A PLASMID-ENCODED RFB(O/54) GENE-CLUSTER IS REQUIRED FOR BIOSYNTHESIS OF THE O/54 ANTIGEN IN SALMONELLA-ENTERICA SEROVAR BORREZE

Previous studies demonstrated that the presence of a 7-8kb plasmid is correlated with expression of the lipopolysaccharide (LPS) O:54 antigen in several Salmonella enterica serovars. In this study, a 6.7kb plasmid from a field isolate of S. enterica serovar Borreze was shown to encode enzymes responsible for the synthesis of the O:54 polysaccharide. Curing the plasmid results in simultaneous loss of smooth O-polysaccharide-substituted LPS molecules and 0:54 serotype. SDS-PAGE analysis of other O:54 isolates indicated that the O:54 O-polysaccharide can be co-expressed with an additional O-polysaccharide, likely encoded by chromosomal genes. The structure of the O:54 polysaccharide was determined by a combination of chemical and nuclear magnetic resonance (NMR) methods and was found to be an unusual homopolymer of N-acetylmannosamine (D-ManNAc) residues. The polysaccharide contained a disaccharide repeating unit with the structure: ->4)-beta-D-ManpNAc-(1-->3)-beta-D-ManpNAc-(1--> This structure does not resemble other O-polysaccharides in S. enterica. To examine the role played by plasmid functions in synthesis of the O:54 polysaccharide, the 6.7 kb plasmid was cloned to produce a hybrid plasmid (pWQ800) in pGEM-7Zf(+). In Escherichia coli K-12 Delta rfb, pWQ800 directed the synthesis of authentic O:54 polysaccharide. Polymerized O:54 polysaccharide was also produced in S. entericaserovar Typhimurium rfb and rfc mutants. From these data, we conclude that pWQ800 carries the rfb(O:54) gene cluster and synthesis of the O:54 polysaccharides does not require host chromosomal rfb functions. However, synthesis of the O:54 polysaccharide requires the function of the rfe and rffE genes which are part of the gene cluster encoding enzymes involved in biosynthesis of enterobacterial common antigen. The rffE gene product synthesizes the O:54 precursor, uridine diphospho-N-acetylmannosamine. This is the first description of a plasmid-encoded rfb gene cluster in Salmonella.