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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.

Characterization of a Pseudomonas aeruginosa Fatty Acid Biosynthetic Gene Cluster: Purification of Acyl Carrier Protein (ACP) and Malonyl-Coenzyme A:ACP Transacylase (FabD)

Alecksandr J. Kutchma,dagger Tung T. Hoang, and Herbert P. Schweizer*

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 beta -ketoacyl-ACP reductase), acpP (encoding ACP), and fabF (encoding beta -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 beta -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.

* Corresponding author. Mailing address: Department of Microbiology, Colorado State University, Fort Collins, CO 80523. Phone: (970) 491-3536. Fax: (970) 491-1815. E-mail: hschweiz{at}cvmbs.colostate.edu.

dagger Present address: Myriad Genetics, Salt Lake City, UT 84108.

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.


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