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Kinetic characterization of the WalRKSpn (VicRK) two-component system of Streptococcus 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 (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.

SUBMITTER: Gutu AD

PROVIDER: S-EPMC2863487 | biostudies-literature | 2010 May

REPOSITORIES: biostudies-literature

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Kinetic characterization of the WalRKSpn (VicRK) two-component system of Streptococcus pneumoniae: dependence of WalKSpn (VicK) phosphatase activity on its PAS domain.

Gutu Alina D AD Wayne Kyle J KJ Sham Lok-To LT Winkler Malcolm E ME

Journal of bacteriology 20100226 9

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 ...[more]

PMID: 20190050

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Project description:The VicRK two-component system of S. pneumoniae (Pneumococcus) is a member of the essential WalRK (YycFG) family found in low G+C gram positive bacteria. Previously, we showed that phosphorylated VicR response regulator (RR) is essential, because of its strong positive regulation of the pcsB gene, which mediates cell division. Little is known about the signals sensed by the VicK or WalK histidine kinases (HK), and it is unknown why pneumococcal VicK is not essential under aerobic growth conditions, whereas other the WalK HKs in other species are essential. VicK contains the only PAS domain in the Pneumococcal genome. PAS domains in other HKs have been shown to sense oxygen or other metabolic signals, such as NAD+, and it has been speculated that VicK may be involved in sensing oxygen in S. pneumoniae. Contrary to previous reports, we found that delta vicK mutants do not grow anaerobically, implying a conditional requirement for the VicK HK. Point mutations and domain deletions in vicK indicated that the autokinase activity of VicK is required for anaerobic growth, whereas the PAS domain and VicK phosphatase activity are not required. These combined results are consistent with the idea that maintenance of VicR~P level is required for anaerobic growth. The inability of delta vicK mutants to grow anaerobically was suppressed by spontaneous high- and low-growth yield mutants. Spontaneous suppressor mutants were still VicR dependent and did not result in bypass of the VicR signaling pathway. The genome of a high yield suppressor was sequenced using the Illumina method, which identified three mutations in the suppressor, two of which are predicted to play roles in phosphate transport (pnpR RR and phosphate ABC transporter). Manual sequencing of regions in additional independently isolated ΔvicK suppressor mutants identified similar mutations, and deletion of the pnpR RR gene in the ΔvicK background fully suppressed the anaerobic growth requirement for the VicK HK. Ongoing experiments will determine whether this suppression requires the PnpS HK or is mediated through changes in signaling. Finally, Western blotting showed that the level of VicK during anaerobic growth is about 3-fold less than that of aerobic cultures. This result suggests that the level of VicRKX operon expression is lower anaerobically, which may prevent crosstalk that occurs during aerobic culture to phosphorylate VicR.

Project description:The VicRK two-component system of S. pneumoniae (Pneumococcus) is a member of the essential WalRK (YycFG) family found in low G+C gram positive bacteria. Previously, we showed that phosphorylated VicR response regulator (RR) is essential, because of its strong positive regulation of the pcsB gene, which mediates cell division. Little is known about the signals sensed by the VicK or WalK histidine kinases (HK), and it is unknown why pneumococcal VicK is not essential under aerobic growth conditions, whereas other the WalK HKs in other species are essential. VicK contains the only PAS domain in the Pneumococcal genome. PAS domains in other HKs have been shown to sense oxygen or other metabolic signals, such as NAD+, and it has been speculated that VicK may be involved in sensing oxygen in S. pneumoniae. Contrary to previous reports, we found that delta vicK mutants do not grow anaerobically, implying a conditional requirement for the VicK HK. Point mutations and domain deletions in vicK indicated that the autokinase activity of VicK is required for anaerobic growth, whereas the PAS domain and VicK phosphatase activity are not required. These combined results are consistent with the idea that maintenance of VicR~P level is required for anaerobic growth. The inability of delta vicK mutants to grow anaerobically was suppressed by spontaneous high- and low-growth yield mutants. Spontaneous suppressor mutants were still VicR dependent and did not result in bypass of the VicR signaling pathway. The genome of a high yield suppressor was sequenced using the Illumina method, which identified three mutations in the suppressor, two of which are predicted to play roles in phosphate transport (pnpR RR and phosphate ABC transporter). Manual sequencing of regions in additional independently isolated ?vicK suppressor mutants identified similar mutations, and deletion of the pnpR RR gene in the ?vicK background fully suppressed the anaerobic growth requirement for the VicK HK. Ongoing experiments will determine whether this suppression requires the PnpS HK or is mediated through changes in signaling. Finally, Western blotting showed that the level of VicK during anaerobic growth is about 3-fold less than that of aerobic cultures. This result suggests that the level of VicRKX operon expression is lower anaerobically, which may prevent crosstalk that occurs during aerobic culture to phosphorylate VicR. Three independent hybridizations using independent RNA preparations from the strain IU1781 (reference strain) and strain IU1896 (a strain with vicK deletion) were performed. Dye swap was performed with the reference strain labeled with Cy3 in two hybridizations and Cy5 in one hybridization.

Dataset Information

Kinetic characterization of the WalRKSpn (VicRK) two-component system of Streptococcus pneumoniae: dependence of WalKSpn (VicK) phosphatase activity on its PAS domain.

Publications

Kinetic characterization of the WalRKSpn (VicRK) two-component system of Streptococcus pneumoniae: dependence of WalKSpn (VicK) phosphatase activity on its PAS domain.

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