EXPRESSION CLONING OF A PSEUDOMONAS GENE ENCODING A HYDROXYDECANOYL-ACYL CARRIER PROTEIN-DEPENDENT UDP-GLCNAC ACYLTRANSFERASE

UDP-N-acetylglucosamine-3-O-acyltransfer (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 IpxA gene of Pseudomonas aeruginosa from a library o f Pseudomonas strain PAO1 expressed in Escherichia coli LE392 (J. Ligh tfoot and J. S. Lam, J. Bacteriol. 173:5624-5630, 1991). Pseudomonas l pxA encodes a 10-carbon-specific UDP-GlcNAc acyltransferase, whereas t he E. coli transferase is selective for a 14-carbon acyl chain. Recomb inant cosmid 1137 enabled production of a 3-hydroxydecanoyl-specific U DP-GlcNAc acyltransferase in E. coli. It was identified by assaying ly sozyme-EDTA lysates of individual members of the library with 3-hydrox ydecanoyl-acyl carrier protein (ACP) as the substrate. Cosmid 1137 con tained a 20-kb insert of P. aeruginosa DNA. The lpxA gene region was l ocalized to a 1.3-kb SalI-PstI fragment. Sequencing revealed that it c ontains one complete open reading frame (777 bp) encoding a new IpxA h omolog. The predicted Pseudomonas LpxA is 258 amino acids long and con tains 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-hydrox ymyristoyl-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'.