Chromosomal-DNA amplification in Bacillus subtilis

Abstract

Tetracycline-resistant (Tetr) mutants RAD1, RAD2, RAD6, and RAD7 were isolated from Bacillus subtilis BC92 after protoplasting, polyethylene glycol treatment, and regeneration on a medium containing tetracycline. The Tetr phenotype in RAD1, RAD2, and RAD6 was very stable with less than 5% loss of resistance after 30 generations of growth in the absence of selection. Of the four isolates, three contained amplified chromosomal DNA closely associated with the Tetr phenotype. The intensity of restriction fragments present in HindIII and EcoRI digests of chromosomal DNA from RAD1, RAD6, and RAD7 indicated the presence of tandemly duplicated DNA. Disparity in the size and number of amplified fragments suggested that the tandemly duplicated DNA is different in all three isolates. The sizes of the duplicated DNA present in RAD1, RAD6, and RAD7 were estimated to be 10, 19, and 20 kilobases, respectively. No amplified DNA was detected in RAD2. Results of transductional-mapping studies with PBS1 showed that the tetracycline resistance (tet) loci of RAD1, RAD2, and RAD6 all mapped near the origin of chromosomal replication and close to the guaA locus. Amplified DNA characteristic of RAD1 and RAD6 was cotransduced with the tet locus. Cotransfer of amplified DNA with the guaA locus or other nearby loci in the absence of tet was not observed. In every case, loss of Tetr was accompanied by loss of amplified DNA. A possible explanation for the occurrence of the amplified DNA is presented.