Deletion mutants from R plasmids with increased copy number

Rms 201-12 and Rms 201-46 are R mutants with increased copy number, and are derived from a conjugative plasmid Rms 201 that encodes resistance to five drugs, ampicillin (Apc), tetracycline (Tc), chloramphenicol (Cm), streptomycin (Sm), and sulfonamides (Sa). The mutants expressed increased levels of resistance to Apc, Sm, and Cm, and a decreased level of resistance to Tc than those of the parent Rms 201 plasmid. When the Rms 201-12+ or Rms 201-46+ cells were inoculated onto plates containing a high concentration of Tc, colonies developed on the plate at a frequency of 10-3 to 10-4 after overnight incubation. The cells grown on the Tc plate carried a tet (Tc gene)-deleted R mutant besides tet-possessing Rms 201-12 or Rms 201-46, and we isolated the tet-deleted R mutant by purifying the R+ cells on drug-free plates. On the other hand, various deletion mutants possessing tet were isolated by prolonged culture of the cells. We have presented a circular gene order of Rms 201 by comparing the genetic markers of all deletion mutants derived from Rms 201, Rms 201-46, and Rms 201-12. The gene(s) regulating the copy number was closely linked to the rep gene. The gene(s) specifying entry exclusion was jointly lost with the tra region.