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.

In the absence of DNA adenine methylase, growth of Salmonella enterica serovar Typhimurium is inhibited by bile. Mutations in any of the mut H, mutL, and mutS genes suppress bile sensitivity in a Dam Ϫ background, indicating that an active MutHLS system renders Dam Ϫ mutants bile sensitive. However, inactivation of the MutHLS system does not cause bile sensitivity. An analogy with Escherichia coli, in which the MutHLS system sensitizes Dam Ϫ mutants to DNA-injuring agents, suggested that bile might cause DNA damage. In support of this hypothesis, we show that bile induces the SOS response in S. enterica and increases the frequency of point mutations and chromosomal rearrangements. Mutations in mut H, mut L, or mutS cause partial relief of virulence attenuation in a Dam Ϫ background (50-to 100-fold by the oral route and 10-fold intraperitoneally), suggesting that an active MutHLS system reduces the ability of Salmonella Dam Ϫ mutants to cope with DNA-damaging agents (bile and others) encountered during the infection process. The DNA-damaging ability of bile under laboratory conditions raises the possibility that the phenomenon may be relevant in vivo, since high bile concentrations are found in the gallbladder, the niche for chronic Salmonella infections.

No abstract

Mismatch repair (MMR) is a near ubiquitous pathway, essential for the maintenance of genome stability. Members of the MutS and MutL protein families perform key steps in mismatch correction. Despite the major importance of this repair pathway, MutS–MutL are absent in almost all Actinobacteria and many Archaea. However, these organisms exhibit rates and spectra of spontaneous mutations similar to MMR-bearing species, suggesting the existence of an alternative to the canonical MutS–MutL-based MMR. Here we report that Mycobacterium smegmatis NucS/EndoMS, a putative endonuclease with no structural homology to known MMR factors, is required for mutation avoidance and anti-recombination, hallmarks of the canonical MMR. Furthermore, phenotypic analysis of naturally occurring polymorphic NucS in a M. smegmatis surrogate model, suggests the existence of M. tuberculosis mutator strains. The phylogenetic analysis of NucS indicates a complex evolutionary process leading to a disperse distribution pattern in prokaryotes. Together, these findings indicate that distinct pathways for MMR have evolved at least twice in nature.

Exposure of Salmonella enterica to sodium cholate, sodium deoxycholate, sodium chenodeoxycholate, sodium glychocholate, sodium taurocholate, or sodium glycochenodeoxycholate induces the SOS response, indicating that the DNA-damaging activity of bile resides in bile salts. Bile increases the frequency of GC / AT transitions and induces the expression of genes belonging to the OxyR and SoxRS regulons, suggesting that bile salts may cause oxidative DNA damage. S. enterica mutants lacking both exonuclease III (XthA) and endonuclease IV (Nfo) are bile sensitive, indicating that S. enterica requires base excision repair (BER) to overcome DNA damage caused by bile salts. Bile resistance also requires DinB polymerase, suggesting the need of SOS-associated translesion DNA synthesis. Certain recombination functions are also required for bile resistance, and a key factor is the RecBCD enzyme. The extreme bile sensitivity of RecB À , RecC À , and RecA À RecD À mutants provides evidence that bile-induced damage may impair DNA replication.

Passage through the digestive tract exposes Salmonella enterica to high concentrations of bile salts, powerful detergents that disrupt biological membranes. Mutations in the wecD or wecA gene, both of which are involved in the synthesis of enterobacterial common antigen (ECA), render S. enterica serovar Typhimurium sensitive to the bile salt deoxycholate. Competitive infectivity analysis of wecD and wecA mutants in the mouse model indicates that ECA is an important virulence factor for oral infection. In contrast, lack of ECA causes only a slight decrease in Salmonella virulence during intraperitoneal infection. A tentative interpretation is that ECA may contribute to Salmonella virulence by protecting the pathogen from bile salts.Bile salts are detergent-like substances that aid in the digestion and absorption of lipids. Bile salts are secreted from the liver, stored in the gall bladder, and released through the bile duct into the intestine during food passage. The most abundant bile salts in humans are cholate and deoxycholate (DOC). Enteric bacteria are intrinsically resistant to bile salts, due both to the low permeability of the outer membrane bilayer to these lipophilic solutes and to active efflux mechanisms. Mutations that impair bile salt resistance in genes for lipopolysaccharide (LPS) synthesis (14), tol genes (16), efflux pump genes (12,22), regulatory genes such as marAB (20) and phoPQ (25), and the DNA adenine methyltransferase gene (8, 17) have been previously described.Here we show that mutations in the wecD and wecA genes of Salmonella enterica cause sensitivity to DOC. The wec gene cluster is required for synthesis of the enterobacterial common antigen (ECA), a glycolipid found in the external leaflet of the outer membrane in all species of the family Enterobacteriaceae (reviewed in reference 18). The ECA biosynthetic pathway is diagrammed in Fig. 1. The polysaccharide portion of ECA consists of a linear trimeric repeat with the following structure: Subsequent steps involve polymerization, transfer of the polymer to a phospholipid aglycone, and translocation to the outer membrane. Below we show that Salmonella mutants unable to synthesize ECA are highly attenuated in oral infections and slightly attenuated in intraperitoneal infections. Hence, we propose that ECA plays a role in Salmonella virulence. Such a role can be tentatively correlated with bile salt resistance.MudJ mutagenesis identifies wecD as a locus necessary for resistance to DOC. In an attempt to identify new genes potentially involved in bile salt resistance in S. enterica serovar Typhimurium, we performed a screen for mutants sensitive to sodium deoxycholate in strain 14028, which is virulent in mice. The strains used in this study are described in Table 1. MudJ mutagenesis was achieved by the method of Hughes and Roth (9). Five thousand Km r colonies from 10 independent mutagenesis trials were patched in grids onto Luria-Bertani (LB) plates containing 1% DOC. Chromosomal DNA from one DOC s P22-sensitive mutant was prepared as pr...