Chimeric streptococcal plasmids and their use as molecular cloning vehicles in Streptococcus sanguis (Challis)

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Abstract
Chimeric plasmids, which were useful as cloning vehicles in a S. sanguis (Challis) host vector system, were constructed. Using 3 different strategies of restriction endonuclease digestion and ligation, a DNA fragment bearing an erythromycin resistance determinant was ligated in vitro to a phenotypically cryptic plasmid from S. ferus. Recombinant plasmids could be recovered after transformation of S. sanguis (Challis) with these preparations. Three useful chimeras were constructed. pVA680, 5.5 megadaltons in size, contained a single KpnI site into which passenger DNA may be spliced. pVA736, 5.0 megadaltons in size, contained single EcoRI, HindIII and KpnI sites into which passenger DNA may be spliced. The EcoRI and KpnI sites of pVA736 may be used in combination with one another when ligating DNA into this plasmid. pVA738, 3.7 megadaltons in size, contained single HindIII and AvaI sites into which passenger DNA may be spliced. pVA680, pVA736 and pVA738 were stably maintained as multicopy plasmids in S. sanguis (Challis). None of them continued to replicate (amplify) in chloramphenicol-treated cells. Using pVA736 as a vector, a chloramphenicol resistance determinant obtained from a large, conjugative streptococcal R plasmid was cloned. Chromosomal DNA sequences from S. mutans were inserted into pVA736 using the KpnI-EcoRI site combination.