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‐acetyl‐glucosamine phosphate to the carrier lipid unde‐caprenol 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. Copyright © 1993, Wiley Blackwell. All rights reserved