Present address: Department of Plant Pathology, University of Kaiserslautern, Kaiserslautern, Germany.
Department of Microbiology, University of Kaiserslautern, D-67663 Kaiserslautern, Germany.
Microbiology (Reading). 2020 Apr;166(4):411-421. doi: 10.1099/mic.0.000894.
The two-component regulatory system CiaRH of affects a large variety of physiological processes including ß-lactam resistance, competence development, maintenance of cell integrity, bacteriocin production, but also host colonization and virulence. The response regulator CiaR is active under a wide variety of conditions and the cognate CiaH kinase is not always needed to maintain CiaR activity. Using tetracycline-controlled expression of and variants, acetyl phosphate was identified as the alternative source of CiaR phosphorylation in the absence of CiaH. Concomitant inactivation of and the acetate kinase gene led to very high levels of CiaR-mediated promoter activation. Strong transcriptional activation was accompanied by a high phosphorylation status of CiaR as determined by Phos-tag gel electrophoresis of cell extracts. Furthermore, AckA acted negatively upon acetyl phosphate-dependent phosphorylation of CiaR. Experiments using the two-hybrid system based on adenylate cyclase reconstitution indicated binding of AckA to CiaR and therefore direct regulation. Subsequent CiaR phosphorylation experiments confirmed observations. Purified AckA was able to inhibit acetyl phosphate-dependent phosphorylation. Inhibition required the presence of ADP. AckA-mediated regulation of CiaR phosphorylation is the first example for a regulatory connection of acetate kinase to a response regulator besides controlling acetyl phosphate levels. It will be interesting to see if this novel regulation applies to other response regulators in or even in other organisms.
双组分调节系统 CiaRH 影响多种生理过程，包括β-内酰胺抗性、感受态发育、细胞完整性维持、细菌素产生，但也包括宿主定植和毒力。响应调节蛋白 CiaR 在多种条件下都是活跃的，而同源的 CiaH 激酶并不总是需要维持 CiaR 的活性。通过四环素控制表达 和变体，鉴定出乙酰磷酸是 CiaH 缺失时 CiaR 磷酸化的替代来源。同时失活 和乙酸激酶基因 导致 CiaR 介导的启动子激活水平非常高。强烈的转录激活伴随着 CiaR 的高磷酸化状态，这可以通过细胞提取物的 Phos-tag 凝胶电泳来确定。此外，AckA 对乙酰磷酸依赖的 CiaR 磷酸化有负作用。使用基于腺苷酸环化酶重组的双杂交系统的实验表明 AckA 与 CiaR 结合，因此直接调节。随后的 CiaR 磷酸化实验证实了 观察结果。纯化的 AckA 能够抑制乙酰磷酸依赖性磷酸化。抑制需要 ADP 的存在。AckA 介导的 CiaR 磷酸化调节是除了控制乙酰磷酸水平之外，乙酸激酶与响应调节蛋白之间的第一个调控连接的例子。有趣的是，看看这种新的调节是否适用于 或甚至其他生物体中的其他响应调节蛋白。
