Efficient insertional mutagenesis in group A streptococci mediated by Tn917 transposon

The replication-conditional thermosensitive vector pTV1-OK (repATs, Kan^r) harbouring the transposon Tn917 (Em^r) was successfully used to mutagenise a clinical Streptococcus pyogenes isolate (CS101). In the initial studies, conditions were established for electrotransformation of the pTV1-OK vector into CS101. Transformants selected on media containing Kan at 29°C were shown to become Kan^s at 39°C and to carry the transposon-linked Em^r marker. One such transformant was chosen for transposition studies. Upon temperature shift, transposition was achieved with a frequency of approximately 0.01% with a plasmid curing efficiency of close to 100%. Southern blot analysis demonstrated that the majority of mutants contained a single copy of Tn917 and showed no evidence for preferential sites of integration (“hot spots”). Screening of Tn917 libraries of S. pyogenes has led to the identification of mutants lacking either haemolysing or plasminogen activating activity. Mutants defective in each of these activities were identified at a frequency of approximately one in 1000 to 4000 colonies. These findings suggest that the pTV1-OK vector can be used for transposon mutagenesis of S. pyogenes and that this strategy will be valuable for identifying virulence factors and regulatory mechanisms in these bacteria.