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 carrier protein (ACP) reductase, was cloned and sequenced. Nucleotide sequence analysis revealed that fabI is probably the last gene in a transcriptional unit that includes a gene encoding an ATP-binding protein of an ABC transporter of unknown function. The FabI protein was similar in size and primary sequence to other bacterial enoyl-ACP reductases, and it contained signature motifs for the FAD-dependent pyridine nucleotide reductase and glucose/ribitol dehydrogenase families, respectively. The chromosomal fabI gene was disrupted, and the resulting mutant was viable but possessed only 62% of the total enoyl-ACP reductase activity found in wild-type cell extracts. The fabI-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 purified and characterized. Purified FabI (i) could use NADH but not NADPH as a cofactor; (ii) used both crotonyl-coenzyme A and crotonyl-ACP as substrates, although it was sixfold more active with crotonyl-ACP; and (iii) was efficiently inhibited by low concentrations of triclosan. A FabI Gly95-to-Val active-site amino acid substitution was generated by site-directed mutagenesis, and the mutant protein was purified. The mutant FabI protein retained normal enoyl-ACP reductase activity but was highly triclosan resistant. When coupled to FabI, purified P. aeruginosa N-butyryl-L-homoserine lactone (C4-HSL) synthase, RhlI, could synthesize C4-HSL from crotonyl-ACP and S-adenosylmethionine. This reaction was NADH dependent and inhibited by triclosan. The levels of C4-HSL and N-(3-oxo)-dodecanoyl-L-homoserine lactones were reduced 50% in a fabI mutant, corroborating the role of FabI in acylated homoserine lactone synthesis in vivo.
Hoang
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Department of Microbiology, Colorado State University, Fort Collins, Colorado 80523, USA.
Schweizer
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D015107:4-Butyrolactone / Q000031:analogs & derivatives* / Q000096:biosynthesis; D000215:Acylation; D000891:Anti-Infective Agents, Local / Q000494:pharmacology*; D001483:Base Sequence; D003001:Cloning, Molecular; D004269:DNA, Bacterial; D050753:Enoyl-(Acyl-Carrier-Protein) Reductase (NADH); D005798:Genes, Bacterial; D016680:Genome, Bacterial; D006714:Homoserine / Q000031:analogs & derivatives* / Q000096:biosynthesis; D008969:Molecular Sequence Data; D010088:Oxidoreductases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism / Q000502:physiology*; D011550:Pseudomonas aeruginosa / Q000201:enzymology* / Q000235:genetics; D017422:Sequence Analysis, DNA; D014260:Triclosan / Q000494:pharmacology*
D000891:Anti-Infective Agents, Local; D004269:DNA, Bacterial; C109860:N-(3-oxododecanoyl)homoserine lactone; C092312:N-butyrylhomoserine lactone; D014260:Triclosan; D006714:Homoserine; D010088:Oxidoreductases; D050753:Enoyl-(Acyl-Carrier-Protein) Reductase (NADH); D015107:4-Butyrolactone