Adaptation to Overflow Metabolism by Mutations That Impair tRNA Modification in Experimentally Evolved Bacteria

When microbes grow in foreign nutritional environments, selection may enrich mutations in unexpected pathways connecting growth and homeostasis. An evolution experiment designed to identify beneficial mutations in Burkholderia cenocepacia captured six independent nonsynonymous substitutions in the essential gene tilS, which modifies tRNA(Ile2) by adding a lysine to the anticodon for faithful AUA recognition. Further, five additional mutants acquired mutations in tRNA(Ile2), which strongly suggests that disrupting the TilS-tRNA(Ile2) interaction was subject to strong positive selection. Mutated TilS incurred greatly reduced enzymatic function but retained capacity for tRNA(Ile2) binding. However, both mutant sets outcompeted the wild type by decreasing the lag phase duration by ~3.5 h. We hypothesized that lysine demand could underlie fitness in the experimental conditions. As predicted, supplemental lysine complemented the ancestral fitness deficit, but so did the additions of several other amino acids. Mutant fitness advantages were also specific to rapid growth on galactose using oxidative overflow metabolism that generates redox imbalance, not resources favoring more balanced metabolism. Remarkably, 13 tilS mutations also evolved in the long-term evolution experiment with Escherichia coli, including four fixed mutations. These results suggest that TilS or unknown binding partners contribute to improved growth under conditions of rapid sugar oxidation at the predicted expense of translational accuracy.