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 lipopolysaccharide (LPS), called A‐band and B‐band. As an attempt to understand the molecular biology of the synthesis and regulation of these LPS antigens, a recombinant plasmid, pFV3, containing genes for A‐band expression was isolated previously. In the present study, P. aeruginosa strain PAO1 was mutagenized with transposon Tn5‐751 and yielded a B‐band‐deficient mutant, called ge6. This mutant was mated with a PAO1 genomic library, and transconjugants were screened for complementation of B‐band using B‐band‐specific monoclonal antibody MF15‐4. Recombinant plasmid pFV100 was subsequently isolated by its ability to complement B‐band expression in ge6. SDS‐PAGE analysis of LPS from ge6 and ge6(pFV100) revealed that ge6 was deficient in expression of B‐band, while ge6(pFV100) had an LPS profile similar to that of the parent strain PA01. With A‐band and B‐band genes cloned in separate plasmids, pFV3 and pFV100 respectively, we were able to determine the map location of these LPS genes on the P. aeruginosa PAO1 chromosome using pulsed‐field gel electrophoresis. A‐band genes mapped at 5.75 to 5.89 Mbp (Spel fragment SpK; Dpnl fragment DpF2), while genes involved with expression of B‐band LPS mapped at 1.9 Mbp (Spel fragments SpC, Spl and SpAl; Dpnl fragment DpD) on the 5.9 Mbp chromosome. We also performed initial characterization of a gene involved with synthesis of A‐band present on pFV3. We previously reported that recombinant plasmid pFV3 and subcloned plasmid pFV36 complemented A‐band synthesis in rd7513, an A− mutant derived from A+ strain AK1401. pFV36 was mutagenized with transposon Tn1000 to reveal a one‐kilobase region capable of complementing the expression of A‐band in the A− strain rd7513. This region was subcloned as a 1.6 kb Kpnl fragment into plasmid vector pAK1900 and the resulting clone named pFV39. Labelling of proteins encoded by pAK1900 and pFV39 in Escherichia coli maxicells revealed a single unique polypeptide of approximately 37kDa expressed by pFV39. Supernatants from disrupted cells of rd7513(pFV39) and AK1401 converted 14C‐labelled‐guanosine diphospho (GDP)‐D‐mannose to GDP‐rhamnose, while supernatants from rd7513 did 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 37kDa protein is involved in this conversion.