Small plasmids were isolated from type strains of Clostridium butyricum. Strain NCIB 7423 carries one plasmid (pCBU1) of 6.4 kb, whereas strain NCTC 7423 carries two unrelated plasmids of 6.3 kb (pCBU2) and 8.4 kb (pCBU3). Cleavage sites for 18 restriction endonucleases have been mapped on these plasmids and detailed physical maps are presented. For the purpose of developing vector plasmids for gene cloning in saccharolytic clostridia these cryptic C. butyricurn plasmids were joined to a selectable marker that will likely be expressed in clostridia. This was achieved by cloning the clostridial plasmids into the E. coli vector pBR322 carrying the chloramphenicol acetyltransferase (CAT) gene from the Staphylococcus aureus plasmid pC194. The recombinant plasmids were tested for their ability to confer chloramphenicol resistance to Bacillus subtilis. Hybrid plasmids (pilL105, pilL1051) derived from pCBU2 were identified, which are capable of replication and expression of the S. aureus drug resistance marker in both E. coli and B. subtilis. No structural instability was detected upon retransformation of pilL105 from B. subtilis into E. coli. The recombinant plasmids might thus be useful as shuttle vectors for the gene transfer between E. coli and a wide range of bacilli and clostridia.
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