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Involvement of WalK (VicK) phosphatase activity in setting WalR (VicR) response regulator phosphorylation level and limiting cross-talk in Streptococcus pneumoniae D39 cells.

ABSTRACT: WalRK (YycFG) two-component systems (TCSs) of low-GC Gram-positive bacteria play critical roles in regulating peptidogylcan hydrolase genes involved in cell division and wall stress responses. The WalRK (VicRK) TCSs of Streptococcus pneumoniae (pneumococcus) and other Streptococcus species show numerous differences with those of other low-GC species. Notably, the pneumococcal WalK sensor kinase is not essential for normal growth in culture, unlike its homologues in Bacillus and Staphylococcus species. The WalK sensor kinase possesses histidine autokinase activity and mediates dephosphorylation of phosphorylated WalR?P response regulator. To understand the contributions of these two WalK activities to pneumococcal growth, we constructed and characterized a set of walK kinase and phosphatase mutants in biochemical reactions and in cells. We identified an amino acid substitution in WalK that significantly reduces phosphatase activity, but not other activities. Comparisons were made between WalRK regulon expression levels and WalR?P amounts in cells determined by Phos-tag SDS-PAGE. Reduction of WalK phosphatase activity resulted in nearly 90% phosphorylation to WalR?P, consistent with the conclusion that WalK phosphatase is strongly active in exponentially growing cells. WalK phosphatase activity was also shown to depend on the WalK PAS domain and to limit cross-talk and the recovery of WalR?P from walK(+) cells.

SUBMITTER: Wayne KJ

PROVIDER: S-EPMC3638944 | biostudies-literature | 2012 Nov

REPOSITORIES: biostudies-literature

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Involvement of WalK (VicK) phosphatase activity in setting WalR (VicR) response regulator phosphorylation level and limiting cross-talk in Streptococcus pneumoniae D39 cells.

Wayne Kyle J KJ Li Shuo S Kazmierczak Krystyna M KM Tsui Ho-Ching T HC Winkler Malcolm E ME

Molecular microbiology 20120927 3

WalRK (YycFG) two-component systems (TCSs) of low-GC Gram-positive bacteria play critical roles in regulating peptidogylcan hydrolase genes involved in cell division and wall stress responses. The WalRK (VicRK) TCSs of Streptococcus pneumoniae (pneumococcus) and other Streptococcus species show numerous differences with those of other low-GC species. Notably, the pneumococcal WalK sensor kinase is not essential for normal growth in culture, unlike its homologues in Bacillus and Staphylococcus sp ...[more]

PMID: 23013245

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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.

Project description:UNLABELLED:The FtsEX protein complex has recently been proposed to play a major role in coordinating peptidoglycan (PG) remodeling by hydrolases with the division of bacterial cells. According to this model, cytoplasmic FtsE ATPase interacts with the FtsZ divisome and FtsX integral membrane protein and powers allosteric activation of an extracellular hydrolase interacting with FtsX. In the major human respiratory pathogen Streptococcus pneumoniae (pneumococcus), a large extracellular-loop domain of FtsX (ECL1FtsX) is thought to interact with the coiled-coil domain of the PcsB protein, which likely functions as a PG amidase or endopeptidase required for normal cell division. This paper provides evidence for two key tenets of this model. First, we show that FtsE protein is essential, that depletion of FtsE phenocopies cell defects caused by depletion of FtsX or PcsB, and that changes of conserved amino acids in the FtsE ATPase active site are not tolerated. Second, we show that temperature-sensitive (Ts) pcsB mutations resulting in amino acid changes in the PcsB coiled-coil domain (CCPcsB) are suppressed by ftsX mutations resulting in amino acid changes in the distal part of ECL1FtsX or in a second, small extracellular-loop domain (ECL2FtsX). Some FtsX suppressors are allele specific for changes in CCPcsB, and no FtsX suppressors were found for amino acid changes in the catalytic PcsB CHAP domain (CHAPPcsB). These results strongly support roles for both ECL1FtsX and ECL2FtsX in signal transduction to the coiled-coil domain of PcsB. Finally, we found that pcsBCC(Ts) mutants (Ts mutants carrying mutations in the region of pcsB corresponding to the coiled-coil domain) unexpectedly exhibit delayed stationary-phase autolysis at a permissive growth temperature. IMPORTANCE:Little is known about how FtsX interacts with cognate PG hydrolases in any bacterium, besides that ECL1FtsX domains somehow interact with coiled-coil domains. This work used powerful genetic approaches to implicate a specific region of pneumococcal ECL1FtsX and the small ECL2FtsX in the interaction with CCPcsB. These findings identify amino acids important for in vivo signal transduction between FtsX and PcsB for the first time. This paper also supports the central hypothesis that signal transduction between pneumococcal FtsX and PcsB is linked to ATP hydrolysis by essential FtsE, which couples PG hydrolysis to cell division. The classical genetic approaches used here can be applied to dissect interactions of other integral membrane proteins involved in PG biosynthesis. Finally, delayed autolysis of the pcsBCC(Ts) mutants suggests that the FtsEX-PcsB PG hydrolase may generate a signal in the PG necessary for activation of the major LytA autolysin as pneumococcal cells enter stationary phase.

Dataset Information

Involvement of WalK (VicK) phosphatase activity in setting WalR (VicR) response regulator phosphorylation level and limiting cross-talk in Streptococcus pneumoniae D39 cells.

Publications

Involvement of WalK (VicK) phosphatase activity in setting WalR (VicR) response regulator phosphorylation level and limiting cross-talk in Streptococcus pneumoniae D39 cells.

Similar Datasets

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