In vitro transposition as a tool for mutagenesis in viridans streptococci

A cornerstone of bacterial genetics has long been the creation of random mutations. Several transposons have been employed for this purpose in gram-negative enteric bacteria, but species-specific differences in plasmid replication and transposition activity have hindered the use of these transposons in the oral streptococci. These impediments could be avoided by employing in vitro transposition. Objectives: To develop in vitro transposition as a tool for random mutagenesis in the viridans streptococci. Methods: The pEMCat plasmid carrying a mini-transposon of the mariner family was obtained from Andrew Camilli at Tufts University, and a plasmid containing a modified transposase gene was obtained from David Lampe at Duquesne University. The bla gene was removed from pEMCat to avoid introduction of beta lactam resistance into viridans streptococci. A second version of the transposon was also created by replacing the transposon-borne chloramphenicol resistance (cat) gene with the aphA3 gene encoding resistance to kanamycin. Both versions were evaluated for suitability in creating random mutations in the naturally transformable species, Streptococcus sanguis. Results: Both transposons were used for in vitro transposition with chromosomal DNA as a target. Following transformation, which allows for introduction of transposons into the genome by homologous recombination of flanking sequences, both transposons produced similar numbers of antibiotic-resistant mutants. Insertions created by the cat-containing transposon were characterized by Southern blot analysis and no evidence of insertion site bias was obtained. Similar results were found when the target for mutagenesis was a 2-kb PCR amplicon. There was also no evidence of insertion instability when mutants were passaged without antibiotic selection in broth culture or in an animal model of infective endocarditis. Conclusions: In vitro transposition can be used to create random and stable mutations in Streptococcus sanguis and potentially other transformable viridans streptococci. Supported by AHA grant B98466V and NIH grant AI47841.