Identification of Restriction Fragments from Two Cryptic Clostridium butyricum Plasmids That Promote the Establishment of a Replication-defective Plasmid in Bacillus subtilis

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Abstract
C. butyricum NCIB 7423 carries 2 cryptic plasmids, pCB101 (6-05 kbp) and pCB102 (7.8 kbp [kilobase]). Sites for the restriction enzymes EcoRI, EcoRV, HindIII, ClaI and PstI were found in 1 or both of these plasmids and their relative positions determined. Restriction fragments from both plasmids were inserted into a vector plasmid (pJAB1) that is able to replicate is Escherichia coli but not in B. subtilis and the recombinant plasmids were established in E. coli. A 3.3 kbp Sau3A fragment of pCB101 conferred upon the vector the ability to transform both Rec+ and Rec- strains of B. subtilis. Plasmid pRB1, a representative chimera carrying only the 3.3 kbp Sau3A fragment of pCB101, was successfully transferred from B. subtilis back to E. coli. Plasmid pRB1 was readily lost from B. subtilis in the absence of selection. This evidence, together with the results of hybridization experiments, suggests that pRB1 is present as a weakly replicating autonomous element in B. subtilis. A recombinant plasmid carrying a 2.0 kpb Sau3A fragment of pCB102 underwent integration into the B. subtilis chromosome.