Construction and characterization of isogenic O‐antigen variants of Salmonella typhi

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Abstract
A 7.5 kb Kpnl‐generated fragment, from within the rfb cluster of Salmonella typhimurium LT2 that encodes abequose synthase (the rfbJ gene) which is necessary for O4 antigen synthesis, and flanking sequences, was inserted into a suicide vector. Using allelic exchange techniques, these rfb sequences of S. typhimurium were integrated into the rfb clusters of wild‐type Salmonella typhi Vi‐positive strain ISP 1820 (i.e. serotype 09,12; Vi+ H‐d), S. typhi Vi‐negative strain H400 (i.e. serotype 09,12; Vi−; H‐d), and a double aro 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 (LPS) purified from H325, H404 and CVD 906‐O4 demonstrated that these 8trains express the 04 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 and CVD 906‐O4 confirmed that a precise recombination event within sequences flanking rfbSE of S. typhi (which encodes the enzymes necessary for cytidine diphosphate‐tyvelose synthesis) resulted in replacement of rfbSE with rfbJ (which encodes abequose synthase and is necessary for O4 synthesis) of S. typhimurium in strains H325, H404 and CVD 906‐O4. The resistance of each strain to the bactericidal effects of guinea‐pig serum (GPC) was assessed. Whereas ISP 1820, H325 and H404 exhibit similar resistance patterns in GPC, strain H400 is sensitive to the bactericidal 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