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

ABSTRACT 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. WalR Spn is phosphorylated by the WalK Spn (VicK) histidine kinase. Little is known about the signals sensed by WalK histidine kinases. To gain information about WalK Spn signal transduction, we performed a kinetic characterization of the WalRK Spn autophosphorylation, phosphoryltransferase, and phosphatase reactions. We were unable to purify soluble full-length WalK Spn . Consequently, these analyses were performed using two truncated versions of WalK Spn lacking its single transmembrane domain. The longer version (Δ35 amino acids) contained most of the HAMP domain and the PAS, DHp, and CA domains, whereas the shorter version (Δ195 amino acids) contained only the DHp and CA domains. The autophosphorylation kinetic parameters of Δ35 and Δ195 WalK Spn were similar [ K m (ATP) ≈ 37 μM; k cat ≈ 0.10 min −1 ] and typical of those of other histidine kinases. The catalytic efficiency of the two versions of WalK Spn ∼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 WalK Spn for WalR Spn ∼P. Deletion and point mutations confirmed that optimal WalK Spn phosphatase activity depended on the PAS domain as well as residues in the DHp domain. In addition, these WalK Spn DHp domain and ΔPAS mutations led to attenuation of virulence in a murine pneumonia model.