To facilitate molecular genetic studies on the virulence of the periodontopathogenActinobacillus actinomycetemcomitans, we have extended the use ofTn5transposon mutagenesis to this organism. A suicide plasmid was used to deliver a spectinomycin‐resistantTn5derivative [mini‐Tn5(Sp)] via conjugation fromEscherichia colitoA. actinomycetemcomitans.Spectinomycin‐resistant exconjugants were found at a frequency of about 1 × 10‐7per recipient cell. Southern blot analysis showed that the transposon had moved into theA. actinomycetemcomitansgenome, 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 ofA. actinomycetemcomitansin studies of its virulence factors an
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