Kinetic Characterization of the WalRKSpn (VicRK) Two-Component System of Streptococcus pneumoniae: Dependence of WalKSpn (VicK) Phosphatase Activity on Its PAS Domain

The WalRK two-component system plays important roles in maintaining cell wall homeostasis and responding to antibiotic stress in low-GC Gram-positive bacteria. In the major human pathogen, Streptococcus pneumoniae, phosphorylated WalR(Spn) (VicR) response regulator positively controls the transcription of genes encoding the essential PcsB division protein and surface virulence factors. WalRSpn is phosphorylated by the WalK(Spn) (VicK) histidine kinase. Little is known about the signals sensed by WalK histidine kinases. To gain information about WalKSpn signal transduction, we performed a kinetic characterization of the WalRK(Spn) autophosphorylation, phosphoryltransferase, and phosphatase reactions. We were unable to purify soluble full-length WalKSpn. Consequently, these analyses were performed using two truncated versions of WalKSpn lacking its single transmembrane domain. The longer version (Delta 35 amino acids) contained most of the HAMP domain and the PAS, DHp, and CA domains, whereas the shorter version (Delta 195 amino acids) contained only the DHp and CA domains. The autophosphorylation kinetic parameters of Delta 35 and Delta 195 WalKSpn were similar [K-m(ATP) approximate to 37 mu M; k(cat) approximate to 0.10 min(-1)] and typical of those of other histidine kinases. The catalytic efficiency of the two versions of WalK(Spn) similar to P were also similar in the phosphoryltransfer reaction to full-length WalR(Spn). In contrast, absence of the HAMP-PAS domains significantly diminished the phosphatase activity of WalKSpn for WalR(Spn)similar to P. Deletion and point mutations confirmed that optimal WalKSpn phosphatase activity depended on the PAS domain as well as residues in the DHp domain. In addition, these WalKSpn DHp domain and Delta PAS mutations led to attenuation of virulence in a murine pneumonia model.