Construction of shuttle vector plasmid between Clostridium acetobutylicum and Escherichia coli

A high copy number plasmid pCS86 (3.0 kb) was isolated from Clostridium acetobutylicum strain No. 86. For developing the vector plasmid for gene cloning of saccharolytic clostridia, two hybrid plasmids were constructed by joining pCS86 to the E. coli promoter probe vector pKK232-8 carrying the promoterless gene of chloramphenicol acetyltransferase (CAT). One of the hybrid plasmids designated as pTY10 replicated and expressed the CAT gene in E. coli, and transformed a plasmid-free strain, C. acetobutylicum strain No. 220, as a shuttle vector. This is the first shuttle vector constructed from C. acetobutylicum and E. coli plasmids. Reciprocal transformation was possible between E. coli and C. acetobutylicum, though deletion occurred in the sequence originated from pKK232-8. A deleted plasmid pTY101 only replicated in Clostridium, though it showed a high copy number.