title: 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.
authors.affiliation: Department of Biology, Indiana University Bloomington, 1001 East Third Street, Bloomington, IN, 47405, USA.
authors.forename: Kyle J
authors.initials: KJ
authors.lastname: Wayne
authors.forename: Shuo
authors.initials: S
authors.lastname: Li
authors.forename: Krystyna M
authors.initials: KM
authors.lastname: Kazmierczak
authors.forename: Ho-Ching T
authors.initials: HC
authors.lastname: Tsui
authors.forename: Malcolm E
authors.initials: ME
authors.lastname: Winkler
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial; D010744:Phosphoric Monoester Hydrolases / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D018085:Regulon; D015398:Signal Transduction; D013296:Streptococcus pneumoniae / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; C119939:YycF protein, Bacteria; D010744:Phosphoric Monoester Hydrolases