Structure and Function of the Bacterial Genome E-Book

Charles J. Dorman

• Herausgeber: John Wiley & Sons
• Kategorie: Wissenschaft und neue Technologien
• Sprache: Englisch

Seitenzahl: 971

Veröffentlichungsjahr: 2020

Beschreibung

Presents an integrated view of the expression of bacterial genetic information, genome architecture and function, and bacterial physiology and pathogenesis This book blends information from the very latest research on bacterial chromosome and nucleoid architecture, whole-genome analysis, cell signaling, and gene expression control with well-known gene regulation paradigms from model organisms (including pathogens) to give readers a picture of how information flows from the environment to the gene, modulating its expression and influencing the competitive fitness of the microbe. Structure and Function of the Bacterial Genome explores the governance of the expression of the genes that make a bacterium what it is, and updates the basics of gene expression control with information about transcription promoter structure and function, the role of DNA as a regulatory factor (in addition to its role as a carrier of genetic information), small RNAs, RNAs that sense chemical signals, ribosomes and translation, posttranslational modification of proteins, and protein secretion. It looks at the forces driving the conservation and the evolution of the dynamic genome and offers chapters that cover DNA replication, DNA repair, plasmid biology, recombination, transposition, the roles of repetitive DNA sequences, horizontal gene transfer, the defense of the genome by CRISPR-Cas, restriction enzymes, Argonaute proteins and BREX systems. The book finishes with a chapter that gives an integrated overview of genome structure and function. * Blends knowledge of gene regulatory mechanisms with a consideration of nucleoid structure and dynamics * Offers a 'DNA-centric' approach to considering transcription control * Views horizontal gene transfer from a gene regulation perspective * Assesses the opportunities and limitations of designing synthetic microbes or rewiring existing ones Structure and Function of the Bacterial Genome is an ideal book for graduate and undergraduate students studying microbial cell biology, bacterial pathogenesis, gene regulation, and molecular microbiology. It will also appeal to principal investigators conducting research on these and related topics and researchers in synthetic biology and other arms of biotechnology.

Leseprobe

Table of Contents

Cover

Dedication

Preface

1 The Bacterial Genome – Where the Genes Are

1.1 Genome Philosophy

1.2 The Bacterial Chromosome

1.3 Chromosome Replication: Initiation

1.4 Chromosome Replication: Elongation

1.5 Chromosome Replication: Termination

1.6 Replication Produces Physically Connected Products

1.7 Decatenating the Sister Chromosomes

1.8 Resolving Chromosome Dimers

1.9 Segregating the Products of Chromosome Replication

1.10 Polar Tethering of Chromosome Origins

1.11 Some Bacterial Chromosomes Are Linear

1.12 Some Bacteria Have More than One Chromosome

1.13 Plasmids

1.14 Plasmid Replication

1.15 Plasmid Segregation

1.16 The Nucleoid

1.17 The Chromosome Has Looped Domains

1.18 The Macrodomain Structure of the Chromosome

1.19 The Chromosome Displays Spatial Arrangement Within the Cell

1.20 SeqA and Nucleoid Organisation

1.21 MukB, a Condensin‐Like Protein

1.22 MatP, the matS Site and Ter Organisation

1.23 MaoP and the maoS Site

1.24 SlmA and Nucleoid Occlusion

1.25 The Min System and Z Ring Localisation

1.26 DNA in the Bacterial Nucleoid

1.27 DNA Topology

1.28 DNA Topoisomerases: DNA Gyrase

1.29 DNA Topoisomerases: DNA Topoisomerase IV

1.30 DNA Topoisomerases: DNA Topoisomerase I

1.31 DNA Topoisomerases: DNA Topoisomerase III

1.32 DNA Replication and Transcription Alter Local DNA Topology

1.33 Transcription and Nucleoid Structure

1.34 Nucleoid‐associated Proteins (NAPs) and Nucleoid Structure

1.35 DNA Bending Protein Integration Host Factor (IHF)

1.36 HU, a NAP with General DNA‐binding Activity

1.37 The Very Versatile FIS Protein

1.38 FIS and the Early Exponential Phase of Growth

1.39 FIS and the Stringent Response

1.40 FIS and DNA Topology

1.41 Ferritin‐Like Dps and the Curved‐DNA‐binding Protein CbpA

1.42 The H‐NS Protein: A Silencer of Transcription

1.43 StpA: A Paralogue of H‐NS

1.44 H‐NS Orthologues Encoded by Plasmids and Phage

1.45 H‐NSB/Hfp and H‐NS2: H‐NS Homologues of HGT Origin

1.46 A Truncated H‐NS‐Like Protein

1.47 Hha‐like Proteins

1.48 Other H‐NS Homologues: The Ler Protein from EPEC

1.49 H‐NS Functional Homologues

1.50 H‐NS Functional Homologues: Rok from Bacillus spp.

1.51 H‐NS Functional Homologues: Lsr2 from Actinomycetes

1.52 H‐NS Functional Homologues: MvaT from Pseudomonas spp.

1.53 The Leucine‐responsive Regulatory Protein, LRP

1.54 Small, Acid‐soluble Spore Proteins, SASPs

2 Conservation and Evolution of the Dynamic Genome

2.1 Disruptive Influences: Mutations

2.2 Repetitive Sequences in the Chromosome and Their Influence on Genetic Stability

2.3 Contingency Loci and the Generation of Microbial Variety

2.4 Rhs: Rearrangement Hotspots

2.5 REP Sequences

2.6 RIB/RIP, BIME‐1, and BIME‐2 Elements

2.7 ERIC Sequences

2.8 Repeat‐Mediated Rearrangements: Mechanisms and Frequency

2.9 Site‐specific Recombination and Phenotypic Variety

2.10 Site‐Specific Recombination: Bacteriophage Lambda

2.11 The Lambda Lysis/Lysogeny Decision

2.12 Tyrosine Integrases

2.13 Serine Invertases

2.14 Large Serine Recombinases

2.15 Transposition and Transposable Elements

2.16 Pathways of Transposition

2.17 Peel‐and‐paste Transposition

2.18 Control of Transposition

2.19 Host Factors and Transposition

2.20 Integrative and Conjugative Elements (ICE)

2.21 Integrons

2.22 Introns

2.23 Horizontal Gene Transfer

2.24 Distinguishing Self from Non‐self

2.25 Distinguishing Self and Non‐self: CRISPR‐Cas Systems

2.26 Distinguishing Self and Non‐self: Argonaute Proteins

2.27 Distinguishing Self and Non‐self: Restriction Enzymes/Methylases

2.28 Distinguishing Self and Non‐self: BREX

2.29 Self‐sacrifice and Other Behaviours Involving Toxin—antitoxin Systems

2.30 Conservative Forces: DNA Repair and Homologous Recombination

2.31 The RecA Protein

2.32 RecA, LexA, and the SOS Response

2.33 Holliday Junction Resolution

2.34 Mismatch Repair

2.35 Non‐homologous End Joining

3 Gene Control: Transcription and Its Regulation

3.1 Transcription: More Than Just Transcribing Genetic Information

3.2 RNA Polymerase

3.3 The Core Enzyme

3.4 The Sigma Factors (and Anti‐Sigma Factors)

3.5 Promoter Architecture

3.6 Stringently Regulated Promoters

3.7 Transcription Factors and RNA Polymerase

3.8 Transcription Initiation

3.9 Transcription Elongation

3.10 Transcription Termination: Intrinsic and Rho‐Dependent Terminators

3.11 Rho and Imported Genes

3.12 Rho, R‐Loops, and DNA Supercoiling

3.13 Rho and Antisense Transcripts

3.14 Anti‐Termination: Insights from Phage Studies

3.15 Transcription Occurs in Bursts

4 Gene Control: Regulation at the RNA Level

4.1 Antisense Transcripts and Gene Regulation incis

4.2 RNA that Regulates intrans

4.3 DsrA and the RpoS/H‐NS Link

4.4 sRNA Turnover

4.5 DEAD‐box Proteins

4.6 RNA Chaperone Proteins

4.7 StpA, H‐NS, and RNA Binding

4.8 Degradation of mRNA

4.9 RNA Folding and Gene Regulation

4.10 Transcription Attenuation

4.11 Riboswitches

4.12 RNA as a Structural Component in the Nucleoid

5 Gene Control: Regulation at the Protein Level

5.1 Control Beyond DNA and RNA

5.2 Translation Machinery and Control: tRNA and rRNA

5.3 Translation Machinery and Control: The Ribosome

5.4 Translation Initiation

5.5 Translation Elongation

5.6 Elongation Factor P (EF‐P)

5.7 Translation Termination

5.8 Protein Secretion

5.9 Protein Secretion: The Sec Pathway

5.10 The Twin Arginine Translocation (Tat) Pathway of Protein Secretion

5.11 Type 1 Secretion Systems (T1SS)

5.12 Type 2 Secretion Systems (T2SS)

5.13 Type 3 Secretion Systems (T3SS)

5.14 Type 4 Secretion Systems (T4SS)

5.15 Type 5 Secretion Systems (T5SS): The Autotransporters

5.16 Type 6 Secretion Systems (T6SS)

5.17 Protein Secretion in Gram‐Positive Bacteria: SecA1, SecA2, and SrtA

5.18 Type 7 Secretion Systems (T7SS)

5.19 Protein Modification: Acetylation

5.20 Protein Modification: Glycosylation

5.21 Protein Modification: Phosphorylation

5.22 Protein Splicing

5.23 Small Proteins

5.24 Selenocysteine and Pyrrolysine: The 21st and 22nd Amino Acids

6 Gene Control and Bacterial Physiology

6.1 The Bacterial Growth Cycle

6.2 Physiology Changes Throughout the Growth Cycle

6.3 Generating Physiological Variety from Genetic Homogeneity

6.4 Bacterial Economics – Some Basic Principles

6.5 Carbon Sources and Metabolism

6.6 Gene Control and Carbon Source Utilisation

6.7 Anaerobic Respiration

6.8 ArcA, Mobile Genetic Elements, and HGT

6.9 Stress and Stress Survival in Bacterial Life

6.10 Oxygen Stress

6.11 Iron Starvation

6.12 Siderophores and Iron Capture

6.13 TonB‐Dependent Transporters

6.14 Gene Regulation and Iron Transport

6.15 Iron Storage and Homeostasis

6.16 Osmotic Stress and Water Relations in Bacteria

6.17 Signal Molecules and Stress

6.18 The Stringent Response

6.19 Regulation of the Acid Stress Response

6.20 Alkaline pH Stress Response

6.21 Motility and Chemotaxis

6.22 Quorum Sensing

6.23 Biofilms

6.24 ‘Cheating’ as a Lifestyle Strategy

6.25 Thermal Regulation

6.26 Epigenomics and Phasevarions

6.27 Some Unifying Themes

7 Gene Control: Global Regulation by H‐NS

7.1 H‐NS Is a Global Regulator

7.2 H‐NS and Foreign DNA

7.3 H‐NS and Xenogenic Silencing: Three Case Studies

7.4 The H‐NS Virulence Regulon in Vibrio cholerae

7.5 HGT in V. cholerae: The CTXφ Phage and the VPI1 Island

7.6 The ToxRS, ToxT, TcpPH Regulatory Network

7.7 Control by VpsR, VpsT, and HapR

7.8 Quorum Sensing and Cholera

7.9 Chitin and HGT

7.10 The H‐NS Virulence Regulon in Shigella flexneri

7.11 Shigella Infection

7.12 The VirF AraC‐Like Transcription Factor

7.13 VirB: A Recruit from a Plasmid‐Partitioning System

7.14 The Shigella Virulence Plasmid

7.15 The Salmonella H‐NS Virulence Gene Regulon

7.16 Salmonella's Pathogenicity Islands (SPI)

7.17 SlyA, PhoP/Q, and SPI Gene Expression

7.18 Gene Control in SPI1 and SPI2

8 An Integrated View of Genome Structure and Function

8.1 Networks versus Hierarchies

8.2 Regulons, Stimulons, and Heterarchies/Netarchies

8.3 Transcription Burstiness and Regulatory Noise

8.4 The Significance of Gene Position

8.5 Messenger RNA May Not Be Free to Diffuse Far in Bacteria

8.6 RNA Polymerase Activity and Genome Organisation

8.7 Gene–Gene Interactions in the Folded Chromosome

8.8 DNA Supercoiling as a Global Regulator

8.9 Modelling the Nucleoid

8.10 Synthetic Biology

References

Index

End User License Agreement

List of Tables

Chapter 1

Table 1.1 The topoisomerases of

E. coli

.

Chapter 2

Table 2.1 The members of the LexA regulon in

E. coli

.

Chapter 3

Table 3.1 Subunit composition of

Escherichia coli

RNA polymerase holoenzyme (with...

Table 3.2 RNA polymerase sigma factors

(Escherichia coli

).

List of Illustrations

Chapter 1

Figure 1.1 The macrodomain structure of the

E. coli

chromosome. Shaded segment...

Figure 1.2 Structure of

oriC

on the

E. coli

chromosome. The ATP‐dependent DnaA...

Figure 1.3 Structure of the

E. coli

replisome in chromosome replication. The r...

Figure 1.4 The structure of the DNA polymerase III subassembly (Pol III*). The...

Figure 1.5 The control of DnaA production and activity. The SeqA and DnaA prot...

Figure 1.6 The genetic neighbourhood of

oriC

in

E. coli

. Filled arrows represe...

Figure 1.7 Termination of chromosome replication in

E. coli

. (a) The moving re...

Figure 1.8 Resolution of a chromosome dimer by XerCD‐mediated recombination at...

Figure 1.9 The choreography of chromosome movement in

E. coli

during the cell ...

Figure 1.10 MukBEF structure and function. (a) The Dimeric MukB protein (inter...

Figure 1.11 Spatiotemporal dispositions of chromosomes in model bacteria other...

Figure 1.12 Theta model of plasmid replication. (a) The structure of the origi...

Figure 1.13 Rolling circle plasmid replication. A circular plasmid using the r...

Figure 1.14 The topological consequences of replisome activity. The replicatio...

Figure 1.15 The topological consequences of transcription. This is the twin su...

Figure 1.16 R‐loop formation. When RNA polymerase reads a G+C‐rich template, s...

Figure 1.17 Integration of bacteriophage lambda at the lambda attachment site ...

Figure 1.18 The interactions of the nucleoid‐associated proteins FIS, HU, and ...

Figure 1.19 The multifaceted stringent response. A summary is shown of the pro...

Figure 1.20 The stress and stationary phase sigma factor, RpoS. The

rpoS

gene ...

Figure 1.21 The vast H‐NS regulon. The H‐NS protein controls the expression of...

Figure 1.22 The dimerisation and oligomerisation of H‐NS. The amino‐terminal d...

Figure 1.23 The DNA‐binding modes of H‐NS. The H‐NS monomer is shown as two li...

Figure 1.24 The genetic switch controlling LEE virulence gene expression in EP...

Chapter 2

Figure 2.1 Simple sequence repeats (SSRs) and phase‐variable gene expression. ...

Figure 2.2 The Hin invertasome and flagella phase variation in

Salmonella

. (a)...

Figure 2.3 The structure of the composite transposon Tn

5

. This mobile element ...

Figure 2.4 The structure of the composite transposon Tn

10

. Tn

10

is superficial...

Figure 2.5 Formation of a hybrid promoter in the IS

911

circle transposition in...

Figure 2.6 Control of transposase expression in insertion sequence IS

911

. The ...

Figure 2.7 The replicative pathway of transposition. The transposon is represe...

Figure 2.8 The structure of transposon Tn

3

and Tn

3‐

family member Tn

501

. ...

Figure 2.9 Cointegrate resolution in Tn

3

transposition. The cointegrate circle...

Figure 2.10 The cut‐and‐paste (or non‐replicative) pathway of transposition. T...

Figure 2.11 Peel and paste transposition. (a) Structure of IS

200

showing the p...

Figure 2.12 Transposon Tn

7

and

att

Tn

7

. The 14‐kb transposon Tn

7

has a left (L)...

Figure 2.13 The CTnDOT Integrative Conjugative Element (ICE). (a) The rectangl...

Figure 2.14 Integron structure. The basic platform of the integron consists of...

Figure 2.15 CRISPR‐

cas

structure. Summary structures are provided for class I ...

Figure 2.16 RecBCD activity and Holliday junction resolution. (a) The RecBCD c...

Figure 2.17 The SOS response. (a) DNA damage interrupts chromosome replication...

Figure 2.18 DNA mismatch repair. (a) A mismatched base pair at position ‘X’ ca...

Figure 2.19 Double‐stranded break repair. In a growing bacterium, when a doubl...

Chapter 3

Figure 3.1 RNA polymerase and transcription initiation. (a) This gives a summa...

Figure 3.2 Structures of an RpoD‐dependent transcription promoter and of the R...

Figure 3.3 Domain structure of RpoN (σ

N

, σ

54

, sigma‐54). The 477‐amino‐acid σ

5

...

Figure 3.4 Transcription regulation at initiation, elongation, termination. (a...

Figure 3.5 Backtracking of RNA polymerase during transcript elongation. The RN...

Chapter 4

Figure 4.1 Gene control at the RNA level. (a) Cleavage of a polycistronic mRNA...

Figure 4.2 CsrA regulon. The CsrA RNA binding protein co‐purifies with hundred...

Figure 4.3 Envelope stress, the RpoE sigma factor and sRNAs. The RpoE sigma fa...

Chapter 5

Figure 5.1 Translation initiation. Clockwise, from top left: Initiation factor...

Figure 5.2 Translation elongation. Clockwise, from the top: the 70S ribosome w...

Figure 5.3 SecA‐SecYEG‐dependent protein secretion. The SecYEG complex in the ...

Figure 5.4 SRP‐SecYEG‐dependent protein secretion. (a) The signal recognition ...

Figure 5.5 The twin arginine translocation (Tat) pathway. The Gram‐negative ve...

Figure 5.6 Type 1 secretion system (T1SS). The system has three components: an...

Figure 5.7 Type 2 secretion system (T2SS). The Gsp protein names refer to the ...

Figure 5.8 Type 3 secretion system. The proteins are given the names of counte...

Figure 5.9 Type 4 secretion system. (a) A summary of the structure of the T4SS...

Figure 5.10 Type 5 secretion system (T5SS). The unfolded autotransporter prote...

Figure 5.11 Type 6 secretion system (T6SS). The system consists of a contracti...

Chapter 6

Figure 6.1 The bacterial growth cycle. The graph shows an idealised growth cur...

Figure 6.2 Generation of proton motive force and the operation of the F

1

F

0

ATP...

Figure 6.3 A simple chemiosmotic circuit. The cytoplasmic membrane is impervio...

Figure 6.4 Iron‐mediated gene regulation via the RyhB sRNA. In iron‐restricted...

Figure 6.5 TonB‐dependent transport systems. The TonB dimer contra‐rotates wit...

Figure 6.6 Operation of the osmotic stress response during upshock. The bacter...

Figure 6.7 Operation of the ProU uptake system in osmotically stressed bacteri...

Figure 6.8 Synthesis of the alarmone (p)ppGpp. In nutrient‐poor conditions, Re...

Figure 6.9 The Acid Fitness Island (AFI) of

Escherichia coli

. The locations, o...

Chapter 7

Figure 7.1 The H‐NS regulon of

V. cholerae

. (a) The genes involved in the colo...

Figure 7.2 The infection of the small intestinal epithelium by

Vibrio cholerae

Figure 7.3 The control of virulence, motility, and biofilm expression in

V. ch

...

Figure 7.4 Chitin influences DNA uptake by

V. cholera

. The

dns

gene encodes th...

Figure 7.5 Invasion of the human large intestinal epithelium by

Shigella flexn

...

Figure 7.6 The H‐NS regulon of

Shigella flexneri

. The Entry Region of the larg...

Figure 7.7 The macrodomain structure of the

Salmonella

chromosome (

S. enterica

Figure 7.8 The H‐NS virulence regulon of

Salmonella enterica

serovar Typhimuri...

Chapter 8

Figure 8.1 Evolutionarily co‐located genes in the

E. coli

chromosome. The chro...

Guide

Cover

Table of Contents

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Structure and Function of the Bacterial Genome

Charles J. Dorman

Department of Microbiology, Moyne Institute of Preventive Medicine, Trinity College Dublin, Dublin 2, Ireland

Copyright

This edition first published 2020

© 2020 John Wiley & Sons, Inc.

All rights reserved. No part of this publication may be reproduced, stored in a retrieval system, or transmitted, in any form or by any means, electronic, mechanical, photocopying, recording or otherwise, except as permitted by law. Advice on how to obtain permission to reuse material from this title is available at http://www.wiley.com/go/permissions.

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Library of Congress Cataloging‐in‐Publication Data

Names: Dorman, Charles J., author.

Title: Structure and function of the bacterial genome / Charles J. Dorman.

Description: Hoboken, NJ : Wiley-Blackwell, 2020. | Includes

bibliographical references and index.

Identifiers: LCCN 2019049741 (print) | LCCN 2019049742 (ebook) | ISBN

9781119308799 (hardback) | ISBN 9781119309673 (adobe pdf) | ISBN

9781119309680 (epub)

Subjects: MESH: Genome, Bacterial–physiology | Gene Expression Regulation,

Bacterial | Transcription, Genetic–physiology | Genome

Components–physiology

Classification: LCC QH450 (print) | LCC QH450 (ebook) | NLM QW 51 | DDC

572.8/65–dc23

LC record available at https://lccn.loc.gov/2019049741

LC ebook record available at https://lccn.loc.gov/2019049742

Cover Design: Wiley

Cover Images: © Pinghung Chen/EyeEm/Getty Images, Courtesy of Charles Dorman

This book is dedicated to my wife, Dr Niamh Ní Bhriain and to our sons, Andrew Dorman and Matthew Dorman.

Preface

The bacterial genome is the software that contains the information for running the cell. The information resides in the genetic material of the bacterium, metaphorically, in both digital (nucleotide sequence) and analogue (DNA topology) forms. From this information emerges the operational hardware, in the form of RNA and proteins, which compose and regulate the pathways for the building of the cell, for the operation of the processes that maintain cell function and which repair and replicate the cell and its contents. Expressing the information that is contained in the cellular software requires sophisticated molecular machines and involves complex processes. Over many decades, molecular microbiology has revealed the details of these machines and processes and has greatly deepened our understanding of the genome itself. By now, the breadth and depth of the information available about these topics can be intimidating, even to experienced investigators. This book will attempt to organize its most important features in ways that allow the reader to grasp the ‘big picture’.

Specialists who focus on ‘the nucleoid’, ‘the cell cycle’, ‘bacterial metabolism’, ‘gene regulation’, ‘transport’, etc. have often studied these bacterial cellular systems and processes in isolation from one another for the very good reason that each is an enormous subject, capable of occupying a whole scientific career. This compartmentalisation of information is valuable because it helps to organize information in discrete packets under clear headings. It is also consistent with the idea of science as an organized body of knowledge. However, even a cursory reading of the literature under the standard headings will reveal that there is tremendous overlap between distinct cellular systems in terms of their components and their governance architecture.

Recently, there has been a great growth in the quantity of data coming from whole‐genome studies of bacterial cells. Experienced investigators may feel that there is just too much information to absorb and the literature is now too vast to read and assimilate. Students entering the field are at an even greater disadvantage and can be forgiven for being discouraged by the mountain of facts. If the discipline of molecular microbiology is to thrive in the future, it is essential that new entrants to the field, and established investigators, have the ability to navigate the sea of information safely. It is the purpose of books like this one to be islands of meaning in this sea of information. This will be done by highlighting the most important components and processes of the bacterial cell, by providing context for cellular operations and by pointing out connections between the different systems and operations. The objective is to provide the reader with a unified picture of the bacterial genome at the structural and functional levels.

Detailed descriptions of the genome's container (the cell envelope) and of the metabolic processes and components that build and maintain it are not within the scope of this book. It will not be possible to provide comprehensive listings of all of the genes and gene products involved in every process that is included because there are just too many, and to attempt this would simply confirm in the reader's mind the impression that this is all just too complicated. Similarly, organism‐to‐organism comparisons will only be made to illustrate important principles; for the most part the narrative will be concerned with the model bacterium Escherichia coli and its close relatives. To attempt wider coverage of the prokaryotic world would simply create an unwieldy book and would defeat its main purposes.

The material in the book is based on the lectures I have delivered to my Junior and Senior Sophister (penultimate and final year) BA students in the Microbiology Moderatorship at Trinity College Dublin over the past 25 years. Although the Microbiology Moderatorship degree at Trinity College is an undergraduate degree, in terms of international comparisons, its advanced content and the demands it places on students are equivalent to taught MSc degrees elsewhere. The lectures seek to introduce the students to the complexities of global gene regulation in model bacteria and how these relate to the structure of the bacterial nucleoid. They are informed by my research in these fields since 1981 and by a close reading of the development of understanding in those fields over the intervening years. This work has been supported by grants from Science Foundation Ireland since 2003. I am grateful to Steve Busby and Jayaraman Gowrishankar for comments on the draft manuscript; the responsibility for any remaining errors of fact or of omission is mine alone.

Dublin 2019

Charles J. Dorman

1The Bacterial Genome – Where the Genes Are

1.1 Genome Philosophy

The genome of a bacterium consists of its entire collective of genes, and these can be located in a chromosome (or chromosomes) and on extra‐chromosomal autonomous replicons such as plasmids. Chromosomes and plasmids replicate, copying the genes that they carry, with the replicon copies being segregated into the daughter cells at cell division. This process drives the vertical transmission of genetic information from one generation to the next and its fidelity determines the stability of the genetic information in the genome. If non‐lethal errors occur during the replication of the genome the resulting mutations will be transmitted to the next generation.

The vertical transmission of genetic copying errors is one of the driving forces of evolution in all types of organisms. Bacterial cells are also prone to the evolutionary influence that is horizontal gene transfer (HGT). Here, foreign genes are transmitted to the bacterium, adding to its genetic complement. Evolution through HGT is much less subtle than evolution through the vertical transmission of copying errors which often involves the gradual accumulation of single nucleotide changes to the genome over many generations. Other types of mutation that are transmitted vertically are inversions, insertions, and deletions of genomic DNA. In HGT a bacterium can acquire entire clusters of genes en bloc, resulting in the acquisition of novel capabilities in a single generation. Examples include the arrival of genes that make the bacterium resistant to an antibiotic or to a heavy metal that previously could kill it, or genes that allow the organism to colonise a niche in the environment from which it had previously been excluded.

HGT played a key role in the early research work that led to the mapping of the bacterial genome and to our understanding of the locations of its genes. Among the autonomously replicating plasmids found in bacteria are elements that can promote their own transfer from cell to cell. The fertility, or ‘F’, factor of Escherichia coli was among the first to be studied. F encodes proteins that can build a connecting bridge between the F+ (or male) cell and one that is F− (female). F transfers one of its DNA strands to the F− cell in a process called conjugation. This is the bacterial equivalent of sex. It resembles sex in higher organisms in that the participants are male (F+) and female (F−) but it differs from conventional sex because the process converts the female into an F+ male. The F plasmid has segments of DNA that are identical in sequence, or almost identical, to DNA segments found in the chromosome. Usually these are mobile genetic elements called insertion sequences (IS). The homologous recombination machinery of the cell can recombine the F‐associated region of DNA sequence identity with a chromosomal counterpart, causing F to become fused with that part of the chromosome (Hadley and Deonier 1980). Where this happens is determined by the location of the IS element, and these mobile elements can be found at sites distributed around the chromosome.

Once F has become one with the chromosome, it is replicated as a part of that molecule. It can still engage in conjugation, however. When this happens, the DNA that is transferred to the F− female bacterium consists of chromosomal DNA with F DNA in the vanguard. Strains that can act as DNA donors in these matings are called ‘Hfr’ (high frequency of recombination) (Reeves 1960) and homologous recombination between the incoming, horizontally transferred DNA and the resident chromosome allows the order of the genes on the chromosome to be determined. Experiments, in which chromosomal gene transfer mediated by the F plasmid was monitored as a function of time, allowed a rudimentary genetic map of the E. coli chromosome to be assembled (Bachmann 1983; Brooks Low 1991). Because the mating experiments were allowed to proceed for fixed periods of time before the deliberate breakage of the conjugation bridges by mechanical shearing, these early genetic maps were calibrated in ‘minutes’. It was discovered that it took 100 minutes to transfer the entire E. coli chromosome from one cell to another by conjugation (Bachmann 1983; Brooks Low 1991). Similar experiments were performed for other bacterial species, including the pathogen Salmonella, giving rough approximations of the physical scale of bacterial genomes (Sanderson and Roth 1988). Hfr strains could also mediate gene transfer between E. coli and Salmonella (Schneider et al. 1961). When the F plasmid is excised from the chromosome, genes that had been adjacent to the plasmid can be removed too, becoming part of the autonomously replicating episome. The plasmids are called F‐prime (F′) and have proved to be very useful in genetic analysis. The chromosomal gene ‘cargo’ can be transferred to F‐minus strains by conjugation and this phenomenon can be exploited in genetic complementation experiments. Work of this type provided useful information about gene order and the position and nature of genetic mutations. F‐primes have been used to investigate plasmid stability, incompatibility, and DNA replication: for example, the F′‐lac episome was used extensively to study plasmid replication in E. coli (Davis and Helmstetter 1973; Dubnau and Maas 1968). Experiments with E. coli mutants deficient in Hfr recombination led to the discovery of important genes involved in homologous recombination: for example, recA (Clark and Margulies 1965), recB, and recC (Barbour and Clark 1970; Willetts et al. 1969; Youngs and Bernstein 1973).

HGT also provided a means for more refined mapping of genomes. Bacteriophages (often abbreviated to ‘phages’) are viruses that replicate in bacterial cells. Some phages package bacterial DNA in their viral heads as they exit the bacterial host and this DNA is transferred to the next bacterium that they manage to infect in a process known as transduction. The length of the DNA segment that a phage head can accommodate is finite and known in the cases of the viruses most commonly used for generalised transduction in E. coli (P1, 100–115 kb) and Salmonella (P22, 42 kb) (Sternberg and Maurer 1991). Therefore, genes that are co‐transduced must be within a distance of one another that is compatible with being co‐packaged by the phage. Very sophisticated experiments with transducing phage allowed not only gene‐to‐gene distance relationships to be determined but also the measurement of the physical relationships between features of individual genes, such as their regulatory elements.

Thus, HGT has driven bacterial evolution and microbial geneticists have exploited it to assemble the first genetic maps of bacterial genomes. Genetic engineers have also used HGT to build novel variants of bacterial genomes in the lab. Cloning experiments using vectors based on natural or engineered plasmids rely on the HGT process known as transformation to move new DNA sequences into bacterial cells. A bacterium that is susceptible to transformation is said to be ‘competent’ and competence can be induced chemically or by electric shock (Hanahan et al. 1991). In addition, many bacterial species are naturally competent and therefore open to the uptake of foreign DNA from the environment. Knowledge and application of the HGT processes of transformation, transduction, and conjugation have revolutionised our understanding of bacterial genomes in a matter of decades. Genome sequencing has extended and deepened this knowledge.

Foreign DNA entering bacterial cells may undergo surveillance. Much attention has been focused on clustered regularly interspaced short palindromic repeat (CRISPR) systems both in their natural roles as systems that identify and destroy ‘non‐self’ DNA, and as a result of their promise as agents of genome editing (Barrangou et al. 2007; Brouns et al. 2008; Garneau et al. 2010). Restriction endonucleases and their associated DNA methylases represent another mechanism for defending the bacterial cell from foreign DNA. Here, the methylases chemically modify the newly synthesised DNA of the bacterial genome so that it matches the ‘approved’ pattern: incoming DNA that lacks this methylation pattern is cut into pieces by the restriction endonucleases. These DNA surveillance mechanisms help to control the access of foreign DNA to established genomes.

The concepts of ‘foreign DNA’ and ‘established genomes’ can also be expressed by the terms ‘accessory genome’ and ‘core genome’, respectively. ‘Accessory’ implies that portion of the genome is not essential for the life of the bacterium, and that may be true in the artificial environment of the laboratory. The name also suggests that some form of value is added to the life of the organism, but that this is conditional. In fact, the same can be said of any gene or portion of the genome. In some cases, the essential nature of a genome component is made obvious because the bacterium dies if this component is eliminated. However, this can apply to a portion of the accessory genome just as much as to one of the core genome, depending on the circumstances of the bacterium. A facile example concerns the presence in a bacterium of a gene encoding resistance to penicillin. The gene is not a part of the core genome (it may even be located on a plasmid and not on the chromosome) and it is not essential unless penicillin‐class antibiotics appear in the neighbourhood. In the absence of this gene during periods of cell wall peptidoglycan synthesis, the bacterium dies if penicillin‐class antibiotics are introduced; despite being a part of the accessory genome, the resistance gene is now an essential gene.

The accessory genome is distinguished from the core genome in being of more recent arrival in the cell. It is not a monolithic entity but a mosaic of imported genetic components that have arrived through HGT, possibly over a very long period of time. In this context, it is very important to realise that genome evolution is not only a process of gene acquisition: gene loss is equally important. A gene may be lost safely if another member of the genome can supply its function, if the cell can acquire the lost product from an exogenous source, or if the selective pressure to retain the gene has been removed.

The core genome consists of those genes that are essential for the life and reproduction of the cell and that are widely shared by other organisms, even those that are only distantly related. Thus, the analysis of genome composition using computers to compare and contrast the genes possessed by thousands of bacteria has helped to identify those genes that are truly almost universally present in bacteria. These designations of essentiality have been supported in some cases by experiments in which the genes have been removed and the impact of their loss on the survival of the bacterium has been measured (Baba et al. 2006; Gerdes et al. 2003; Goodall et al. 2018; Rousset et al. 2018). Essential genes include those whose products replicate the genome, transcribe the genes, translate the messages, and operate the principal metabolic pathways of the cell. In many cases, redundancies are revealed where more than one gene can contribute to an essential process. For example, E. coli has seven operons that contribute to the building of ribosomes, so losing one is not life threatening. On the other hand, the loss of even an apparently redundant gene might impose a fitness cost when a bacterium that has lost this gene competes with one that has not (Condon et al. 1995a).

An essential gene may cease to be essential if another microbe can supply the missing function. This phenomenon is easily illustrated in the laboratory by cross feeding of the mutant by a strain lacking the mutation, but it is not confined to metabolic functions. For example, the absence of an apparently essential virulence gene in one pathogen during infection can be compensated by a function encoded by a second, co‐infecting pathogen (Ibberson et al. 2017).

Loss of competitive fitness arises when a change to the genome (a mutation) renders the bacterium unable to compete with an otherwise genetically identical counterpart. While this can result from the loss of a gene it can also be caused by gene acquisition. Indeed, the negative effect even may arise simply due to the process of expressing the new gene, and not to the effect on the cell of the new gene product (Stoebel et al. 2008a). This illustrates the subtle nature of the causes of competitive fitness differences and their relationships to genome composition and structure.

We will begin by considering genome composition and structure in the model bacterium E. coli and some others where useful data are available. This survey will provide information about any discernable rules governing these important aspects of microbial cell biology.

1.2 The Bacterial Chromosome

E. coli K‐12 has played a central role in the history of bacterial genetics and bacterial physiology. The original K‐12 isolate came from a stool sample from a human patient suffering from diphtheria and was cultured in Palo Alto, California, USA, in 1922 (Bachmann 1996). This isolate was the ancestor of W1485 from the Joshua Lederberg laboratory, the isolate that was named MG1655 by Mark Guyer (hence ‘MG’). The first E. coli chromosome to be sequenced came from this intensively studied MG1655 strain (Blattner et al. 1997). However, this was not the first bacterial chromosome to have its complete nucleotide sequence determined: that honour belongs to Haemophilus influenza (Fleischmann et al. 1995).

The Blattner lab chose MG1655 because it has undergone relatively little genetic manipulation and is considered a good representative of wild‐type E. coli. It has been cured of bacteriophage lambda and of the F plasmid and has few genetic lesions. An ilvG mutation deprives it of acetohydroxy acid synthase II, making it prone to valine‐dependent isoleucine starvation (Lawther et al. 1981, 1982) and there is an IS5 insertion in the rfb locus that interferes with O‐antigen synthesis (Liu and Reeves 1994). If this mutation is repaired, the bacterium has its lipopolysaccharide expression reinstated and it becomes pathogenic in an infection model based on the worm Caenorhabditis elegans (Browning et al. 2013). Strain MG1655 displays mild starvation for pyrimidine arising from poor expression of its pyrE gene: the cause is a frameshift mutation at the end of the rph locus (rph‐1) (Jensen 1993). Interestingly, genome sequence analysis shows that MG1655 is closely related to NCTC 86, the bacterium originally named Bacillus coli by Theodor Escherich in 1885, isolated before the antibiotic era (Dunne et al. 2017).

The E. coli K‐12 chromosome is a single, covalently closed, circular, double‐stranded DNA molecule of 4 639 221 bp (Blattner et al. 1997). Although chromosome circularity is the norm in E. coli, cells in which the chromosome is artificially linearised (with the ends closed by hairpin turns) are viable, show few alterations in gene expression, have normal nucleoid structure, and do not display growth defects (Cui et al. 2007). Thus, the circular nature of the chromosome is not essential for its functionality or for its ability to be replicated and to be segregated at cell division.

The E. coli chromosome was visualised originally in the early 1960s by autoradiography of cells fed with tritiated thymidine in a classic experiment that also revealed the existence of the moving replication fork (Cairns 1963a,b). The chromosome undergoes bi‐directional replication from its oriC locus (Kaguni 2011), creating two replichores: Left and Right (Figure 1.1) (Lesterlin et al. 2005; Wang, X., et al. 2006). Through a process of semi‐conservative DNA replication, the bacterium acquires a second copy of its chromosome prior to cell division. In rapidly growing bacteria, one or more additional rounds of chromosome replication are initiated before the first one is completed, creating multiple copies of those chromosomal sequences that lie closest to oriC (Figure 1.1) (Cooper and Helmstetter 1968). Genes in the oriC‐proximal zones of the E. coli chromosome will be present in higher copy numbers than genes in Ter, the region of the chromosome where replication terminates. In slower‐growing bacterial populations, gene copy numbers are more in balance around the chromosome with only a twofold difference in copy number between genes close to oriC and those near Ter.

Figure 1.1 The macrodomain structure of the E. coli chromosome. Shaded segments represent the Ori, Right, Ter, and Left macrodomains, and the Left and Right non‐structured regions. The curved arrows outside the circular chromosome represent the Left (anticlockwise) and Right (clockwise) replichores. (a) The positions of genes that encode NAPs, chromosome organisation factors, topoisomerases, proteins involved in the process of transcription, the Hfq RNA chaperone are indicated around the periphery of the chromosome. (b) The positions of the seven rrn operons and genes encoding transcription regulators that are discussed in the text are shown. The positions of the lac operon and the bacteriophage lambda attachment site (attλ) are also indicated.

Most of our knowledge about chromosome replication and segregation comes from studying a handful of model organisms: E. coli, Caulobacter crescentus, Vibrio cholerae, and Bacillus subtilis. The focus in this chapter will be on E. coli, with comparisons to other organisms where this is useful.

1.3 Chromosome Replication: Initiation

Chromosome replication, segregation, and cell division are complex processes that must be coordinated to ensure the successful replication of the cell (Reyes‐Lamothe et al. 2012). The nutritional status of the cell and its metabolic flux are very influential in achieving this coordination and they have a direct bearing on the growth rate of the culture (Wang and Levin 2009).

Replication of the E. coli chromosome begins at a specific site, oriC, which has a number of important DNA sequence elements called DnaA boxes that make up the DnaA Oligomerisation Region, DOR (Figure 1.2) (Fuller et al. 1984; Jameson and Wilkinson 2017; Katayama et al. 2017). These boxes are bound by DnaA, an adenosine triphosphate (ATP)‐dependent initiator protein (Schaper and Messer 1995; Sutton and Kaguni 1997), which then forms a right‐handed helical protein oligomer along the DNA that unwinds the duplex at an A+T‐rich element known as the DNA Unwinding Element, DUE (Bramhill and Kornberg 1988a; Kowalski and Eddy 1989) (Figure 1.2). The DnaA oligomerisation process is assisted by another protein called DiaA (Ishida et al. 2004). The DUE has an A‐rich and a T‐rich DNA strand; once it is unwound, the T‐rich strand binds to the DnaA oligomers at the DOR. A helicase loader known as DnaC then loads the DnaB helicase onto the single‐stranded DNA (Koboris and Kornberg 1982). This helicase then recruits in turn the DnaG primase and DnaN, the DNA polymerase beta‐clamp (Fang et al. 1999). When fully assembled, this complex is known as the replisome (Figures 1.3 and 1.4).

Figure 1.2 Structure of oriC on the E. coli chromosome. The ATP‐dependent DnaA protein binds to sites throughout oriC and oligomerises in the DnaA Oligomerisation Region (DOR), driving DNA unwinding at the A+T‐rich DNA Unwinding Element (DUE). Single‐stranded T‐rich DNA in DUE binds to the DnaA oligomers at DOR. High‐affinity sites bind DnaA‐ATP or DnaA‐ADP; low affinity sites bind just DnaA‐ATP. Binding sites for the NAPs FIS and IHF are also shown: FIS and IHF modulate the process of replication initiation negatively and positively, respectively. The Dam methylase methylates oriC at several 5′‐GATC‐3′ sites (indicated by vertical arrows): hemimethylated sites bind SeqA, excluding DnaA and preventing untimely re‐initiation of chromosome replication.

Figure 1.3 Structure of the E. coli replisome in chromosome replication. The replisome is made up of the two cores of DNA Polymerase III, a gamma (γ) complex (or clamp loader) and the beta clamp together with a hexameric helicase, the DnaG primase, and the single‐stranded binding protein, SSB. The DnaB helicase uses energy from ATP hydrolysis to translocate along the lagging strand, unwinding the DNA duplex. The two Polymerase III cores, linked by the tau subunits (Figure 1.4), are each dedicated to coordinated and simultaneous replication of the leading and lagging template strands of the replication fork. The ring‐like beta (β) clamp (DnaN), or processivity factor, encircles DNA and is attached to the replisome via the alpha (α) subunit. The β clamp stabilises the moving replication machine on its template, allowing it to operate with a high degree of processivity. A single‐stranded DNA bubble is formed by the unwinding action of the replisome and SSB protein coats the ssDNA. The DnaG primase interacts with the helicase to generate RNA primers that are used to prime Okazaki fragment synthesis.

Figure 1.4 The structure of the DNA polymerase III subassembly (Pol III*). The core is made up of the alpha (α), epsilon (ɛ), and theta (θ) subunits and the holoenzyme contains two cores. The tau (τ) subunit (two copies) links the cores together, ensuring simultaneous replication of the leading and lagging strands. The function of the cores is DNA synthesis on the leading and lagging strands (Figure 1.3). The clamp loader (or gamma complex) is made up of the chi (χ), delta (δ), delta‐prime (δ′), gamma (γ), and psi (ψ) subunits. The gamma subunit loads the beta clamp onto the DNA that is primed for de novo DNA synthesis. The arrival of the beta (β) clamp (processivity factor) converts the Pol III* subassembly into the Pol III holoenzyme (Figure 1.3).

In oriC of E. coli, the DnaA boxes are of variable affinity for the DnaA protein (Blaesing et al. 2000) (Figure 1.2). Boxes with high affinity bind DnaA that is in a complex with either ATP or ADP, whereas weak boxes bind only DnaA that has bound ATP (Grimwade et al. 2007). Binding of DnaA to oriC is cooperative, with DnaA‐ATP that has bound to strong boxes facilitating the subsequent binding of DnaA‐ATP to the weaker sites, promoting the formation of the DnaA oligomer at the origin of replication (Miller et al. 2009; Kaur et al. 2014). The activity of DnaA may also be controlled by reversible acetylation at lysine residues: of the 13 lysine amino acids in DnaA, acetylation of residues K178 and K243 seems to be especially important in promoting the initiation of chromosome replication (Li et al. 2017; Zhang, Q., et al. 2016).

The availability of DnaA‐ATP is a rate‐limiting factor for the initiation of chromosome replication. A protein called Hda converts active DnaA‐ATP into inactive DnaA‐ADP through ATP hydrolysis (Kato and Katayama 2001). This conversion also requires DnaN, the DNA polymerase beta‐clamp, linking ATP hydrolysis to the elongation phase of DNA synthesis (Takata et al. 2004).

The nucleoid‐associated proteins (NAPs) Integration Host Factor (IHF) and the Factor for Inversion Stimulation (FIS) are DNA‐binding and ‐bending proteins that are thought to play important architectural roles at the origin of replication (Figure 1.2) (Kasho et al. 2014; Ryan et al. 2004). IHF has a positive role at oriC where it binds to a specific DNA sequence, introducing a DNA bend that encourages DnaA binding and oligomer formation; it can also redistribute DnaA on supercoiled DNA (Grimwade et al. 2000). While some work has not found a major role for FIS in regulating events at oriC (Weigel et al. 2001) data from other investigations show that, in contrast to IHF, the role of FIS is inhibitory to DNA replication: when it binds to oriC it interferes with the binding of IHF and DnaA, blocking unwinding of the DUE sequence (Ryan et al. 2004).

The many 5′‐GATC‐3′ sites found throughout oriC (Figure 1.2) are hemimethylated in the period immediately following the initiation of chromosome replication (Lu et al. 1994). The SeqA protein binds to these hemimethylated sites, preventing immediate and untimely re‐initiation of chromosome replication by DnaA: SeqA also downregulates the expression of the negatively autoregulated dnaA gene (Campbell and Kleckner 1990; Waldminghaus and Skarstad 2009). Dam‐mediated methylation of the 5′‐GATC‐3′ sites is inhibitory to SeqA binding and re‐admits DnaA to oriC (Lu et al. 1994).

E. coli uses clusters of DnaA binding sites that are located outside oriC to modulate the initiation of chromosome replication (Figure 1.5). One of these is the 183‐bp datA site, located next to the vjeV gene on the E. coli chromosome. The datA site is made up of five high‐affinity DnaA binding sites (Kitagawa et al. 1996); datA also binds IHF (Nozaki et al. 2009). The interaction of IHF with datA occurs immediately after the initiation of chromosome replication and this facilitates the binding of DnaA‐ATP to datA (Nozaki et al. 2009). DnaA‐ATP bound to datA undergoes ATP hydrolysis, reducing the size of the pool of DnaA‐ATP that is available for binding to oriC (Ogawa et al. 2002). This IHF‐dependent process has a negative influence on the frequency with which chromosome replication is initiated at oriC (Kasho and Katayama 2012).

Figure 1.5 The control of DnaA production and activity. The SeqA and DnaA proteins regulate expression of the dnaA gene negatively. DnaA‐ATP is generated at the DnaA Reactivating Sequences DARS1 and DARS2, and is converted to DnaA‐ADP by ATP hydrolysis (i) at the datA site stimulated by binding of IHF in a process called datA‐dependent DnaA‐ATP Hydrolysis (DDAH) and (ii) by Regulatory Inactivation of DnaA (RIDA) in which the DnaA inhibitor protein Hda catalyses the hydrolysis of DnaA‐bound ATP to ADP, yielding DnaA‐ADP. Hda activation in RIDA follows interaction with the DNA polymerase clamp on newly synthesised DNA. The relative locations of datA (4.39 Mb), DARS1 (0.81 Mb) and DARS2 (2.97 Mb) with respect to the oriC and dif sites on the 4.6 Mb E. coli chromosome are shown (inset). Black lettering: generation of DnaA‐ATP, grey lettering: conversion of DnaA‐ATP to DnaA‐ADP.

Conversion of DnaA‐ADP to DnaA‐ATP has been associated with two so‐called DnaA Reactivation Sites, DARS1 and DARS2 (Fujimitsu et al. 2009) (Figure 1.5). DARS1 is 103 bp in length, has three DnaA binding sites, and is located upstream of uvrB in E. coli. The DARS2 site is more sophisticated. It is 455 bp in length and is located upstream of the mutH gene in E. coli. DARS2 binds IHF and FIS in addition to DnaA. Binding of these NAPs to DARS2 stimulates the conversion of DnaA‐ADP to DnaA‐ATP. IHF binding is cell cycle determined while FIS binding is growth phase determined: FIS binds in rapidly growing cells and this is consistent with the observation that FIS stimulates DNA replication in rapidly growing E. coli (Flåtten and Skarstad 2013; Kasho et al. 2014). The chromosomal locations of datA and the DARS elements seem to be important for their function: if they are repositioned, the chromosome replication control is disrupted (Frimodt‐Møller et al. 2016).

The oriC locus is found between two highly conserved genes, mioC and gidA (Figure 1.6). The mioC gene is transcribed towards oriC while gidA is transcribed away from it. The two genes exhibit reciprocal transcription patterns that are functions of the cell cycle: mioC transcription is maximal midway through chromosome replication while gidA transcription is minimal at that point; maximal expression of gidA coincides with the onset of septation and cell division (Lies et al. 2015). MraZ, a protein possibly involved in cell division control, binds and represses the mioC promoter (Eraso et al. 2014) and this promoter is also stringently regulated, linking mioC transcription to growth rate (Chiaramello and Zyskind 1989). The biological function of MioC is not firmly established, although it has been reported to be involved in biotin metabolism (Birch et al. 2001). The GidA protein contributes to tRNA modification, working in association with MnmE (GidA is also known as MnmG) (Bregeon et al. 2001). Neither protein is thought to have DNA‐binding activity. Transcription of mioC is repressed by DnaA acting at a DnaA box in the promoter. The initiation of chromosome replication displaces DnaA and de‐represses mioC, with the return of DnaA being delayed as the protein is recruited by the new DnaA binding sites generated by replication (Bogan and Helmstetter 1996). Transcription of gidA is repressed by SeqA when this protein binds to the 5′‐GATC‐3′ sites at the promoter that become hemimethylated following DNA replication (Bogan and Helmstetter 1997). The process of transcribing gidA and mioC is important for the initiation of chromosome replication at oriC (Bramhill and Kornberg 1988b; Theisen et al. 1993), at least under some circumstances (Asai et al. 1998; Bates et al. 1997; Lies et al. 2015).

Figure 1.6 The genetic neighbourhood of oriC in E. coli. Filled arrows represent the genes and an open rectangle indicates the position of oriC. DnaA represses the gidA gene transcriptionally through DnaA boxes that overlap the gidA promoter. The mioC gene is repressed by SeqA binding to hemimethylated versions of 5′‐GATC‐3′ sites at the promoter that are generated by DNA replication. The mioC promoter is also subject to stringent control via the (p)ppGpp alarmone and it is repressed by MraZ, a protein that has been linked to the control of cell division. The rsmG gene encodes a methyltransferase for the modification of 16S rRNA (see Benítez‐Páez et al. 2012). The asnC gene encodes a HTH‐motif‐containing transcription regulator that is related to LRP and controls genes involved in asparagine metabolism (see Kölling and Lother 1985; Willins et al. 1991). Termination of transcription extending from asnC to mioC is dependent on a DnaA‐DNA complex at the asnC terminator, as described by Schaefer and Messer (1988).

1.4 Chromosome Replication: Elongation

Once replication has been initiated, the replisome is responsible for progressive DNA synthesis during the elongation phase of chromosome replication. This large complex is composed of a pentameric clamp loader, the DNA polymerase clamp (DnaN), the three‐subunit DNA primase (DnaG), and the hexameric helicase DnaB (Bailey et al. 2007; Reyes‐Lamothe et al. 2010) (Figure 1.4). The helicase uses ATP hydrolysis to unwind the DNA duplex, moving along the lagging strand of the DNA as it does so. Single‐stranded DNA‐binding protein (SSB) coats the separated ssDNA strands, thus preventing reformation of the duplex by religation and attack by nucleases (Beattie and Reyes‐Lamothe 2015).

The primase, DnaG, possesses a central RNA polymerase domain where the RNA primers used in DNA synthesis are manufactured (Corn et al. 2008). The primer emerges from the DnaG‐DnaB complex and is transferred to DNA polymerase and SSB (Corn et al. 2008). DNA Polymerase III works with the beta‐clamp protein (DnaN) to extend the primer, creating a new DNA strand at a rate of 1000 bases per second (Beattie and Reyes‐Lamothe 2015). It is advantageous to have DnaN as a component of the replisome because a beta‐clamp must be reloaded for the synthesis of each lagging strand Okazaki fragment (Beattie and Reyes‐Lamothe 2015). If the replication fork stalls or breaks, replication can be restarted through a DnaA‐independent mechanism. Here, the PriA helicase, in association with accessory proteins such as PriB, PriC, and DnaT, binds to the gapped replication fork and loads DnaBC. In some cases, the restart may be associated with a strong transcription promoter that generates an R‐loop where PriA can introduce DnaBC on the displaced DNA strand (Heller and Marians 2006; Kogoma 1997). Of the approximately 300 copies of DNA gyrase that are bound to the E. coli chromosome at any one time, about 12 accompany each moving replication fork to manage the DNA topological disturbance that is associated with fork migration (Stracy et al. 2019).

1.5 Chromosome Replication: Termination

Termination of DNA synthesis occurs within Ter, located at a point that is diametrically opposite oriC on the chromosome (Hill et al. 1987) (Figure 1.7). The Ter region has five copies of a 23‐bp DNA element on each flank and the 36‐kDa Tus protein binds to these sequences (Neylon et al. 2005). The Tus binding sites are asymmetric and have a permissive and a non‐permissive orientation (Figure 1.7). Replication forks can pass the Tus‐Ter nucleoprotein complexes when the DNA sequences are in the permissive orientation, but fork movement becomes arrested when the sequences are oriented in the non‐permissive direction. The mechanism of replication fork passage at Ter sites that are in the permissive orientation involves displacement of Tus by the DnaB helicase; when in the non‐permissive orientation, Tus prevents DnaB, and the replication fork, from translocating past that point (Bastia et al. 2008; Berghuis et al. 2015; Mulcair et al. 2006). Single‐molecule experiments performed in vitro have shown that the DNA also plays a critical role: in the non‐permissive orientation, the unwinding of the DNA by the approaching replication fork creates a powerful lock at the Tus‐Ter site that is an effective roadblock to further translocation by the fork; in the permissive orientation the lock does not operate and the fork can proceed (Berghuis et al. 2015).

Figure 1.7 Termination of chromosome replication in E. coli. (a) The moving replisome encounters an appropriately oriented Tus/Ter nucleoprotein complex and the interaction between Tus and the DnaB helicase halts replisome movement, leading to the termination of chromosome replication. (b) The 4.6 Mb chromosome of E. coli