INACTIVATION OF THE ESCHERICHIA-COLI B41 (O101-K99/F41) RFB GENE ENCODING AN 80-KDA POLYPEPTIDE RESULTS IN THE SYNTHESIS OF AN ANTIGENICALLY ALTERED LIPOPOLYSACCHARIDE IN ESCHERICHIA-COLI K-12

The genetic organisation of the rfb region from Escherichia coli B41 (O101:K99/F41) which determines the biosynthesis of the O101 O-antigen of the lipopolysaccharide (LPS) has been,examined in E. coli K-12. Maxicell analysis of the plasmid-encoded proteins facilitated the construction of a physical map of the rfb region, consisting of six proteins, designated A (87 kDa), B (80 kDa), C (49 kDa), D (38 kDa), E (36.5 kDa) and F (27 kDa). Proteins E and F are not required for O-antigen biosynthesis. The introduction of frameshift mutations within the region encoding protein B resulted in the synthesis of an antigenically altered LPS which is shorter than the wild-type LPS, as assessed by reaction to antisera in colony and Western immunoblots, and by silver staining of LPS separated on sodium dodecyl sulfate-polyacrylamide-gel electrophoresis. The results demonstrate that protein B has a novel role in O-antigen biosynthesis associated with both the control of LPS chain length and antigenic structure. The nucleotide sequence of the rfb gene encoding protein B has been determined, confirming it to be a 697-amino acid protein of 78.9 kDa predicted to be located in the cytoplasmic membrane.