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 26kb insert, is able to restore O-antigen synthesis in se rotype 05 rough mutants of Pseudomonas aeruginosa. Mobilization of pFV 100 into two P. aeruginosa semi-rough (SR) mutants, AK1401 and rd7513, resulted in O-antigen expression, indicating that pFV100 may contain an O-polymerase (rfc) gene, pFV.TK6, a subclone of pFV100 that contain s a 5.6 kb chromosomal insert, was able to complement O-antigen expres sion in these SR mutants. Mutagenesis of pFV.TK6 using Tn 1000 exposed a 1.5 kb region that was essential for complementing O-antigen expres sion in AK1401. PI 2.0 kb Xhol-Hindlll fragment, containing this regio n, was cloned into vector pUCP26 and the resulting plasmid called pFV. TK8. In Southern analysis of the 20 P. aeruginosa serotypes using a pr obe generated from the 1.5kb Xhol fragment of pFV.TK8, the rfc probe h ybridized to a common fragment of the crossreactive O2-O5-O16-O18-O20 serogroup, suggesting that these serotypes may share a common O-polyme rase gene. In functional studies of the rfc gene, the PAO1 (serotype O 5) chromosomal rfc was mutated using a gene-replacement strategy. Thes e knockout mutants expressed the SR lipopolysaccharide (LPS) phenotype , which indicated that they were no longer producing a functional O-po lymerase 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 RNA and protein database s, no significant homology was found. However, the deduced structure o f the P. aeruginosa Rfc protein indicated that it is very hydrophobic and contains 11 putative membrane-spanning domains. Therefore, the pre dicted structure is similar to that of other reported Rfc proteins, Fu rthermore, comparison of the amino acid composition and codon usage of the P. aeruginosa Rfc with other Rfc proteins revealed significant si milarity between them.