ISOLATION AND CHARACTERIZATION OF 2 GENES, WAAC (RFAC) AND WAAF (RFAF), INVOLVED IN PSEUDOMONAS-AERUGINOSA SEROTYPE O5 INNER-CORE BIOSYNTHESIS

Recent studies have provided evidence to implicate involvement of the core oligosaccharide region of Pseudomonas aeruginosa lipopolysacchari de (LPS) in adherence to host tissues. To better understand the role p layed by LPS in the virulence of this organism, the aim of the present study was to clone and characterize genes involved in core biosynthes is. The inner-core regions of P. aeruginosa and Salmonella enterica se rovar Typhimurium are structurally very similar; both contain two main chain residues of heptose linked to lipid A-Kdo, (kdo is 3-deoxy-D-ma nno-octulosonic acid). By electrotransforming a P. aeruginosa PAO1 lib rary into Salmonella waaC and waaF (formerly known as rfaC and rfaF, r espectively) mutants, we were able to isolate the homologous heptosylt ransferase I and II genes of P. aeruginosa. Two plasmids, pCOREc1 and pCOREc2, which restored smooth LPS production in the waaC mutant, were isolated. Similarly, plasmid pCOREf1 was able to complement the Salmo nella waaF mutant. Sequence analysis of the DNA insert of pCOREc2 reve aled one open reading frame (ORF) which could code for a protein of 39 .8 kDa. The amino acid sequence of the deduced protein exhibited 53% i dentity with the sequence of the WaaC protein of S. enterica serovar T yphimurium. pCOREf1 contained one ORF capable of encoding a 38.4-kDa p rotein. The sequence of the predicted protein was 49% identical to the sequence of the Salmonella WaaF protein. Protein expression by the Ma xicell system confirmed that a 40-kDa protein was encoded by pCOREc2 a nd a 38-kDa protein was encoded by pCOREf1. Pulsed-field gel electroph oresis was used to determine the map locations of the cloned waaC and waaF genes, which were found to lie between 0.9 and 6.6 min on the PAO 1 chromosome. Using a gene-replacement strategy, we attempted to gener ate P. aeruginosa waaC and waaF null mutants. Despite multiple attempt s to isolate true knockout mutants, all transconjugants were identifie d as merodiploids.