Department of Microbiology, University of Colorado School of Medicine, Aurora, Colorado, USA.
J Bacteriol. 2013 Dec;195(24):5499-515. doi: 10.1128/JB.00726-13. Epub 2013 Oct 4.
AlgR is a key Pseudomonas aeruginosa transcriptional response regulator required for virulence. AlgR activates alginate production and twitching motility but represses the Rhl quorum-sensing (QS) system, including rhamnolipid production. The role of AlgR phosphorylation is enigmatic, since phosphorylated AlgR (AlgR-P) is required for twitching motility through the fimU promoter but is not required for the activation of alginate production. In order to examine the role of AlgR phosphorylation in vivo, a PAO1 algRD54E strain (with algR encoding a D-to-E change at position 54), which constitutively activates fimU transcription and exhibits twitching motility, was created. A corresponding PAO1 algRD54N strain (with algR encoding a D-to-N change at position 54) that does not activate fimU or twitching motility was compared to PAO1, PAO1 algRD54E, PAO1 ΔalgZ (deletion of the algZ [fimS] gene, encoding a putative histidine kinase), and PAO1 ΔalgR for swarming motility, rhamnolipid production, and rhlA transcription. PAO1 and PAO1 algRD54E produced approximately 2-fold-higher levels of rhamnolipids than PAO1 algRD54N and PAO1 ΔalgZ, thereby indicating that phosphorylated AlgR is required for normal rhamnolipid production. Examination of purified AlgR, AlgR-P, AlgR D54N, and AlgR D54E showed that AlgR-P and AlgR D54E bound preferentially to the fimU and rhlA promoters. Additionally, AlgR-P bound specifically to two sites within the rhlA promoter that were not bound by unphosphorylated AlgR. Taken together, these results indicate that phosphorylated AlgR-P has increased affinity for the rhlA promoter and is required for the coordinate activation of twitching motility, rhamnolipid production, and swarming motility in P. aeruginosa.
AlgR 是铜绿假单胞菌毒力所必需的关键转录反应调节剂。AlgR 激活藻酸盐产生和蠕动运动，但抑制 Rhl 群体感应（QS）系统，包括鼠李糖脂产生。AlgR 磷酸化的作用是神秘的，因为磷酸化的 AlgR（AlgR-P）通过 fimU 启动子是蠕动运动所必需的，但不是藻酸盐产生的激活所必需的。为了研究 AlgR 磷酸化在体内的作用，创建了一个 PAO1 algRD54E 菌株（algR 编码第 54 位的 D 到 E 变化），该菌株持续激活 fimU 转录并表现出蠕动运动。比较了相应的 PAO1 algRD54N 菌株（algR 编码第 54 位的 D 到 N 变化）与 PAO1、PAO1 algRD54E、PAO1 ΔalgZ（algZ[fimS]基因缺失，编码假定的组氨酸激酶）和 PAO1 ΔalgR 用于群体运动、鼠李糖脂产生和 rhlA 转录。PAO1 和 PAO1 algRD54E 产生的鼠李糖脂水平比 PAO1 algRD54N 和 PAO1 ΔalgZ 高约 2 倍，这表明磷酸化的 AlgR 是正常鼠李糖脂产生所必需的。对纯化的 AlgR、AlgR-P、AlgR D54N 和 AlgR D54E 的检查表明，AlgR-P 和 AlgR D54E 优先结合到 fimU 和 rhlA 启动子上。此外，AlgR-P 特异性结合到 rhlA 启动子内的两个未被非磷酸化的 AlgR 结合的位点。综上所述，这些结果表明，磷酸化的 AlgR-P 对 rhlA 启动子具有更高的亲和力，并且是铜绿假单胞菌协调激活蠕动运动、鼠李糖脂产生和群体运动所必需的。
