Abstract
WalKR (YycFG) is the only essential two-component regulator in the human pathogen Staphylococcus aureus. WalKR regulates peptidoglycan synthesis, but this function alone does not explain its essentiality. Here, to further understand WalKR function, we investigate a suppressor mutant that arose when WalKR activity was impaired; a histidine to tyrosine substitution (H271Y) in the cytoplasmic Per-Arnt-Sim (PASCYT) domain of the histidine kinase WalK. Introducing the WalKH271Y mutation into wild-type S. aureus activates the WalKR regulon. Structural analyses of the WalK PASCYT domain reveal a metal-binding site, in which a zinc ion (Zn2+) is tetrahedrally-coordinated by four amino acids including H271. The WalKH271Y mutation abrogates metal binding, increasing WalK kinase activity and WalR phosphorylation. Thus, Zn2+-binding negatively regulates WalKR. Promoter-reporter experiments using S. aureus confirm Zn2+ sensing by this system. Identification of a metal ligand recognized by the WalKR system broadens our understanding of this critical S. aureus regulon.
Highlights
WalKR (YycFG) is the only essential two-component regulator in the human pathogen Staphylococcus aureus
The mutation occurred in the cytoplasmic Per-Arnt Sim (PAS) domain of WalK, wherein histidine 271 was replaced by tyrosine (WalKH271Y)
In the absence of Zn2+, the dihedral angle between the PASCYTO and CAT domains in each monomer was ~136° when viewed down the central axis of the kinase, while the average distance between the upper and lower helices of the cytoplasmic domains was 21.6 Å (Fig. 5a)
Summary
WalKR (YycFG) is the only essential two-component regulator in the human pathogen Staphylococcus aureus. WalKR regulates peptidoglycan synthesis, but this function alone does not explain its essentiality. To further understand WalKR function, we investigate a suppressor mutant that arose when WalKR activity was impaired; a histidine to tyrosine substitution (H271Y) in the cytoplasmic Per-Arnt-Sim (PASCYT) domain of the histidine kinase WalK. The WalKH271Y mutation abrogates metal binding, increasing WalK kinase activity and WalR phosphorylation. Staphylococcus aureus is a major human pathogen that causes a wide range of hospital- and community-acquired opportunistic infections[1]. The WalR/WalK system ( called YycFG and VicRK) was first identified from temperature-sensitive mutants in Bacillus subtilis[8] and S. aureus[9], with the system essential in both genera. In an impressive recent study, Villanueva et al.[14] deleted all 15 TCSs from two different S. aureus backgrounds and confirmed that
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