Function of the rfb gene cluster and the rfe gene in the synthesis of O antigen by Shigella dysenteriae 1.
A plasmid that included both an 8.9 kb chromosomal DNA insert containing genes from the rfb cluster of Shigella dysenteriae 1 and a smaller insert containing the rfp gene from a S. dysenteriae 1 multicopy plasmid resulted in efficient expression of O antigen in an rfb-deleted strain of Escherichia coli K-12. Eight genes were identified in the rfb fragment: the rfbB-CAD cluster which encodes dTDP-rhamnose synthesis, rfbX which encodes a hydrophobic protein involved in assembly of the O antigen, rfc which encodes the O antigen polymerase, and two sugar transferase genes. The production of an O antigen also required the E. coli K-12 rfe gene, which is known to encode a transferase which adds N-acetylglucosamine phosphate to the carrier lipid undecaprenol phosphate. Thus Rfe protein appears to function as an analogue of the Salmonella RfbP protein to provide the first sugar of the O unit. Functional analysis of the other genes was facilitated by the fact that partial O units of one, two or three sugars were efficiently transferred to the lipopolysaccharide core. This analysis indicated that the plasmid-encoded Rfp protein is the transferase that adds the second sugar of the O unit while the two rfb transferases add the distal sugars to make an O antigen whose structure is (Rha-Rha-Gal-GlcNAc)n. The use of the rfe gene product as the transferase that adds the first sugar of an O unit is a novel mechanism which may be used for the synthesis of other enteric O antigens.
Klena
J D
JD
Department of Microbiology, Arizona State University, Tempe 85287.
Schnaitman
C A
CA
D050260:Carbohydrate Metabolism; D002240:Carbohydrate Sequence; D029968:Escherichia coli Proteins; D015964:Gene Expression Regulation, Bacterial; D005798:Genes, Bacterial; D006031:Glycosylation; D008969:Molecular Sequence Data; D005810:Multigene Family; D019081:O Antigens; D010641:Phenotype; D010802:Phylogeny; D010957:Plasmids; D011110:Polymorphism, Genetic; D011135:Polysaccharides, Bacterial / Q000096:biosynthesis*; D012486:Salmonella typhimurium / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D012762:Shigella dysenteriae / Q000235:genetics* / Q000276:immunology / Q000378:metabolism; D013045:Species Specificity; D017855:Transferases (Other Substituted Phosphate Groups) / Q000235:genetics* / Q000378:metabolism
D029968:Escherichia coli Proteins; D019081:O Antigens; D011135:Polysaccharides, Bacterial; D017855:Transferases (Other Substituted Phosphate Groups); C083617:wecA protein, E coli