A Central Role for Magnesium Homeostasis during Adaptation to Osmotic Stress

Abstract

Osmotic stress is a significant physical challenge for free-living cells. Cells from all three domains of life maintain viability during osmotic stress by tightly regulating the major cellular osmolyte potassium (K1) and by import or synthesis of compatible solutes. It has been widely established that in response to high salt stress, many bacteria transiently accumulate high levels of K1, leading to bacteriostasis, with growth resuming only when compatible solutes accumulate and K1 levels are restored to biocompatible levels. Using Bacillus subtilis as a model system, we provide evidence that K1 fluxes perturb Mg21 homeostasis: import of K1 upon osmotic upshift is correlated with Mg21 efflux, and Mg21 reimport is critical for adaptation. The transient growth inhibition resulting from hyperosmotic stress is coincident with loss of Mg21 and a decrease in protein translation. Conversely, the reimport of Mg21 is a limiting factor during resumption of growth. Furthermore, we show the essential signaling dinucleotide cyclic di-AMP fluctuates dynamically in coordination with Mg21 and K1 levels, consistent with the proposal that cyclic di-AMP orchestrates the cellular response to osmotic stress. IMPORTANCE Environments with high concentrations of salt or other solutes impose an osmotic stress on cells, ultimately limiting viability by dehydration of the cytosol. A very common cellular response to high osmolarity is to immediately import high levels of potassium ion (K1), which helps prevent dehydration and allows time for the import or synthesis of biocompatible solutes that allow a resumption of growth. Here, using Bacillus subtilis as a model, we demonstrate that concomitant with K1 import there is a large reduction in intracellular magnesium (Mg21) mediated by specific efflux pumps. Further, it is the reimport of Mg21 that is rate-limiting for the resumption of growth. These coordinated fluxes of K1 and Mg21 are orchestrated by cyclic-di-AMP, an essential second messenger in Firmicutes. These findings amend the conventional model for osmoadaptation and reveal that Mg21 limitation is the proximal cause of the bacteriostasis that precedes resumption of growth.