UDP-
N-acetylglucosamine-3-
O-acyltransferase (UDP-GlcNAc acyltransferase) catalyzes the first step of lipid A biosynthesis (M. S. Anderson and C. R. H. Raetz, J. Biol. Chem. 262:5159–5169, 1987). We here report the isolation of the
lpxA gene of
Pseudomonas aeruginosa from a library of
Pseudomonas strain PAO1 expressed in
Escherichia coli LE392 (J. Lightfoot and J. S. Lam, J. Bacteriol. 173:5624–5630, 1991).
Pseudomonas lpxA encodes a 10-carbon-specific UDP-GlcNAc acyltransferase, whereas the
E. coli transferase is selective for a 14-carbon acyl chain. Recombinant cosmid 1137 enabled production of a 3-hydroxydecanoyl-specific UDP-GlcNAc acyltransferase in
E. coli. It was identified by assaying lysozyme-EDTA lysates of individual members of the library with 3-hydroxydecanoyl-acyl carrier protein (ACP) as the substrate. Cosmid 1137 contained a 20-kb insert of
P. aeruginosa DNA. The
lpxA gene region was localized to a 1.3-kb
SalI-
PstI fragment. Sequencing revealed that it contains one complete open reading frame (777 bp) encoding a new
lpxA homolog. The predicted
Pseudomonas LpxA is 258 amino acids long and contains 21 complete hexapeptide repeating units, spaced in approximately the same manner as the 24 repeats of
E. coli LpxA. The
P. aeruginosa UDP-GlcNAc acyltransferase is 54% identical and 67% similar to the
E. coli enzyme. A plasmid (pGD3) containing the 1.3-kb
SalI-
PstI fragment complemented
E. coli RO138, a temperature-sensitive mutant harboring
lpxA2. LpxA assays of extracts of this construct indicated that it is >1,000-fold more selective for 3-hydroxydecanoyl-ACP than for 3-hydroxymyristoyl-ACP. Mass spectrometry of lipid A isolated from this strain by hydrolysis at pH 4.5 revealed [M-H]
− 1,684.5 (versus 1,796.5 for wild-type lipid A), consistent with 3-hydroxydecanoate rather than 3-hydroxymyristate at positions 3 and 3′.
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