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 (K⁺) 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 K⁺, leading to bacteriostasis, with growth resuming only when compatible solutes accumulate and K⁺ levels are restored to biocompatible levels. Using Bacillus subtilis as a model system, we provide evidence that K⁺ fluxes perturb Mg²⁺ homeostasis: import of K⁺ upon osmotic upshift is correlated with Mg²⁺ efflux, and Mg²⁺ reimport is critical for adaptation. The transient growth inhibition resulting from hyperosmotic stress is coincident with loss of Mg²⁺ and a decrease in protein translation. Conversely, the reimport of Mg²⁺ is a limiting factor during resumption of growth. Furthermore, we show the essential signaling dinucleotide cyclic di-AMP fluctuates dynamically in coordination with Mg²⁺ and K⁺ levels, consistent with the proposal that cyclic di-AMP orchestrates the cellular response to osmotic stress. <b>IMPORTANCE</b> 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 (K⁺), 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 K⁺ import there is a large reduction in intracellular magnesium (Mg²⁺) mediated by specific efflux pumps. Further, it is the reimport of Mg²⁺ that is rate-limiting for the resumption of growth. These coordinated fluxes of K⁺ and Mg²⁺ are orchestrated by cyclic-di-AMP, an essential second messenger in <i>Firmicutes</i>. These findings amend the conventional model for osmoadaptation and reveal that Mg²⁺ limitation is the proximal cause of the bacteriostasis that precedes resumption of growth.