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Thiolactomycin [(4S)(2E,5E)-2,4,6-trimethyl-3-hydroxy-2,5,7-octatriene-4-thiolide] (TLM) is a unique antibiotic structure that inhibits dissociated type II fatty acid synthase systems but not the multifunctional type I fatty acid synthases found in mammals. We screened an Escherichia coli genomic library for recombinant plasmids that impart TLM resistance to a TLM-sensitive strain of E. coli K-12. Nine independent plasmids were isolated, and all possessed a functional I8-ketoacyl-acyl carrier protein synthase I gene (fabB) based on their restriction enzyme maps and complementation of the temperature-sensitive growth of a fabBIS(Ts) mutant. A plasmid (pJTB3) was constructed that contained only the fabB open reading frame. This plasmid conferred TLM resistance, complemented thefabB(Ts) mutation, and directed the overproduction of synthase I activity. TLM selectively inhibited unsaturated fatty acid synthesis in vivo; however, synthase I was not the only TLM target, since supplementation with oleate to circumvent the cellular requirement for an active synthase I did not confer TLM resistance. Overproduction of the FabB protein resulted in TLM-resistant fatty acid biosynthesis in vivo and in vitro. These data show that ,I-ketoacyl-acyl carrier protein synthase I is a major target for TLM and that increased expression of this condensing enzyme is one mechanism for acquiring TLM resistance. However, extracts from a TLM-resistant mutant (strain CDM5) contained normal levels of TLM-sensitive synthase I activity, illustrating that there are other mechanisms of TLM resistance.The 3-ketoacyl-acyl carrier protein (ACP) synthases are key regulators of dissociated (type II) fatty acid synthase systems typified by the Escherichia coli system (for reviews, see references 5 and 28). ,-Ketoacyl-ACP synthase I is required for a critical step in the elongation of unsaturated acyl-ACP, and mutants (fabB) lacking synthase I activity are unable to synthesize either palmitoleic or cis-vaccenic acid and require supplementation with unsaturated fatty acids for growth (6, 30). 13-Ketoacyl-ACP synthase II is responsible for the temperature-dependent regulation of fatty acid composition (for a review, see reference 7). Mutants (fabF) lacking synthase II activity are deficient in cis-vaccenic acid but grow normally (11,12). ,B-Ketoacyl-ACP synthase III selectively catalyzes the formation of acetoacetyl-ACP in vitro (17). The role of this third condensing enzyme remains to be established, but its position at the beginning of the biosynthetic pathway suggests that it plays a role in governing the total rate of fatty acid production.Thiolactomycin [(4S)(2E,5E)-2,4,6-trimethyl-3-hydroxy-2,5,7-octatriene-4-thiolide] (TLM) is a unique antibiotic structure that inhibits type II (bacterial and plant) but not type I (yeast and mammalian) fatty acid synthases (14,15,25,26,31). The antibiotic is not toxic to mice and affords significant protection against urinary tract and intraperitoneal bacterial infections (23). Understanding the mechanism of TLM ac...

Thiolactomycin (TLM) and cerulenin are antibiotics that block Escherichia coli growth by inhibiting fatty acid biosynthesis at the 1-ketoacyl-acyl carrier protein synthase I step. Both TLM and cerulenin trigger the accumulation of intracellular malonyl-coenzyme A coincident with growth inhibition, and the overexpression of synthase I protein confers resistance to both antibiotics. Strain CDM5 was derived as a TLM-resistant mutant but remained sensitive to cerulenin. TLM neither induced malonyl-coenzyme A accumulation nor blocked fatty acid production in vivo; however, the fatty acid synthase activity in extracts from strain CDM5 was sensitive to TLM inhibition. The TLM resistance gene in strain CDM5 was mapped to 57.5 min of the chromosome and was an allele of the emrB gene. Disruption of the emrB gene converted strain CDM5 to a TLM-sensitive strain, and the overexpression of the emrAB operon conferred TLM resistance to sensitive strains. Thus, activation of the emr efflux pump is the mechanism for TLM resistance in strain CDM5.The 3-ketoacyl-acyl carrier protein (0-ketoacyl-ACP) synthases are key regulators of dissociated (type II) fatty acid synthase systems typified by Escherichia coli (for reviews, see references 6 and 16). P-Ketoacyl-ACP synthase I is required for a critical step in the elongation of unsaturated acyl-ACP, and fabB mutants lacking synthase I activity synthesize neither palmitoleic nor cis-vaccenic acids and require supplementation with unsaturated fatty acids for growth (29). 3-Ketoacyl-ACP synthase II is responsible for the temperature-dependent regulation of fatty acid composition (for a review, see reference 9). Mutants (fabF) lacking synthase II activity are deficient in cis-vaccenic acid but do not have a growth phenotype (12, 13). O-Ketoacyl-ACP synthase III selectively catalyzes the formation of acetoacetyl-ACP in vitro (19). Synthase III possesses acetyl coenzyme A (acetyl-CoA):ACP transacylase activity (34); however, it is unknown whether synthase III accounts for all of the acetyl transacylase activity. The role of this third condensing enzyme remains to be firmly established, but its position at the beginning of the biosynthetic pathway suggests that it plays a role in governing the rate of fatty acid initiation.Thiolactomycin [(4S)(2E,5E)-2,4,6-trimethyl-3-hydroxy-2,5,7-octatriene-4-thiolide] (TLM) is a unique antibiotic structure that inhibits type II (bacterial and plant) but not type I (Saccharomyces cerevisiae and mammalian) fatty acid synthases (14,15,23,24,26,27,31). The antibiotic is not toxic to mice and affords significant protection against urinary tract and intraperitoneal bacterial infections (23). Understanding the mechanism of TLM action is important to the development of more-effective antibiotics that exhibit selective action against type II bacterial fatty acid synthases. synthase suggests that the 3-ketoacyl-ACP synthase and the acetyl-CoA:ACP transacylase activities are the only individual enzymes inhibited by TLM in vitro (24). The findings that malonyl-ACP pr...

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Plasmids that corrected the temperature-sensitive unsaturated fatty acid auxotrophy of strain M6 [fabA6 (Ts)] were isolated from an Escherichia coli genomic library. Subcloning and physical mapping localized the new gene (called sfa for suppressor of fabA) at 1,070 kb on the E. coli chromosome. DNA sequencing revealed the presence of a 227-bp open reading frame which directed the synthesis of a peptide of approximately 8 kDa, which correlated with the correction of the fabA6(Ts) phenotype. However, the sfa gene was an allele-specific suppressor since plasmids harboring the sfa gene corrected the growth phenotype of fabA6(Ts) mutants but did not correct the growth of fabA2(Ts) or fabB15(Ts) unsaturated fatty acid auxotrophs. Overexpression of the sfa gene in fabA6(Ts) mutants restored unsaturated fatty acid content at 42؇C, and overexpression in wild-type cells resulted in a substantial increase in the unsaturated fatty acid content of the membrane. Thus, the suppression of the fabA6(Ts) mutation by sfa was attributed to its ability to increase the biosynthesis of unsaturated fatty acids.Unsaturated fatty acids are absolutely required for the normal growth of Escherichia coli (9), and the investigation of the enzymes responsible for the formation of these fatty acids and their regulation remains an area of active research (21). In E. coli, there are two gene products known to be required for unsaturated fatty acid synthesis. The product of the fabA gene, ␤-hydroxydecanoyl-acyl carrier protein (ACP) dehydratase, catalyzes the dehydration of ␤-hydroxydecanoyl-ACP to a mixture of trans-2-decenoyl-ACP and cis-3-decenoyl-ACP, thus introducing the cis double bond into the growing fatty acid chain. The trans-2 isomer is the normal intermediate in saturated fatty acid synthesis and is converted to saturated fatty acids following reduction of the double bond by enoyl-ACP reductase (fabI) (14). The double bond in the cis-3 intermediate is preserved, and the 10-carbon intermediate is elongated to form the unsaturated fatty acids. The essential nature of FabA is clear from the isolation of mutants defective in this enzyme activity (26). These mutants cannot make unsaturated fatty acids, although saturated fatty acid synthesis is not affected. The nucleotide sequence of the fabA gene is known (10), and the regulation of fabA expression involves the transcriptional activator, FadR, which also functions as a repressor of fatty acid ␤-oxidation genes (15, 16). The first indication of a second enzyme required for unsaturated fatty acid biosynthesis was that the unsaturated fatty acid auxotrophs could be divided into two complementation groups (8). The fabA gene maps to minute 21.9 on the E. coli chromosome (11), whereas the second mutation, termed fabB, maps to minute 52.6 (5). The second essential enzyme is ␤-ketoacyl-ACP synthase I, the product of the fabB gene (24). This condensing enzyme carries out an essential step in unsaturated fatty acid synthesis, which is most likely the elongation of cis-3-decenoyl-ACP. The fabB gene ...