Response of Bacillus subtilis to cerulenin and acquisition of resistance

Cerulenin is a fungal mycotoxin that potently inhibits fatty acid synthesis by covalent modification of the active site thiol of the chain-elongation subtypes of beta-ketoacyl-acyl carrier protein (ACP) synthases. The Bacillus subtilis fabF (yjaY) gene (fabF(b)) encodes an enzyme that catalyzes the condensation of malonyl-ACP with acyl-ACP to extend the growing acyl chain by two carbons. There were two mechanisms by which B. subtilis adapted to exposure to this antibiotic. First, reporter gene analysis demonstrated that transcription of the operon containing the fabF gene increased eightfold in response to a cerulenin challenge. This response was selective for the inhibition of fatty acid synthesis, since triclosan, an inhibitor of enoyl-ACP reductase, triggered an increase in fabF reporter gene expression while nalidixic acid did not. Second, spontaneous mutants arose that exhibited a 10-fold increase in the MIC of cerulenin. The mutation mapped at the B. subtilis fabF locus, and sequence analysis of the mutant fabF allele showed that a single base change resulted in the synthesis of FabF(b)[I108F]. The purified FabF(b) and FabF(b)[I108F] proteins had similar specific activities with myristoyl-ACP as the substrate. FabF(b) exhibited a 50% inhibitory concentration (IC(50)) of cerulenin of 0.1 microM, whereas the IC(50) for FabF(b)[I108] was 50-fold higher (5 microM). These biochemical data explain the absence of an overt growth defect coupled with the cerulenin resistance phenotype of the mutant strain.