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 × 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.