Molecular cloning of a pIP501 derivative yields a model replicon for the study of streptococcal conjugation

We have previously constructed a derivative of the broad host range streptococcal plasmid pIP501, a conjugative plasmid designated pVA797, that confers chloramphenicol resistance and contains a unique EcoRI site in a non-essential region of the plasmid molecule. pVA797 (30.7 kb) when cloned in toto as an EcoRI fragment into the positive selection vector pOP203(A2+) gave a recombinant, pVA904 (37.7 kb), which was able to replicate in Escherichia coli and in streptococcal species. It can be phenotypically monitored in either genus by specific drug resistance markers (chloramphenicol resistance in streptococci, tetracycline resistance in E. coli). pVA904 segregates into E. coli minicells where it specifies the production of at least 13 polypeptides. Many of the polypeptides are missing in minicells containing a transfer-defective, deletion derivative of pVA904. pVA904 is an ideal model replicon for the study of streptococcal conjugation because it is a shuttle plasmid thus enabling manipulation using procedures established for E. coli. Specifically, it should be possible to define the genetic basis of streptococcal conjugation by coupling mutagenesis protocols and minicell protein analyses in E. coli with evaluation of transfer function in streptococci.