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Identification and molecular characterization of the Mg2+ stimulon of Escherichia coli.

ABSTRACT: Transcription profile microarray analysis in Escherichia coli was performed to identify the member genes of the Mg(2+) stimulon that respond to the availability of external Mg(2+) in a PhoP/PhoQ two-component system-dependent manner. The mRNA levels of W3110 in the presence of 30 mM MgCl(2), WP3022 (phoP defective), and WQ3007 (phoQ defective) were compared with those of W3110 in the absence of MgCl(2). The expression ratios of a total of 232 genes were <0.75 in all three strains (the supplemental data are shown at http://www.nara.kindai.ac.jp/nogei/seiken/array.html), suggesting that the PhoP/PhoQ system is involved directly or indirectly in the transcription of these genes. Of those, 26 contained the PhoP box-like sequences with the direct repeats of (T/G)GTTTA within 500 bp upstream of the initiation codon. Furthermore, S1 nuclease assays of 26 promoters were performed to verify six new Mg(2+) stimulon genes, hemL, nagA, rstAB, slyB, vboR, and yrbL, in addition to the phoPQ, mgrB, and mgtA genes reported previously. In gel shift and DNase I footprinting assays, all of these genes were found to be regulated directly by PhoP. Thus, we concluded that the phoPQ, mgrB, mgtA, hemL, nagA, rstAB, slyB, vboR, and yrbL genes make up the Mg(2+) stimulon in E. coli.

SUBMITTER: Minagawa S

PROVIDER: S-EPMC161583 | biostudies-literature | 2003 Jul

REPOSITORIES: biostudies-literature

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Identification and molecular characterization of the Mg2+ stimulon of Escherichia coli.

Minagawa Shu S Ogasawara Hiroshi H Kato Akinori A Yamamoto Kaneyoshi K Eguchi Yoko Y Oshima Taku T Mori Hirotada H Ishihama Akira A Utsumi Ryutaro R

Journal of bacteriology 20030701 13

Transcription profile microarray analysis in Escherichia coli was performed to identify the member genes of the Mg(2+) stimulon that respond to the availability of external Mg(2+) in a PhoP/PhoQ two-component system-dependent manner. The mRNA levels of W3110 in the presence of 30 mM MgCl(2), WP3022 (phoP defective), and WQ3007 (phoQ defective) were compared with those of W3110 in the absence of MgCl(2). The expression ratios of a total of 232 genes were <0.75 in all three strains (the supplement ...[more]

PMID: 12813061

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Project description:UnlabelledThe vacuolating autotransporter toxin (Vat) contributes to uropathogenic Escherichia coli (UPEC) fitness during systemic infection. Here, we characterized Vat and investigated its regulation in UPEC. We assessed the prevalence of vat in a collection of 45 UPEC urosepsis strains and showed that it was present in 31 (68%) of the isolates. The isolates containing the vat gene corresponded to three major E. coli sequence types (ST12, ST73, and ST95), and these strains secreted the Vat protein. Further analysis of the vat genomic locus identified a conserved gene located directly downstream of vat that encodes a putative MarR-like transcriptional regulator; we termed this gene vatX The vat-vatX genes were present in the UPEC reference strain CFT073, and reverse transcriptase PCR (RT-PCR) revealed that the two genes are cotranscribed. Overexpression of vatX in CFT073 led to a 3-fold increase in vat gene transcription. The vat promoter region contained three putative nucleation sites for the global transcriptional regulator histone-like nucleoid structuring protein (H-NS); thus, the hns gene was mutated in CFT073 (to generate CFT073 hns). Western blot analysis using a Vat-specific antibody revealed a significant increase in Vat expression in CFT073 hns compared to that in wild-type CFT073. Direct H-NS binding to the vat promoter region was demonstrated using purified H-NS in combination with electrophoresis mobility shift assays. Finally, Vat-specific antibodies were detected in plasma samples from urosepsis patients infected by vat-containing UPEC strains, demonstrating that Vat is expressed during infection. Overall, this study has demonstrated that Vat is a highly prevalent and tightly regulated immunogenic serine protease autotransporter protein of Enterobacteriaceae (SPATE) secreted by UPEC during infection.ImportanceUropathogenic Escherichia coli (UPEC) is the major cause of hospital- and community-acquired urinary tract infections. The vacuolating autotransporter toxin (Vat) is a cytotoxin known to contribute to UPEC fitness during murine sepsis infection. In this study, Vat was found to be highly conserved and prevalent among a collection of urosepsis clinical isolates and was expressed at human core body temperature. Regulation of vat was demonstrated to be directly repressed by the global transcriptional regulator H-NS and upregulated by the downstream gene vatX (encoding a new MarR-type transcriptional regulator). Additionally, increased Vat-specific IgG titers were detected in plasma from corresponding urosepsis patients infected with vat-positive isolates. Hence, Vat is a highly conserved and tightly regulated urosepsis-associated virulence factor.

Dataset Information

Identification and molecular characterization of the Mg2+ stimulon of Escherichia coli.

Publications

Identification and molecular characterization of the Mg2+ stimulon of Escherichia coli.

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