Short tandem gene duplications as potential agents of genetic memory

Tandem gene duplications are spontaneous mutations that occur frequently but whose significance is often overlooked because they are genetically unstable and can be difficult to detect by sequencing. Here, we studied the spontaneous loss, amplification, and phenotypic effects of engineered duplications giving rise to antibiotic resistance in <i>Pseudomonas aeruginosa</i>. The duplications, ranging from 2.6 to 497 kb in length, were constructed using phage lambda <i>red</i> recombination of PCR products and carry resistance markers at their novel junctions. The genetic behaviors of the duplications depended on segment length: long duplications were readily lost during growth without selection and showed limited amplification potential, whereas short duplications were lost slowly and could amplify to high segment copy numbers. The shortest duplications analyzed (2.6 and 3.3 kb) persisted during extended growth without selection and are predicted to be stable for thousands of generations. Following growth without selection, the duplications retained their ability to amplify and generate extreme antibiotic resistance. The remarkable stability of short duplications combined with their capacity to amplify suggests that they can regenerate gene amplifications which were previously selected and then lost, a novel form of genetic memory. This feature could be particularly significant in adaptation to intermittent stresses, such as during cycled antibiotic treatment of chronic infection.IMPORTANCEUnstable mutations, such as tandem gene duplications, contribute significantly to the evolution of antibiotic resistance and other traits, but their importance is often underestimated. In this study, we characterized duplication genetic behavior in the ESKAPE pathogen <i>Pseudomonas aeruginosa</i>. The work helps define the genetic behavior of duplications in <i>P. aeruginosa</i> and provides a framework for evaluating their potential roles in the organism's well-known capacity to adapt to new environments.