Role of the Histone-Like Nucleoid Structuring Protein in the Regulation of rpoS and RpoS-Dependent Genes in Vibrio cholerae

Abstract

Production of the Zn-metalloprotease hemagglutinin (HA)/protease by Vibrio cholerae has been reported to enhance enterotoxicity in rabbit ileal loops and the reactogenicity of live cholera vaccine candidates. Expression of HA/protease requires the alternate sigma factor S (RpoS), encoded by rpoS. The histone-like nucleoid structuring protein (H-NS) has been shown to repress rpoS expression in Escherichia coli. In V. cholerae strains of the classical biotype, H-NS has been reported to silence virulence gene expression. In this study we examined the role of H-NS in the expression of HA/protease and motility in an El Tor biotype strain by constructing a ⌬hns mutant. The ⌬hns mutant exhibited multiple phenotypes, such as production of cholera toxin in nonpermissive LB medium, reduced resistance to high osmolarity, enhanced resistance to low pH and hydrogen peroxide, and reduced motility. Depletion of H-NS by overexpression of a dominant-negative allele or by deletion of hns resulted in diminished expression of HA/protease. Epistasis analysis of HA/protease expression in ⌬hns, ⌬rpoS, and ⌬hns ⌬rpoS mutants, analysis of RpoS reporter fusions, quantitative reverse transcription-PCR measurements, and ectopic expression of RpoS in ⌬rpoS and ⌬rpoS ⌬hns mutants showed that H-NS posttranscriptionally enhances RpoS expression. The ⌬hns mutant exhibited a lower degree of motility and lower levels of expression of flaA, flaC, cheR-2, and motX mRNAs than the wild type. Comparison of the mRNA abundances of these genes in wild-type, ⌬hns, ⌬rpoS, and ⌬hns ⌬rpoS strains revealed that deletion of rpoS had a more severe negative effect on their expression. Interestingly, deletion of hns in the rpoS background resulted in higher expression levels of flaA, flaC, and motX, suggesting that H-NS represses the expression of these genes in the absence of S . Finally, we show that the cyclic AMP receptor protein and H-NS act along the same pathway to positively affect RpoS expression.

Interaction of the Histone-Like Nucleoid Structuring Protein and the General Stress Response Regulator RpoS at Vibrio cholerae Promoters That Regulate Motility and Hemagglutinin/Protease Expression

“…Consistent with this role, H-NS has been shown to silence virulence gene expression in V. cholerae by acting at different levels of the ToxR regulatory cascade, which includes the toxT, tcpA, and ctxA promoters (37). Further, V. cholerae hns mutants have been reported to exhibit diminished motility and intestinal colonization capacity (18,27,49,53). There are numerous evidences indicating that repression by H-NS can be relieved in response to environmental stimuli that activate the expression of other regulators whose binding site overlaps with that of H-NS.…”

“…Consistently, microarray studies have shown that rpoS mutants express reduced levels of multiple motility and chemotaxis genes, suggesting that rpoS could act at an early stage of the motility regulatory cascade (35). In a previous study, we addressed the role of H-NS in the regulation of V. cholerae RpoS and RpoS-dependent genes that affect motility and HA/protease production (49). We found that H-NS posttranscriptionally affects RpoS expression in a positive manner, which in turn enhances motility and HA/protease production (49).…”

“…V. cholerae rpoS mutants are more sensitive to starvation, high osmolarity, and oxidative stresses, are less motile than the wild type (WT), and do not express HA/protease (35,49,58). We recently reported that RpoS diminishes the cellular concentration of c-di-GMP, an inhibitor of flagellar motility (57).…”

Integration of Global Regulatory Mechanisms Controlling Vibrio Cholerae Behavior

“…V. cholerae rpoS mutants are more sensitive to starvation, high osmolarity, and oxidative stresses, are less motile than their wild-type precursors (Fig. 13), and do not express hapA (Nielsen et al, 2006;Silva et al, 2008;Yildiz & Schoolnik, 1998;Silva & Benitez, 2004). We have recently shown that, similar to HapR, expression of RpoS acts to diminish the c-di-GMP pool .…”

“…In V. cholerae, hns mutants form small colonies, are incapable of using -glucosides as a carbon source, exhibit diminished motility (Fig. 12) and intestinal colonization capacity, and show altered responses to environmental stresses (Ghosh et al, 2006;Krishnan et al, 2004;Tending et al, 2000;Silva et al 2008). An emerging function of H-NS is the transcriptional silencing of horizontally acquired genes (Lucchini et al, 2006;Navarre et al, 2006;Oshima et al, 2006).…”