Use of transposon Tn916 to inactivate and isolate a mutacin-associated gene from Streptococcus mutans

Abstract

Among the attributes thought to contribute to the virulence of Streptococcus mutans is its ability to elaborate bacteriocinlike substances, which may provide a selective force enhancing its colonization potential. One such inhibitory substance, mutacin II, is produced by certain plasmid-containing strains of S. mutans. We introduced insertional mutations into a mutacin II-producing strain of S. mutans (UA96) by transformation with a plasmid carrying Tn916, resulting in transformants bearing single inserts of the transposon at different sites within the chromosome. The insertions identify five different EcoRI fragments required for production of mutacin II (Bac phenotype; bac-1 to bac-5 genotypes). The EcoRI fragments, containing bac-1::Tn916 was ligated into a cosmid vector, pJC74, and transduced into Escherichia coli DH1, where Tn916 is known to be unstable. The loss of Tn916 resulted in a 30-kb plasmid, pPC974, containing approximately 15 kb of S. mutans DNA. A Bac-associated DNA fragment was then subcloned into the streptococcus-E. coli shuttle vector pVA838 and transformed into S. mutants, where it was capable of complementing the bac mutation in the Bac- parent. These findings suggest that we have isolated at least one gene associated with mutacin production.