Characterization of H-box region mutants of WalK inert to the action of waldiomycin in <i>Bacillus subtilis</i>

<p>The WalK/WalR two-component system is essential for cell wall metabolism and thus for cell growth in <i>Bacillus subtilis</i>. Waldiomycin was previously isolated as an antibiotic that targeted WalK, the cognate histidine kinase (HK) of the response regulator, WalR, in <i>B. subtilis</i>. To gain further insights into the action of waldiomycin on WalK and narrow down its site of action, mutations were introduced in the H-box region, a well-conserved motif of the bacterial HKs of WalK. The half-maximal inhibitory concentrations (IC₅₀s) of waldiomycin against purified WalK protein with triple substitutions in the H-box region, R377M/R378M/S385A and R377M/R378M/R389M, were 26.4 and 55.1 times higher than that of the wild-type protein, respectively, indicating that these residues of WalK are crucial for the inhibitory effect of waldiomycin on its kinase activity. Surprisingly, this antibiotic severely affected cell growth in a minimum inhibitory concentration (MIC) assay, but not transcription of WalR-regulated genes or cell morphology in <i>B. subtilis</i> strains that harbored the H-box triple substitutions on the bacterial chromosome. We hypothesized that waldiomycin targets other HKs as well, which may, in turn, sensitize <i>B. subtilis</i> cells with the H-box triple mutant alleles of the <i>walK</i> gene to waldiomycin. Waldiomycin inhibited other HKs such as PhoR and ResE, and, to a lesser extent, CitS, whose H-box region is less conserved. These results suggest that waldiomycin perturbs multiple cellular processes in <i>B. subtilis</i> by targeting the H-box region of WalK and other HKs.</p>