Impact of changed c-di-AMP levels and hypoosmotic stress on the transcriptome of Haloferax volcanii and on RCK domain-containing proteins.

We investigated the role of cyclic di-adenosine monophosphate (c-di-AMP) in the halophilic archaeon Haloferax volcanii by analysing transcriptomic changes in a strain with lowered c-di-AMP levels and by characterizing the function of key RCK (regulator-of-conductance-of-K⁺) domain proteins. The c-di-AMP-reduced mutant showed elevated expression of cell division genes and metabolic enzymes, whereas a Na⁺/H⁺ antiporter and an aspartate aminotransferase were strongly repressed. These patterns reveal previously unknown links between this messenger and both cell division and osmolyte homeostasis. To probe downstream effectors, we created deletion mutants of four RCK domain proteins and observed distinct phenotypes under potassium or sodium limitation. Deleting the primary RCK protein, linked to a high-affinity potassium importer, abolished growth under potassium limitation and caused extreme cell enlargement under hypoosmotic conditions, underscoring its essential role in potassium uptake and cell volume control. Removing a secondary transporter-associated RCK protein caused only mild defects, mainly under low sodium, indicating an auxiliary potassium acquisition system. Two stand-alone RCK proteins (unlinked to transporters) were dispensable for normal growth yet critical during osmotic stress: one knockout alleviated excessive swelling of c-di-AMP-reduced cells, whereas the other caused hypersensitivity to low-salt conditions. Biochemical assays revealed that only transporter-associated RCK proteins bound c-di-AMP, suggesting direct control of potassium transport, while stand-alone RCK proteins mediate osmotic adaptation through c-di-AMP-independent mechanisms. These findings define a novel osmotic stress regulatory network in H. volcanii integrating second-messenger signalling with ion homeostasis, highlighting the broader importance of cyclic nucleotide signalling in archaeal stress adaptation.