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 -ketoatyl-acyl carrier protein (ACP) synthases, The Bacill us subtilis fabF (yiaY) 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 tha t transcription of the operon containing the fabF gene increased eightfold in response to a cerulenin challenge, This response was selective for the i nhibition of fatty acid synthesis, since triclosan, an inhibitor of enoyl-A CP 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, sub tlis fabF locus, and sequence analysis of the mutant fabF allele showed tha t a single base change resulted in the synthesis of FabF(b)[I108F]. The pur ified FabF(b) and FabF(b)[I108F] proteins had similar specific activities w ith myristoyl-ACP as the substrate. FabF(b) exhibited a 50% inhibitory conc entration (IC50) of cerulenin of 0.1 muM, whereas the IC50 for FabF(b)[I180 ] was 50-fold higher (5 muM). These biochemical data explain the absence of an overt growth defect coupled with the cerulenin resistance phenotype of the mutant strain.