Molecular cloning and characterization of the rfc gene of Pseudomonas aeruginosa (serotype 05).

Previous work from our laboratory has shown that cosmid clone pFV100, containing a 26 kb insert, is able to restore O-antigen synthesis in serotype 05 rough mutants of Pseudomonas aeruginosa. Mobilization of pFV100 into two P. aeruginosa semi-rough (SR) mutants, AK1401 and rd7513, resulted in O-antigen expression, indicating that pFV100 may contain an 0-polymerase (rfc) gene. pFV.TK6, a subclone of pFV100 that contains a 5.6kb chromosomal insert. was able to complement O-antigen expression in these SR mutants. Mutagenesis of pFV.TK6 using Tn 1000 exposed a 1.5 kb region that was essential for complementing O-antigen expression in AK1401. A 20 kb Xhol-Hindlll fragment, containing this region, was cloned into vector pUCP26 and the resulting plasmid called pFV.TK8. In Southern analysis of the 20 P. aeruginosa serotypes using a probe generated from the 1.5 kb Xhol fragment of pFV.TK8, the rfc probe hybridized to a common fragment of the cross-reactive 02-05-016-018-020 serogroup, suggesting that these serotypes may share a common O-polymerase gene. In functional studies of the rfc gene, the PA01 (serotype 05) chromosomal rfc was mutated using a gene-replacement strategy. These knockout mutants expressed the SR lipopolysaccharide (LPS) phenotype, which indicated that they were no longer producing a functional 0-polymerase enzyme. Nucleotide sequence analysis of the insert DNA of pFV.TK8 revealed one open reading frame (ORF), designated ORF48.9, which could code for a 48.9 kDa protein. In comparisons of the P. aeruginosa rfc nucleotide and amino acid sequences with DNA and protein databases, no significant homology was found. However, the deduced structure of the P. aeruginosa Rfc protein indicated that it is very hydro-phobic and contains 11 putative membrane-spanning domains. Therefore, the predicted structure is similar to that of other reported Rfc... [ABSTRACT FROM AUTHOR]