Dataset Information

Binding interface between the Salmonella ?(S)/RpoS subunit of RNA polymerase and Crl: hints from bacterial species lacking crl.

ABSTRACT: In many Gram-negative bacteria, including Salmonella enterica serovar Typhimurium (S. Typhimurium), the sigma factor RpoS/?(S) accumulates during stationary phase of growth, and associates with the core RNA polymerase enzyme (E) to promote transcription initiation of genes involved in general stress resistance and starvation survival. Whereas ? factors are usually inactivated upon interaction with anti-? proteins, ?(S) binding to the Crl protein increases ?(S) activity by favouring its association to E. Taking advantage of evolution of the ?(S) sequence in bacterial species that do not contain a crl gene, like Pseudomonas aeruginosa, we identified and assigned a critical arginine residue in ?(S) to the S. Typhimurium ?(S)-Crl binding interface. We solved the solution structure of S. Typhimurium Crl by NMR and used it for NMR binding assays with ?(S) and to generate in silico models of the ?(S)-Crl complex constrained by mutational analysis. The ?(S)-Crl models suggest that the identified arginine in ?(S) interacts with an aspartate of Crl that is required for ?(S) binding and is located inside a cavity enclosed by flexible loops, which also contribute to the interface. This study provides the basis for further structural investigation of the ?(S)-Crl complex.

SUBMITTER: Cavaliere P

PROVIDER: S-EPMC4559669 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

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Binding interface between the Salmonella σ(S)/RpoS subunit of RNA polymerase and Crl: hints from bacterial species lacking crl.

Cavaliere Paola P Sizun Christina C Levi-Acobas Fabienne F Nowakowski Mireille M Monteil Véronique V Bontems François F Bellalou Jacques J Mayer Claudine C Norel Françoise F

Scientific reports 20150904

In many Gram-negative bacteria, including Salmonella enterica serovar Typhimurium (S. Typhimurium), the sigma factor RpoS/σ(S) accumulates during stationary phase of growth, and associates with the core RNA polymerase enzyme (E) to promote transcription initiation of genes involved in general stress resistance and starvation survival. Whereas σ factors are usually inactivated upon interaction with anti-σ proteins, σ(S) binding to the Crl protein increases σ(S) activity by favouring its associati ...[more]

PMID: 26338235

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Project description:The rpoS gene encodes the alternative sigma factor sigma(S) (RpoS) and is required for survival of bacteria under starvation and stress conditions. It is also essential for Salmonella virulence in mice. Most work on the RpoS regulon has been in the closely related enterobacterial species Escherichia coli. To characterize the RpoS regulon in Salmonella, we isolated 38 unique RpoS-activated lacZ gene fusions from a bank of Salmonella enterica serovar Typhimurium mutants harboring random Tn5B21 mutations. Dependence on RpoS varied from 3-fold to over 95-fold, and all gene fusions isolated were regulated by growth phase. The identities of 21 RpoS-dependent fusions were determined by DNA sequence analysis. Seven of the fusions mapped to DNA regions in Salmonella serovar Typhimurium that do not match any known E. coli sequence, suggesting that the composition of the RpoS regulon differs markedly in the two species. The other 14 fusions mapped to 13 DNA regions very similar to E. coli sequences. None of the insertion mutations in DNA regions common to both species appeared to affect Salmonella virulence in BALB/c mice. Of these, only three (otsA, katE, and poxB) are located in known members of the RpoS regulon. Ten insertions mapped in nine open reading frames of unknown function (yciF, yehY, yhjY, yncC, yjgB, yahO, ygaU, ycgB, and yeaG) appear to be novel members of the RpoS regulon. One insertion, that in mutant C52::H87, was in the noncoding region upstream from ogt, encoding a O(6)-methylguanine DNA methyltransferase involved in repairing alkylation damage in DNA. The ogt coding sequence is very similar to the E. coli homolog, but the ogt 5' flanking regions were found to be markedly different in the two species, suggesting genetic rearrangements. Using primer extension assays, a specific ogt mRNA start site was detected in RNAs of the Salmonella serovar Typhimurium wild-type strains C52 and SL1344 but not in RNAs of the mutant strains C52K (rpoS), SL1344K (rpoS), and C52::H87. In mutant C52::H87, Tn5B21 is inserted at the ogt mRNA start site, with lacZ presumably transcribed from the identified RpoS-regulated promoter. These results indicate that ogt gene expression in Salmonella is regulated by RpoS in stationary phase of growth in rich medium, a finding that suggests a novel role for RpoS in DNA repair functions.

Dataset Information

Binding interface between the Salmonella ?(S)/RpoS subunit of RNA polymerase and Crl: hints from bacterial species lacking crl.

Publications

Binding interface between the Salmonella σ(S)/RpoS subunit of RNA polymerase and Crl: hints from bacterial species lacking crl.

Similar Datasets

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