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Resistance to Cerulenin Unbalance Fatty Acid Biosynthesis in Bacillus subtilis Gustavo E. Schujman, Silvia Altabe and Diego de Mendoza Instituto de Biología Molecular y Celular de Rosario (IBR-CONICET), Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Suipacha 531, 2000-Rosario, Argentina. E-mail: schujman@ibr.gov.ar Drug resistance in infectious organisms has become a serious medical problem, and fatty acid synthesis has emerged as a promising target for the development of novel therapeutic agents. Lipid synthesis is not only essential to cell viability, but specificity for bacteria and other infectious organisms can be achieved by taking advantage of the structural and regulatory differences that exist in the fatty acid synthetic systems of diverse organisms. In the biosynthesis of fatty acids, the b-ketoacyl-acyl carrier protein (ACP) synthases, or condensing enzymes, catalyze chain elongation by the addition of two-carbon units derived from malonyl-ACP to an acyl group bound to either ACP or CoA. These enzymes (found in bacteria and plants) are the targets for the fungal mycotoxin cerulenin. This antibiotic potently inhibits fatty acid synthesis by covalently modification of the active site thiol of the chain elongation subtype b-ketoacyl-ACP synthases. The expression of the condensing enzymes, as of most of the genes involved in fatty acid biosynthesis, is regulated by the transcriptional repressor FapR in many Gram-positive bacteria. The activity of FapR responds, in turn, to the intracellular levels of malonyl-CoA, a precursor in fatty acid biosynthesis. The fabF gene of Bacillus subtilis codifies for the only condensing enzyme able to elongate medium and long chain acyl-ACPs and its product is the target of cerulenin. We have previously characterized a spontaneous mutant of B. subtilis, named GS77, resistant to cerulenin, and the mutation mapped to the fabF gene. A point mutation resulted in the expression of FabF[I108F], which presented a 50-fold higher IC50 (50% inhibitory concentration) than the wild type enzyme. In this work we present studies showing that the single base change in fabF gene of strain GS77, that confers resistance to cerulenin, has a profound impact on the expression of the fatty acid synthase, on the lipid metabolism and on the membrane lipid composition of the bacterium. Microarray analysis revealed that most of the genes involved in fatty acid biosynthesis and in the transfer of fatty acids into phospholipids are over-expressed in the mutant strain, what mimics the fapR- phenotype. Expression of wild type fapR in trans did not restore transcription of the fap regulon to its normal level, but it did so the expression of wild type fabF. We determined in vitro that FabFI108F does not efficiently synthesize long-chain acyl-ACPs and accumulates short-chain intermediates. This result is consistent with shortening of the chain length of membrane fatty acids. We established that the over-expression of the fap regulon is due to the accumulation of malonyl-CoA, what releases the FapR repressor from its operators. This result indicates the existence of a feedback regulation loop exerted by the acyl-ACPs intermediates on the condensing enzymes. We also determined that high levels of fabFI108F expression are necessary for cell viability. Altogether, our results suggest that the selection of altered versions of FabF in response to antibiotics targeting this enzyme, might cause cross-resistance to other antibiotics affecting the fatty acid biosynthetic pathway, as most of the enzymes involved are overexpressed. This observation could be extended to many pathogens that regulate the expression of their fatty acid synthethase via FapR.