AMPLIFICATION OF A CHROMOSOMAL REGION IN BACILLUS-SUBTILIS

The amplification in B. subtilis of a defined DNA sequence after exposure of the bacteria to increasing levels of antibiotic is reported. The experimental system consisted of transformation of competent cells with a plasmid (pRHA39) unable to replicate in the host and carrying the .alpha.-amylase gene derived from B. subtilis. Selection of transformants resistant to 5 .mu.g of chloramphenicol/ml resulted in the isolation of strains with the plasmid integrated into the chromosome at the site of homology, by a Campbell type mechanism. Starting from such a nontandem duplication, amplification was achieved by growing the bacteria in increasing concentrations of chloramphenicol. By dilution, Southern blotting and hybridization to a radioactive probe, one estimated a copy number of .apprx. 10 for the amplified sequence of samples grown in the presence of 50 .mu.g of chloramphenicol/ml. No free plasmid could be detected in the amplified strains. The extent of the amplified region was the same for all transformants and the endpoints appeared to be the same in all isolates. As a consequence of the amplification, there was a noticeable increase in amylase production and the amount of enzyme produced correlated with gene dosage. The amplification did not occur in a recE genetic background.