Insertion mutations in Helicobacter pylori flhA reveal strain differences in RpoN-dependent gene expression.
Insertion mutations in Helicobacter pylori flhA reveal strain differences in RpoN-dependent gene expression.
复制标题
幽门螺杆菌 flhA 中的插入突变揭示了 RpoN 依赖性基因表达的菌株差异。
DOI：
10.1099/mic.0.059063-0
复制
发表时间：
2013
期刊：
影响因子：
--
通讯作者：
Hoover,TimothyR
中科院分区：
文献类型：
--
作者：
Tsang,Jennifer;Smith,ToddG;Pereira,LaraE;Hoover,TimothyR
胃病原体幽门螺杆菌中的鞭毛生物发生涉及利用在该细菌中发现的所有三种σ因子（RpoD、RpoN和FliA）的转录层级。RpoN依赖性基因的转录需要传感激酶FlgS和反应调节因子FlgR。据认为，FlgS感觉到一些细胞的线索，以调节RpoN依赖性鞭毛基因的转录，但这种信号尚未被确定。先前的研究表明，转录的RpoN依赖性基因的抑制突变inflhA，它编码的鞭毛蛋白输出装置的膜结合组件。我们发现这取决于H。pylori菌株中，插入突变inflhA对RpoN依赖基因的表达有不同的影响。基因突变会影响hAinH。通过插入氯霉素抗性盒以有效消除基因（ΔflhA）的表达，或在密码子77（命名为flhA 77）或密码子454（命名为flhA 454）之后的基因内，产生幽门螺杆菌菌株B128和ATCC 43504（模式菌株），这可以允许表达截短的FlhA蛋白。所有三个flhA突变都严重抑制RpoN依赖基因flaBandflgEinH的转录。pyloriB128.相反，在H.携带flhA 77或flhA 454但不携带ΔflhA的pyloriATCC 43504的表达水平为野生型水平的约60%。 FlhA 454变异体在从H. pyloriATCC 43504而不是H. pyloriB 128的突变，这可能是两株菌株flhA突变表型差异的原因。总之，这些发现表明，只有N-末端区域的FlhA是需要转录的RpoN调节子。有趣的是，anflaB′-′ xylE报告基因在H.携带flhA 77等位基因的pyloriATCC 43504菌株的表达比具有野生型等位基因的菌株高约8倍，表明flaB的表达不仅在转录水平上受到调节，而且在转录后水平上受到调节。
Flagellar biogenesis in the gastric pathogenHelicobacter pyloriinvolves a transcriptional hierarchy that utilizes all three sigma factors found in this bacterium (RpoD, RpoN and FliA). Transcription of the RpoN-dependent genes requires the sensor kinase FlgS and response regulator FlgR. It is thought that FlgS senses some cellular cue to regulate transcription of the RpoN-dependent flagellar genes, but this signal has yet to be identified. Previous studies showed that transcription of the RpoN-dependent genes is inhibited by mutations inflhA, which encodes a membrane-bound component of the flagellar protein export apparatus. We found that depending on theH. pyloristrain used, insertion mutations inflhAhad different effects on expression of RpoN-dependent genes. Mutations inflhAinH. pyloristrains B128 and ATCC 43504 (the type strain) were generated by inserting a chloramphenicol resistance cassette so as to effectively eliminate expression of the gene (ΔflhA), or within the gene following codon 77 (designatedflhA77) or codon 454 (designatedflhA454), which could allow expression of truncated FlhA proteins. All threeflhAmutations severely inhibited transcription of the RpoN-dependent genesflaBandflgEinH. pyloriB128. In contrast, levels offlaBandflgEtranscripts inH. pyloriATCC 43504 bearing eitherflhA77orflhA454, but not ΔflhA, were ~60 % of wild-type levels. The FlhA454variant was detected in membrane fractions prepared fromH. pyloriATCC 43504 but notH. pyloriB128, which may account for the phenotypic differences in theflhAmutations of the two strains. Taken together, these findings suggest that only the N-terminal region of FlhA is needed for transcription of the RpoN regulon. Interestingly, expression of anflaB′-′xylEreporter gene inH. pyloriATCC 43504 bearing theflhA77allele was about eightfold higher than that of a strain with the wild-type allele, suggesting that expression offlaBis not only regulated at the level of transcription but also regulated post-transcriptionally.