texas atDallas, Richardson, TX 75083-0688

Barbara H. Iglewski, Department of Micro-biology and Immunology, University of Ro-chester School of Medicine, Rochester, NY14642

Teresa de Kievit, Department of Microbiol-ogy and Immunology, University of Roches-ter School of Medicine, Rochester, NY 14642

Elizabeth M. Kutter, The Evergreen StateCollege, Olympia, WA 98505

J. Eugene LeClerc, Molecular Biology Div-ision, Center for Food Safety and AppliedNutrition, US Food and Drug Administra-tion, Washington, DC 20204

Stephen D. Levene, Department of Molecularand Cell Biology, University of Texas atDallas, Richardson, TX 75083-0688

Thomas S. Livinghouse, Department ofChemistry and Biochemistry, and Depart-ment of Land Resources and EnvironmentalSciences, Montana State University, Boze-man, MT 59717

xiii

XIV CONTRIBUTORS

Timothy R. McDermott, Department of LandResources and Environmental Sciences, Mon-tana State University, Bozeman, MT 59717

Charles P. Moran Jr., Department of Micro-biology and Immunology, Emory UniversitySchool of Medicine, Atlanta, GA 30322

Urs A. Ochsner, Department of Microbiol-ogy, University of Colorado Health SciencesCenter, Denver, CO 80262

Matthew R. Parsek, Department of Civil En-gineering, Northwestern University, Evan-ston, IL 60208

Luciano Passador, Department of Microbiol-ogy and Immunology, University of Roches-ter, School of Medicine, Rochester, NY 14642

Michael H. Perlin, Department of Biology,University of Louisville, Louisville, KY 40292

Ronald D. Porter, Department of Biochemis-try and Molecular Biology, The PennsylvaniaState University, University Park, PA 16802

Walt Ream, Department of Microbiology,Oregon State University, Corvallis, OR 97331

John J. Rowe, Department of Biology, Uni-versity of Dayton, Dayton, OH 45469

Abigail A. Salyers, Department of Micro-biology, University of Illinois, Urbana, IL61801

Uldis N. Streips, Department of Micro-biology and Immunology, School of Medi-cine, University of Louisville, Louisville, KY40292

George M. Weinstock, Department of Bio-chemistry and Molecular Biology, Universityof Texas Medical School, Houston, TX77225

Jeffrey A. Whitsett, Division of PulmonaryBiology, Children's Hospital Medical Center,Cincinatti, OH 45229-3039

Gabrielle Whittle, Department of Micro-biology, University of Illinois, Urbana, IL61801

Ronald E. Yashin, Program in MolecularBiology, University of Texas at Dallas, Ri-chardson, TX 75083

Section I: DMA METABOLISM

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IProkaryotic DNA Replication

WILLLIAM FIRSHEINDepartment of Molecular Biology and Biochemistry, Wesleyan University, Middletown,

Connecticut 06459

I. Introduction 3II. General Concepts of DNA Replication 4

A. Semiconservative Synthesis 4B. The Replicon Model 5

III. Replication Operations 6A. Initiation 6B. Elongation 7

1. Fine Details of Elongation 8C. Termination 12D. Precursors in DNA Replication 16

1. Introduction 162. Types of Metabolic Pathways 163. Multienzyme Complexes 18

IV. The Replicon Membrane Interaction 18A. Introduction 18B. Specific Organisms 19

1. E. coli 192. B. subtilis 203. Plasmid RK2 21

V. General Conclusions 22

|. INTRODUCTION the success was made possible after Wat-T T I ^ - ^ 1 TVKTA * 4. u j son *&& Crick (1953) proposed that theUltimately DNA structure must be under- A A ^ T ^ - V T A • \ j j 1.1 ^ \-, . J

e. r . ,. . structure of DNA existed as a double helixstood in terms of its function just as function . u u * u u * *u u *, , , r * * T- i r °f sugar-phosphates held together by tworequires knowledge of structure. Each func- . , • • _ , • , • / •A. , 1 j j j • punne and pynmidme base pairs, adenine-tion must be resolved and reconstituted in f. _f . . . ,. ,, , . , . , . thymine a n d guanme-cytosine, respectively,complete detail in order to connect it to a ' ,, f ,, u. ,. „ T A, c TX-VTA It was the sequence of these base pairsstructure in the cell. In the case of DNA, Al A , ^ - l ^ 4 •*• r, , • 1 • i r- • f that determined the exact composition ofthree hierarchical functions—storage of ,, T^XT. , , , ,, , , ,. . r . ,. . r i l_. . r the DNA molecule and the molecular struc-genetic information, replication of this infor- x ril / A - 4- • rto . r \- A x - j ture of the gene (storage of genetic informa-mation from generation to generation, and . ,ultimate control of the functions of cellularactivities—have been elucidated in exquisitedetail, although OUr Understanding Of , nj.

M°drn MIcrobM Genetics, Second Edition Edited by' fc to UldisN. Streips and Ronald E.Yasbin. ISBN 0-471-38665-0

those details is far from complete. Much of copyright © 2002 wney-uss, inc.

4 FIRSHEIN

Replication of the double helix was pro- the beginning (origin) of replication (oriQposed by Watson and Crick to be based for this and other organisms such as Bacillusupon the separation of two helices which subtilis (Kornberg and Baker, 1992; Moriyaacted as templates for the precise copying et al., 1994).of complementary strands to form twoprogeny double helices according to the se- ||. GENERAL CONCEPTS OFquence of the base pairs (termed semi- DNA REPLICATIONconservative replication). However, in attemp-ting to identify the components (enzymes, A' Semiconservative Synthesiscontrol factors) responsible for this precise How could Watson and Crick's model beduplication, it became obvious that the pro- proven that replication occurred in a semi-cess was interdependent with other related conservative manner? In fact two additionalphenomena such as repair and recombin- possibilities existed besides such a mechan-ation of DNA. Some of the enzymes could ism. These included conservative (bothbe used for all of the processes. In fact there strands replicated simultaneously) or disper-is a growing body of knowledge that not sive (each strand was fragmented, copiedonly are the pathways intimately related, and joined to form a completely new paren-but many of the proteins may be part of a tal and progeny strand),"superfamily" in which all of them share a The most important and definitive experi-highly conserved DNA-binding motif as de- ments that proved that DNA was replicatedtermined by X-ray crystallography or elec- semiconservatively were carried out by Me-tron microscopy (Engelman, 2000). selson and Stahl (1958). They adapted E. coli

The difficulty (and complexity) of eluci- to a growth medium containing N15H4C1dating these interactions is further under- ensuring that every molecule in the cell con-scored by two additional characteristics of taining nitrogen (including DNA) wouldthe replicative process. First, unlike RNA have the N15 heavy density label. When theseand protein synthesis, DNA replication cells were shifted to a medium containingoccurs at discrete times during the cell cycle, the normal light density N14H4C1, theThe many components involved must be as- resulting progeny double helices after onesembled and disassembled after each round generation consisted of a hybrid densityof replication. Second, unlike the organelle DNA species containing presumably oneinvolved in protein synthesis (the ribosome) strand of N15-DNA and one strand of N14-which is held together with strong forces, DNA. After a second generation in lightthose that maintain the DNA replisome density medium, the double helices consisted(the components involved in DNA replica- equally of both the hybrid density speciestion) involve weak electrostatic forces which and a complete light density species. This iscan be dissociated under mild salt condi- seen in Figure 1 where the various DNAtions. Thus in vitro studies that have formed species are separated by centrifugation in athe bases for understanding many of the in- neutral cesium chloride equilibrium densitytricacies of replication are subject to artifacts gradient.because extraction of the replisome from The other hypotheses could not be sup-cells may be disruptive and not represent ported by these results. Further proof ofthe in vivo condition as fully as possible. the mechanism was obtained by separating

Nevertheless, much has been revealed by the hybrid density species in an alkalineclassic in vitro studies of prokaryotes using cesium chloride density gradient which de-single stranded DNA viruses that infect Es- natured the DNA into two single strandedcherichia coli and sequester many of the forms on the gradient, one consisting of N15-host's components (Kornberg and Baker, DNA, the other of N14-DNA (Meselson and1992) and recombinant plasmids containing Stahl, 1958).

PROKARYOTIC DMA REPLICATION 5

Fig. I. Semiconservative replication of £ coli DNA I5N (heavy) parental, I4N (light) progeny and hybridfirst-generation DMAs are separated by sedimentation in a cesium chloride equilibrium density gradient.(Reproduced from Kornberg and Baker, 1992, with permission of the publisher.)

B. The Replicon Model

DNA replication is divided into three partsor phases. These include initiation (or begin-ning of replication), elongation, and termin-ation. Early studies had demonstrated thatinitiation required the synthesis of new pro-teins (Maaloe and Hanawalt, 1961) whileelongation did not. However, it was theautoradiographic studies of Cairns (1963)with E. coli and the genetic studies of Yoshi-kawa and Sueoka (1963) with B. subtilis thatdemonstrated that bacterial chromosomeshad a fixed origin of replication. Cairnsresults further confirmed earlier genetic stud-ies (Hayes, 1962) that the E. coli chromo-some consisted of one double helical speciesof DNA without a free end, namely a circu-lar molecule. During replication this mol-ecule was split into two "replicating" forksthat traveled along the template in oppositedirections migrating away from the sitewhere elongation began, namely the origin,until they met, approximately 180° oppositethe origin where elongation terminated.

Based on these studies, Jacob et al. (1963)proposed a model that envisioned one circu-lar chromosome as a genetic unit of replica-tion (the replicon) in which replication beganat a defined small region on the chromo-some, the origin, and proceeded, usually bi-directionally to the terminus via tworeplication forks seen in the theta circles ofCairns. This model is depicted in Figure 2.

A number of control features wereproposed based on a positive activation

Fig. 2. Bidirectional replication of the £ coli chro-mosome. The thin black arrows identify the advan-cing replication forks. The micrograph is of abacterial chromosome in the process of replication,comparable to the figure next to it. (Modified fromKlug and Cummings, 2000, with permission of thepublisher.)

mechanism in which a series of genes nearthe origin region controlled the synthesis ofinitiation proteins (the initiator) that acti-vated the "replicator" (the origin region),which then replicated (elongated) any DNAthat was part of the replicon. The repliconwas thought to be anchored to a specific site

6 FIRSHEIN

in the cells (probably the membrane) wherethe various enzymes and initiator proteinscould be sequestered and where segregationof the newly synthesized chromosome couldoccur.

Even after 35 years, the replicon modelstill represents a good conceptual framework(with modifications) to explain these import-ant events.

III. REPLICATIONOPERATIONS

A. Initiation

Many complex problems must be resolved toreplicate a complete chromosome. Amongthe first of these is the localized unwindingof the duplex at a specific site (the origin) inorder that each stand can begin its functionas a template. In addition this open configur-ation must be stabilized so that synthesis canactually occur. The process is called initi-ation and is at the heart of the entire replica-tive process.

Although a number of origin regions havebeen studied, the most complete analysis todate has been that of the E. coli origin (ororiC). It was detected and localized by sev-eral different strategies, among them its in-ability to be deleted from the chromosomewithout destroying the cell, gene dosage ex-periments in which genes near the originwere replicated first, and the constructionof an oriC plasmid which required the samefunctions as replication of the entire chromo-some including bidirectional replication (Birdet al., 1972; Von Meyenburg et al., 1979;Meijer and Messer, 1980).

The minimum oriC region (absolutely re-quired for in vitro synthesis) (Oka et al.,1980) consists of 245 base pairs in a negativesupercoil state that can be subdivided on thebasis of function into two parts. The firstpart contains four repeating sequences of 9base pairs (9 mers), while the second partcontains three repeating sequences of 13base pairs (13 mers). The latter are highlyAT rich, while the former contains sequences(consensus 5"-TTAT C/A CAC/AA-3') that

recognize an important 52 kDa initiationprotein (DnaA) (Fuller and Kornberg, 1983).This protein is not only a central player ininitiation of oriC in E. coli but in the initi-ation of many other bacteria (Zyskind et al.,1983; Zyskind and Smith, 1986; Bramhilland Kornberg, 1988) and even of some plas-mids (Masai et al., 1987; Konieczny andHelsinski, 1997), suggesting its ubiquity andfulfilling one of the tenets of the repliconmodel (Jacob et al., 1963).

The remarkable structure of the originpredicted how the localized denaturationwas effected, and a variety of experimentalapproaches (genetic, biochemical, and ultra-structural) confirmed the predictions (Fun-nell et al., 1987; Bramhill and Kornberg,1988; Echols, 1990). The process was initi-ated in E. coli by the specific binding of theDnaA protein (20-40 monomers) to the 9base pair DnaA boxes.

As a result of this binding and in the pres-ence of ATP, a basic instability of the AT-rich region is heightened and approximately45 base pairs are denatured to mark it forrecruitment of other essential proteins intothe bubble (e.g., DnaB and DnaC) that fur-ther open and destabilize the complex (seenext section). Additional accessory proteinsalso are involved in the process. These in-clude the HU protein, a small doublestranded DNA-binding protein (Rouviere-Yaniv and Gros, 1975) that may be involvedin bending the DNA, and the single-strandedDNA-binding protein (SSB) that may stabil-ize the single-stranded regions when they arepresent (Meyer and Laine, 1990).

Figure 3 a, b depicts the initiation processas currently envisaged with electron micro-graphs that visualize the complex.

There are additional important aspectsconcerning the regulation and activity ofthe DnaA initiation protein in the initiationprocess that point to its probable location(and that of the replicon) in the cell, namelythe cell membrane. DnaA functions primar-ily in a membrane environment (Yung andKornberg, 1988), is activated by anionicphospholipids (Sekimizu and Kornberg,

PROKARYOTIC DMA REPLICATION 7

Fig. 3. a: A scheme for initiation at oriC. The DnaA protein binds the four 9 mers, organizing oriC around aprotein core to form the initial complex. The three 13 mers are then melted serially by DnaA protein tocreate the open complex. The DnaB-DnaC complex can now be directed to the 13-mer region to extend theduplex opening and generate a preprimng complex, which unwinds the template for priming and replication.(Modified from Kornberg and Baker, 1992, with permission of the publisher.) b: Electron micrographs ofprotein complexes at oriC. Initial complexes (above) were formed on a supercoiled oricC plasmid with DnaAprotein only. Prepriming complexes (below) were formed with DnaA, DnaB, DnaC, and HU proteins.Complexes were cross-linked and the DMA was cut with a restriction endonuclease. Protein complexesare seen at the oriC site asymmetrically situated on the DMA fragments. (Taken from Kornberg and Baker,1992, with permission of the publisher.)

1988), and has been found in living cells to belocated at the cell membrane (Newman andCrooke, 2000). This will be discussed furtherin a separate section (Section IV).

B. Elongation

The elongation of DNA from the initiationsite bubble is truly a remarkable and efficient

process. Enzymes (see below) interact witheach template strand polymerizing newDNA at approximately 6 x 104 base pairs/min, completing a typical prokaryotic chro-mosome of approximately 4.8 megabases bi-directionally in 40 minutes (Helmstetter andLeonard, 1987). The amazing aspect of thisfeat is that there are many other essential

8 FIRSHEIN

proteins (more than 40 at the latest count)acting in coordination and that each strandis elongated in opposite directions, althoughit appears that they are being synthesizedsimultaneously. At the heart of the elongationprocess are the activities of two out of thethree known DNA polymerases (I and III)that are absolutely required for elongation.The latter polymerase is the principal replica-tive enzyme in most prokaryotes, consistingof at least 10 distinct subunits organized astwo complete units, one for each strand (seebelow).

Three inherent problems embody the com-plexity of the process. The first is that thetwo DNA strands of the helix are mirrorimages of each other (or antiparallel) andthat one strand runs in the 5' to 3' direction,while the other strand runs in the oppositedirection 3' to 5'. These notations refer to thechemical structure of each DNA strand asshown in Figure 4a.

The second is that all DNA polymerases,as far as is known, extend a growing DNAchain by the addition of a deoxyribonucleo-side triphosphate precursor (dNTP) to anopen 3' OH group in a 5' to 3' directionas shown in Figure 4b. Thus only one strand(termed the leading strand) can be extendedcontinuously in the same direction as thereplication fork. The other strand (termedthe lagging strand) can not be extendedin this way because there is no open 3' OHgroup at the same end as its complementarystrand (see Figs. 4a, b above). Therefore itis necessary to elongate DNA, literally, inthe direction opposite from that of theleading strand and replication fork, at leastfor a short distance, in order for it toappear as if elongation of both strands isoccurring simultaneously in the same direc-tion.

The third is that no DNA polymerase iscapable of starting a DNA chain de novo. Itcan only extend a chain already initiated.Therefore another mechanism is required toprovide a "primer" with an open 3' OHgroup for extension on both the leadingand lagging strands).

After considerable genetic and biochemicalanalysis of these three problems (summarizedin Kornberg and Baker, 1992; Marians,1992, 1996; and Ogawa and Okazaki, 1980)the concept of continuous and discontinuoussynthesis was proposed in which one strand(3'-5') serves as a template for continuousDNA synthesis (leading strand) while theother strand (5'-3') serves as a template fordiscontinuous DNA synthesis (the laggingstrand). In the former only one point of initi-ation is required whereas in the latter manyseparate initiation points are necessary.Involvement of RNA as the primer forDNA chain extension was inferred initiallyfrom the sensitivity of such replication to ri-fampicin, an inhibitor of RNA polymeraseactivity (Brutlag et al., 1971). However, itappears that this particular requirement isrelated to the phenomenon of transcriptionalactivation of regions upstream from the initi-ation site which aid the DnaA protein toopen the DNA duplex (Baker and Kornberg,1988). Instead, the primer that does consistof a short 10 to 15 bp segment of RNAis synthesized by another type of polymeriz-ing enzyme, the primase, which actuallycan polymerize both DNA and RNA precur-sors (see next section). The existence ofan RNA-DNA single-stranded moleculeduring elongation was demonstrated by anumber of techniques, among them the detec-tion of a covalent phosphodiester bond be-tween a deoxynucleotide (DNA precursor)and a ribonucleotide (RNA precursor)(described in Kornberg and Baker, 1992).The entire process is illustrated in Figures 5aand 5b.

I. Fine details of elongation

Knowledge of the details of elongation is stillemerging, and although there is generalagreement concerning the basic mechanisms,much still remains to be settled. Genetic an-alysis has played a leading role in explainingmost of what is known. One of the classicmutations (polAl) revealed that the first andmost abundant DNA polymerase discovered(DNA pol I; Lehman et al., 1958) was not

PROKARYOTIC DMA REPLICATION 9

Fig. 4. a: (I) The linkage of two nucleotides by the formation of a C-3'-5' (3'-5') phosphodiester bond,producing a dinucleotide. (2) A shorthand notation for a polynucleotide chain. (Reproduced from Klug andCummings, 2000, with permission of the publisher.) b: Demonstration of 5' to 3' synthesis of DMA.

10 FIRSHEIN

Fig. 5. a: A conceptual diagram of the initiation ofDMA synthesis. A complementary RNA primer isfirst synthesized to which DMA is added. All synthe-sis is in the 5'-3' direction. (Reproduced from Klugand Cummings, 2000, with permission of the pub-lisher.) b: Illustration of the opposite polarity ofDMA synthesis along the two strands, necessarybecause the two strands of DMA run antiparallel toone another and DMA polymerase III synthesisoccurs only in one direction (5'-3'). On the laggingstrand, synthesis must be discontinuous. On theleading strand, synthesis is continuous. RNA primersare used to initiate synthesis on both strands. (Re-produced from Klug and Cummings, 2000, with per-mission of the publisher.)

the only polymerase present in bacteria butalso was not the true replicative polymerasesince polAl mutants could still replicateDNA (Delucia and Cairns, 1969). Neverthe-less, an essential function of DNA pol I wasrevealed because of defects in the mutant'sability to repair DNA. Since then, a variety

of other mutants, in particular, conditionalmutants (those whose products are inhibitedunder restrictive, but not permissive, condi-tions, e.g., high [42 °C] and low [30 °C] tem-peratures respectively) provided significantinsights into whether a particular component(enzyme or control protein) was present andactive in a particular complex. Table 1 depictsa number of genes that were discovered in thisand other ways involved in many aspects ofDNA replication or repair in E. coli. Al-though such genes are presumably present inother organisms, only B. subtilis has beeninvestigated to any great extent and thereare some significant differences between thesetwo organisms as well (Yoshikawa andWake, 1993; Imai et al, 2000).

The steps in elongation of E. coli can be des-cribed best as a series of points (summarizedin Kornberg and Baker, 1992; Marians, 1992,1996, 2000; Kelman and O'Donnell, 1995).

1. After the melting of the AT rich regionsby the DnaA-DNA complex in the origin toprovide a 45 base pair bubble during initi-ation, the separated single-stranded DNAsare coated with SSB (Meyer and Laine,1990) to (a) prevent their degradation, (b)keep each strand rigid, and (c) possibly directpriming of DNA synthesis to specific un-bound regions in the open bubble.

2. The DnaA protein directs theDnaB-DnaC protein complex into the openregion at oriC to extend the duplex openingand generate a prepriming complex by aprotein-protein interaction with DnaB(Marszalek and Kaguni, 1994). DnaB per-forms two functions. It acts as a helicase tounwind the duplex in front of the replicationfork (in the presence of ATP) (see step 4below) and as a marker or activator for theprimase (DnaG) that begins the synthesis ofthe RNA primers both on the leading andlagging strands (Rowen and Kornberg,1978). Because replication is bidirectional,two DnaB-DnaC complexes are positionedat the beginning of each replication fork.The DnaC protein also has two functions(Kobori and Kornberg, 1982; Allen and

PROKARYOTIC DNA REPLICATION I I

TABLE 1. A Partial List of Genes Involved in DNA Replication and Repair of E. coliGene ProteinpolApolBdnaE, N, Q, X, holA, holB,

holC, holD, holEdnaGpriA, priB, priC, dnaT

dnaB, CdnaAgyrA, BHgssbrnha

DNA polymerase I (repair and replication)DNA polymerase II (repair of UV damage)DNA polymerase III subunits (main replicative

enzyme)Primase (initiates RNA primers)subunits of the primosome (with DnaG,

DnaB and DnaC)Helicase and helicase binder, respectivelyInitiationGyrase subunits (relaxes supercoils)DNA ligase (joining enzyme)Single-stranded binding proteinsRibonuclease H (degrades single-stranded RNA in

RNA-DNA hybrid molecule)Sources: Compiled from Kornberg and Baker, 1992; Marians, 1992, 1996, 2000.

Kornberg, 1991). It keeps the DnaB proteinin an inactive state until the latter is pos-itioned via a cryptic DnaC-DNA bindingsite onto the SSB-free denatured DNA.Once binding occurs, DnaC is dissociatedfrom oriC to allow for DnaB function as ahelicase.

3. At this instant, two different but inter-acting complexes form at each replicationfork. One contains the DNA polymerase IIIten subunit replicase (called the holoenzyme)that synthesizes both nascent leading and lag-ging strands in a coordinated manner (oneholoenzyme for each strand), and the othercontains the primosome. The latter consistsof a seven subunit multienzyme complex thatis positioned along the lagging strand tem-plate and unwinds both the parental templateand synthesizes the RNA primers for multipleinitiations of the small 2 kb DNA fragments(termed Okazaki fragments, after the scientistwho discovered them) that characterize dis-continuous DNA synthesis (Ogawa and Oka-zaki, 1980; Marians, 1992). The leadingstrand presumably only requires one primingevent with the DnaG protein, and so synthe-sis is continuous.

4. As unwinding mediated by the DnaBhelicase proceeds, supercoiling intensifies

ahead of the replication fork; that is, thedouble helix becomes twisted more tightly.Such supercoiling must be relieved and thatis accomplished by the action of specialenzymes called topoisomerases (in bacteria,DNA gyrase} (Wang, 1987). This bacterialenzyme acts by nicking the supercoil onboth strands to "relax" the supercoil andthen acts to reseal the nick or nicks aheadof the DnaB helicase (all of this occurring inthe presence of ATP).

5. DNA pol III is the principal replica-tion enzyme required for elongation of aduplex template in E. coli and probablymost other prokaryotes although differentsubunits might replace some of the E. colicomponents in these other organisms. Pre-sent only in very small amounts (10-20 mol-ecules/cell with a molecular weight of 900kDa), it is a remarkably efficient "replicatingmachine" (Kelman and O'Donnell, 1995).How the holoenzyme is actually recruitedto the initial replication forks is unknown.It may not be due to any specific protein toprotein interaction but rather to an ex-tremely efficient recognition of a primer ter-minus (O'Donnell, personal communication),which then acts to bind the holoenzyme asfollows: One of the subunits is a complex of

12 FIRSHEIN

six monomers ((3-subunit) that encircles theDNA as a clamp, while another of the sub-units acts to load the clamp on to the DNA(clamp loader). The clamploader is quitecomplex and consists of five different pro-teins (-/-complex). A two-stage process isenvisaged in which the -/-complex first recog-nizes a primed template (on the leading orlagging strand) and in the presence of ATPassembles the clamp on to the template. In asecond step the catalytic core of the polymer-ase (consisting of three subunits including a3'-5' endonuclease [and its stimulator] toexcise the occasional mismatched nucleotidein base pairing [proofreading] and the poly-merizing [catalytic] enzyme that recognizesthe correct deoxyribonucleoside triphos-phate precursor as well as the correct tem-plate base to which it will be paired) isassembled behind the clamp. Although itwas originally assumed that the clamp slidalong the DNA template in a processivemanner (i.e., the maintenance of enzyme ac-tivity over a relatively long sequence of tem-plate for both the leading and laggingstrands), it is most probable, instead, thatthe template migrates through a fixed holo-enzyme site (factory model; Lemon andGrossman, 1998) and that this site is prob-ably the cell membrane (Firshein, 1989, Fir-

shein and Kim, 1997) (see Section IV). Otherimportant features of this remarkableprocess include the dimerization of the cata-lytic core by the dnaX gene. Most of thesepoints are depicted in Table 2 and Figures 6to 10.

C. Termination

The most significant event to occur afterelongation is initiated in a bidirectionalmanner from a fixed origin on a circularchromosome, is termination approximately180° from the initiation site. The movementof the replication forks, like two expresstrains, must be inhibited from "crashing"into each other, and at the same time somemechanism must exist to separate the twochromosomes after they have been com-pleted. Only two bacterial species, E. coliand B. subtilis, have been examined in detailin this respect, and although the overall fea-tures are similar (inhibition of replicationforks, and separation of completed chromo-somes), many differences exists in specificdetails (for reviews, see Yoshikawa andWake, 1993; and Baker, 1995). In both or-ganisms the main target for inhibition maybe the DNA helicases (DnaB in E. coli;DnaC in B. subtilis} (Lee et al., 1989; Khatriet al., 1989; Imai et al., 2000).

Fig. 6. Two-stage assembly of a processive polymerase. The -/-complex recognizes a primed template andcouples hydrolysis of ATP to assemble p on DNA. The "/-complex easily dissociates from DNA and canresume its action in loading (3 clamps on other DNA templates. In a second step, core assembly with the (3clamp to form a processive polymerase. (Taken from Kelman and O'Donnell, 1995, with permission of thepublisher.)

TABLE 2. DNA Polymerase III Holoenzyme Subunits and Subassemblies

Subunitas.4>

T

•y85'Xijr

P

GenednaEdnaQ, mutDholE

dnatf

dnaX]

holAholBholCholD

dnaN

Mass(kDa)129.927.5

8.6

71.1

47.538.736.916.615.2

40.6

FunctionDNA polymerase ^Proofreading 3'-5' exonucleaseStimulates e exonuclease )

Dimerizes core. DNA-dependent

Binds ATPBinds to (3

Subassembly

, \core

ATPase /

pol III'

\

Cofactor for -y ATPase and stimulates clamp loadingBinds SSBBridge between x and -y

Clamp on DNA

/

\

•^-complex

/

pol III*

Note:1' This gene contains two coding sequences due to a frameshift.Source: (modified from Kelman and O'Donnell, 1995, with permission of the publisher).

14 FIRSHEIN

Fig. 7. The molecular structure of the (3 clamp. A"doughnut" structure consisting of a head to taildimer containing six domains with a central openinglarge enough to accommodate duplex DMA. (Takenfrom Kelman and O'Donnell, 1995, with permissionof the publisher.)

The terminus regions (Ter) of both organ-isms contain multiple DNA replication ter-minators consisting of short DNA sequencesthat bind specific terminator proteins (repli-cation terminator protein—RTF, in B. sub-tills and terminus utilization substance; Tus,in E. coli). Thus far, 9 such terminators havebeen detected in B. sub tills and 10 in E. coll(Coskun-Ari and Hill, 1997; Griffiths et al.,1998). There is no relationship between theproteins involved or the sequences in the

terminator regions of both organisms(Baker, 1995). Thus, in E. coli, Ter sites are22 bp in length, while the B. subtilis Ter sitesconsist of 30 bp imperfect inverted repeats.The E. coli regions are recognized by amonomer of the Tus proteins (MW 36,000),while the B. subtilis terminators are recog-nized by two dimers of the RTF (MW14,500). The Ter sites in E. coli are spreadover a long distance on the chromosome(approximately 50 kb), while the Ter sites inB. subtilis encompass only 59 bp.

These multiple terminator sites have beenthought to act as a series of trip wires to slowdown the replication forks, with the outerregions acting as backups to those more cen-trally located within the terminus region(Griffith and Wake, 2000). However, it isimportant to point out that some of the Tersites are oriented to stop the clockwise repli-cation fork, while others are oriented to in-hibit the anticlockwise replication fork inboth organisms. For example, in E. coli, theclockwise fork passes through three Ter sitesthat are in an inactive orientation until itcontacts one oriented in the right direction(Baker, 1995).

A model of helicase inactivation by theTer complexes presupposes that there is an

Fig. 8. Scheme of polymerase cycling on the lagging strand. Pol III holoenzyme is held to DNA by the Pclamp for continuous (processive) extension of Okazaki fragment (left). At the end of polymerization, pol III(every subunit except the clamp) is dissociated from the fragment and reattaches to a new RNA primer(right) with the original clamp remaining on the finished Okazaki fragment. (Taken from Stukenberg et al.,1994, with permission of publisher.)

PROKARYOTIC DMA REPLICATION 15

Fig. 9. Illustration of how concurrent DMA synthesis may be achieved on both the leading and laggingstrands at a single replication fork. The lagging template strand is "looped" in order to invert the physicaldirection of synthesis, but not the biochemical direction. The enzyme functions as a dimer with each coreenzyme achieving synthesis on one or the other strands, a: Conceptual diagram (taken from Klug andCummings, 2000, with permission of the publisher), b: Two pol III cores interacting with dnaX which alsointeracts with the ^-complex clamp loader (taken from Kelman and O'Donnell, 1995, with permission of thepublisher).

Fig. 10. Conceptual model of DMA replication fork without looping of the tagging strand.