CHROMOSOMAL MAPPING, EXPRESSION AND SYNTHESIS OF LIPOPOLYSACCHARIDE IN PSEUDOMONAS-AERUGINOSA - A ROLE FOR GUANOSINE DIPHOSPHO (GDP)-D-MANNOSE

Pseudomonas aeruginosa can express two distinct forms of lipopolysacch aride (LPS), called A-band and B-band. As an attempt to understand the molecular biology of the synthesis and regulation of these LPS antige ns, a recombinant plasmid, pFV3, containing genes for A-band expressio n was isolated previously. In the present study, P. aeruginosa strain PAO1 was mutagenized with transposon Tn5-751 and yielded a B-band-defi cient mutant, called ge6. This mutant was mated with a PAO1 genomic li brary, and transconjugants were screened for complementation of B-band using B-band-specific monoclonal antibody MF1 5-4. Recombinant plasmi d pFV100 was subsequently isolated by its ability to complement B-band expression in ge6. SDS-PAGE analysis of LPS f rom ge6 and ge6(pFV100) revealed that ge6 was deficient in expression of B-band, while ge6(pF V100) had an LPS profile similar to that of the parent strain PAO1. Wi th A-band and B-band genes cloned in separate plasmids, pFV3 and pFV10 0 respectively, we were able to determine the map location of these LP S genes on the P. aeruginosa PAO1 chromosome using pulsed-field gel el ectrophoresis. A-band genes mapped at 5.75 to 5.89 Mbp (SpeI fragment SpK; DpnI fragment DpF2), while genes involved with expression of B-ba nd LPS mapped at 1.9 Mbp (SpeI fragments SpC, SpI and SpAI; DpnI fragm ent DpD) on the 5.9 Mbp chromosome. We also performed initial characte rization of a gene involved with synthesis of A-band present on pFV3. We previously reported that recombinant plasmid pFV3 and subcloned pla smid pFV36 complemented A-band synthesis in rd7513, an A- mutant deriv ed from A+ strain AK1401. pFV36 was mutagenized with transposon Tn 100 0 to reveal a one-kilobase region capable of complementing the express ion of A-band in the A- strain rd7513. This region was subcloned as a 1.6 kb KpnI fragment into plasmid vector pAK1900 and the resulting clo ne named pFV39. Labelling of proteins encoded by pAK1900 and pFV39 in Escherichia coli maxicells revealed a single unique polypeptide of app roximately 37 kDa expressed by pFV39. Supernatants from disrupted cell s of rd7513(pFV39) and AK1401 converted C-14-labelled-guanosine diphos pho (GDP)-D-mannose to GDP-rhamnose, while supernatants from rd7513 di d not show synthesis of GDP-rhamnose. The data therefore suggest that conversion of GDP-D-mannose to GDP-rhamnose is required for synthesis of A-band LPS, and that a 37 kDa protein is involved in this conversio n.