A GENERAL-METHOD FOR THE CONSECUTIVE INTEGRATION OF SINGLE COPIES OF A HETEROLOGOUS GENE AT MULTIPLE LOCATIONS IN THE BACILLUS-SUBTILIS CHROMOSOME BY REPLACEMENT RECOMBINATION

We have devised a two-step procedure by which multiple copies of a het erologous gene can be consecutively integrated into the Bacillus subti lis 168 chromosome without the simultaneous integration of markers (an tibiotic resistance). The procedure employs the high level of transfor mability of B. subtilis 168 strains and makes use of the observation t hat thymine-auxotrophic mutants of B. subtilis are resistant to the fo lic acid antagonist trimethoprim (Tmp(r)), whereas thymine prototrophs are sensitive. First, a thymine-auxotrophic B. subtilis mutant is tra nsformed to prototrophy by integration of a thymidylate synthetase enc oding gene at the desired chromosomal locus. In a second step, the mut ant strain is transformed with a DNA fragment carrying the heterologou s gene and Tmp(r) colonies are selected. Approximately 5% of these app ear to be thymine auxotrophic and contain a single copy of the heterol ogous gene at the chromosomal locus previously carrying the thymidylat e synthetase-encoding gene. Repetition of the procedure at different l ocations on the bacterial chromosome allows the isolation of strains c arrying multiple copies of the heterologous gene. The method was used to construct B. subtilis strains carrying one, two, and three copies o f the Bacillus stearothermophilus branching enzyme gene (glgB) in thei r genomes.