Abstract
Gene amplification mutations enlarge the genome by adding extra gene copies to the genome to enhance gene expression. The increased expression can be found in antibiotic resistant microbes and cancer cells, but the formation of these mutations is not fully known in any system. To study the processes of gene amplification formation, the Ben reversion system in Acinetobacter baylyi was used because it exclusively selects gene amplification mutants when cells are introduced to minimum benzoate media. Two theories are proposed to explain the origins of these amplification mutants: growth inhibition (stress) induced or pre-existing (non-stress induced). To verify either of these theories are plausible explanations, lawn growth measurements were performed to test whether mutant accumulation on selective media could be explained by a fixed mutation rate during residual growth of non-mutant cells. This study found lawn growth could not explain the amplification mutant colony accumulation rate, therefore, allowing the debate to continue. To further test these two theories, mutants were screened via replica plating assays to identify amplification precursor (intermediate) mutants during non-selective growth conditions. This study found the presence of precursor mutants before selection at frequencies higher than measured amplification mutant frequencies during selection, thereby supporting the pre-existing theory and gene copy accumulation occurring through multiple steps driven by natural selection. Genomic analysis performed by Pulse-Field Gel Electrophoresis of precursors, lawn revertant, and full revertant mutants indicated high-copy amplification mutants are derived from intermediate mutants carrying relatively few copies of large amplicons. Consistent with a multi-step process, these intermediate large amplicons must remodel to decrease amplicon size, thereby reducing their fitness cost, and allowing cells to selectively gain higher amplicon copy numbers.