Genetic analysis of essential plasmid determinants of pathogenicity in Yersinia pestis

The role of the Yersinia pestis virulence-associated plasmid, pYV019, in the expression of Ca++ dependence, virulence, and the production of the V antigen was investigated. Derivatives of bacteriophage P1 were used to deliver the transposon Tn5 into Y pestis strain EV76. Ca++-independent mutants in which transposon Tn5 had been inserted into plasmid pYV019 were isolated, the resulting plasmids--pYV019::Tn5--were transformed into an Escherichia coli K12 derivative, and the site of insertion of transposon Tn5 was mapped with restriction endonucleases. The plasmids were then transduced by bacteriophage P1 into avirulent strain 195-P1 of Y pestis. The transductants were analyzed for expression of Ca++ dependence, virulence in mice, and the expression of the V antigen. Introduction of plasmid pYV019 with insertions outside of the Ca++ dependence loci restored strain 195-P1 to full virulence, while disruption of plasmid genes associated with Ca++ dependence led to loss of virulence. Using Western blotting analysis and E coli minicells, it was shown that genes specific for the V antigen are plasmid encoded.