LIPOPOLYSACCHARIDE WITH AN ALTERED O-ANTIGEN PRODUCED IN ESCHERICHIA-COLI K-12 HARBORING MUTATED, CLONED SHIGELLA-FLEXNERI RFB GENES

Cloning of the rfb genes of Shigella flexneri 2a into Escherichia coli K-12 strain DH1 results in the synthesis of lipopolysaccharides (LPS) with an O-antigen chain having type antigen IV and group antigens 3,4 . During genetic studies of these rfb genes in E. coil K-12, we observ ed that strains harbouring plasmids with certain mutations (inversion and transposon insertions) which should have blocked O-antigen synthes is nevertheless still produced LPS with O-antigen chains. These LPS mi grated differently on silver-stained SDS-polyacrylamide gels, compared with the LPS produced by wild-type rfb genes, and the group 3,4 antig ens were barely detectable, suggesting that the O-antigen was altered, Investigation of the genetic determinants for production of the alter ed O-antigen/LPS indicated that: (i) these LPS are produced as a resul t of mutations which are either polar on rfbF or inactivate rfbF; (ii) the rfbX gene product (or a similar protein in the E. coli K-12 rfb r egion) is needed for production of the altered O-antigen in the form o f LPS; (iii) the rfbG gene product is required for the production of b oth the parental and altered LPS; (iv) the dTDP-rhamnose biosynthesis genes are required, Additionally, an E. coil K-12 gene product(s) enco ded outside the rfb region also contributes to production of the O-ant igen of the altered LPS. An antiserum raised to the altered LPS from s train DH1(pPM2217 (rfbX::tn 1725)) was found to cross-react with nearl y all S. flexneri serotypes, and with the altered LPS produced by othe r DH1 strains harbouring plasmids with different rfb mutations, as des cribed above. The reactivity of the altered LPS with a panel of monocl onal antibodies specific for various S. flexneri O-antigen type and gr oup antigens demonstrated that their O-antigen components were closely related to that of S. flexneri serotype 4, The RfbF and RfbG proteins were shown to have similarity to rhamnose transferases, and we identi fied a motif common to the N-termini of 6-deoxy-hexose nucleotide suga r transferases, We propose that the E, coil K-12 strains harbouring th e mutated S. flexneri rfb genes produce LPS with a hybrid O-antigen as a consequence of inactivation of RfbF and complementation by an E. co il K-12 gene product, Analysis of the genetic and immunochemical data suggested a possible structure for the O-antigen component of the alte red LPS.