Characterization of Pseudomonas aeruginosa Enoyl-Acyl carrier protein reductase (FabI): a target for the antimicrobial triclosan and its role in acylated homoserine lactone synthesis
Tt. Hoang et Hp. Schweizer, Characterization of Pseudomonas aeruginosa Enoyl-Acyl carrier protein reductase (FabI): a target for the antimicrobial triclosan and its role in acylated homoserine lactone synthesis, J BACT, 181(17), 1999, pp. 5489-5497
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.