Nucleoid-associated proteins (NAPs) are global regulators of gene expression in Escherichia coli, which affect DNA conformation by bending, wrapping and bridging the DNA. Two of these—H-NS and Fis—bind to specific DNA sequences and structures. Because of their importance to global gene expression, the binding of these NAPs to the DNA was previously investigated on a genome-wide scale using ChIP-chip. However, variation in their binding profiles across the growth phase and the genome-scale nature of their impact on gene expression remain poorly understood. Here, we present a genome-scale investigation of H-NS and Fis binding to the E. coli chromosome using chromatin immunoprecipitation combined with high-throughput sequencing (ChIP-seq). By performing our experiments under multiple time-points during growth in rich media, we show that the binding regions of the two proteins are mutually exclusive under our experimental conditions. H-NS binds to significantly longer tracts of DNA than Fis, consistent with the linear spread of H-NS binding from high- to surrounding lower-affinity sites; the length of binding regions is associated with the degree of transcriptional repression imposed by H-NS. For Fis, a majority of binding events do not lead to differential expression of the proximal gene; however, it has a significant indirect effect on gene expression partly through its effects on the expression of other transcription factors. We propose that direct transcriptional regulation by Fis is associated with the interaction of tandem arrays of Fis molecules to the DNA and possible DNA bending, particularly at operon-upstream regions. Our study serves as a proof-of-principle for the use of ChIP-seq for global DNA-binding proteins in bacteria, which should become significantly more economical and feasible with the development of multiplexing techniques.

IHF and HU are two heterodimeric nucleoid-associated proteins (NAP) that belong to the same protein family but interact differently with the DNA. IHF is a sequence-specific DNA-binding protein that bends the DNA by over 160°. HU is the most conserved NAP, which binds non-specifically to duplex DNA with a particular preference for targeting nicked and bent DNA. Despite their importance, the in vivo interactions of the two proteins to the DNA remain to be described at a high resolution and on a genome-wide scale. Further, the effects of these proteins on gene expression on a global scale remain contentious. Finally, the contrast between the functions of the homo- and heterodimeric forms of proteins deserves the attention of further study. Here we present a genome-scale study of HU- and IHF binding to the Escherichia coli K12 chromosome using ChIP-seq. We also perform microarray analysis of gene expression in single- and double-deletion mutants of each protein to identify their regulons. The sequence-specific binding profile of IHF encompasses ∼30% of all operons, though the expression of <10% of these is affected by its deletion suggesting combinatorial control or a molecular backup. The binding profile for HU is reflective of relatively non-specific binding to the chromosome, however, with a preference for A/T-rich DNA. The HU regulon comprises highly conserved genes including those that are essential and possibly supercoiling sensitive. Finally, by performing ChIP-seq experiments, where possible, of each subunit of IHF and HU in the absence of the other subunit, we define genome-wide maps of DNA binding of the proteins in their hetero- and homodimeric forms.

DnA cytosine methylation regulates gene expression in mammals. In bacteria, its role in gene expression and genome architecture is less understood. Here we perform high-throughput sequencing of bisulfite-treated genomic DnA from Escherichia coli K12 to describe, for the first time, the extent of cytosine methylation of bacterial DnA at single-base resolution. Whereas most target sites (C m CWGG) are fully methylated in stationary phase cells, many sites with an extended CC m CWGG motif are only partially methylated in exponentially growing cells. We speculate that these partially methylated sites may be selected, as these are slightly correlated with the risk of spontaneous, non-synonymous conversion of methylated cytosines to thymines. microarray analysis in a cytosine methylation-deficient mutant of E. coli shows increased expression of the stress response sigma factor Rpos and many of its targets in stationary phase. Thus, DnA cytosine methylation is a regulator of stationary phase gene expression in E. coli.

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HighlightsE. coli cyclic-AMP receptor protein (CRP) is a paradigm of gene regulation.Comparison of CRPs reveals differences in their affinity of cAMP.A range of dependency on cAMP for DNA-binding exists.CRPs have adapted to function in the specific niches occupied by the bacteria.

Chromatin immunoprecipitation identified 191 binding sites of Mycobacterium tuberculosis cAMP receptor protein (CRPMt) at endogenous expression levels using a specific α-CRPMt antibody. Under these native conditions an equal distribution between intragenic and intergenic locations was observed. CRPMt binding overlapped a palindromic consensus sequence. Analysis by RNA sequencing revealed widespread changes in transcriptional profile in a mutant strain lacking CRPMt during exponential growth, and in response to nutrient starvation. Differential expression of genes with a CRPMt-binding site represented only a minor portion of this transcriptional reprogramming with ∼19% of those representing transcriptional regulators potentially controlled by CRPMt. The subset of genes that are differentially expressed in the deletion mutant under both culture conditions conformed to a pattern resembling canonical CRP regulation in Escherichia coli, with binding close to the transcriptional start site associated with repression and upstream binding with activation. CRPMt can function as a classical transcription factor in M. tuberculosis, though this occurs at only a subset of CRPMt-binding sites.

Background: Mycobacterium tuberculosis LexA is thought to repress the expression of a small number of genes.Results: 25 in vivo binding sites were identified by ChIP-seq, including nine novel sites.Conclusion: M. tuberculosis LexA also shows examples of positive regulation, binding without apparent regulation, and binding to genes encoding small RNAs.Significance: This investigation identified new aspects of LexA regulation in M. tuberculosis.

Mycobacterium tuberculosis (MTb) is the causative agent of pulmonary tuberculosis (TB). MTb colonizes the human lung, often entering a non-replicating state before progressing to life-threatening active infections. Transcriptional reprogramming is essential for TB pathogenesis. In vitro, Cmr (a member of the CRP/FNR super-family of transcription regulators) bound at a single DNA site to act as a dual regulator of cmr transcription and an activator of the divergent rv1676 gene. Transcriptional profiling and DNA-binding assays suggested that Cmr directly represses dosR expression. The DosR regulon is thought to be involved in establishing latent tuberculosis infections in response to hypoxia and nitric oxide. Accordingly, DNA-binding by Cmr was severely impaired by nitrosation. A cmr mutant was better able to survive a nitrosative stress challenge but was attenuated in a mouse aerosol infection model. The complemented mutant exhibited a ∼2-fold increase in cmr expression, which led to increased sensitivity to nitrosative stress. This, and the inability to restore wild-type behaviour in the infection model, suggests that precise regulation of the cmr locus, which is associated with Region of Difference 150 in hypervirulent Beijing strains of Mtb, is important for TB pathogenesis.