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Mutations in rpoA affect expression of anaerobically regulated genes in Salmonella typhimurium.

ABSTRACT: oxrB8, a mutation that diminishes the anaerobic induction of pepT and other anaerobically regulated, oxrA (fnr)-dependent Salmonella typhimurium genes, is an allele of rpoA, the gene for the alpha subunit of RNA polymerase. Four additional rpoA mutations that affect anaerobic pepT expression have been isolated after localized mutagenesis of the rpoA region. All but one of these rpoA mutations appear to have relatively specific effects on genes that require the OxrA (FNR) protein, a positive transcriptional regulator of a family of anaerobically expressed genes. All of these mutations lead to amino acid substitutions in the C-terminal region of the alpha subunit. These results taken with a number of previous observations suggest a role for the alpha subunit in the interaction between RNA polymerase and positive transcriptional regulatory proteins. They also suggest that the C-terminal region of alpha is important for these interactions.

SUBMITTER: Lombardo MJ

PROVIDER: S-EPMC212517 | biostudies-other | 1991 Dec

REPOSITORIES: biostudies-other

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Mutations in rpoA affect expression of anaerobically regulated genes in Salmonella typhimurium.

Lombardo M J MJ Bagga D D Miller C G CG

Journal of bacteriology 19911201 23

oxrB8, a mutation that diminishes the anaerobic induction of pepT and other anaerobically regulated, oxrA (fnr)-dependent Salmonella typhimurium genes, is an allele of rpoA, the gene for the alpha subunit of RNA polymerase. Four additional rpoA mutations that affect anaerobic pepT expression have been isolated after localized mutagenesis of the rpoA region. All but one of these rpoA mutations appear to have relatively specific effects on genes that require the OxrA (FNR) protein, a positive tran ...[more]

PMID: 1938946

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Project description:Salmonella enterica serovar Typhimurium (S. Typhimurium) is a Gram-negative pathogen that must successfully adapt to the broad fluctuations in the concentration of dissolved dioxygen encountered in the host. In Escherichia coli, ArcA (Aerobic Respiratory Control) helps the cells to sense and respond to the presence of dioxygen. The global role of ArcA in E. coli is well characterized; however, little is known about its role in anaerobically grown S. Typhimurium.We compared the transcriptional profiles of the virulent wild-type (WT) strain (ATCC 14028s) and its isogenic arcA mutant grown under anaerobic conditions. We found that ArcA directly or indirectly regulates 392 genes (8.5% of the genome); of these, 138 genes are poorly characterized. Regulation by ArcA in S. Typhimurium is similar, but distinct from that in E. coli. Thus, genes/operons involved in core metabolic pathways (e.g., succinyl-CoA, fatty acid degradation, cytochrome oxidase complexes, flagellar biosynthesis, motility, and chemotaxis) were regulated similarly in the two organisms. However, genes/operons present in both organisms, but regulated differently by ArcA in S. Typhimurium included those coding for ethanolamine utilization, lactate transport and metabolism, and succinate dehydrogenases. Salmonella-specific genes/operons regulated by ArcA included those required for propanediol utilization, flagellar genes (mcpAC, cheV), Gifsy-1 prophage genes, and three SPI-3 genes (mgtBC, slsA, STM3784). In agreement with our microarray data, the arcA mutant was non-motile, lacked flagella, and was as virulent in mice as the WT. Additionally, we identified a set of 120 genes whose regulation was shared with the anaerobic redox regulator, Fnr.We have identified the ArcA regulon in anaerobically grown S. Typhimurium. Our results demonstrated that in S. Typhimurium, ArcA serves as a transcriptional regulator coordinating cellular metabolism, flagella biosynthesis, and motility. Furthermore, ArcA and Fnr share in the regulation of 120 S. Typhimurium genes.

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.

Project description:The Ferric uptake regulator (Fur) is a transcriptional regulator that controls iron homeostasis in bacteria. Although the regulatory role of Fur in Escherichia coli is well characterized, most of the studies were conducted under routine culture conditions, i.e., in ambient oxygen concentration. To reveal potentially novel aspects of the Fur regulon in Salmonella enterica serovar Typhimurium under oxygen conditions similar to that encountered in the host, we compared the transcriptional profiles of the virulent wild-type strain (ATCC 14028s) and its isogenic ?fur strain under anaerobic conditions.Microarray analysis of anaerobically grown ?fur S. Typhimurium identified 298 differentially expressed genes. Expression of several genes controlled by Fnr and NsrR appeared to be also dependent on Fur. Furthermore, Fur was required for the activity of the cytoplasmic superoxide disumutases (MnSOD and FeSOD). The regulation of FeSOD gene, sodB, occurred via small RNAs (i.e., the ryhB homologs, rfrA and rfrB) with the aid of the RNA chaperone Hfq. The transcription of sodA was increased in ?fur; however, the enzyme was inactive due to the incorporation of iron instead of manganese in SodA. Additionally, in ?fur, the expression of the gene coding for the ferritin-like protein (ftnB) was down-regulated, while the transcription of the gene coding for the nitric oxide (NO·) detoxifying flavohemoglobin (hmpA) was up-regulated. The promoters of ftnB and hmpA do not contain recognized Fur binding motifs, which indicated their probable indirect regulation by Fur. However, Fur activation of ftnB was independent of Fnr. In addition, the expression of the gene coding for the histone-like protein, H-NS (hns) was increased in ?fur. This may explain the observed down-regulation of the tdc operon, responsible for the anaerobic degradation of threonine, and ftnB in ?fur.This study determined that Fur is a positive factor in ftnB regulation, while serving to repress the expression of hmpA. Furthermore, Fur is required for the proper expression and activation of the antioxidant enzymes, FeSOD and MnSOD. Finally, this work identified twenty-six new targets of Fur regulation, and demonstrates that H-NS repressed genes are down-regulated in ?fur.

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Mutations in rpoA affect expression of anaerobically regulated genes in Salmonella typhimurium.

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Mutations in rpoA affect expression of anaerobically regulated genes in Salmonella typhimurium.

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