A MEDIUM-COPY-NUMBER PLASMID FOR INSERTIONAL MUTAGENESIS OF STREPTOCOCCUS-MUTANS

We have constructed a plasmid useful for insertional mutagenesis in St reptococcus mutans. The molecule, pSU20Erm, is based on a derivative o f pACYC184 known as pSU20. The plasmid described here is approximately 3.7 kb in size and has the following properties: it replicates in Esc herichia coil, does not replicate in S, mutans, contains an erythromyc in-resistance marker which can be selected in E. coli or the streptoco cci, contains a multiple cloning site with few restriction sites in th e remainder of the molecule, and can be screened on X-Gal-containing m edium for the presence of insertions into the multiple cloning site. W e 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 c hromosome via homologous recombination, thereby interrupting native ge nes. The plasmid has been used to clone part of a homologue of the E. coil 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. (C) 1998 Academic Press.