Histone-like nucleoid structuring protein (H-NS) is a modular protein that is associated with the bacterial nucleoid. We used chromatin immunoprecipitation to determine the binding sites of H-NS and RNA polymerase on the Salmonella enterica serovar Typhimurium chromosome. We found that H-NS does not bind to actively transcribed genes and does not co-localize with RNA polymerase. This shows that H-NS principally silences gene expression by restricting the access of RNA polymerase to the DNA. H-NS had previously been shown to preferentially bind to curved DNA in vitro. In fact, at the genomic level we discovered that the level of H-NS binding correlates better with the AT-content of DNA. This is likely to have evolutionary consequences because we show that H-NS binds to many Salmonella genes acquired by lateral gene transfer, and functions as a gene silencer. The removal of H-NS from the cell causes un-controlled expression of several Salmonella pathogenicity islands, and we demonstrate that this has deleterious consequences for bacterial fitness. Our discovery of this novel role for H-NS may have implications for the acquisition of foreign genes by enteric bacteria.

The Escherichia coli chromosome is condensed into an ill-defined structure known as the nucleoid. Nucleoid-associated DNA-binding proteins are involved in maintaining this structure and in mediating chromosome compaction. We have exploited chromatin immunoprecipitation and high-density microarrays to study the binding of three such proteins, FIS, H-NS and IHF, across the E.coli genome in vivo. Our results show that the distribution of these proteins is biased to intergenic parts of the genome, and that the binding profiles overlap. Hence some targets are associated with combinations of bound FIS, H-NS and IHF. In addition, many regions associated with FIS and H-NS are also associated with RNA polymerase.

Fis is a key DNA-binding protein involved in nucleoid organization and modulation of many DNA transactions, including transcription in enteric bacteria. The regulon of genes whose expression is influenced by Fis in Salmonella enterica serovar Typhimurium (S. typhimurium) has been defined by DNA microarray analysis. These data suggest that Fis plays a central role in coordinating the expression of both metabolic and type III secretion factors. The genes that were most strongly up-regulated by Fis were those involved in virulence and located in the pathogenicity islands SPI-1, SPI-2, SPI-3 and SPI-5. Similarly, motility and flagellar genes required Fis for full expression. This was shown to be a direct effect as purified Fis protein bound to the promoter regions of representative flagella and SPI-2 genes. Genes contributing to aspects of metabolism known to assist the bacterium during survival in the mammalian gut were also Fis-regulated, usually negatively. This category included components of metabolic pathways for propanediol utilization, biotin synthesis, vitamin B 12 transport, fatty acids and acetate metabolism, as well as genes for the glyoxylate bypass of the tricarboxylic acid cycle. Genes found to be positively regulated by Fis included those for ethanolamine utilization. The data reported reveal the central role played by Fis in coordinating the expression of both housekeeping and virulence factors required by S. typhimurium during life in the gut lumen or during systemic infection of host cells. INTRODUCTIONSalmonella enterica serovar Typhimurium (S. typhimurium) is the most common and best studied of the S. enterica serovars that infect humans (Finlay & Brumell, 2000). It is able to infect a range of animal species, including chicken, cattle and mice, and intensive study of this organism is providing important insights into key processes involved in bacterial pathogenesis. In the mouse, S. typhimurium is a facultative intracellular pathogen capable of invading epithelial cells and it has the ability to survive and proliferate within macrophages. The bacterium can be manipulated genetically with relative ease and the complete genome sequence is available (McClelland et al., 2001), allowing a combination of genetic analysis, cell biology and animal infection studies. This multidisciplinary approach has provided a picture of the major events involved when S. typhimurium infects the murine host. Following ingestion and passage through the stomach, the bacteria cross the lining of the intestine by invading the intestinal epithelium, predominantly via M cells. The Salmonellae are subsequently phagocytosed by macrophages before entering the blood stream and establishing a systemic infection (Finlay & Brumell, 2000; Galán, 2001;Groisman & Mouslim, 2000;Holden, 2002;Scherer & Miller, 2001).S. typhimurium is dependent upon the products of a large number of genes (up to 200) to cause infection (Finlay & Brumell, 2000). Some of the virulence genes are located on a 90 kb pathogenicity plasmid, of which the spv gen...

Background Escherichia coli can experience a multifaceted life, in some cases acting as a commensal while in other cases causing intestinal and/or extraintestinal disease. Several studies suggest enteroaggregative E. coli are the predominant cause of E. coli-mediated diarrhea in the developed world and are second only to Campylobacter sp. as a cause of bacterial-mediated diarrhea. Furthermore, enteroaggregative E. coli are a predominant cause of persistent diarrhea in the developing world where infection has been associated with malnourishment and growth retardation.MethodsIn this study we determined the complete genomic sequence of E. coli 042, the prototypical member of the enteroaggregative E. coli, which has been shown to cause disease in volunteer studies. We performed genomic and phylogenetic comparisons with other E. coli strains revealing previously uncharacterised virulence factors including a variety of secreted proteins and a capsular polysaccharide biosynthetic locus. In addition, by using Biolog™ Phenotype Microarrays we have provided a full metabolic profiling of E. coli 042 and the non-pathogenic lab strain E. coli K-12. We have highlighted the genetic basis for many of the metabolic differences between E. coli 042 and E. coli K-12.ConclusionThis study provides a genetic context for the vast amount of experimental and epidemiological data published thus far and provides a template for future diagnostic and intervention strategies.

Temperature is a primary environmental stress to which micro-organisms must be able to adapt and respond rapidly. Whereas some bacteria are restricted to specific niches and have limited abilities to survive changes in their environment, others, such as members of the Enterobacteriaceae, can withstand wide fluctuations in temperature. In addition to regulating cellular physiology, pathogenic bacteria use temperature as a cue for activating virulence gene expression. This work confirms that the nucleoid-associated protein H-NS (histone-like nucleoid structuring protein) is an essential component in thermoregulation of Salmonella. On increasing the temperature from 25 to 37 degrees C, more than 200 genes from Salmonella enterica serovar Typhimurium showed H-NS-dependent up-regulation. The thermal activation of gene expression is extremely rapid and change in temperature affects the DNA-binding properties of H-NS. The reduction in gene repression brought about by the increase in temperature is concomitant with a conformational change in the protein, resulting in the decrease in size of high-order oligomers and the appearance of increasing concentrations of discrete dimers of H-NS. The present study addresses one of the key complex mechanisms by which H-NS regulates gene expression.

SummaryDps is a nucleoid-associated protein that plays a major role in condensation of the Escherichia coli chromosome in stationary phase. Here we show that two other nucleoid-associated proteins, Fis and H-NS, can bind at the dps gene promoter and downregulate its activity. Both Fis and H-NS selectively repress the dps promoter, preventing transcription initiation by RNA polymerase containing s 70 , the housekeeping s factor, but not by RNA polymerase containing s 38 , the stationary-phase s factor. Fis represses by trapping RNA polymerase containing s 70 at the promoter. In contrast, H-NS functions by displacing RNA polymerase containing s 70 , but not RNA polymerase containing s 38 . Dps levels are known to be very low in exponentially growing cells and rise sharply as cells enter stationary phase. Conversely, Fis levels are high in growing cells but fall to nearly zero in stationary-phase cells. Our data suggest a simple model to explain how the Dps-dependent supercompaction of the folded chromosome is triggered as cell growth ceases.

The abundance and diversity of intermediate filaments (IFs) in the C. elegans intestine indicate important contributions to intestinal function and organismal wellbeing. fluorescent if reporters localize below the actin-rich brush border and are highly enriched in the lumen-enveloping endotube, which is attached to the C. elegans apical junction. Mapping intestinal viscoelasticity by contact-free Brillouin microscopy reveals that the IF-rich endotube is positioned at the interface between the stiff brush border and soft cytoplasm suggesting a mechanical buffering function to deal with the frequent luminal distortions occurring during food intake and movement. In accordance, depletion of IFB-2, IFC-2 and IFD-2 leads to intestinal lumen dilation although depletion of IFC-1, IFD-1 and IFP-1 do not. Ultrastructural analyses of loss of function mutants further show that IFC-2 mutants have a rarefied endotube and IFB-2 mutants lack an endotube altogether. Remarkably, almost all IFB-2-and IFC-2-deficient animals develop to fertile adults. But developmental retardation, reduced brood size, altered survival and increased sensitivity to microbial toxin, osmotic and oxidative stress are seen in both mutants albeit to different degrees. Taken together, we propose that individual intestinal IF polypeptides contribute in different ways to endotube morphogenesis and cooperate to cope with changing environments.

Virulence gene expression in enteropathogenic Escherichia coli (EPEC) is governed by a combination of environmental factors and virulence regulators. These factors control the expression of the bundle‐forming pili (BFP), intimin, the type III secretion apparatus and the secreted proteins EspA, EspB, EspD and Tir. Expression of the bfp genes occurs for a short period in early exponential phase during growth in tissue culture medium. The nucleoid‐associated regulator protein, Fis, is also expressed transiently during this period. To determine whether Fis was responsible for the growth phase‐dependent expression of bfp, fis was deleted from the EPEC strain E2348/69S. Paradoxically, the Δfis mutant retained the ability to colonize HEp‐2 cells in a characteristic localized adherence pattern, and Fis was found negatively to regulate the expression of BFP. However, the Δfis mutant failed to induce the accretion of filamentous actin, which is associated with attaching and effacing lesions. Using a combination of Western blotting and a novel multiplex primer extension assay (MPEA), we showed that, although the expression of intimin and Tir was not affected, transcription of the LEE4 operon encoding espADB and the virulence activator, Ler, were found to be Fis dependent.