Dataset Information

PpGpp regulation of RpoS degradation via anti-adaptor protein IraP.

ABSTRACT: IraP is a small protein that interferes with the delivery of sigma(S) (RpoS) to the ClpXP protease by blocking the action of RssB, an adaptor protein for sigma(S) degradation. IraP was previously shown to mediate stabilization of sigma(S) during phosphate starvation. Here, we show that iraP is transcribed in response to phosphate starvation; this response is mediated by ppGpp. The iraP promoter is positively regulated by ppGpp, dependent on the discriminator region of the iraP promoter. Sensing of phosphate starvation requires SpoT but not RelA. The results demonstrate a target for positive regulation by ppGpp and suggest that the cell use of ppGpp to mediate a variety of starvation responses operates in part by modulating sigma(S) levels.

SUBMITTER: Bougdour A

PROVIDER: S-EPMC1937563 | biostudies-other | 2007 Jul

REPOSITORIES: biostudies-other

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ppGpp regulation of RpoS degradation via anti-adaptor protein IraP.

Bougdour Alexandre A Gottesman Susan S

Proceedings of the National Academy of Sciences of the United States of America 20070719 31

IraP is a small protein that interferes with the delivery of sigma(S) (RpoS) to the ClpXP protease by blocking the action of RssB, an adaptor protein for sigma(S) degradation. IraP was previously shown to mediate stabilization of sigma(S) during phosphate starvation. Here, we show that iraP is transcribed in response to phosphate starvation; this response is mediated by ppGpp. The iraP promoter is positively regulated by ppGpp, dependent on the discriminator region of the iraP promoter. Sensing ...[more]

PMID: 17640895

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Project description:UnlabelledRpoS (?(S)), the general stress response sigma factor, directs the expression of genes under a variety of stressful conditions. Control of the cellular ?(S) concentration is critical for appropriately scaled ?(S)-dependent gene expression. One way to maintain appropriate levels of ?(S) is to regulate its stability. Indeed, ?(S) degradation is catalyzed by the ClpXP protease and the recognition of ?(S) by ClpXP depends on the adaptor protein RssB. Three anti-adaptors (IraD, IraM, and IraP) exist in Escherichia coli K-12; each interacts with RssB and inhibits RssB activity under different stress conditions, thereby stabilizing ?(S). Unlike K-12, some E. coli isolates, including uropathogenic E. coli strain CFT073, show comparable cellular levels of ?(S) during the logarithmic and stationary growth phases, suggesting that there are differences in the regulation of ?(S) levels among E. coli strains. Here, we describe IraL, an RssB anti-adaptor that stabilizes ?(S) during logarithmic phase growth in CFT073 and other E. coli and Shigella strains. By immunoblot analyses, we show that IraL affects the levels and stability of ?(S) during logarithmic phase growth. By computational and PCR-based analyses, we reveal that iraL is found in many E. coli pathotypes but not in laboratory-adapted strains. Finally, by bacterial two-hybrid and copurification analyses, we demonstrate that IraL interacts with RssB by a mechanism distinct from that used by other characterized anti-adaptors. We introduce a fourth RssB anti-adaptor found in E. coli species and suggest that differences in the regulation of ?(S) levels may contribute to host and niche specificity in pathogenic and nonpathogenic E. coli strains.ImportanceBacteria must cope with a variety of environmental conditions in order to survive. RpoS (?(S)), the general stress response sigma factor, directs the expression of many genes under stressful conditions in both pathogenic and nonpathogenic Escherichia coli strains. The regulation of ?(S) levels and activity allows appropriately scaled ?(S)-dependent gene expression. Here, we describe IraL, an RssB anti-adaptor that, unlike previously described anti-adaptors, stabilizes ?(S) during the logarithmic growth phase in the absence of additional stress. We also demonstrate that iraL is found in a large number of E. coli and Shigella isolates. These data suggest that strains containing iraL are able to initiate ?(S)-dependent gene expression under conditions under which strains without iraL cannot. Therefore, IraL-mediated ?(S) stabilization may contribute to host and niche specificity in E. coli.

Project description: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 sigma(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 Deltahns mutant. The Deltahns 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 Deltahns, DeltarpoS, and Deltahns DeltarpoS mutants, analysis of RpoS reporter fusions, quantitative reverse transcription-PCR measurements, and ectopic expression of RpoS in DeltarpoS and DeltarpoS Deltahns mutants showed that H-NS posttranscriptionally enhances RpoS expression. The Deltahns 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, Deltahns, DeltarpoS, and Deltahns DeltarpoS 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 sigma(S). Finally, we show that the cyclic AMP receptor protein and H-NS act along the same pathway to positively affect RpoS expression.

Dataset Information

PpGpp regulation of RpoS degradation via anti-adaptor protein IraP.

Publications

ppGpp regulation of RpoS degradation via anti-adaptor protein IraP.

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