Abstract
Gene amplification mutations are strong contributors to the formation of cancer cells and building resistance to antibiotics in many pathogens, however the factors regulating their formation and their underlying mechanisms are widely unknown. Recent studies suggest gene amplification mutations are induced by environmental stress imposed by growth limiting conditions. In contrast, an alternative theory suggests these mutations spontaneously occur without stress induction. In this study, both theories were tested in the genetically tractable bacteria Acinetobacter baylyi using a model system where amplification mutants are exclusively selected and continually accumulate under prolonged growth-limiting selective conditions in benzoate minimum media. To test whether the stress caused by this growth-limiting selective environment causes the formation of these gene amplification mutations two independent tests were conducted: reconstruction experiments and replica plating assays. The results from these tests indicate amplification is generated through a multi-step process, whereby low-copy amplification precursor cells formed before selection was imposed. During selection, these low-copy mutants grow slowly, but outcompete the haploid population. This growth provides a subpopulation of cells favored by selection, further driving multiple secondary steps to higher amplification, generating high-copy amplification cells able to form visible colonies without stress induction. Thus, a natural selection process can explain the formation of amplification within the Acinetobacter baylyi system rather than a stress induced mechanism. Additionally, the mutation frequency of these low-copy precursor cells in unselected populations was estimated to be 10-6, which is higher than the estimated mutation frequency of 10-8 for high-copy mutant colonies under selective conditions within a two-week period. Therefore, this suggests there are a sufficient number of precursors in the unselected population to account for the high-copy mutants that arise under selective conditions further indicating that stressful environments do not induce gene amplification.