title: 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 beta-ketoacyl-acyl carrier protein synthase I gene (fabB) based on their restriction enzyme maps and complementation of the temperature-sensitive growth of a fabB15(Ts) mutant. A plasmid (pJTB3) was constructed that contained only the fabB open reading frame. This plasmid conferred TLM resistance, complemented the fabB(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 beta-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.
authors.affiliation: Department of Biochemistry, St. Jude Children's Research Hospital, Memphis, Tennessee 38101.
authors.forename: J T
authors.initials: JT
authors.lastname: Tsay
authors.forename: C O
authors.initials: CO
authors.lastname: Rock
authors.forename: S
authors.initials: S
authors.lastname: Jackowski
mesh_terms: D000217:Acyltransferases / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D002352:Carrier Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D004352:Drug Resistance, Microbial / Q000235:genetics; D004926:Escherichia coli / Q000187:drug effects / Q000201:enzymology / Q000235:genetics*; D064429:Fatty Acid Synthases / Q000037:antagonists & inhibitors; D005231:Fatty Acids, Unsaturated / Q000096:biosynthesis; D013876:Thiophenes / Q000494:pharmacology
chemical_list: D002352:Carrier Proteins; D005231:Fatty Acids, Unsaturated; D013876:Thiophenes; C035068:thiolactomycin; D000217:Acyltransferases; C052639:acetoacetyl-acyl carrier protein synthase; D064429:Fatty Acid Synthases