EXPRESSION OF THE O9 POLYSACCHARIDE OF ESCHERICHIA-COLI - SEQUENCING OF THE ESCHERICHIA-COLI O9 RFB GENE-CLUSTER, CHARACTERIZATION OF MANNOSYL TRANSFERASES, AND EVIDENCE FOR AN ATP-BINDING CASSETTE TRANSPORT-SYSTEM

The rfb gene cluster of Escherichia coil O9 directs the synthesis of t he O9-specific polysaccharide which has the structure pha-Man-(1-->3)- alpha-Man-(1-->3)-alpha-Man-(1-->. The E. coli O9 rfb cluster has been sequenced, and six genes, in addition to the previously described rfb K and rfbM, were identified. They correspond to six open reading frame s (ORFs) encoding polypeptides of 261, 431, 708, 815, 381, and 274 ami no acids. They are all transcribed in the counter direction to those o f the his operon. No gene was found between rfb and his. A higher G+C content indicated that E. coli O9 rfb evolved independently of the rfb clusters from other E. coil strains and from Shigella and Salmonella spp. Deletion mutagenesis, in combination with analysis of the in vitr o synthesis of the O9 mannan in membranes isolated from the mutants, s howed that three genes (termed mtfA, -B, and -C, encoding polypeptides of 815, 381, and 274 amino acids, respectively) directed alpha-mannos yl transferases. MtfC (from ORF274), the first mannosyl transferase, t ransfers a mannose to the endogenous acceptor. It critically depended on a functional rfe gene (which directs the synthesis of the endogenou s acceptor) and initiates the growth of the polysaccharide chain. MtfB (from ORF381) then transfers two mannoses into the 3 position of the previous mannose, and MtfA (from ORF815) transfers three mannoses into the 2 position. Further chain growth needs only the two transferases MtfA and MtfB. Thus, there are fewer transferases needed than the numb er of sugars in the repeating unit. Analysis of the predicted amino ac id sequence of the ORF261 and ORF431 proteins indicated that they func tion as components of an ATP binding cassette transport system. A poss ible correlation between the mechanism of polymerization and mode of m embrane translocation of the products is discussed.