VIRULENCE GENE DELETION FREQUENCY IS INCREASED IN SHIGELLA-FLEXNERI FOLLOWING CONJUGATION, TRANSDUCTION, AND TRANSFORMATION

Some commonly used methods for introducing DNA provoke spontaneous los s of expression of a virulence gene located on the high molecular mass plasmid in Shigella flexneri. The introduction of plasmid DNA by calc ium chloride-mediated transformation in strains harbouring wild-type o r mutated copies of regulatory genes resulted in the loss of expressio n of an mxiC-lacZ reporter gene at high frequency, approaching 100% in some cases. Lac(-) segregants arose whether or not the introduced pla smids harboured S. flexneri virulence gene sequences. Conjugation and generalised transduction with bacteriophage P1 were also found to prov oke the appearance of Lac(-) mutants at high frequency. The Lac(-) mut ants described in this report had deletions of the regulatory genes vi rF or virB, or mon extensive deletions which included structural genes . The frequency of mutation was greatly reduced when electroporation w as used to introduce DNA into the strains used in this study, suggesti ng that this is the best method to use when transforming them.