Characterization of a Pseudomonas aeruginosa fatty acid biosynthetic gene cluster: Purification of acyl carrier protein (ACP) and malonyl-coenzyme A : ACP transacylase (FabD)

A DNA fragment containing the Pseudomonas aeruginosa fabD (encoding malonyl -coenzyme A [CoA]:acyl carrier protein [ACP] transacylase), fabG (encoding beta-ketoacyl-ACP reductase), acpP (encoding ACP), and fabF (encoding beta- ketoacyl-ACP synthase II) genes was cloned and sequenced. This fab gene clu ster is delimited by the pLsX (encoding a poorly understood enzyme of phosp holipid metabolism) and pabC (encoding 4-amino-4-deoxychorismate lyase) gen es; the fabF and pabC genes seem to be translationally coupled. The fabH ge ne (encoding beta-ketoacyl-ACP synthase III), which in most gram-negative b acteria is located between plsX and fabD, is absent from this gene cluster. A chromosomal temperature-sensitive fabD mutant was obtained by site-direc ted mutagenesis that resulted in a W258Q change. A chromosomal fabF inserti on mutant was generated, and the resulting mutant strain contained substant ially reduced levels of cis-vaccenic acid. Multiple attempts aimed at disru ption of the chromosomal fabG gene were unsuccessful. We purified FabD as a hexahistidine fusion protein (H-6-FabD) and ACP in its native form via an ACP-intein-chitin binding domain fusion protein, using a novel expression a nd purification scheme that should be applicable to ACP from other bacteria . Matrix-assisted laser desorption-ionization spectroscopy, native polyacry lamide electrophoresis, and amino-terminal sequencing revealed that (i) mos t of the purified ACP was properly modified with its 4'-phosphopantetheine functional group, (ii) it was not acylated, and (iii) the amino-terminal me thionine was removed. In an in vitro system, purified ACP functioned as acy l acceptor and H-6-FabD exhibited malonyl-CoA:ACP transacylase activity.