| Two component systems (TCS) are widely used by bacteria to sense and respond to environmental changes. A typical TCS consists of a sensor histidine kinase (HK) located in the cell membrane and a cytoplasm response regulator (RR). After stimulated, HK could autophosphorylate itself at a highly conserved Histidine residue, and then transfer the phosphoryl group to the RR. Phosphorylated RR becomes active and affects gene expression by interacting with DNA promoter region or other regulator proteins. In Streptococcus pneumoniae, 13 pairs of TCSs and an orphan RR have been genetically identified. WalRK is the only essential TCS in Streptococcus pneumoniae as it positively regulates the essential pcsB gene. Previous study has shown that DeltaWalK strain can survive in aerobic culture conditions, and the mechanism is still under studying. Another kinetics study has also shown that WalK is a bi-functional enzyme that processes both kinase and phosphatase activity on WalR and the phosphatase activity greatly depends on the presence of the PAS domain. The mechanism of the phosphates activity and how important the phosphatase activity is remains unclear. In this study we measured the crosstalk from other non-cognate histidine kinases to WalR. We found that PnpS and HK08 could phosphorylate WalR, but kinetically they are not significant. We also identified a Kinase+Phosphatase- WalK mutant, WalKT222A, which lost the phosphatase activity but remains the kinase activity. This mutant suggests the importance of the highly conserved Thr residue in phosphatase activity. In vivo study shows that in WalKT222A mutant strain, WalR~P can be accumulated, and the transcription level of WalRK regulon was increased. These data suggest a working model that the phosphatase activity of WalK is the ON/OFF switch for WalRK TCS pathway. |
