Characterization of Pseudomonas aeruginosa Enoyl-Acyl carrier protein reductase (FabI): a target for the antimicrobial triclosan and its role in acylated homoserine lactone synthesis

The Pseudomonas aeruginosa fabI structural gene, encoding enoyl-acyl carrie r protein (ACP) reductase, was cloned and sequenced. Nucleotide sequence an alysis revealed that fabI is probably the last gene in a transcriptional un it that includes a gene encoding an ATP-binding protein of an ABC transport er of unknown function, The FabI protein was similar in size and primary se quence to other bacterial enoyl-ACP reductases, and it contained signature motifs for the FAD-dependent pyridine nucleotide reductase and glucose/ribi tol dehydrogenase families, respectively. The chromosomal fabI gene was dis rupted, and the resulting mutant was viable but possessed only 62% of the t otal enoyl-ACP reductase activity found in wild-type cell extracts. The fab I-encoded enoyl-ACP reductase activity was NADH dependent and inhibited by triclosan; the residual activity in the fabI mutant was also NADH dependent but not inhibited by triclosan. An polyhistidine-tagged FabI protein was p urified and characterized. Purified FabI (i) could use NADH but not NADPH a s a cofactor; (ii) used both crotonyl-coenzyme A and crotonyl-ACP as substr ates, although it was sixfold more active with crotonyl-ACP; and (iii) was efficiently inhibited by low concentrations of triclosan, A FabI Gly(95)-to -Val active-site amino acid substitution was generated by site-directed mut agenesis, and the mutant protein was purified. The mutant FabI protein reta ined normal enoyl-ACP reductase activity but was highly triclosan resistant . When coupled to FabI, purified P. aeruginosa N-butyryl-L-homoserine lacto ne (C-4-HSL) synthase, RhlI, could synthesize C-4-HSL from crotonyl-ACP and S-adenosylmethionine. This reaction was NADH dependent and inhibited by tr iclosan. The levels of C-4-HSL and N-(3-oxo)-dodecanoyl L-homoserine lacton es were reduced 50% in a fabI mutant, corroborating the role of FabI in acy lated homoserine lactone synthesis in vivo.