Overproduction of beta-ketoacyl-acyl carrier protein synthase I imparts thiolactomycin resistance to Escherichia coli K-12

Abstract

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...

A Genetic Screen for the Identification of Thiamin Metabolic Genes

“…For instance, recombinant cells with increased doses of murZ, the E. coli gene encoding UDP-GlcNAc enolpyruvate transferase, displayed phosphomycin resistance (8). Clones resistant to thiolactomycin were found to contain increased levels of fabB, which encodes ␤-ketoacyl-acyl carrier protein synthase I, the cellular target of thiolactomycin (9). Increased levels of prolipoprotein signal peptidase rendered cells resistant to globomycin, a specific inhibitor of that enzyme (10).…”

An array of Escherichia coli clones over-expressing essential proteins: A new strategy of identifying cellular targets of potent antibacterial compounds

“…Turnowsky and colleagues [39] demonstrated that overexpression of EnvM (FabI) protein from the plasmid-borne gene conferred resistance to diazaborine, a specific inhibitor for FabI. Tsay and coworkers [40] screened a thiolactomycin sensitive E. coli strain against an E. coli genomic library for recombinant plasmids that conferred resistance to thiolactomycin, an inhibitor for FabB protein (β-ketoacyl-acyl carrier protein synthase I). They isolated nine plasmids, all of which contain a functional fabB gene.…”

“…They isolated nine plasmids, all of which contain a functional fabB gene. Over-expression of FabB protein appeared to lead to increased resistance to thiolactomycin in E. coli [40]. Apfel and colleagues [41] demonstrated that over-expression of peptide deformylase in a cell reduced its susceptibility to inhibitors specific for the essential target, peptide deformylase.…”

Differences in photoelectrocatalytic inactivation processes between E. coli and its isogenic single gene knockoff mutants: Destruction of membrane framework or associated proteins?

“…The fabH encodes ␤-ketoacyl-acyl carrier protein synthase III (KAS III), which catalyzes the condensation reaction during the initial step of fatty acid biosynthesis [23]. Previous studies found that the KAS III deficient E. coli strain had a higher UFA proportion compared with the wild type strain, especially the monounsaturated fatty acid with 18 carbons (18:1) [24]. The bacterial strains were stored in 25% sterilized glycerin at −80 • C. To prepare the bacterial suspension for PEC inactivation, the individual bacterial strain was streaked on a nutrient agar plate and incubated to acquire isolated colonies.…”

“…The FA profiles of three E. coli strains were first determined to confirm the target mutations; Table S2 shows the results. Before PEC treatment, the parental strain E. coli BW25113 showed a typical E. coli fatty acid profile [16,24], with palmitic acid (16:0) as the dominant congener (45.6%), followed by monounsaturated fatty acids such as hexadecenoic acid (16:1, 12.1%) and octadecenoic acid (18:1, 22.9%). Compared with the parental strain, the mutants showed no difference in aspect of the total fatty acid content, but with different composition profiles.…”