A Cloning Vector Derived From an Indigenous Small Plasmid of Rhizobium Meliloti - Construction and Mobilization Into the Rhizobiaceae

Detection and isolation of a small (MW ~ 7.3 kb) cryptic plasmid from a strain of Rhizobium meliloti (M19S) opened up the possibility of using it as a cloning vector for studies on bacterial genes involved in symbiotic nitrogen fixation. Restriction endonuclease analyses revealed that pRme 19a contains five cleavage sites for ClaI, two for Bc1I and an unique site for Sa1I. The latter allowed the in vitro insertion of pRme 19a into the narrow-host-range cloning vector pBR322 (1). The resulting ampicillin-resistance-conferring hybrid plasmid (pBRRM 1) was introduced by transformation into Escherichia coli strain C600 where it replicated stably and was amplifiable by chloramphenicol. pBRRM 1 was then found to exhibit two unexpected properties: it was mobilized efficiently by pRK2013 (2) in triparental matings, it coded for a kanamycine resistance.