Journal of Bacteriology, September 1999, p. 5498-5504, Vol. 181, No. 17
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

Department of Microbiology, Colorado State University, Fort Collins, Colorado 80523
Received 8 April 1999/Accepted 14 June 1999
A DNA fragment containing the Pseudomonas aeruginosa
fabD (encoding malonyl-coenzyme A [CoA]:acyl carrier protein
[ACP] transacylase), fabG (encoding
-ketoacyl-ACP
reductase), acpP (encoding ACP), and fabF
(encoding
-ketoacyl-ACP synthase II) genes was cloned and sequenced.
This fab gene cluster is delimited by the plsX (encoding a poorly understood enzyme of phospholipid metabolism) and
pabC (encoding 4-amino-4-deoxychorismate lyase) genes; the fabF and pabC genes seem to be translationally
coupled. The fabH gene (encoding
-ketoacyl-ACP synthase
III), which in most gram-negative bacteria is located between
plsX and fabD, is absent from this gene
cluster. A chromosomal temperature-sensitive fabD mutant was obtained by site-directed mutagenesis that resulted in a W258Q change. A chromosomal fabF insertion mutant was generated,
and the resulting mutant strain contained substantially reduced levels of cis-vaccenic acid. Multiple attempts aimed at disruption
of the chromosomal fabG gene were unsuccessful. We purified
FabD as a hexahistidine fusion protein (H6-FabD) and ACP in
its native form via an ACP-intein-chitin binding domain fusion protein,
using a novel expression and purification scheme that should be
applicable to ACP from other bacteria. Matrix-assisted laser
desorption-ionization spectroscopy, native polyacrylamide
electrophoresis, and amino-terminal sequencing revealed that (i) most
of the purified ACP was properly modified with its
4'-phosphopantetheine functional group, (ii) it was not acylated, and
(iii) the amino-terminal methionine was removed. In an in vitro system,
purified ACP functioned as acyl acceptor and H6-FabD
exhibited malonyl-CoA:ACP transacylase activity.
Present address: Myriad Genetics, Salt Lake City, UT 84108.
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