Identification of RpoS (σS)-regulated genes in Salmonella enterica serovar Typhimurium
Identification of RpoS (σS)-regulated genes in Salmonella enterica serovar Typhimurium
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DOI：
10.1128/jb.182.20.5749-5756.2000
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发表时间：
2000-10-01
影响因子：
3.2
通讯作者：
Norel, F
中科院分区：
文献类型：
--
作者：
Ibanez-Ruiz, M;Robbe-Saule, V;Norel, F
rpoS 基因编码替代 sigma 因子 sigma(S) (RpoS)，是细菌在饥饿和应激条件下生存所必需的。它对于小鼠沙门氏菌的毒力也至关重要。大多数关于 RpoS 调节子的工作都是在密切相关的肠杆菌种大肠杆菌中进行的。为了表征沙门氏菌中的 RpoS 调节子，我们从一组含有随机 Tn5B21 突变的肠沙门氏菌鼠伤寒血清型突变体中分离出 38 个独特的 RpoS 激活的 lacZ 基因融合体。对 RpoS 的依赖性从 3 倍到超过 95 倍不等，并且所有分离的基因融合体均受生长阶段的调节。通过 DNA 序列分析确定了 21 个 RpoS 依赖性融合体的身份。其中 7 个融合体映射到鼠伤寒沙门氏菌血清型的 DNA 区域，与任何已知的大肠杆菌序列都不匹配，这表明两个物种的 RpoS 调节子的组成存在显着差异。其他 14 个融合体映射到 13 个 DNA 区域，与大肠杆菌序列非常相似。这两个物种共有的 DNA 区域中的插入突变似乎都不会影响 BALB/c 小鼠中的沙门氏菌毒力。其中，只有三个（otsA、katE 和 poxB）位于 RpoS 调节子的已知成员中。映射到九个功能未知的开放阅读框中的十个插入（yciF、yehY、yhjY、yncC、yjgB、yahO、ygaU、ycgB 和 yeaG）似乎是 RpoS 调节子的新成员。突变体 C52::H87 中的一个插入位于 ogt 上游的非编码区，编码 O-6-甲基鸟嘌呤 DNA甲基转移酶参与修复DNA中的烷基化损伤。 OGT 编码序列与大肠杆菌同源物非常相似，但两个物种的 OGT 5' 侧翼区域明显不同，表明存在遗传重排。使用引物延伸测定，在鼠伤寒沙门氏菌野生型菌株 C52 和 SL1344 的 RNA 中检测到特定的 ogt mRNA 起始位点，但在突变菌株 C52K (rpoS)、SL1344K (rpoS) 和 C52::H87 的 RNA 中未检测到。在突变体 C52::H87 中，Tn5B21 插入到 ogt mRNA 起始位点，lacZ 可能是从已识别的 RpoS 调节启动子转录而来。这些结果表明，沙门氏菌中的ogt基因表达在丰富培养基中的生长稳定期受到RpoS的调节，这一发现表明RpoS在DNA修复功能中具有新的作用。
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.