Isolation of a tetracycline-resistance plasmid excised from a chromosomal DNA sequence in Bacillus subtilis

When Bacillus subtilis GSY908 (recE4-) (H. C. Spatz and T. A. Trautner, 1971, Mol. Gen. Genet. 113, 174-190) protoplasts were infected with Staphylococcus aureus plasmid pNS1 specifying tetracycline resistance (Tcr) (N. Noguchi et al., 1983, Gene 21, 105-112), which was modified such that it either could not replicate or did not carry a functional Tcr gene, a plasmid with a molecular weight of 3.1 X 10(6) (4.9 kb) was generated in Tcr phenotypes. This plasmid, named Tcr pNS1981, exhibited completely different restriction endonuclease cleavage patterns to pNS1 and showed only negligible sequence homology in hybridization experiments. Southern hybridization experiments revealed that pNS1981 arises by excision of a B. subtilis chromosomal DNA sequence. No sequence corresponding to pNS1 was detectable on the chromosome of pNS1981-maintaining B. subtilis. The production of pNS1981 was also observed in B. subtilis RM125 (r-Mm-Mrec+) (T. Uozumi et al., 1977, Mol. Gen. Genet. 152, 65-69.) with almost the same frequency as B. subtilis GSY908. Since the recipient B. subtilis Marburg 168 derivatives stated above are sensitive to Tc, the results indicate that information essential for Tcr is under negative regulatory control in the integrated state on the chromosome. Restriction endonuclease analysis suggested that pNS1981 is essentially the same as pBC16, formerly found in B. cereus (K. Bernhard, H. Schrempf, and W. Goebel, 1978, J. Bacteriol. 133, 897-903).