PhoP/Q regulated genes inSalmonella typhi: identification of melittin sensitive mutants

Many of the genes (pags (phoPactivatedgenes) andprgs (phoPrepressedgenes) ) regulated by the PhoP and PhoQ proteins (PhoP/Q) are necessary for survival ofSalmonella typhimuriumin murine macrophages and pathogenesis in mice. Although a great deal is known about theS. typhimuriumphoP/Qregulon, little has been done with the human specific pathogenS. typhi, prompting us to investigateS. typhiphoP/Qregulated genes. IsogenicphoP12(null) andphoP24(constitutive) strains were constructed inS. typhiTy2 andS. typhimuriumC5 strains. Comparison of whole cell proteins from these strains by SDS-PAGE showed differences in both the number and molecular mass of PhoP/Q regulated proteins. This suggested thatS. typhiandS. typhimuriummay have different PhoP/Q regulated proteins and/or that their regulation may be different. A genetic procedure was developed to isolate mutations in PhoP/Q regulated genes. This involved random MudJ transposon mutagenesis of aphoP12mutant, creatinglacZ-gene fusions, and screening for Lac⁺ or Lac^- colonies. A mobilizable plasmid carrying thephoP24mutant gene was conjugated into these insertion mutants. Those that changed from Lac^- to Lac⁺ were inferred to bepag::MudJ insertions and those that changed from Lac⁺ to Lac^- were inferred to beprg::MudJ insertions. Five mutants with PhoP/Q regulated MudJ fusions were found by this scheme. The mutations were termedpqa(PhoPQ activated) andpqr(PhoPQ repressed) to distinguish them from other PhoP/Q regulated genes. Thepqa/pqr::MudJ mutations were transduced intoS. typhiphoP⁺ andphoP24strains by Vi-I phage transduction. Characterization of the mutants (Southern blot analysis, β-galactosidase activity on indicator plates and in liquid cultures) strongly suggested that their MudJ insertion mutations were in five different genes. Further characterization involved determining cationic peptide sensitivity and mouse virulence. Two mutants were found to be sensitive to the antimicrobial peptide melittin.