TRANSPOSON TN5 MUTAGENESIS OF ACTINOBACILLUS-ACTINOMYCETEMCOMITANS VIA CONJUGATION

To facilitate molecular genetic studies on the virulence of the periodontopathogen Actinobacillus actinomycetemcomitans, we have extended the use of Tn5 transposon mutagenesis to this organism. A suicide plasmid was used to deliver a spectinomycin-resistant Tn5 derivative [mini-Tn5(Sp)] via conjugation from Escherichia coli to A. actinomycetemcomitans. Spectinomycin-resistant exconjugants were found at a frequency of about 1 x 10(-7) per recipient cell. Southern blot analysis showed that the transposon had moved into the A. actinomycetemcomitans genome, and each exconjugant had a transposon at a single location. Importantly, the mini-Tn5(Sp) appeared to transpose into relatively random sites: there were 32 different sites of integration among the 33 exconjugants examined by Southern blot analysis. Finally, the mini-Tn5(Sp) was stably integrated, since all of the exconjugants were still spectinomycin-resistant after 7 passages on non-selective media. Thus, the mini-Tn5(Sp) system is a useful tool for the mutagenesis of A. actinomycetemcomitans in studies of its virulence factors and their regulation.