Parallel Evolution in Predatory sp. NC01 during Long-Term Coculture with a Single Prey Strain.

Experimental evolution provides a powerful tool for examining how evolves in response to unique selective pressures associated with its predatory lifestyle. We tested how sp. NC01 adapts to long-term coculture with Pseudomonas sp. NC02, which is less susceptible to predation compared to other Gram-negative bacteria. Analyzing six replicate populations across six time points spanning 40 passages and 2,880 h of coculture, we detected 30 to 40 new mutations in each population that exceeded a frequency of 5%. Nonsynonymous substitutions were the most abundant type of new mutation, followed by small indels and synonymous substitutions. After completing the final passage, we detected 20 high-frequency (>75%) mutations across all six evolved populations. Eighteen of these alter protein sequences, and most increased in frequency rapidly. Four genes acquired a high-frequency mutation in two or more evolved populations, reflecting parallel evolution and positive selection. The genes encode a sodium/phosphate cotransporter family protein (Bd2221), a metallophosphoesterase (Bd0054), a TonB family protein (Bd0396), and a hypothetical protein (Bd1601). Tested prey range and predation efficiency phenotypes did not differ significantly between evolved populations and the ancestor; however, all six evolved populations demonstrated enhanced starvation survival compared to the ancestor. These results suggest that, instead of evolving improved killing of Pseudomonas sp. NC02, evolved to better withstand nutrient limitation in the presence of this prey strain. The mutations identified here point to genes and functions that may be important for adaptation to the different selective pressures of long-term coculture with Pseudomonas. attack and kill Gram-negative bacteria, including drug-resistant pathogens of animals and plants. This lifestyle is unusual among bacteria, and it imposes unique selective pressures on . Determining how evolve in response to these pressures is valuable for understanding the mechanisms that govern predation. We applied experimental evolution to test how sp. NC01 evolved in response to long-term coculture with a single Pseudomonas strain, which NC01 can kill, but with low efficiency. Our experimental design imposed different selective pressures on the predatory bacteria and tracked the evolutionary trajectories of replicate populations. Using genome sequencing, we identified genes that acquired high-frequency mutations in two or more populations. Using phenotype assays, we determined that evolved populations did not improve their ability to kill Pseudomonas, but rather are better able to survive starvation. Overall, our results point to functions that may be important for adaptation.