Isolation and characterization of antibiotic-resistance plasmids in thermophilic bacilli

Four antibiotic-resistance plasmids isolated from thermophilic bacilli were characterized in detail. Three tetracycline-resistance (Tc1) plasmids were designated as pTHT9 (7.7 kilobases (kb], pTHT15 (4.5 kb) and pTHT22 (8.4 kb). From the results of restriction endonuclease analysis and the subsequent Southern hybridization, these were found to possess extensive genetic homology in the regions that include the replication origin and the Tcr gene. Detailed restriction maps of the smallest Tcr plasmid pTHT15 and a kanamycin-resistance (Kmr) plasmid pTHN1 (4.8 kb) were constructed. The positions of antibiotic-resistance loci and regions essential for plasmid replication were determined by cloning plasmid fragments in Bacillus subtilis. These four plasmids were found to replicate and express the resistance genes stably in both B. subtilis and B. stearothermophilus.