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 tRNAIle2 by adding a lysine to the anticodon for faithful AUA recognition. Further, five additional mutants acquired mutations in tRNAIle2, which strongly suggests that disrupting the TilS-tRNAIle2 interaction was subject to strong positive selection. Mutated TilS incurred greatly reduced enzymatic function but retained capacity for tRNAIle2 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. IMPORTANCE There is growing evidence that the fundamental components of protein translation can play multiple roles in maintaining cellular homeostasis. Enzymes that interact with transfer RNAs not only ensure faithful decoding of the genetic code but also help signal the metabolic state by reacting to imbalances in essential building blocks like free amino acids and cofactors. Here, we present evidence of a secondary function for the essential enzyme TilS, whose only prior known function is to modify tRNAIle(CAU) to ensure accurate translation. Multiple nonsynonymous substitutions in tilS, as well as its cognate tRNA, were selected in evolution experiments favoring rapid, redox-imbalanced growth. These mutations alone decreased lag phase and created a competitive advantage, but at the expense of most primary enzyme function. These results imply that TilS interacts with other factors related to the timing of exponential growth and that tRNA-modifying enzymes may serve multiple roles in monitoring metabolic health.

Evolved resistance to a novel cationic peptide antibiotic requires high mutation supply.

Background and Objectives: A key strategy for resolving the antibiotic resistance crisis is the development of new drugs with antimicrobial properties. The engineered cationic antimicrobial peptide WLBU2 (also known as PLG0206) is a promising broad-spectrum antimicrobial compound that has completed Phase I clinical studies. It has activity against Gram-negative and Gram-positive bacteria including infections associated with biofilm. No definitive mechanisms of resistance to WLBU2 have been identified. Methodology: Here, we used experimental evolution under different levels of mutation supply and whole genome sequencing (WGS) to detect the genetic pathways and probable mechanisms of resistance to this peptide. We propagated populations of wild-type and hypermutator Pseudomonas aeruginosa in the presence of WLBU2 and performed WGS of evolved populations and clones. Results: Populations that survived WLBU2 treatment acquired a minimum of two mutations, making the acquisition of resistance more difficult than for most antibiotics, which can be tolerated by mutation of a single target. Major targets of resistance to WLBU2 included the orfN and pmrB genes, previously described to confer resistance to other cationic peptides. More surprisingly, mutations that increase aggregation such as the wsp pathway were also selected despite the ability of WLBU2 to kill cells growing in a biofilm. Conclusions and implications: The results show how experimental evolution and WGS can identify genetic targets and actions of new antimicrobial compounds and predict pathways to resistance of new antibiotics in clinical practice.