Transcriptional analysis of the Pseudomonas aeruginosa toxA regulatory gene ptxR
Transcriptional analysis of the Pseudomonas aeruginosa toxA regulatory gene ptxR
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DOI：
10.1139/w05-138
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发表时间：
2006-04-01
影响因子：
2.8
通讯作者：
Hamood, Abdul N.
中科院分区：
文献类型：
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作者：
Colmer-Hamood, Jane A.;Aramaki, Hironori;Hamood, Abdul N.
外毒素A基因(ToxA)在铜绿假单胞菌中的表达是一个复杂的过程，涉及多个调控因子，其中包括ptxR，它能使ToxA的表达增加4-5倍。现有证据表明，ptxR由两个不同的启动子P1和P2表达。以前的证据表明，在ptxR上游区域，存在几种调节蛋白的结合位点，包括ptxS，它负向调节ptxR的表达。我们利用嵌套缺失和体外转录分析来检测ptxR表达的调节。嵌套缺失分析的结果表明，在缺铁的有氧条件下，ptxR的表达遵循只涉及P2启动子的双相曲线。铁消除了ptxR表达的第二个高峰，但不影响P2启动子的表达。在微氧条件下，铁抑制了生长早期和后期仅携带PI或P2的亚克隆的ptxR表达。在厌氧条件下，ptxR的表达显著增加。此外，我们的结果表明，ptxR上游区域的不同片段在ptxR表达中起着特定的作用；它们的缺失导致了ptxR表达水平和模式的变化。我们的结果还表明，PtxS对ptxR表达的负调控不是通过ptxR-ptxS基因间隔区的PtxS结合部位发生的。使用Sigma(70)重组的铜绿假单胞菌RNA聚合酶进行体外转录分析产生了一个与T1非常相似的转录本，表明P1被Sigma(70)识别。用RPOS或ALGU重组的RNA聚合酶不产生转录本。然而，RpoH-重组RNA聚合酶产生了转录本。
The expression of the exotoxin A gene (toxA) in Pseudomonas aeruginosa is a complicated process that involves several regulators, including ptxR, which enhances toxA expression by 4- to 5-fold. Available evidence suggests that ptxR is expressed from two separate promoters, P1 and P2. Previous evidence indicated the presence, within the ptxR upstream region, of binding sites for several regulatory proteins, including PtxS, which negatively regulates ptxR expression. We utilized nested deletion and in vitro transcription analyses to examine the regulation of ptxR expression. The results from nested deletion analysis suggest that under aerobic conditions in iron-deficient medium, ptxR expression follows a biphasic curve that involves the P2 promoter only. Iron eliminated the second peak of ptxR expression but did not affect expression from the P2 promoter. Under microaerobic conditions, iron represses ptxR expression from subclones that carry PI alone or P2 alone at both early and late stages of growth. Under anaerobic conditions, ptxR expression increases considerably. In addition, our results suggest that different segments of the ptxR upstream region play specific roles in ptxR expression; their deletion caused variations in the level as well as the pattern of ptxR expression. Our results also indicate that negative regulation of ptxR expression by PtxS does not occur through the PtxS binding site within the ptxR-ptxS intergenic region. In vitro transcription analysis using sigma(70)-reconstituted P. aertiginosa RNA polymerase produced one transcript that closely resembles T1, indicating that P1 is recognized by sigma(70). RNA polymerase reconstituted with either RpoS or AlgU produced no transcripts. However, a transcript was produced by RpoH-reconstituted RNA polymerase.