Virulence gene deletion frequency is increased in Shigella flexneri following conjugation, transduction, and...

Some commonly used methods for introducing DNA provoke spontaneous loss of expression of a virulence gene located on the high molecular mass plasmid in Shigella flexneri. The introduction of plasmid DNA by calcium chloride-mediated transformation in strains harbouring wild-type or mutated copies of regulatory genes resulted in the loss of expression of an mxiC-lacZ reporter gene at high frequency, approaching 100% in some cases. Lac- segregants arose whether or not the introduced plasmids harboured S. flexneri virulence gene sequences. Conjugation and generalised transduction with bacteriophage P1 were also found to provoke the appearance of Lac- mutants at high frequency. The Lac- mutants described in this report had deletions of the regulatory genes virF or virB, or more extensive deletions which included structural genes. The frequency of mutation was greatly reduced when electroporation was used to introduce DNA into the strains used in this study, suggesting that this is the best method to use when transforming them.