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Overproduction of exopolysaccharides by an Escherichia coli K-12 rpoS mutant in response to osmotic stress.

ABSTRACT: The yjbEFGH operon is implicated in the production of an exopolysaccharide of an unknown function and is induced by osmotic stress and negatively regulated by the general stress response sigma factor RpoS. Despite the obvious importance of RpoS, negative selection for rpoS has been reported to take place in starved cultures, suggesting an adaptive occurrence allowing the overexpression of RpoD-dependent uptake and nutrient-scavenging systems. The trade-off of the RpoS-dependent functions for improved nutrient utilization abilities makes the bacterium more sensitive to environmental stressors, e.g., osmotic stress. In this work, we addressed the hypothesis that overinduction of genes in rpoS-deficient strains indicates their essentiality. Using DNA microarrays, real-time PCR, and transcriptional fusions, we show that genes of the wca operon, implicated in the production of the colanic acid exopolysaccharide, previously shown to be induced by osmotic stress, are also negatively controlled by RpoS. Both exopolysaccharides in the synthesis of which yjb and wca are involved are overproduced in an rpoS mutant during osmotic stress. We also show that both operons are essential in an rpoS-deficient strain but not in the wild type; promoters of both operons are constitutively active in yjb rpoS mutants; this strain produces extremely mucoid colonies, forms long filaments, and exhibits a reduced growth capability. In addition, the wca rpoS mutant's growth is inhibited by osmotic stress. These results indicate that although induced in the wild type, both operons are much more valuable for an rpoS-deficient strain, suggesting that the overproduction of both exopolysaccharides is an adaptive action.

SUBMITTER: Ionescu M

PROVIDER: S-EPMC2620720 | biostudies-literature | 2009 Jan

REPOSITORIES: biostudies-literature

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Overproduction of exopolysaccharides by an Escherichia coli K-12 rpoS mutant in response to osmotic stress.

Ionescu Michael M Belkin Shimshon S

Applied and environmental microbiology 20081107 2

The yjbEFGH operon is implicated in the production of an exopolysaccharide of an unknown function and is induced by osmotic stress and negatively regulated by the general stress response sigma factor RpoS. Despite the obvious importance of RpoS, negative selection for rpoS has been reported to take place in starved cultures, suggesting an adaptive occurrence allowing the overexpression of RpoD-dependent uptake and nutrient-scavenging systems. The trade-off of the RpoS-dependent functions for imp ...[more]

PMID: 18997023

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Project description:The alternative sigma factor RpoS is a central regulator of many stress responses in Escherichia coli The level of functional RpoS differs depending on the stress. The effect of these differing concentrations of RpoS on global transcriptional responses remains unclear. We investigated the effect of RpoS concentration on the transcriptome during stationary phase in rich media. We found that 23% of genes in the E. coli genome are regulated by RpoS, and we identified many RpoS-transcribed genes and promoters. We observed three distinct classes of response to RpoS by genes in the regulon: genes whose expression changes linearly with increasing RpoS level, genes whose expression changes dramatically with the production of only a little RpoS ("sensitive" genes), and genes whose expression changes very little with the production of a little RpoS ("insensitive"). We show that sequences outside the core promoter region determine whether an RpoS-regulated gene is sensitive or insensitive. Moreover, we show that sensitive and insensitive genes are enriched for specific functional classes and that the sensitivity of a gene to RpoS corresponds to the timing of induction as cells enter stationary phase. Thus, promoter sensitivity to RpoS is a mechanism to coordinate specific cellular processes with growth phase and may also contribute to the diversity of stress responses directed by RpoS.IMPORTANCE The sigma factor RpoS is a global regulator that controls the response to many stresses in Escherichia coli Different stresses result in different levels of RpoS production, but the consequences of this variation are unknown. We describe how changing the level of RpoS does not influence all RpoS-regulated genes equally. The cause of this variation is likely the action of transcription factors that bind the promoters of the genes. We show that the sensitivity of a gene to RpoS levels explains the timing of expression as cells enter stationary phase and that genes with different RpoS sensitivities are enriched for specific functional groups. Thus, promoter sensitivity to RpoS is a mechanism that coordinates specific cellular processes in response to stresses.

Project description:Experimental evolution of Escherichia coli K-12 W3110 by serial dilutions for 2,200 generations at high pH extended the range of sustained growth from pH 9.0 to pH 9.3. pH 9.3-adapted isolates showed mutations in DNA-binding regulators and envelope proteins. One population showed an IS1 knockout of phoB (encoding the positive regulator of the phosphate regulon). A phoB::kanR knockout increased growth at high pH. phoB mutants are known to increase production of fermentation acids, which could enhance fitness at high pH. Mutations in pcnB [poly(A) polymerase] also increased growth at high pH. Three out of four populations showed deletions of torI, an inhibitor of TorR, which activates expression of torCAD (trimethylamine N-oxide respiration) at high pH. All populations showed point mutations affecting the stationary-phase sigma factor RpoS, either in the coding gene or in genes for regulators of RpoS expression. RpoS is required for survival at extremely high pH. In our microplate assay, rpoS deletion slightly decreased growth at pH 9.1. RpoS protein accumulated faster at pH 9 than at pH 7. The RpoS accumulation at high pH required the presence of one or more antiadaptors that block degradation (IraM, IraD, and IraP). Other genes with mutations after high-pH evolution encode regulators, such as those encoded by yobG (mgrB) (PhoPQ regulator), rpoN (nitrogen starvation sigma factor), malI, and purR, as well as envelope proteins, such as those encoded by ompT and yahO Overall, E. coli evolution at high pH selects for mutations in key transcriptional regulators, including phoB and the stationary-phase sigma factor RpoS.IMPORTANCEEscherichia coli in its native habitat encounters high-pH stress such as that of pancreatic secretions. Experimental evolution over 2,000 generations showed selection for mutations in regulatory factors, such as deletion of the phosphate regulator PhoB and mutations that alter the function of the global stress regulator RpoS. RpoS is induced at high pH via multiple mechanisms.

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Dataset Information

Overproduction of exopolysaccharides by an Escherichia coli K-12 rpoS mutant in response to osmotic stress.

Publications

Overproduction of exopolysaccharides by an Escherichia coli K-12 rpoS mutant in response to osmotic stress.

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