Evolutionary Engineering of Sexual and Asexual Escherichia coli Strains for Osmotic Stress Tolerance

Osmotic stress can significantly impact growth and productivity.  In order to better understand the osmotic stress tolerance phenotype, we evolved sexual (capable of in situ DNA exchange) and asexual Escherichia coli strains under sodium chloride (NaCl) stress.  Isolates from end point populations all exhibited significant improvements in growth under NaCl stress; and could grow in up to 0.80 M NaCl, a concentration that completely inhibits the growth of the unevolved parental strains.  Whole genome resequencing revealed frequent mutations in genes controlling n-acetylglucosamine catabolism (nagC, nagA), cell shape (mrdA, mreB), osmoprotectant uptake (proV), and motility (fimA).  N-acetylglucosamine catabolism and cell shape control have not been previously associated with osmotic stress tolerance.  Possible epistatic interactions between nagC, nagA, fimA, and proV deletions were also detected when reconstructed as defined mutations.  Transcriptional analysis also revealed significant changes in ompACGL porin expression, and increased transcription of sulfonate uptake systems in the evolved mutants.  These findings expand our current knowledge of the osmotic stress phenotype, and will be useful for the rational engineering of osmotic tolerance.