IDENTIFICATION OF THE O-ANTIGEN POLYMERASE (RFC) GENE IN ESCHERICHIA-COLI O4 BY INSERTIONAL MUTAGENESIS USING A NONPOLAR CHLORAMPHENICOL RESISTANCE CASSETTE

Computer analysis of the O4 polysaccharide gene cluster of Escherichia coli revealed the presence of two open reading frames (ORFs) encoding strongly hydrophobic polypeptides. O antigen polymerase, which is enc oded by the rfc gene, is a potential membrane protein and therefore sh ould be hydrophobic, To identify the rfc gene, these two ORFs were sub jected to insertional mutagenesis. A chloramphenicol resistance casset te was designed which, when properly inserted, does not cause a polar effect in downstream genes, Each of two ORFs, cloned into a plasmid ve ctor, was inactivated with this cassette, Two types of mutants bearing chromosomal insertions of the cassettes in each ORF were constructed by homologous recombination, These mutants were characterized by PCR, Southern blotting, and transverse-alternating-field electrophoresis, O nly one class of mutants exhibited the expected O polymerase-deficient phenotype; they produced O4-specific, semirough lipopolysaccharide, T herefore, this ORF was identified as the rfc gene, The chromosomal rfc mutation was complemented in trans by the rfc gene expressed from a p lasmid vector.