Adaptive laboratory evolution of E. coli to magnesium chloride stress reveals noncanonical routes to broad chaotropic salt resistance.

Extensive research on model microorganisms under kosmotropic sodium chloride stress has established much of what is known about the genetic and molecular mechanisms of osmoregulation. However, genetic adaptation to chaotropic salts such as magnesium chloride remains poorly defined. Chaotropes destabilize biomolecules and can constrain biomanufacturing, underscoring the need to enhance stress tolerance to improve production efficiency. Environmentally, the exceptional chaotropic activity of magnesium chloride brines offers rare opportunities to probe the limits of life, with major implications for bioprospecting and astrobiology. To explore mechanisms of adaptation to high chaotropicity, we used adaptive laboratory evolution (ALE) to isolate Escherichia coli strains with improved growth under magnesium chloride stress. Two isolates were obtained which exhibited significantly enhanced growth when exposed to magnesium chloride and other chaotropic salts, but not to sodium chloride. Whole-genome sequencing, label-free quantification, and genetic analysis implicated mutations in the sugar phosphotransferase system, the stringent response, and envelope remodeling as key contributors to magnesium chloride resistance. Mutations in ptsI, a sugar phosphotransferase, and spoT, encoding the bifunctional (p)ppGpp hydrolase/synthetase, were essential for improved growth under magnesium chloride stress. Interestingly, activation of the regulator of capsular polysaccharide synthesis (Rcs) system, a critical regulator of the osmotic stress response, was required for magnesium chloride tolerance in the ancestral strain but became dispensable in the evolved strains. Our report identifies multiple strategies used by E. coli to overcome magnesium chloride stress.