Previous work from this laboratory has shown that a 26-kb insert in cosmid clone pFV100, isolated from a Pseudomonas aeruginosa gene library, contained genes that could restore serotype-specific B-band lipopolysaccharide (LPS) expression in rough mutant ge6. In this study, subclones from pFV100 were made to identify genes responsible for B-band LPS synthesis. Transformation of Escherichia coli HB101 with cosmid clone pFV100 resulted in expression of P. aeruginosa serotype O5 B-band LPS, indicating the presence of an rfb cluster in pFV100. Expression of P. aeruginosa LPS could not be achieved in E. coli HB101 transformed with any of the subclones. Complementation studies of well-characterized rough mutants of P. aeruginosa PAO1 deficient in B-band LPS biosynthesis were performed with the various subclones. Subclone pFV110, containing a 1.4-kb XbaI-HindIII insert, restored B-band LPS biosynthesis in mutant AK44 (A+B-; complete core). Probing chromosomal DNA from the 20 International Antigenic Typing Scheme serotypes with the 1.4-kb insert from pFV110 in Southern hybridizations revealed a positive reaction to restriction fragments in serotypes O2, O5, O16, O20, and O18. LPS of serotypes O2, O5, O16, and O20 were shown earlier to have a similar backbone structure in their O antigen. The insert in pFV110 was sequenced, and the deduced amino acid sequence was compared with sequences of protein databases. No significant homology could be detected with any sequences in the database. Open reading frame analysis identified one region, ORF303, which could encode a 33-kDa protein. Using E. coli maxicells for protein expression, orf303 mediated the expression of a unique polypeptide with an apparent molecular mass of 32.5 kDa. The deficiency in the synthesis of B-band LPS biosynthesis in mutant AK44 is apparently complemented by the 33-kDa protein encoded by orf303. We have designated this ORF rfbA. This investigation is the first report on cloning and sequencing of an rfb gene involved specifically in O-antigen biosynthesis in P. aeruginosa PAO1
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