ABSTRACT:
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, including
Bacillus subtilis
,
Staphylococcus aureus
,
Enterococcus faecalis
, and
Streptococcus mutans
. In this study, we found the first antibiotic that functions as a WalK inhibitor (signermycin B) by screening 10,000
Streptomyces
extracts. The chemical structure (C
23
H
35
NO
4
; 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-resistant
Staphylococcus aureus
and vancomycin-resistant
Enterococcus 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 of
Bacillus 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. In
Bacillus subtilis
and
Staphylococcus 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.
