Kinetic Characterization of the WalRKSpn(VicRK) Two-Component System ofStreptococcus pneumoniae: Dependence of WalKSpn(VicK) Phosphatase Activity on Its PAS Domain
- 1 May 2010
- journal article
- Published by American Society for Microbiology in Journal of Bacteriology
- Vol. 192 (9), 2346-2358
- https://doi.org/10.1128/jb.01690-09
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 (Delta35 amino acids) contained most of the HAMP domain and the PAS, DHp, and CA domains, whereas the shorter version (Delta195 amino acids) contained only the DHp and CA domains. The autophosphorylation kinetic parameters of Delta35 and Delta195 WalK(Spn) were similar [K(m)(ATP) approximately 37 microM; k(cat) approximately 0.10 min(-1)] and typical of those of other histidine kinases. The catalytic efficiency of the two versions of WalK(Spn) approximately 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) approximately 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 DeltaPAS mutations led to attenuation of virulence in a murine pneumonia model.Keywords
This publication has 103 references indexed in Scilit:
- MzrA: a novel modulator of the EnvZ/OmpR two‐component regulonMolecular Microbiology, 2009
- Kinetic Buffering of Cross Talk between Bacterial Two-Component SensorsJournal of Molecular Biology, 2009
- Roles of relSpn in stringent response, global regulation and virulence of serotype 2 Streptococcus pneumoniae D39Molecular Microbiology, 2009
- Cross‐talk suppression between the CpxA–CpxR and EnvZ–OmpR two‐component systems in E. coliMolecular Microbiology, 2008
- A sensor histidine kinase co‐ordinates cell wall architecture with cell division in Bacillus subtilisMolecular Microbiology, 2008
- Two variable active site residues modulate response regulator phosphoryl group stabilityMolecular Microbiology, 2008
- Rewiring the Specificity of Two-Component Signal Transduction SystemsCell, 2008
- An essential sensor histidine kinase controlled by transmembrane helix interactions with its auxiliary proteinsProceedings of the National Academy of Sciences of the United States of America, 2008
- Discovery of a novel class of highly conserved vaccine antigens using genomic scale antigenic fingerprinting of pneumococcus with human antibodiesThe Journal of Experimental Medicine, 2007
- Using circular dichroism spectra to estimate protein secondary structureNature Protocols, 2006