PHOP Q REGULATED GENES IN SALMONELLA-TYPHI - IDENTIFICATION OF MELITTIN SENSITIVE MUTANTS/

Many of the genes (pags (phoP activated genes) and prgs (phoP represse d genes)) regulated by the PhoP and PhoQ proteins (PhoP/Q) are necessa ry for survival of Salmonella typhimurium in murine macrophages and pa thogenesis in mice. Although a great deal is known about the S. typhim urium phoP/Q regulon, little has been done with the human specific pat hogen S. typhi, prompting us to investigate S. typhi phoP/Q regulated genes. Isogenic phoP12 (null) and phoP24 (constitutive) strains were c onstructed in S. typhi Ty2 and S. typhimurium C5 strains. Comparison o f whole cell proteins from these strains by SDS-PAGE showed difference s in both the number and molecular mass of PhoP/Q regulated proteins. This suggested that S. typhi and S. typhimurium may have different Pho P/Q regulated proteins and/or that their regulation may be different. A genetic procedure was developed to isolate mutations in PhoP/Q regul ated genes. This involved random MudJ transposon mutagenesis of a phoP 12 mutant, creating lacZ-gene fusions, and screening for Lac(+) or Lac (-) colonies. A mobilizable plasmid carrying the phoP24 mutant gene wa s conjugated into these insertion mutants. Those that changed from Lac (-) to Lac(+) were inferred to be pag::MudJ insertions and those that changed from Lac(+) to Lac(-) were inferred to be prg::MudJ insertions . Five mutants with PhoP/Q regulated MudJ fusions were found by this s cheme. The mutations were termed pqa (PhoPQ activated) and pqr (PhoPQ repressed) to distinguish them from other PhoP/Q regulated genes. The pqa/pqr::MudJ mutations were transduced into S. typhi phoP(+) and phoP 24 strains by Vi-I phage transduction. Characterization of the mutants (Southern blot analysis, beta-galactosidase activity on indicator pla tes and in liquid cultures) strongly suggested that their MudJ inserti on mutations were in five different genes. Further characterization in volved determining cationic peptide sensitivity and mouse virulence. T wo mutants were found to be sensitive to the antimicrobial peptide mel ittin. (C) 1997 Academic Press Limited.