Isolation and Characterization of Signermycin B, an Antibiotic That Targets the Dimerization Domain of Histidine Kinase WalK

The WalK (histidine kinase)/WalR (response regulator) two-component signal transduction system is a master regulatory system for cell wall metabolism and growth. This system is conserved in low G+C Gram-positive bacteria, includingBacillus subtilis ,Staphylococcus aureus ,Enterococcus faecalis , andStreptococcus mutans . In this study, we found the first antibiotic that functions as a WalK inhibitor (signermycin B) by screening 10,000Streptomyces extracts. The chemical structure (C23 H35 NO4 ; molecular weight, 389.5) comprises a tetramic acid moiety and a decalin ring. Signermycin B exhibited antimicrobial activity, with MIC values ranging from 3.13 μg/ml (8 μM) to 6.25 μg/ml (16 μM) against Gram-positive bacteria that possess the WalK/WalR two-component signal transduction system, including the drug-resistant bacteria methicillin-resistantStaphylococcus aureus and vancomycin-resistantEnterococcus faecalis . The half-maximal inhibitory concentrations of signermycin B against WalK in these organisms ranged from 37 to 61 μM. To determine the mechanism of action of signermycin B, surface plasmon resonance response analysis with the two WalK domains ofBacillus subtilis and competition assay with ATP were performed. The results showed that signermycin B binds to the dimerization domain but not the ATP-binding domain of WalK. In the presence of the cross-linker glutaraldehyde, signermycin B did not cause protein aggregation but interfered with the cross-linking of WalK dimers. These results suggest that signermycin B targets the conserved dimerization domain of WalK to inhibit autophosphorylation. InBacillus subtilis andStaphylococcus aureus , signermycin B preferentially controlled the WalR regulon, thereby inhibiting cell division. These phenotypes are consistent with those of cells starved for the WalK/WalR system.