Tetracycline tolerance mediated by gene amplification in Bacillus subtilis

Bacillus subtilis can acquire a higher tolerance to tetracycline by increasing the gene dosage of its resistance gene tetB. In this study, we estimated the multiplication effect of tetB on tetracycline tolerance. Cells harbouring multiple copies of tetB were found to comprise approximately 30 % of the total tetracycline-resistant cell population when selected on medium containing 10 µg tetracycline ml(-1). Disruption of recA resulted in a significant decrease in the frequency of tetB amplification. Although four direct repeats exist around tetB, the majority of tetB amplicons were found to be flanked by non-homologous sequences, indicating that the initial duplication of tetB can occur largely through RecA-independent recombination. The correlation between the tetB copy number and the MIC values for tetracycline indicated that more than three copies of tetB were required for tolerance to 10 µg tetracycline ml(-1). Thus, the RecA-dependent expansion step appears to be necessary for developing significant tetracycline tolerance mediated by tetB amplification.