Department of Biology, Indiana University Bloomington, 1001 East Third Street, Bloomington, IN, 47405, USA.
Mol Microbiol. 2012 Nov;86(3):645-60. doi: 10.1111/mmi.12006. Epub 2012 Sep 27.
WalRK (YycFG) two-component systems (TCSs) of low-GC Gram-positive bacteria play critical roles in regulating peptidogylcan hydrolase genes involved in cell division and wall stress responses. The WalRK (VicRK) TCSs of Streptococcus pneumoniae (pneumococcus) and other Streptococcus species show numerous differences with those of other low-GC species. Notably, the pneumococcal WalK sensor kinase is not essential for normal growth in culture, unlike its homologues in Bacillus and Staphylococcus species. The WalK sensor kinase possesses histidine autokinase activity and mediates dephosphorylation of phosphorylated WalR∼P response regulator. To understand the contributions of these two WalK activities to pneumococcal growth, we constructed and characterized a set of walK kinase and phosphatase mutants in biochemical reactions and in cells. We identified an amino acid substitution in WalK that significantly reduces phosphatase activity, but not other activities. Comparisons were made between WalRK regulon expression levels and WalR∼P amounts in cells determined by Phos-tag SDS-PAGE. Reduction of WalK phosphatase activity resulted in nearly 90% phosphorylation to WalR∼P, consistent with the conclusion that WalK phosphatase is strongly active in exponentially growing cells. WalK phosphatase activity was also shown to depend on the WalK PAS domain and to limit cross-talk and the recovery of WalR∼P from walK(+) cells.
低 GC 革兰氏阳性菌的 WalRK（YycFG）双组分系统（TCS）在调节与细胞分裂和细胞壁应激反应相关的肽聚糖水解酶基因方面发挥着关键作用。肺炎链球菌（肺炎球菌）和其他链球菌种的 WalRK（VicRK）TCS 与其他低 GC 种有许多不同。值得注意的是，肺炎球菌 WalK 传感器激酶对于正常生长在培养物中不是必需的，不像其在芽孢杆菌和葡萄球菌种中的同源物。WalK 传感器激酶具有组氨酸自体激酶活性，并介导磷酸化 WalR∼P 反应调节子的去磷酸化。为了了解这两种 WalK 活性对肺炎球菌生长的贡献，我们在生化反应和细胞中构建并表征了一组 walK 激酶和磷酸酶突变体。我们鉴定了 WalK 中的一个氨基酸取代，该取代显著降低了磷酸酶活性，但不影响其他活性。通过 Phos-tag SDS-PAGE 测定细胞中 WalRK 调控子表达水平和 WalR∼P 含量进行了比较。WalK 磷酸酶活性的降低导致 WalR∼P 的磷酸化率接近 90%，这与 WalK 磷酸酶在指数生长期细胞中具有强烈活性的结论一致。还表明 WalK 磷酸酶活性依赖于 WalK PAS 结构域，并限制了交叉对话和 WalR∼P 从 walK(+) 细胞中的恢复。
