CONSTRUCTION AND CHARACTERIZATION OF ISOGENIC O-ANTIGEN VARIANTS OF SALMONELLA-TYPHI

A 7.5kb Kpnl-generated fragment, from within the rfb cluster of Salmon ella typhimurium LT2 that encodes abequose synthase (the rfbJ gene) wh ich is necessary for O4 antigen synthesis, and flanking sequences, was inserted into a suicide vector. Using allelic exchange techniques, th ese rfb sequences of S. typhimurium were integrated into the rfb clust ers of wild-type Salmonella typhi Vi-positive strain ISP 1820 (i.e. se rotype O9,12; Vi(+); H-d), S. typhi Vi-negative strain H400 (i.e. sero type O9,12; Vi(-); H-d), and a double are mutant of S. typhi ISP 1820, strain CVD 906, resulting in the isolation of strains H325, H404 and CVD 906-O4, respectively. Immunoblot analysis of lipopolysaccharide (L PS) purified from H325, H404 and CVD 906-O4 demonstrated that these st rains express the O4 antigen (an abequose residue) in place of the O9 antigen (a tyvelose residue) in the LPS molecule. Hence, the serotype of H325 is O4,12; Vi(+); H-d and the serotype of H404 is O4,12; Vi(-); H-d. DNA hybridization analysis of chromosomal DNA from H325, H404 an d CVD 906-O4 confirmed that a precise recombination event within seque nces flanking rfbSE of S. typhi (which encodes the enzymes necessary f or cytidine diphosphate-tyvelose synthesis) resulted in replacement of rfbSE with rfbJ (which encodes abequose synthase and is necessary far O4 synthesis) of S. typhimurium in strains H325, H404 and CVD 906-O4. The resistance of each strain to the bactericidal effects of guinea-p ig serum (GPC) was assessed. Whereas ISP 1820, H325 and H404 exhibit s imilar resistance patterns in GPC, strain H400 is sensitive to the bac tericidal effects of GPC. The results suggest that the development of the O-antigen serotype diversity of Salmonella is probably the result of both sequence divergence and recombination