"V体育ios版" Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The . gov means it’s official. Federal government websites often end in . gov or . mil. Before sharing sensitive information, make sure you’re on a federal government site. VSports app下载.

Https

The site is secure. The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely V体育官网. .

. 2000 Oct;182(20):5749-56.
doi: 10.1128/JB.182.20.5749-5756.2000.

Identification of RpoS (sigma(S))-regulated genes in Salmonella enterica serovar typhimurium

M Ibanez-Ruiz  1 , V Robbe-Saule, D Hermant, S Labrude, F Norel
Affiliations

Identification of RpoS (sigma(S))-regulated genes in Salmonella enterica serovar typhimurium

M Ibanez-Ruiz et al. J Bacteriol. 2000 Oct.

VSports app下载 - Abstract

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 VSports手机版. 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. .

PubMed Disclaimer

Figures (VSports)

FIG. 1
FIG. 1
Strategy used to select Tn5B21 insertion mutants of serovar Typhimurium carrying ςS-dependent promoter-lacZ fusions. (A) Transposon Tn5B21 (35) and primers used for DNA sequencing. Restriction sites: B, BamHI; E, EcoRI; H, HindIII; P, PstI. (B) Transposon Tn5B21 insertion mutagenesis of the Smr Kmr serovar Typhimurium rpoS mutant C52K-Smr is described in Materials and Methods. Mutants containing poorly expressed lacZ gene fusions were identified as having a pale blue colony phenotype after growth in LB agar containing X-Gal. Such strains were used to inoculate 96-well enzyme-linked immunosorbent assay (ELISA) plates (0.2 ml of LB per well) and grown overnight at 37°C. Conjugation between these mutant strains and E. coli strain S17-1 carrying the mobilizable plasmid pVKRpoS (Cmr) was carried out on LB agar plates for 4 to 5 h at 37°C (donor and recipient cells were mixed in a 1:1 ratio). Serovar Typhimurium transconjugants carrying pVKRpoS were selected in LB broth supplemented with 20 μg of tetracycline per ml 25 μg of kanamycin per ml, and 30 μg of chloramphenicol per ml (LBTetKmCm). β-Galactosidase activity of serovar Typhimurium Tn5B21 mutants with and without pVKRpoS (plates II and plate I, respectively) was assayed using 96-well ELISA plates and the method of Miller (24). An automated ELISA plate reader (Labsystems Multiskan RC) was used to measure A420 after 1 h of incubation at room temperature. Mutant candidates showing increased gene fusion activity on acquisition of plasmid pVKRpoS were selected for further studies.
FIG. 2
FIG. 2
Genomic organization of the ogt region and mapping of the ςS-regulated promoter of ogt. (A) Comparison of the gene order near ogt in serovar Typhimurium and E. coli. (B) DNA sequence in the ydaL-ogt region in serovar Typhimurium. The 5′ ends of the ydaL and ogt genes are shown in bold. The nucleotide sequence was that from serovar Typhimurium C52 (this study). The position of the putative RpoS-dependent transcriptional start site of ogt (+1) is indicated by asterisks. The −10 and −35 regions of the rpoS-regulated promoter of ogt (ogtp1) are underlined. The position of insertion of Tn5B21 in mutant C52::H87 is indicated by a broken arrow. (C) Mapping of the 5′ end of ogt mRNA in serovar Typhimurium. A 5′ 32P-labeled primer complementary to the ogt coding strand was annealed to total RNAs isolated from LB-grown stationary-phase cultures of serovar Typhimurium wild-type (C52 and SL1344) and rpoS mutant (C52K and SL1344K) strains and of the Tn5B21 insertion mutant C52::H87. The primer was extended with reverse transcriptase, and the products were resolved by electrophoresis on a sequencing gel. The DNA sequencing ladder (lanes A, C, G, and T) was prepared using the primer as used to sequence pUCogt1. The major extended product is indicated by an arrow. Lane 1, C52; lane 2, C52K; lane 3, SL1344; lane 4, SL1344K; lane 5; C52::H87.
See this image and copyright information in PMC

References

    1. Bearson B L, Wilson L, Foster J W. A low pH-inducible, PhoPQ-dependent acid tolerance response protects Salmonella typhimurium against inorganic acid stress. J Bacteriol. 1998;180:2409–2417. - PMC - PubMed
    1. Blattner F R, Plunkett III G, Bloch C A, Perna N T, Burland V, Riley M, Collado-Vides J, Glasner J D, Rode C K, Mayhew G F, Gregor J, Davis N W, Kirkpatrick A, Goeden M A, Rose D J, Mau B, Shao Y. The complete genome sequence of Escherichia coli K-12. Science. 1997;277:1453–1462. - PubMed (VSports在线直播)
    1. Casadaban M, Cohen S N. Analysis of a gene control signal by DNA fusion and cloning in Escherichia coli. J Mol Biol. 1980;138:179–207. - PubMed
    1. Chen C Y, Buchmeier N A, Libby S, Fang F C, Krause M, Guiney D G. Central regulatory role for the RpoS sigma factor in expression of Salmonella dublin plasmid virulence genes. J Bacteriol. 1995;177:5303–5309. - "VSports最新版本" PMC - PubMed
    1. Collinson S K, Clouthier S C, Doran J L, Banser P A, William W K. Salmonella enteritidis agfBAC operon encoding thin, aggregative fimbriae. J Bacteriol. 1996;178:662–667. - PMC - PubMed

"VSports最新版本" Publication types

MeSH terms