Expression cloning of a Pseudomonas gene encoding a hydroxydecanoyl-acyl carrier protein-dependent UDP-GlcNAc acyltransferase

Abstract

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'.

FIG. 1

Role of LpxA in lipid A biosynthesis in E. coli. LpxA catalyzes the first step of lipid A biosynthesis (24, 25). The transfer of an R-3-hydroxyacyl group from R-3-hydroxyacyl–ACP to UDP-GlcNAc is very selective for R-3-hydroxymyristoyl–ACP in the case of E. coli LpxA (3, 33). UDP-GlcNAc acyltransferases from other gram-negative bacteria have specificity for ACP thioesters of different acyl chain lengths (33). Biosynthetic intermediates and known genes encoding the enzymes of the rest of the E. coli pathway are shown (24, 25). Chemical hydrolysis of lipopolysaccharide or of whole cells at pH 4.5 in the presence of sodium dodecyl sulfate cleaves the 3-deoxy-d-manno-octulosonic acid (Kdo)-lipid A linkage without disturbing the phosphates (7, 8). The released lipid product is designated lipid A throughout this paper. The 10-carbon-specific Pseudomonas LpxA can replace the 14-carbon-specific E. coli enzyme in living cells without interfering with the functioning of the other enzymes of the pathway.

FIG. 2

Expression cloning of a 3-hydroxydecanoyl-specific UDP-GlcNAc acyltransferase from a P. aeruginosa PAO1 DNA library in E. coli LE392 assayed in pools of four. When [α-³²P]UDP-GlcNAc is acylated, it migrates off the origin in this thin-layer system, as indicated. Acylation with 3-hydroxymyristate results in a product that migrates slightly faster than does the product of acylation with 3-hydroxydecanoate. Extracts of wild-type P. aeruginosa acylate [α-³²P]UDP-GlcNAc efficiently in the presence of 3-hydroxydecanoyl–ACP (lane B) but not at all in the presence of 3-hydroxymyristoyl–ACP (lane A). Conversely, strains of E. coli that do not harbor the P. aeruginosa lpxA gene acylate [α-³²P]UDP-GlcNAc in the presence of 3-hydroxymyristoyl–ACP (lane E) but not in the presence of 3-hydroxydecanoyl–ACP (lane C). The more rapidly migrating compounds in lanes E and F represent further metabolites of the lipid A pathway that are derived in E. coli extracts from acylated-UDP-GlcNAc (1, 3). Lanes C to F show the assay results obtained with pools of four lysates (prepared as described in Materials and Methods), each derived from distinct E. coli colonies harboring different P. aeruginosa DNA inserts. With 3-hydroxydecanoyl–ACP as the substrate, only lysate pool G9 (of the 288 pools assayed) catalyzed measurable acylation of [α-³²P]UDP-GlcNAc (lane D). With 3-hydroxymyristoyl–ACP as the substrate (lane F), pool G9 was comparable in activity to all other pools in the collection (such as C11 in lane E), indicating that the protein concentrations of G9 and C11 were about the same.

FIG. 3

Identification of a single P. aeruginosa library clone in E. coli LE392 expressing a 3-hydroxydecanoyl-specific UDP-GlcNAc acyltransferase. Assays of lysozyme-EDTA lysates of the individual colonies that were used to make pool G9 were carried out as described for Fig. 2. Only extracts of the E. coli strain harboring P. aeruginosa cosmid 1137 catalyzed 3-hydroxydecanoyl–ACP-dependent acylation of [α-³²P]UDP-GlcNAc.

FIG. 4

DNA sequence of the P. aeruginosa lpxA gene and its flanking regions. The predicted protein sequences of LpxA and of the N-terminal part of LpxB are indicated.

FIG. 5

Comparison of the amino acid sequences of P. aeruginosa and E. coli LpxA. The overall sequence identity is 54%, and the sequence similarity is 67%. The aliphatic residues that denote the beginning of each hexapeptide repeat are in bold print. The sequence has been arranged to indicate the stacking of the hexads on top of each other in the formation of the left-handed β-helix seen in the E. coli LpxA crystal structure (28). The positions of two loops (residues 72 to 83 and 102 to 108) that can be recognized as interruptions of the contiguous hexad repeats in the primary sequences are located in the same places in both enzymes. One hexad is truncated at the N terminus of Pseudomonas LpxA, and valine 171 of the last complete hexad of E. coli is replaced by serine (italicized) in Pseudomonas LpxA. The significance of these differences to the overall protein fold and acyl chain selectivity of the Pseudomonas enzyme remains to be established. PSEUD, P. aeruginosa; ECOLI, E. coli.

FIG. 6

Acyl-ACP specificity of Pseudomonas UDP-GlcNAc acyltransferase determined in cytosolic extracts of RO138/pGD3. Cells were grown to late log phase at 42°C in LB in the presence of ampicillin, and assays of supernatants centrifuged at 100,000 × g were performed at 30°C, as described in the Materials and Methods. The extract concentration was 0.1 mg/ml. Results at the 2- and 5-min time points are shown for each reaction.

FIG. 7

Mass spectrum of lipid A isolated from strain RO138 complemented at 42°C with pGD3. There is no peak at m/z 1,796 in the lipid A isolated from RO138/pGD3, demonstrating the absence of residual E. coli LpxA function in living cells under these conditions.