Bacterial evolution in high osmolarity environments

Abstract

Bacteria must maintain a cytosolic osmolarity higher than that of their environment in order to take up water. High osmolarity environments therefore present a formidable stress to bacteria. To explore the evolutionary mechanisms by which bacteria adapt to high osmolarity environments, we selected Escherichia coli in media with a variety of osmolytes and concentrations for 250 generations. Adaptation was osmolyte-dependent, with sorbitol stress generally resulting in increased fitness in conditions with higher osmolarity, while selection in high concentrations of proline resulted in increased fitness specifically on proline. Consistent with these phenotypes, sequencing of the evolved populations showed that passaging in proline resulted in specific mutations in an associated metabolic pathway that increases the ability to utilize proline for growth, while evolution in sorbitol resulted in mutations in many different genes that generally improve growth in high osmolarity conditions at the expense of growth at low osmolarity. High osmolarity decreased growth rate but increased mean cell volume compared with growth on proline as the sole carbon source, demonstrating that osmolarity-induced changes in growth rate and cell size follow an orthogonal relationship from the classical Growth Law relating cell size and nutrient quality. Isolates from a sorbitol-evolved population that capture the likely temporal sequence of mutations revealed by metagenomic sequencing demonstrate a tradeoff between growth at high and low osmolarity. Our study highlights the utility of experimental evolution for dissecting complex cellular networks and environmental interactions, particularly in the case of behaviors that can involve both specific and general metabolic stressors. ImportanceFor bacteria, maintaining higher internal solute concentrations than the environment allows cells to take up water. As a result, survival is challenging in high osmolarity environments. To investigate how bacteria adapt to high osmolarity environments, we evolved Escherichia coli in a variety of high osmolarity solutions for hundreds of generations. We found that evolved populations adopted different strategies to improve their growth depending on the osmotic passaging condition, either generally adapting to high osmolarity conditions or better metabolizing the osmolyte as carbon source. Single-cell imaging demonstrated that enhanced fitness was coupled to faster growth, and metagenomic sequencing revealed mutations that reflect growth tradeoffs across osmolarities. Our study demonstrates the utility of long-term evolution experiments for probing adaptation during environmental stress.