Characterization of gram-positive broad host-range plasmids carrying a thermophilic replicon

The cryptic plasmid pBC1 (1.6 kb) isolated from Bacillus coagulans Zu1961 was genetically marked with the genes for chloramphenicol and ampicillin resistance (CmR and ApR) from the Escherichia coli plasmid pJH101. The recombinant vector obtained (pCP49, 7.0 kb) replicated and expressed CmR in B. subtilis and CmR and ApR in E. coli. Different shuttle vectors for Gram+ bacteria were also constructed by inserting pBC1 into the Staphylococcus aureus plasmid pC194. The smallest of these, pLM6 (2.8 kb), containing essentially pBC1 and the chloramphenicol acetyl transferase gene from pC194, replicated in B. subtilis at a copy number of 60. By electroporation, these plasmids were introduced and stably maintained in B. subtilis, B. amyloliquefaciens, S. aureus, S. carnosus and Lactobacillus reuteri.