Evolution of streamlined genome variants of Acinetobacter baylyi ADP1

Organelles and endosymbionts have evolved dramatically reduced genome sizes compared to their free-living ancestors. Synthetic biologists have engineered streamlined bacterial genomes to create more efficient cellular chassis and to define the minimal components of cellular life. Deletions of many non-essential genes in combination during natural or engineered genome streamlining often reduces bacterial fitness for idiosyncratic or unknown reasons. We investigated how and to what extent laboratory evolution could overcome these defects in six variants of the transposon-free Acinetobacter baylyi strain ADP1-ISx that each had a different 20-40 kb deletion. We evolved replicate populations of ADP1-ISx and each reduced-genome strain for ~300 generations in either minimal medium or rich medium and sequenced the genomes of endpoint clonal isolates. Mutations in three genes and a large (>30 kb) duplication were associated with ancestors that had specific deletions. Post-transcriptional regulators, including rnd (RNase D), csrA (RNA-binding carbon storage regulator), and hfq (RNA-binding protein and chaperone), were frequently mutated in all evolved strains, though the effects of these mutations on gene function and fitness varied across genetic backgrounds and environments. Mutations in this regulatory network seem to compensate for how deletion of a transposon in the ADP1-ISx ancestor restored wild-type csrA function, highlighting an unexpected interaction between genome streamlining and prior evolution that likely occurred during laboratory domestication. More generally, our results demonstrate that fitness lost during genome streamlining can usually be regained rapidly through laboratory evolution.