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

摘要

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 WalRSpn (VicR) response regulator positively controls the transcription of genes encoding the essential PcsB division protein and surface virulence factors. WalRSpn is phosphorylated by the WalKSpn (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 WalRKSpn 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 (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 WalKSpn were similar [Km(ATP) ≈ 37 μM; kcat ≈ 0.10 min−1] and typical of those of other histidine kinases. The catalytic efficiency of the two versions of WalKSpn∼P were also similar in the phosphoryltransfer reaction to full-length WalRSpn. In contrast, absence of the HAMP-PAS domains significantly diminished the phosphatase activity of WalKSpn for WalRSpn∼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 ΔPAS mutations led to attenuation of virulence in a murine pneumonia model.