Leveraging adaptive evolution to diagnose and combat genomic instability in Acinetobacter baylyi ADP1

Assessing evolutionary stability is an important Test parameter in the Design-Build-Test cycle for biological engineering. Adaptive long-term evolution (ALE) experiments can provide vital parameters regarding evolutionary stability, garnering information about mutational processes, selection pressures, and other factors that can ‘break’ strains. Such experiments can provide actionable insights to improve stain stability and functionality. Here, we studied Acinetobacter baylyi ADP1, a naturally transformable bacterium that has been found to lose its capacity for DNA uptake during laboratory culture. We conducted a 1000 generation ALE experiment with ADP1 and leveraged next generation sequencing (NGS) along with phenotypic characterizations to determine how competence was lost and to characterize overall genomic instability. NGS revealed that the sole mobile genetic element in ADP1, IS1236, was responsible for the majority of mutations in many evolved clones and was the primary genetic driver behind competence loss. Using this insight, we created an IS-less strain of ADP1 (ISx), which we found to have markedly increased genetic stability as well as other beneficial strain characteristics. Unexpectedly, we also discovered the emergence of a novel filamentous competence reducing Acinetobacter phage (CRAϕ) from the ADP1 genome during the evolution experiment. Loss of competence mutations in ADP1 were found to provide resistance to CRAϕ, indicating that this phage likely drives the selection towards reduced competence in ADP1 populations. In total, our studies demonstrate the utility of ALE in diagnosing the forces driving strain instability and show how these insights can guide genome engineering efforts to prevent undesirable evolution.