CHARACTERIZATION OF DEFINED OMPR MUTANTS OF SALMONELLA-TYPHI - OMPR IS INVOLVED IN THE REGULATION OF VI POLYSACCHARIDE EXPRESSION

The ompB operon, comprising the ompR and envZ genes, was cloned from a Salmonella typhi Ty2 cosmid bank and characterized by DNA sequence an alysis. The S. typhi ompR and envZ genes contained open reading frames encoding proteins of 240 and 451 amino acids, respectively. Compariso n with the Salmonella typhimurium OmpB protein sequences revealed 99.5 % homology. The DNA sequence data were used to identify appropriate re striction sites for generating a defined deletion of 517 bp within the open reading frame of the ompR gene. This deletion was introduced by homologous recombination into the chromosomes of two S. typhi strains which already harbored defined deletions in both the aroC and aroD gen es. The presence of the deletions within ompR was confirmed by Souther n hybridization and sequencing of the DNA fragments surrounding the de leted regions by PCR. The S. typhi ompR mutants displayed a marked dec rease in OmpC and OmpF porin expression as demonstrated by examination of outer membrane preparations. It was also found that S. typhi strai ns harboring the defined ompR deletions no longer agglutinated with Vi antiserum. However, when a functional ompB operon was introduced back into the S. typhi ompR mutants, either on a multicopy plasmid or as a single-copy chromosomal replacement, the Vi(+) phenotype was restored . The levels of Vi synthesis were also found to be sensitive to differ ent concentrations of sodium chloride present in the growth medium, al though the levels of sensitivity varied between different isolates of S. typhi. It is therefore concluded that the ompR-envZ two component r egulatory system plays an important role in the regulation of Vi polys accharide synthesis in S. typhi and that one of the environmental sign als for this regulation may be osmolarity.