Department of Environmental Protection, CSIC-EEZ, Granada, Spain.
PLoS One. 2012;7(7):e39390. doi: 10.1371/journal.pone.0039390. Epub 2012 Jul 23.
Homologs of the transcriptional regulator PtxS are omnipresent in Pseudomonas, whereas PtxR homologues are exclusively found in human pathogenic Pseudomonas species. In all Pseudomonas sp., PtxS with 2-ketogluconate is the regulator of the gluconate degradation pathway and controls expression from its own promoter and also from the P(gad) and P(kgu) for the catabolic operons. There is evidence that PtxS and PtxR play a central role in the regulation of exotoxin A expression, a relevant primary virulence factor of Pseudomonas aeruginosa. We show using DNaseI-footprint analysis that in P. aeruginosa PtxR binds to the -35 region of the P(toxA) promoter in front of the exotoxin A gene, whereas PtxS does not bind to this promoter. Bioinformatic and DNaseI-footprint analysis identified a PtxR binding site in the P(kgu) and P(gad) promoters that overlaps the -35 region, while the PtxS operator site is located 50 bp downstream from the PtxR site. In vitro, PtxS recognises PtxR with nanomolar affinity, but this interaction does not occur in the presence of 2-ketogluconate, the specific effector of PtxS. DNAaseI footprint assays of P(kgu) and P(gad) promoters with PtxS and PtxR showed a strong region of hyper-reactivity between both regulator binding sites, indicative of DNA distortion when both proteins are bound; however in the presence of 2-ketogluconate no protection was observed. We conclude that PtxS modulates PtxR activity in response to 2-ketogluconate by complex formation in solution in the case of the P(toxA) promoter, or via the formation of a DNA loop as in the regulation of gluconate catabolic genes. Data suggest two different mechanisms of control exerted by the same regulator.
转录调节因子 PtxS 的同源物普遍存在于假单胞菌中，而 PtxR 同源物仅存在于人类致病性假单胞菌中。在所有假单胞菌中，带有 2-酮葡萄糖酸的 PtxS 是葡萄糖酸盐降解途径的调节剂，它控制自身启动子以及分解代谢操纵子的 P(gad)和 P(kgu)的表达。有证据表明，PtxS 和 PtxR 在调节外毒素 A 表达中起着核心作用，外毒素 A 是铜绿假单胞菌的一个重要的原始毒力因子。我们通过 DNA 酶 I 足迹分析表明，在铜绿假单胞菌中，PtxR 结合在位于外毒素 A 基因前的 P(toxA)启动子的-35 区域，而 PtxS 不结合该启动子。生物信息学和 DNA 酶 I 足迹分析在 P(kgu)和 P(gad)启动子中鉴定出一个 PtxR 结合位点，该位点与-35 区域重叠，而 PtxS 操纵子位点位于 PtxR 位点下游 50bp 处。在体外，PtxS 以纳摩尔亲和力识别 PtxR，但这种相互作用不会在 2-酮葡萄糖酸存在下发生，2-酮葡萄糖酸是 PtxS 的特异性效应物。带有 PtxS 和 PtxR 的 P(kgu)和 P(gad)启动子的 DNA 酶 I 足迹分析显示，在两个调节剂结合位点之间存在一个强烈的超反应区域，表明当两个蛋白结合时 DNA 发生扭曲；然而，在 2-酮葡萄糖酸存在下，没有观察到保护。我们得出结论，PtxS 通过在 P(toxA)启动子的情况下形成溶液中的复合物，或者通过形成 DNA 环，来响应 2-酮葡萄糖酸调节 PtxR 活性，而不是通过形成 DNA 环来调节葡萄糖酸盐分解代谢基因。数据表明，同一个调节剂发挥了两种不同的控制机制。
