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

We investigated the signaling role of cyclic di-adenosine monophosphate in the halophilic archaeon Haloferax volcanii by analyzing global transcriptomic changes in a cyclic di-adenosine monophosphate–depleted strain and characterizing the functions of key RCK (regulator-of-conductance-of-K?) domain proteins. Notably, the cyclic di-adenosine monophosphate 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 second messenger and both cell division and osmolyte homeostasis. To probe downstream effectors, we generated 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 hypo-osmotic conditions, underscoring its essential role in potassium uptake and cell volume control. In contrast, removing a secondary transporter-associated RCK protein caused only mild defects (notably under low sodium), indicating an auxiliary potassium acquisition system. Two stand-alone RCK domain proteins (unlinked to any transporter) were dispensable for normal growth yet critical during osmotic stress: one knockout alleviated the excessive cell swelling of cyclic di-AMP–reduced cells, whereas the other knockout caused hypersensitivity to low-salt conditions. Biochemical assays revealed that only the transporter-associated RCK proteins bound cyclic di-adenosine monophosphate, suggesting direct second-messenger control of potassium transport, while the stand-alone RCK proteins mediate osmotic adaptation through alternate, cyclic di-adenosine monophosphate–independent mechanisms. Together, these findings define a novel osmotic stress regulatory network in H. volcanii that integrates second-messenger signaling with ion homeostasis, highlighting the broader importance of cyclic nucleotide signaling in archaeal stress adaptation.