A Medium-Copy-Number Plasmid for Insertional Mutagenesis ofStreptococcus mutans

We have constructed a plasmid useful for insertional mutagenesis in Streptococcus mutans. The molecule, pSU20Erm, is based on a derivative of pACYC184 known as pSU20. The plasmid described here is approximately 3.7 kb in size and has the following properties: it replicates in Escherichia coli, does not replicate in S. mutans, contains an erythromycin-resistance marker which can be selected in E. coli or the streptococci, contains a multiple cloning site with few restriction sites in the remainder of the molecule, and can be screened on X-Gal-containing medium for the presence of insertions into the multiple cloning site. We have used the plasmid to construct a library of S. mutans DNA in E. coli and show that the clones can be reintegrated into the S. mutans chromosome via homologous recombination, thereby interrupting native genes. The plasmid has been used to clone part of a homologue of the E. coli drpA gene, encoding a global regulatory element for RNA synthesis. Further, we have identified an element closely linked to drpA in S. mutans with high homology to IS861.