CONSTRUCTION OF RECOMBINANT AROA SALMONELLAE STABLY PRODUCING THE SHIGELLA-DYSENTERIAE SEROTYPE-1 O-ANTIGEN AND STRUCTURAL CHARACTERIZATIONOF THE SALMONELLA SHIGELLA HYBRID LPS/

The TN501 mercury resistant transposon containing the rfp and rfb loci encoding biosynthesis of the O-antigen of Shigella dysenteriae seroty pe 1 lipopolysaccharide (LPS) was constructed and introduced into aroA mutants of Salmonella typhimurium and Salmonella dublin. In five reco mbinant strains, both homologous LPS and hybrid LPS, consisting of Sal monella lipid A-core and Shigella O-antigen, were produced. All deriva tives but one (SL3235) stably inherited the new trait. Immunofluoresce nce microscopy, using mixtures of differentially-labelled antibodies s pecific for either the Salmonella or the Shigella O-antigen, demonstra ted that individual bacteria produced both types of LPS. Qualitative a nd quantitative analysis of polysaccharides obtained by mild hydrolysi s of purified LPS was carried out by methylation analysis and NMR spec troscopy, and revealed that the ratio of Salmonella to Shigella O-anti gen repeating units in the high molecular weight fraction of isolated polysaccharides varied from 1.3: 1 to 8.4:1 as based on the relative p roportions of 1,4,5-tri-O-acetyl-2,3-di-O-methyl-L-rhamnitol (Salmonel la repeating unit) and 1,3,5-tri-O-acetyl-2,4-di-0-methyl-L-rhamnitol (Shigella repeating unit). The attachment site of the Shigella O-antig en to the Salmonella core was investigated by construction of a mutant rfp-rfb gene cluster encoding the synthesis of only one repeat unit o f the Shigella dysenteriae type 1 O-antigen, and its introduction into a rough Salmonella strain. This hybrid organism produced a polysaccha ride with the following structure, [GRAPHICS] Rha(alpha 1-3)Rha(alpha 1-2)Gal(alpha 1-3)GlcNAc(alpha 1- 4)Glc(alpha 1-2)Gal(alpha 1-3)Glc(al pha 1-Shigella dysenteriae type 1 O-antigen demonstrating that the Shi gella dysenteriae type 1 O-antigen is linked at position O-4 of the su bterminal D-glucose unit in the Salmonella core. (C) 1996 Academic Pre ss Limited.