List of Figures ................................................ ix
Acknowledgements ............................................. xiii
1 Introduction: Bacterial Genomes and Gene Expression .......... 1
2 Comparative Genomics in the Era of Sanger Sequencing ......... 4
2.1 Introduction ............................................ 4
2.2 The process of assembling and annotating bacterial
genomes ................................................. 6
2.2.1 Genome assembly and gap closure .................. 7
2.2.2 Genome-scale computational identification of
features ......................................... 8
2.2.3 Annotating genes with functions ................. 13
2.3 Case studies ........................................... 16
2.3.1 The Escherichia coli complex and large-scale
horizontal gene acquisition ..................... 16
2.3.2 Genome reduction in intracellular pathogens,
endosymbionts and marine α-proteobacteria ....... 20
2.3.3 The dynamic genomes of Helicobacter pylori and
Campylobacter jejuni ............................ 26
2.4 Some lessons learnt from studying 2,000 bacterial
genomes ................................................ 27
2.4.1 Genome size ..................................... 28
2.4.2 Coding density .................................. 29
2.4.3 Gene order conservation ......................... 30
2.4.4 Comparative genomics of gene functions:
Systematic annotation ........................... 31
2.4.5 Comparative genomics of gene functions:
Scaling laws .................................... 33
Summary ..................................................... 35
3 Studying Bacterial Genome Variation with Microarrays ........ 36
3.1 Introduction ........................................... 36
3.2 DNA microarrays: The concept ........................... 36
3.3 DNA microarrays: From fluorescence intensities to
information ............................................ 38
3.3.1 Background correction ........................... 39
3.3.2 Normalisation ................................... 41
3.3.3 Differences in signal from the same probe
between two samples ............................. 44
3.4 Comparative genome hybridisation and bacterial
phylogenomics .......................................... 45
3.5 Case studies ........................................... 46
3.5.1 Comparative genome hybridisation studies of
Escherichia coli ................................ 47
3.5.2 Comparative genome hybridisation studies of
Staphylococcus aureus ........................... 52
3.5.3 Comparative genome hybridisation studies of
Helicobacter pylori ............................. 56
Summary ..................................................... 59
4 Studying Bacterial Genomes using Next-Generation
Sequencing .................................................. 60
4.1 Introduction ........................................... 60
4.2 Next-generation sequencing technologies ................ 61
4.2.1 Template preparation strategies ................. 62
4.2.2 Sequencing strategies ........................... 64
4.3 Sequencing data processing for genome sequencing
and re-sequencing ...................................... 66
4.3.1 Genome assembly ................................. 66
4.3.2 Aligning short reads to long genomes ............ 70
4.4 Case studies ........................................... 73
4.4.1 Pyrosequencing-enabled complete genome
sequence of Acinetobacter baumanii .............. 74
4.4.2 On the track of pandemics: The genome of the
aetiological agent of Black Death ............... 75
4.4.3 From community genomes to complete genomes to
single-cell genomes ............................. 80
4.4.4 Bacteria evolving in the laboratory ............. 84
4.4.5 Bacteria evolving in their biotic hosts ......... 86
Summary ..................................................... 88
5 Genome-Scale Analysis of Gene Expression and its
Regulation in Bacteria ...................................... 89
5.1 Introduction ........................................... 89
5.2 The process of transcription and the regulation of
its initiation: An overview ............................ 90
5.3 Measuring gene expression on a genomic scale:
Technologies ........................................... 99
5.4 Next-generation sequencing for gene expression
measurements: Data analysis ........................... 101
5.4.1 Transcriptome assembly ......................... 104
5.4.2 Measuring gene expression levels ............... 110
5.5 Gene expression at high temporal resolution using
fluorescent reporters ................................. 114
5.6 Constructing transcriptional regulatory networks:
СЫР-chip and ChlP-seq ................................. 117
5.7 Case studies .......................................... 122
5.7.1 Experimental annotation of bacterial genomes ... 122
5.7.2 Bioinformatic analysis of bacterial promoters .. 129
5.7.3 DNA topology and its interplay with gene
expression ..................................... 134
5.7.4 RNA polymerase occupancy and the a-factors ..... 145
5.7.5 Transcription factors and transcriptional
regulatory networks ............................ 154
5.7.6 Transcriptional control by the small-molecule
alarmone ppGpp ................................. 186
5.7.7 RNA chaperones and their regulons .............. 188
Summary .................................................... 190
6 DNA Methylation in Bacteria: A Case for Bacterial
Epigenetics ................................................ 191
6.1 Introduction .......................................... 191
6.2 DNA methyltransferases in bacteria: From
restriction-modification systems ...................... 192
6.3 Identifying sites of DNA methylation on a genomic
scale ................................................. 195
6.3.1 Methylated DNA immunoprecipitation ............. 195
6.3.2 Bisulphite sequencing .......................... 195
6.3.3 DNA cytosine methylation in laboratory
E. coli ........................................ 197
6.4 Detecting DNA methylation by single-molecule real-
time sequencing ....................................... 200
6.4.1 DNA adenine methylation in pathogenic E. coli
by SMRT sequencing ............................. 202
6.4.2 Insight into the epigenetic control of
Caulobacter crescentus cell cycle from SMRT
sequencing ..................................... 203
Summary ....................................................... 205
Index ......................................................... 207
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