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

Abstract

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.

This study explores how the second messenger c-di-AMP controls osmotic balance in Haloferax volcanii. Using transcriptomics and RCK-protein knockouts, we show that reduced c-di-AMP disrupts cell division and ion homeostasis, and that the main c-di-AMP–binding RCK protein is essential for potassium uptake and cell-volume control. Stand-alone RCK proteins, although not binding c-di-AMP, uniquely modulate stress responses under low-salt conditions. Together, these results reveal an integrated archaeal network linking c-di-AMP signalling to ion transport and osmotic adaptation.

Introduction

Cyclic di-adenosine monophosphate (c-di-AMP) has emerged as a near-universal second messenger in bacteria and many euryarchaea, with essential roles in cellular physiology and adaptation to environmental changes. This nucleotide-based signalling molecule is involved in the regulation of diverse processes, including the response to DNA damage (Fahmi et al. 2019, He et al. 2020, Yin et al. 2020, Foster et al. 2024), central metabolism (Schuster et al. 2016, Whiteley et al. 2017, Commichau et al. 2018, Yin et al. 2020, Kundra et al. 2021, Foster et al. 2024), cell wall homeostasis (Agostoni et al. 2018, Fahmi et al. 2019, Cereija et al. 2021, Mudgal et al. 2021, Oberkampf et al. 2021), stress adaptation (Holtmann et al. 2003, Corratgé-Faillie et al. 2010, Huynh et al. 2016, Zarrella et al. 2018), potassium transport (Corratgé-Faillie et al. 2010, Bai et al. 2014, Krüger et al. 2021, Cereija et al. 2021), and virulence (Kundra et al. 2021). The mechanisms of c-di-AMP synthesis and hydrolysis are conserved across different species and highlight its fundamental importance; however, the molecular targets and downstream effectors of c-di-AMP often display species-specific characteristics, reflecting evolutionary divergence tailored to distinct ecological niches and lifestyles. The synthesis of c-di-AMP is catalysed by diadenylate cyclase (DAC) domains, which are evolutionarily conserved across various bacterial and archaeal species (Mudgal et al. 2021). Unlike the GGDEF domain responsible for c-di-GMP synthesis (Corrigan et al. 2011), which is often found in multiple copies and diverse domain architectures within a single genome, DAC domains are typically present in low copy numbers and exhibit limited domain combinations (Galperin 2023). This restricted distribution suggests a tightly regulated and specialized function for c-di-AMP within the cell. The formation of this molecule requires the dimerization and precise orientation of two DAC domains, each binding one ATP (adenosine triphosphate) molecule along with a metal ion cofactor to facilitate catalysis (Heidemann et al. 2019). The chemical stability and reactivity of c-di-AMP are influenced by its cyclic phosphate backbone, which confers resistance to many nucleases but also necessitates dedicated phosphodiesterases for its degradation (Yin et al. 2020). These phosphodiesterases play a central role in this process by hydrolyzing c-di-AMP into linear 5′-phosphoadenylyl-(3′,5′)-adenosine (pApA), which can be further degraded into adenosine monophosphate (AMP) (Bowman et al. 2016). The activity and substrate specificity of these PDEs (phosphodiesterases) vary among species and even within different cellular contexts. Both excessive accumulation and depletion of this nucleotide can have detrimental effects on cell growth and viability, earning it the name of an ‘essential poison’ in bacteria (Gundlach et al. 2015, Commichau and Stülke 2018). For example, studies in Bacillus subtilis demonstrate that both elevated levels and complete absence of c-di-AMP disrupt normal growth patterns (Mehne et al. 2013). The majority of intracellular c-di-AMP appears to be protein-bound under physiological conditions, with only a small fraction existing as free molecules (Heidemann et al. 2022). This distribution is likely dynamic and responsive to environmental cues, such as changes in ionic strength or pH, which can alter binding affinities and thus shift the equilibrium between free and bound states (Corrigan and Gründling 2013).

c-di-AMP functions as a second messenger that orchestrates signal transduction in bacteria and archaea by modulating a network of effector proteins and regulatory elements. The mechanisms underlying c-di-AMP-mediated signal transduction are multifaceted, involving direct binding to protein effectors, regulation of transporter activity (Cereija et al. 2021), and modulation of gene expression through riboswitches (Kellenberger et al. 2015, Wang et al. 2019) and transcriptional repressors (Mahmoud et al. 2020). At the core of c-di-AMP signalling is its ability to sense and respond to environmental signals, such as osmotic stress and nutrient availability. The transmission of this signal relies on the interaction between c-di-AMP and specific receptor proteins, many of which contain regulatory domains such as RCK (regulator of conductance for K+) or CBS (cystathionine β-synthase) domains.

Among these, RCK domain-containing proteins have received particular attention due to their central role in potassium ion regulation. Potassium is a major intracellular cation required for numerous physiological processes, including maintenance of turgor pressure, enzyme activation, and pH regulation (Epstein,1986). The regulation of these systems by c-di-AMP has emerged as a central mechanism to ensure proper ion homeostasis under fluctuating environmental conditions. In bacteria such as B. subtilis, c-di-AMP directly influences the activity of potassium transporters and associated regulatory proteins (Herzberg et al. 2023). The primary effectors include RCK domain-containing proteins, which are integral to the function of potassium channels and transporters. These RCK proteins act as direct binding partners for c-di-AMP, modulating their conformation and activity in response to changes in intracellular c-di-AMP concentrations. For instance, the KtrA and KtrC subunits form part of the KtrAB and KtrCD potassium uptake systems (Holtmann et al. 2003), respectively. Binding of c-di-AMP to these RCK domains inhibits transporter activity, thereby reducing potassium influx when intracellular levels are sufficient or excessive. The sensor histidine kinase KdpD represents another key component regulated by c-di-AMP (Rocha et al. 2024). In many bacteria, KdpD controls the expression of the high-affinity ATPase-type potassium uptake system (KdpFABC) through its cognate response regulator KdpE. c-di-AMP binds to the universal stress protein domain within KdpD, which can inhibit its function and thus downregulate expression of the kdp operon under conditions where potassium is not limiting (Price-Whelan et al. 2013, Wang et al. 2019). This regulatory mechanism ensures that cells avoid toxic accumulation of potassium ions while maintaining sufficient levels for metabolic needs. Experimental evidence demonstrates that perturbations in c-di-AMP metabolism have profound effects on potassium homeostasis. Mutants with elevated c-di-AMP levels, often due to loss-of-function mutations in phosphodiesterases, such as GdpP or PgpH, exhibit impaired potassium uptake and associated growth defects (Gundlach et al. 2015, Huynh and Woodward 2016, Wang et al. 2022). These phenotypes underscore the necessity for tight control over both synthesis and degradation of c-di-AMP. For example, B. subtilis strains with mutations in the di-adenylate cyclase cdaA display an impaired potassium ion channel system and weakened cell wall integrity, highlighting the interconnectedness between ion transport and other cellular processes (Rosenberg et al. 2015, Rismondo et al. 2016, Zhu et al. 2016, Pathania et al. 2021). Beyond regulating potassium uptake systems, c-di-AMP has been implicated in broader osmotic stress responses. Suppressor mutations that restore growth in strains with altered c-di-AMP levels are frequently found not only in potassium transporter genes but also in genes encoding compatible solute transporters (Price-Whelan et al. 2013) and amino acid antiporters (Corrigan et al. 2013). These findings indicate that c-di-AMP coordinates ion and osmolyte homeostasis through a complex, interconnected regulatory network.

C-di–AMP is an indispensable second messenger in the euryarchaeon Haloferax volcanii. The single di–adenylate cyclase gene, dacZ, is essential, and its overexpression is lethal, underscoring the necessity for tight intracellular control of the nucleotide (Braun et al. 2019). Reduced c-di-AMP levels lead to slower growth rates and salt–dependent cellular swelling, thereby linking c–di–AMP homeostasis directly to osmoadaptive fitness. Basal c–di–AMP concentrations are at 8–12 ng/mg total protein, with elevated values recorded under hypoosmotic growth, indicating rapid nucleotide accumulation when external salinity diminishes. Comparative genomic surveys revealed a single dacZ orthologue conserved across Halobacteria, Methanococci, and Thermococci. In contrast, it is absent from TACK, DPANN and Asgard lineages, suggesting an early but phylogenetically restricted adoption of the messenger within Euryarchaeota. Biochemical characterization of H. volcanii DacZ (Braun et al. 2019) and Pyrococcus yayanosii PyaDAC (Jin et al. 2023) showed that both catalyse ATP–dependent c-di-AMP synthesis. In H. volcanii, dacZ is co–transcribed with the mechanosensitive channel gene mscS2 (Braun et al. 2019); promoter–replacement studies demonstrate that reduced c–di–AMP provokes uncontrolled osmolyte influx and cell swelling, consistent with a model in which the nucleotide modulates osmotic release systems. The first complete archaeal c–di–AMP metabolic cycle has been delineated in P. yayanosii, where a partner TrkA_N–DHH–DHHA1 phosphodiesterase, PyaPDE, sequentially converts c–di–AMP to pApA and AMP (Jin et al. 2023). Complementary genetic experiments in Thermococcus kodakarensis show that dacZ deletion abolishes nucleotide production, whereas inactivation of the sole PDE triples cellular c–di–AMP yet imposes only modest fitness costs, highlighting lineage–specific tolerance thresholds (Jin et al. 2023). Archaeal phosphodiesterases retain the canonical TrkA_N–DHH–DHHA1 scaffold found in bacteria but display stricter adenine specificity. Collectively, current evidence positions c–di–AMP as a pivotal euryarchaeal signal that integrates osmolarity sensing, potassium homeostasis and, by extension, cell–shape maintenance.

Here we present a study to investigate the role of c-di-AMP signaling in H. volcanii, with a particular focus on its control of potassium transport and osmotic stress responses. First, we performed a transcriptomic analysis of an H. volcanii strain with reduced c-di-AMP levels to capture the scope of cellular pathways influenced by this second messenger. This allowed us to identify genes and processes (from ion transporters to cell division proteins) that are responsive to c-di-AMP perturbation, providing clues to its physiological roles in archaea. Second, we directly examined the functions of the four RCK domain proteins (HVO_1055, HVO_1058, HVO_1885, and HVO_2211) by constructing gene deletion mutants and characterizing their phenotypes under varying ionic conditions (potassium limitation, sodium limitation, and reduced c-di-AMP levels). Concurrently, we assessed the c-di-AMP binding capacity of these RCK proteins in vitro. Below, we report how lowered c-di-AMP levels affects H. volcanii gene expression and morphology, and we describe the distinct roles of each RCK domain protein in potassium homeostasis and cell volume regulation. Our findings not only confirm that c-di-AMP is a pivotal osmotic regulator in archaea, but also reveal new aspects of archaeal cell biology, including a surprising link between c-di-AMP signalling, potassium transport, and cell division that broaden the paradigm of how prokaryotes integrate environmental signals to maintain homeostasis.

Materials and methods

Plasmid construction

All plasmids were created using classical restriction enzyme-based molecular cloning techniques. Inserts were amplified via polymerase chain reaction (PCR) from wild-type H26 genomic DNA. PCR amplification was performed using Phusion polymerase (NEB) and all restriction endonucleases were obtained from NEB. Restriction enzyme digestion and PCR were conducted following the manufacturer’s protocols. Primers, plasmids, and strains are shown in Tables S1–S3. Plasmids used for transformation into H. volcanii were extracted from dam−/dcm− competent Escherichia coli cells (NEB).

Genetic manipulation of H. volcanii and construction of deletion mutants

Transformation and deletion mutant construction in H. volcanii were carried out using uracil-based selection, as previously described (Bitan-Banin et al. 2003). Briefly, deletion constructs were generated using the pTA131 vector, incorporating a pyrE2-cassette along with ∼500 bp upstream and downstream flanking regions of the target gene. These constructs were introduced into the desired H. volcanii strain via transformation. Transformants were plated on selective CA plates and incubated for 5 days. Single pop-in colonies were restreaked on CA plates and incubated for an additional 3 days before inoculation in YPC medium (yeast extract, peptone, and casamino acids) for nonselective growth. This step was repeated three times overnight to relieve selective pressure for the pyrE2 cassette. Selection of pop-out mutants was performed by plating cells on CA medium supplemented with 50 µg/ml 5-fluoroorotic acid (5-FOA) and 10 µg/ml uracil. After 5 days of incubation, up to 100 colonies were transferred to YPC plates and incubated for two additional days. Colony-PCR was performed using cell lysate as the template to screen for successful deletions. PCR products from candidate deletion mutants were compared with wild-type H26 PCR products to confirm the expected deletion size. Selected deletion strains were cultured overnight in YPC medium, and genomic DNA (gDNA) was isolated for further validation. The purified gDNA was used as a template for PCR amplification, and the resulting PCR products were confirmed via sequencing to verify successful gene deletions.

RNA isolation and quality control

Total RNA was extracted from H. volcanii strains H26 (wild-type) and HTQ410 (c-di-AMP–reduced mutant) grown in CAB medium at 42°C with shaking until mid-exponential phase (OD₆₀₀ ≈ 0.4–0.6). Cell pellets were harvested by centrifugation (5000  ×  g, 10 min, 4°C), immediately frozen in liquid nitrogen, and stored at –80°C until further use. RNA isolation was performed using the RNeasy® Plus Mini Kit (Qiagen) according to the manufacturer’s protocol, which includes an on-column gDNA removal step via the gDNA Eliminator spin columns to eliminate contaminating genomic DNA. The RNA concentration and purity were initially assessed using a NanoDrop™ One UV–Vis Spectrophotometer (ThermoFisher Scientific), with A260/A280 and A260/A230 ratios recorded for purity evaluation. For more accurate quantification, RNA concentration was subsequently measured using the Qubit™ RNA High Sensitivity (HS) Assay Kit (ThermoFisher Scientific) on a Qubit™ 4 Fluorometer. To assess RNA integrity, 1 µl of each sample was analysed on an Agilent 2100 Bioanalyzer system using the RNA 6000 Nano Kit (Agilent Technologies), following the manufacturer’s instructions for prokaryotic total RNA. Samples with an RNA Integrity Number ≥ 8.0 were considered suitable for downstream transcriptome analysis. All RNA samples were stored at –80°C and processed further only once confirmed to be of high quality and free from genomic DNA contamination.

RNA library preparation and sequencing

RNA libraries were prepared and sequenced following the Illumina TruSeq Stranded mRNA Sample Preparation Guide, utilizing the NextSeq 500 platform (Illumina, Inc.). Briefly, 100 ng of total RNA was chemically fragmented to 200–400 bp using divalent cations at 94°C for 4 min, omitting mRNA purification with poly-T beads. First-strand cDNA synthesis was carried out using random hexamer primers and reverse transcriptase in the presence of actinomycin D to ensure strand specificity. Second-strand cDNA was synthesized using DNA polymerase I, RNase H, and dUTP, followed by 3' adenylation and adapter ligation. Libraries were PCR-enriched and quantified with the KAPA Library Quantification Kit (Roche). Equimolar libraries were sequenced using two 75-cycle High Output Kits with single-end reads. Base calling and demultiplexing were performed using RTA (v2.4.11) and bcl2fastq v2.18 software. Sequencing was conducted at the Genomics Core Facility ‘KFB—Center of Excellence for Fluorescent Bioanalytics’ (University of Regensburg).

RNAseq data analysis

Raw sequencing reads were quality-assessed using FastQC v0.12.1 (https://www.bioinformatics.babraham.ac.uk/projects/fastqc/). Adapter sequences and low-quality bases were trimmed using fastp v0.23.4 (Chen et al. 2018) with the following parameters: –cut_front, –cut_tail, and a minimum base quality of 30. Trimmed reads were aligned to the H. volcanii DS2 reference genome (RefSeq: GCA_000025685.1) using Bowtie2 v2.5.4 (Langmead and Salzberg 2012) with default parameters in single-end mode. Resulting SAM files were converted to BAM, sorted, and indexed using samtools v1.21 (Li et al. 2009). Strand-specific coverage tracks were generated with bamCoverage (part of deepTools v3.5.5; Ramírez et al. 2014), normalizing to counts per million using a bin size of 1 bp and strand-specific filters. Forward and reverse coverage profiles were saved in BigWig format.

Read counts were generated using featureCounts (Subread v1.5.3) (Liao et al. 2014), using a custom GTF file derived from genome annotations. Gene-level normalization was performed using DESeq2 (v1.42.0) (Love et al. 2014), with variance-stabilized transformation used for principal component analysis (PCA). Transcript abundance was additionally calculated as TPM by length-normalizing read counts and scaling by sample-wise library size.

Differential expression analysis was conducted with DESeq2 (v1.42.0) using the Wald test and shrinkage of log₂ fold changes via the apeglm method (v1.28.0) (Zhu et al. 2019). Genes were considered differentially expressed if |log₂FC| ≥ 1 and adjusted P-value (Benjamini–Hochberg) < .05. Transcripts with fold changes between 0 and 1 were also recorded as ‘moderately regulated’ if statistically significant. Functional enrichment analysis was conducted using goseq (v1.58.0) with arCOG annotations. Gene length bias correction was applied, and enrichment was calculated against the full set of protein-coding transcripts. Enriched arCOG categories were visualized using custom R scripts. All analyses were performed in R (v4.4.2).

16S rRNA phylogenetic tree construction

High-quality, full-length 16S rRNA gene sequences from 55 representative archaeal species were retrieved from the SILVA SSURef database (Quast et al. 2012). Sequences were aligned using MAFFT v7 (Katoh and Standley 2013), followed by manual trimming to remove low-confidence or gapped regions. The resulting alignment was used to infer a maximum-likelihood phylogenetic tree using FastTree v2 (Price et al. 2010) under the default generalized time-reversible model for nucleotide sequences. The final tree was visualized and annotated in iTOL v5 (Letunic and Bork 2021).

Protein homolog detection via reciprocal BLAST

To identify homologs of potassium homeostasis-related proteins, we employed a reciprocal best BLAST hit (RBH) strategy using local BLASTp searches (Miller-Gallacher et al. 2014) against a custom archaeal protein database compiled from NCBI RefSeq (O’Leary et al. 2016). In the forward search, each H. volcanii query protein was aligned against the archaeal proteome database using blastp with the following parameters: e-value ≤ 1e−5, max_target_seqs = 2000, and output format 6 (tab-delimited, including query ID, subject ID, e-value, and bit score). For each species, the top-scoring hit (based on bit score) was selected as the forward candidate. These candidates were then subjected to a reverse BLASTp against the H. volcanii reference proteome using the same parameters. A match was classified as reciprocal best hit if the reverse BLAST retrieved the original query protein as the top hit. Matches that passed the forward search but failed the reverse check were considered as nonreciprocal hits, whereas proteins with no significant forward hit were recorded as absent.

Strains and growth conditions

Haloferax volcanii strains were cultivated in either rich YPC medium or selective CA medium (casamino acids), both supplemented with an expanded trace element solution, hereafter referred to as CAB medium. Cultures were incubated at 45°C in liquid media, with rotation applied for volumes up to 5 ml and shaking conditions used for cultures exceeding 5 ml. Strains carrying auxotrophic marker deletions were grown in CAB medium supplemented with the appropriate selection compound, specifically, 50 µg/ml uracil (Sigma-Aldrich) was added for strains carrying the ΔpyrE2 mutation. Haloferax volcanii strains were precultured in CAB+URA medium following a standardized workflow to investigate the effects of potassium and sodium concentrations on growth dynamics and cell morphology. A 5 ml starter culture was inoculated from a fresh plate into CAB+URA medium and incubated overnight at 45°C. The next day, 50 ml precultures were prepared by inoculating 10 µl of the starter culture into fresh CAB+URA and grown until mid-log phase.

For the main experiment, three conditions were tested: standard (2.5 M NaCl, 55 mM KCl), low-Sodium (1.8 M NaCl, 55 mM KCl), and low potassium (2.5 M NaCl, 5 mM KCl). For each ionic condition (standard: 2.5 M NaCl, 55 mM KCl; low-sodium: 1.8 M NaCl, 55 mM KCl; and low-potassium: 2.5 M NaCl, 5 mM KCl), four independent biological replicates were performed on different days. Each biological replicate comprised three technical replicates (50 ml cultures in separate flasks) inoculated to an initial OD₆₀₀ of 0.01. Cultures were incubated at 45°C under shaking conditions, and OD₆₀₀ was recorded twice daily from Monday through the following Wednesday, covering a total experimental duration of 225 h. During the exponential growth phase, when cells exhibited the highest division rates, samples were collected for microscopic analysis of cell morphology. Growth curves and cell shape distributions were subsequently evaluated to characterize the physiological responses of H. volcanii under different ionic stress conditions.

Phase-contrast microscopy, cell area quantification, and statistical analysis

Growth curves were established with sampling performed at 50 h during the exponential growth phase, when cells exhibited the highest division rates. Cultures were diluted to an OD₆₀₀ of 0.1, and 5 µl were spotted onto agarose pads [0.4% (w/v) agarose in 18% salt water containing 144 g/l NaCl, 21 g/l MgSO₄·7 H₂O, 18 g/l MgCl₂·6 H₂O, 4.2 g/l KCl, 12 mmol/l Tris/HCl, and pH 7.5]. Pads were air-dried and covered with coverslips for imaging. Cells were visualized using an Axio Observer.Z1 inverted phase-contrast microscope (Zeiss) at 1000X magnification, and images from at least 500 cells per biological replicate per strain and condition were captured. Cell area quantification and shape parameters were analysed using FIJI software (Schindelin et al. 2012) with the MicrobeJ plug-in (Ducret et al. 2016). The scale bar represents 5 µm. After microscopy, anomalous data points were manually removed. Subsequently, a dataset comprising ~414 cell measurements per condition was compiled into a long-format data structure via data pivoting, resulting in a total of 12 420 data points. This dataset was then subjected to statistical analysis using SPSS software. Multiple linear regression analysis was employed to evaluate the effects of medium (Normal, HP, and HS), c-di-AMP levels (WT_H26, HTQ410), and different gene deletions (Control, ΔHVO_1055, ΔHVO_1058, ΔHVO_1885, and ΔHVO_2211) on cell area. The overall significance of the model was determined by ANOVA. Visualization of cell area distributions was performed to further interpret the impact of experimental conditions.

Spot-dilution assay of H. volcanii under salt stress

For spot-dilution assays, H. volcanii strains or transformed colonies were grown overnight in 5 ml CAB medium (standard KCl concentration for deletion mutants). Cultures were adjusted to an OD₆₀₀ of 1.0, and 1 ml of each culture was harvested by centrifugation (4500 × g, 10 min). Cell pellets were resuspended in 2.5 M NaCl with 25 mM Tris–HCl (pH 7.5) to normalize cell density. Serial dilutions (10⁻¹–10⁻⁷) were prepared in the same buffer, and 5 µl of each dilution was spotted onto CA plates containing defined with varying concentrations of KCl (0–50 mM) or NaCl (1.6–2.5 M). Plates were allowed to air-dry, incubated at 45°C for 48 h, and subsequently stored at room temperature for an additional 90 h before imaging. Colony-forming capacity was visually assessed across the dilution series, and a semiquantitative survival score was assigned to each strain based on the highest dilution that still exhibited discrete and complete colony formation. Borderline cases (e.g. faint or incomplete colonies at the last dilution) were assigned intermediate scores (e.g. 5.5). Scoring was manually curated and performed consistently across biological replicates.

Heterologous expression of TrkA-domain proteins

N-terminal 6xHis-tagged TrkA-domain proteins (HVO_1055, HVO_1058, HVO_1885, and HVO_2211) were heterologously expressed in E. coli Rosetta cells (Novagen) transformed with expression plasmids (pSVA6228-6231) or empty vector pETDuet™-1 (negative control). Transformed cells were cultured in LB (Lysogeny Broth) medium with ampicillin/chloramphenicol to OD₆₀₀ ∼0.5 and induced with 0.2 mM IPTG at room temperature overnight. Cells were harvested, resuspended in lysis buffer (150 mM NaCl, 1 mM MgCl₂, 50 mM Tris–HCl, and pH 8.0) with 10 µg/ml DNase I (Roche), and lysed by sonication. Cleared lysates were obtained by centrifugation at 48 200 × g (4°C, 25 min). Total protein concentration was measured using the BCA Protein Assay Kit (Serva). Lysates were analysed by SDS-PAGE and mass spectrometry, with peptides identified by matching against the translated H. volcanii proteome (UniProt ID: 309800).

Synthesis and processing of [α-32P]-labelled c-di-AMP

In vitro synthesis of [α-32P]-labelled c-di-AMP was performed using purified di-adenylate cyclase DacZ from Methanococcus maripaludis (MmDacZ). Reactions (50 µl) were carried out in buffer containing 100 mM NaCl, 50 mM KCl, 1 mM DTT, 50 mM Tris–HCl (pH 7.5), 20 µM MmDacZ, and 10 mM MgCl₂. Substrate included 46.9 µM ATP (with [α-32P] ATP at ∼17 nM, ∼3.7 MBq/ml). Reactions proceeded at 37°C overnight and were stopped by heating at 95°C for 5 min. Unconverted ATP was removed by incubation with 10 µM purified Pyrococcus furiosis ArlI (Chaudhury et al. 2018) at 50°C for 1 h, followed by filtration through a 3 kDa centrifugal filter (Nanosep®, Pall Corporation). Conversion efficiency was analysed via thin-layer chromatography using Polygram® CEL 300 PEI-cellulose plates developed in 1:1.5 saturated (NH₄)₂SO₄ and 1.5 M KH₂PO₄ (pH 3.6). Plates were visualized using a phosphor imaging screen (BAS-MS 2040, Fujifilm) and scanned with a Typhoon FLA 9500 system. Quantification was done using Fiji software.

Differential radial capillary action of ligand assay with TrkA-domain proteins

To assess direct c-di-AMP binding, the differential radial capillary action of ligand assay (DRaCALA) was performed following the method described by Roelofs et al. (2011), with minor modifications. This assay relies on differential capillary action to detect protein–ligand complexes on nitrocellulose membranes. Reactions were conducted in a lysis buffer (150 mM NaCl, 1 mM MgCl₂, 50 mM Tris–HCl, and pH 8.0) with a final volume of 25 µl. Cleared E. coli lysates containing TrkA-domain proteins were used at a final protein concentration of 0.5 mg/ml. The assay contained 100 nM total c-di-AMP, including ∼0.41 nM [α-³²P]-labelled c-di-AMP. Reactions were incubated at room temperature for 10 min before spotting 5 µl of the mixture onto Protran® Nitrocellulose membranes (Whatman) and air-drying. Membranes were exposed to a phosphor imaging screen (BAS-MS 2040, Fujifilm) for 1 h and scanned using a Typhoon FLA 9500 (GE Healthcare). For competition assays, cold c-di-AMP (Biolog) was added to final concentrations of 1 µM or 1 mM; ATP (Sigma) was added at 1 mM where indicated.

Results

Global transcriptional changes in response to osmotic stress and lowered c-di-AMP levels

To investigate the effects of osmotic stress and reduced c-di-AMP levels on the transcriptome of H. volcanii, we compared the transcriptional profiles of wild-type and c-di-AMP-reduced strains under both normal (2.5 M NaCl) and hypoosmotic (1.8 M NaCl) conditions (see Fig. S1). Experiments were conducted using the H26 wild-type strain and the HTQ410 strain (c-di-AMP-reduced), in which the dacZ (hvo_1660) gene is placed under the control of a genetically engineered version of the tryptophan-inducible promoter (p.tnaA) of the tryptophanase gene tnaA (hvo_0789) (Braun et al. 2019). This genetic modification results in an approximately three-fold reduction in intracellular c-di-AMP levels (Braun et al. 2019). Indeed, in the c-di-AMP-reduced strain, the expression of dacZ, was significantly lowered compared to the wild type, regardless of osmotic conditions [Fig. 1A; transcripts per million (TPM) values in Table S4). This confirms the successful reduction of dacZ levels and establishes a reference point for assessing its downstream effects. As a control, we note that p.tnaA is not transcriptionally responsive to osmotic stress under the tested conditions as supported by the unchanged expression of dacZ between normal- and hypoosmotic conditions in the HTQ410 strain (Fig. 1A).

Figure 1.

Transcriptomic effects of reduced c-di-AMP levels and osmotic conditions in H. volcanii. (A) Normalized expression levels TPM of the dacZ gene (HVO_1660), encoding di-adenylate cyclase, across wild-type (circles) and c-di-AMP-reduced strains (square) under normal (dark grey)- and hyposalt (light grey) conditions. (B) PCA of transcriptomic profiles under varying c-di-AMP levels and osmotic conditions. Each point represents a biological replication. (C) MA plot showing differential gene expression between wild-type and c-di-AMP strains under normal-salt conditions. Protein-coding transcripts are categorized by significance and fold changes, colour-coded as strongly upregulated (padj < .05 and log2FC ≥ 1, magenta, bold font), upregulated (padj < .05 and log2FC < 1 and log2FC > 0, light magenta normal font), nonregulated genes (NS, padj ≥ .05, grey), strongly downregulated (padj < .05 and log2FC ≤ −1, green, bold font), and downregulated (padj < .05 and log2FC > −1 and log2FC < 0, light green, normal font).

PCA revealed clear separation between the experimental conditions (Fig. 1B). PC1, accounting for 75% of the variance, primarily reflected transcriptional changes driven by osmotic stress, while PC2 (10% variance) distinguished c-di-AMP levels. These findings highlight the combined influence of osmotic stress and c-di-AMP signalling on global gene expression but also showed that the effect of osmotic stress influences more genes than the reduction of c-di-AMP levels.

Under normal-salt conditions in the wild-type strain, differential expression analysis identified 76 genes (1.9%) that were significantly upregulated and 122 genes (3.0%) that were downregulated in the c-di-AMP-reduced strain (Fig. 1C). Among the most upregulated genes, dihydrofolate reductase (hvo_2919) and the cell division proteins cdrS (hvo_0582) and ftsZ 2 (hvo_0581) stood out, coupling folate metabolism and cell division to c-di-AMP levels. Conversely, highly downregulated genes included a Na+/H+ antiporter (hvo_2093) and an aspartate aminotransferase (hvo_2091), indicating regulation of ion homeostasis and synthesis of the compatible solute glutamate in the c-di-AMP-reduced strain. Differential gene expression analysis of the other conditions can be found in Fig. S2.

Differential gene expression across conditions

To systematically assess the proportion of differentially expressed genes (DEGs) under varying conditions, we analysed the distribution of genes based on fold-change thresholds (Fig. 2A, Table S5). Genes were classified as highly upregulated (log₂FC ≥ 1), moderately upregulated (0 < log₂FC < 1), highly downregulated (log₂FC ≤ −1), moderately downregulated (−1 < log₂FC < 0), or nonsignificant. In both strains, hypoosmotic stress induced a greater proportion of upregulated genes compared to strain-dependent differences, indicating that osmotic adaptation involves extensive transcriptional activation independent of c-di-AMP levels.

Figure 2.

Functional categorization and overlap of transcriptional changes under varying c-di-AMP levels and osmotic conditions in H. volcanii. (A) Proportion of DEGs across all conditions. Categories are colour-coded as strongly upregulated (padj < .05 and log2FC ≥ 1, magenta, bold font), upregulated (padj < .05 and log2FC < 1 and log2FC > 0, light magenta normal font), nonregulated genes (NS, padj ≥ .05, grey), strongly downregulated (padj < .05 and log2FC ≤ −1, green, bold font), and downregulated (padj < .05 and log2FC > −1 and log2FC < 0, light green, normal font). Each bar represents a comparison between strains or conditions, highlighting the transcriptional responses to hypoosmotic stress and lower c-di-AMP levels. (B) Overlap of highly up-and downregulated DEGs across experimental conditions visualized with an UpSet plot. The numbers above bars indicate the size of each intersection, with shared and condition-specific DEGs grouped to reveal conserved and distinct transcriptional responses to osmotic stress and c-di-AMP regulation. (C) Functional enrichment analysis of DEGs categorized by arCOG groups. Enriched categories are shown separately for highly upregulated and highly downregulated genes. Circle size represents the number of DEGs within each category, while colour intensity reflects the significance of enrichment.

To distinguish shared from condition-specific responses, we examined the overlap of DEGs across conditions (Fig. 2B). The largest shared group of DEGs (193 highly upregulated, 110 highly downregulated genes) was observed in both wild-type and c-di-AMP-reduced strains under hypoosmotic conditions, representing c-di-AMP-independent mechanisms of osmotic adaptation. In contrast, strain-specific DEGs dominated comparisons between wild-type and c-di-AMP-reduced strains, suggesting distinct regulatory mechanisms under each condition. Notably, 35 genes exhibited opposing expression patterns, being highly upregulated in the wild-type yet highly downregulated in the c-di-AMP-reduced strain under hypoosmotic stress. This subset included genes associated with ion transport and metabolism, along with several hypothetical or uncharacterized proteins (Table 1). The distinct expression profile of these genes suggests a critical role in c-di-AMP-dependent osmotic adaptation mechanisms.

Table 1.

List of 35 genes that are highly upregulated in the wild-type strain and strongly downregulated in the c-di-AMP-reduced strain under hypoosmotic conditions. Genes are sorted by locus tag and include annotations and log2 fold changes under both conditions. This subset highlights c-di-AMP-dependent transcriptional responses during osmotic stress adaptation.

HVO locus tag	Annotation	log2FC (wt normal vs wt hypo)	log2FC (wt hypo vs HTQ hypo)
HVO_0250	Hemolysin family protein	1.11	−1.24
HVO_0258	Tyrosine-type recombinase/integrase	2.15	−3.51
HVO_0386	Hypothetical protein	1.85	−1.82
HVO_1191	Proline dehydrogenase family protein	1.14	−1.14
HVO_1431	Hypothetical protein	2.48	−2.44
HVO_1432	Hypothetical protein	2.32	−2.02
HVO_1433	Hypothetical protein	2.51	−2.69
HVO_1434	Hypothetical protein	2.04	−1.09
HVO_1556	Hypothetical protein	1.44	−1.03
HVO_1561	Hypothetical protein	1.11	−1.56
HVO_2120	Sialidase family protein	2.21	−1.26
HVO_2391	Hypothetical protein	2.46	−2.96
HVO_2424	Hypothetical protein	1.19	−1.17
HVO_2627	DUF4397 domain-containing protein	1.88	−1.61
HVO_C0029	Type II toxin-antitoxin system VapC family toxin	1.56	−1.10
HVO_C0072	ABC transporter permease	4.79	−1.83
HVO_A0068	Hypothetical protein	2.53	−4.16
HVO_A0072	Cdc6/Cdc18 family protein	2.29	−2.25
HVO_A0074	AAA family ATPase	1.24	−2.14
HVO_A0163	Proline racemase family protein	2.03	−1.99
HVO_A0165	Sodium-dependent transporter	1.94	−2.28
HVO_A0168	Helix-turn-helix domain-containing protein	2.16	−1.01
HVO_A0283	Sugar ABC transporter substrate-binding protein	1.54	−1.00
HVO_A0301	Polysaccharide deacetylase	2.16	−1.07
HVO_A0336	ABC transporter ATP-binding protein	1.97	−1.53
HVO_A0337	ABC transporter permease	2.52	−1.83
HVO_A0338	ABC transporter permease	2.85	−1.36
HVO_A0339	ABC transporter substrate-binding protein	2.72	−1.74
HVO_A0350	Hypothetical protein	1.09	−1.41
HVO_A0351	Hypothetical protein	1.13	−1.34
HVO_A0594	Glutamine–fructose-6-phosphate transaminase (isomerizing)	1.39	−1.56
HVO_A0595	Sugar phosphate nucleotidyltransferase	1.49	−1.35
HVO_A0598	PadR family transcriptional regulator	4.48	−2.96
HVO_A0602	Phosphoglucosamine mutase	3.35	−2.29
HVO_A0632	Type IV pilin	3.08	−2.44

To gain deeper insight into the functional implications of these transcriptional changes, we performed functional enrichment analysis using arCOG categories (Fig. 2C, Table S6). The results revealed that transcriptional changes were either osmoregulation-specific or influenced by c-di-AMP levels. Among highly upregulated genes, those involved in amino acid transport and metabolism (category E) and energy production and conversion (category C) were enriched in both strains under hypoosmotic stress, highlighting their essential role in osmotic adaptation. In contrast, genes associated with replication, recombination, and repair (category L) and cell motility (category N) were significantly overrepresented among highly upregulated genes in c-di-AMP-reduced strains, suggesting their relevance to c-di-AMP-dependent regulatory processes. Additionally, many strongly downregulated genes fell into uncharacterized functional categories (category R, category S), indicating a subset of transcriptional responses that remain poorly understood and not well-clustered by strain or condition. These findings underscore the intricate relationship between osmotic stress, c-di-AMP regulation, and unexplored functional pathways.

Regulation of ion transport genes under osmotic stress and c-di-AMP depletion

As no significant enrichment was detected in arCOG categories associated with ion metabolism, we conducted a targeted analysis of the genes possibly involved in c-di-AMP degradation and key ion transporter genes, including mechanosensitive (Msc) channels, potassium transporters, and sodium transporters (Fig. 3).

Figure 3.

Heatmap of c-di-AMP synthesizing and degrading enzymes, Msc channels, and potassium and sodium transporters across four conditions. Colours indicate log2 fold-changes (green: downregulation, magenta: upregulation), with circles marking significant changes. Conditions: wild-type and c-di-AMP-reduced strains under normal- and hypoosmotic conditions.

The genes hvo_0756, hvo_0990 and hvo_1690 encode putative c-di-AMP phosphodiesterases, which might be responsible for c-di-AMP degradation. Remarkably, the transcription levels of these genes do not change under hypoosmotic conditions or when the strain is grown under low salt conditions. While under most conditions, no regulation of these genes is observed, the expressions of hvo_0990 and hvo_1690 are significantly downregulated under hyposalt stress conditions in the c-di-AMP-reduced strain. This suggests a possible mechanism to prevent the further depletion of c-di-AMP under hyposalt stress conditions.

Haloferax volcanii expresses eight Msc channels. These are membrane proteins that open in response to mechanical stress, such as membrane tension, allowing ions and small solutes to exit the cell and prevent lysis under hypoosmotic conditions. The dacZ gene (hvo_1660) is part of an operon alongside mscS2 (hvo_1659), a gene encoding a mechanosensitive channel of small conductance. Remarkably, despite being placed under the lower expressing ptna3 promoter, mscS2 is upregulated in this strain. This suggests an additional layer of regulation acting on mscS2 in the c-di-AMP-reduced background.

The expression of sodium transporters was predominantly modulated in response to hypoosmotic stress, with minimal regulation under the c-di-AMP-reduced conditions. These findings suggest that transcriptional adjustments of sodium transport genes are primarily driven by osmotic shifts rather than by c-di-AMP depletion itself. Notably, one Na⁺/H⁺ antiporter gene (hvo_2093, encoding a NhaC-type antiporter) was strongly downregulated in the c-di-AMP-reduced strain; however, this change was also observed when WT (wild type) cells grown under normal conditions were compared to cell grown under low-salt conditions. This downregulation likely reflects an indirect response to altered ionic conditions—with high internal potassium in the c-di-AMP-reduced strain or under the lowered sodium concentrations under the hyposalt conditions. The cell most likely minimizes sodium efflux to retain essential cations and maintain osmotic balance and the regulation might not be a direct effect of c-di-AMP signalling. hvo_A0154, encoding a member of the SSSF family transport protein was the only gene that was consistently upregulated in the c-di-AMP-reduced levels strain across both osmotic conditions, suggesting that it functions in a c-di-AMP-dependent manner and might be involved in sodium-coupled uptake of amino acids or compatible solutes, under low c-di-AMP conditions (Henriquez et al. 2021).

Finally, the expression levels of potassium transporters were analysed. We identified several potassium transporters in the genome; each associated with canonical regulatory RCK domains, suggesting potential c-di-AMP targets. A prototypical RCK domain consists of two subdomains: an N-terminal RCK_N domain, capable of binding adenine-containing nucleotides such as ATP, ADP, NADH, and NAD⁺; and a C-terminal RCK_C domain, which might bind the second messenger cyclic di-AMP (c-di-AMP). The transporter encoded by hvo_1137 harbours two RCK domains directly fused to its transmembrane domain (TMD). In contrast, hvo_1916 encodes a single RCK domain fused to its TMD. In the case of hvo_1055, which encodes a single, separate RCK domain, the transporter TMD HVO_1056 is encoded within the same operon. Similarly, the transporter TMD hvo_1057 is cotranscribed with hvo_1058, which encodes two distinct RCK domains.

Additionally, the operon comprising hvo_2616–2618 encodes three proteins with a modular organization: one protein features an RCK_N domain fused to a transporter TMD, another encodes a standalone RCK_C domain, and the third possesses an RCK_C domain fused to a transporter TMD. A similar local gene arrangement is also observed for hvo_0051 and hvo_0052: hvo_0051 encodes a membrane protein with a TMD but lacks any associated RCK domains, while hvo_0052 encodes a single RCK_C domain and is not fused to a transporter domain. This organization resembles the split architecture observed in the hvo_2616–2618 operon, albeit with fewer components. We also identified four proteins that are not fused to a TMD nor encoded within an operon containing a transporter domain. Specifically, hvo_1885 and hvo_2211 each encode single RCK domains with both RCK_N and RCK_C subdomains. hvo_2626 encodes a single RCK_N domain, while hvo_0467 encodes a protein that contains two RCK_C domains.

Analysis of the expression levels of these genes showed that for most of them neither significant regulation under hypoosmotic conditions, nor after reduction of the c-di-AMP levels could be observed. hvo_1055, the gene that encodes the RCK domain protein was upregulated in the c-di-AMP-reduced levels strain under hypoosmotic conditions. The genes encoding the two single RCK domain proteins HVO_1885 and HVO_2211 were upregulated under hypoosmotic conditions both in the WT and the c-di-AMP-reduced strain, suggesting that both proteins play a role under hypoosmotic conditions. The role of these single RCK domain proteins is unclear. They might compete for binding to transporter TMD domains or might function as c-di-AMP sinks, to regulate c-di-AMP levels.

To assess the evolutionary distribution of RCK, RCK_N, and RCK_C domain-containing proteins, we conducted a comprehensive forward and reciprocal BLAST analysis using homologs of H. volcanii RCK, RCK_N, and RCK_C domain proteins across 54 representative archaeal genomes (Fig. 4). This analysis revealed that the occurrence of these proteins, along with their associated TMDs, is closely correlated with the presence of a DacZ homolog, further supporting the hypothesis that these proteins may bind the second messenger c-di-AMP.

Figure 4.

Distribution of c-di-AMP-related proteins across archaeal species. The presence or absence of proteins homologous to H. volcanii HVO_1660 (DacZ), three putative phosphodiesterases of the DHH family (HVO_0756, HVO_0990, and HVO_1690), and 15 other c-di-AMP-associated genes involved in potassium homeostasis (e.g. HVO_1055 and HVO_1056) was analysed across 54 archaeal genomes. The top row shows a phylogenetic tree based on 16S rRNA sequences. Each cell represents one BLAST result for a given protein in each organism: Filled shapes indicate a RBH (both forward and reverse BLAST confirmed). Outlined shapes show a forward BLAST hit, but the reverse BLAST did not return the original query as top hit. Empty cells denote no significant hit was found in the forward BLAST. The shape of each marker corresponds to a specific query protein (e.g. red stars for HVO_1660/DacZ). On the right side, the different domains present in the respective protein is indicated: grey = pk_C, red = DAC, pink = RCK_N, green = RCK_C, yellow = transmembrane domain TMD, and blue = DHH/DHHA1.

To further investigate the function of these RCK domain-containing proteins, four candidates were selected: HVO_1055 and HVO_1058, which are proteins containing respectively a single and two RCK domains proteins, and both are encoded in operons together with their predicted transporter TMDs, HVO_1056 and HVO_1057. In addition, HVO_1885 and HVO_2211, which both are single RCK domain proteins which are not associated with a TMD-encoding gene, were also included in the study.

Growth and shape analysis of four deletion mutants of RCK domain-containing proteins

To investigate the physiological roles of HVO_1055, HVO_1058, HVO_1885, and HVO_2211, deletion mutants of the genes encoding these proteins were constructed in both the wild-type H. volcanii H26 strain and the c-di-AMP-reduced background strain HTQ410. The strains were cultured under three different ionic conditions for up to 216 h: standard salt conditions (2.5 M NaCl, 55 mM KCl), low potassium conditions (2.5 M NaCl, 5 mM KCl), and low sodium conditions (1.8 M NaCl, 55 mM KCl). Growth patterns were highly reproducible across the four independent biological replicates. Two representative biological replicates (each with three technical replicates) are shown (Fig. 5) as they reflect the overall trends.

Figure 5.

Growth curves of H. volcanii wild-type and gene knockouts under different salt stress conditions. Strains were cultured in (A) standard salt conditions (2.5 M NaCl and 55 mM KCl), (B) low potassium (2.5 M NaCl and 5 mM KCl), and (C) low sodium (1.8 M NaCl and 55 mM KCl) for up to 216 h. Upper panels show growth of the wild-type strain WT H26 and four single-gene deletion strains (ΔHVO_1055, ΔHVO_1058, ΔHVO_1885, and ΔHVO_2211). Lower panels show the c-di-AMP-reduced strain HTQ410 and its corresponding double knockouts with the same gene deletions. Growth was monitored by measuring OD₆₀₀ at the indicated time points. Data from two representative biological replicates (each with three technical replicates) are shown, reflecting trends observed across four independent experiments. Values are mean ± SD of technical replicates.

Deletion of hvo_1055 resulted in impaired growth, particularly under ionic stress. The mutant showed reduced growth under low sodium conditions, with a more pronounced defect under low potassium conditions. Interestingly, in the c-di-AMP-reduced HTQ410 background, the mutant exhibited enhanced growth and reached a higher final optical density under standard conditions. However, growth was again reduced under low sodium and even more severely under low potassium conditions. The deletion of hvo_1058 led to a mild growth defect under standard conditions, which became more evident under low sodium and low potassium conditions. In the c-di-AMP-reduced background, the mutant displayed a faster growth rate than the wild-type under standard conditions but ultimately reached a similar final OD. Under low sodium conditions, a slight growth reduction was observed, while growth was significantly impaired under low potassium conditions. In contrast, deletions of hvo_1885 and hvo_2211 displayed minimal phenotypes in the wild-type background. Deletion of hvo_1885 had no discernible impact on growth under any of the tested conditions in either the wild-type or c-di-AMP-reduced background. Similarly, deletion of hvo_2211 did not affect growth under standard or low potassium conditions. However, under low sodium conditions, the hvo_2211 deletion mutant exhibited a severe growth defect, with almost no detectable growth.

We previously reported that a strain with reduced c-di-AMP levels exhibited increased cell area in low-salt medium, consistent with impaired osmoregulation (Braun et al. 2019). To assess the impact of individual RCK-domain gene deletions on cell morphology, cells were sampled during exponential growth and analysed microscopically under standard, low-sodium, and low-potassium conditions (Fig. 6). Quantification of cell area (Fig. S3) confirmed that the c-di-AMP-reduced levels strain displayed a marked increase in cell size under standard conditions, which was partially suppressed under potassium-limiting conditions, suggesting that potassium restriction mitigates the effects of unregulated potassium uptake. In contrast, sodium limitation further exacerbated cell enlargement in this strain. The hvo_1055 deletion strain showed an increased cell area under low-potassium conditions compared to wild type, with a further enlargement under low-sodium conditions. In the c-di-AMP-reduced background, hvo_1055 deletion led to a significant increase in cell area across all tested conditions, most notably under sodium limitation. In contrast, the hvo_1058 deletion strain exhibited cell areas similar to wild type under standard and low-potassium conditions but showed substantial enlargement under low-sodium conditions. However, deletion of hvo_1058 in the c-di-AMP-reduced background did not significantly alter the cell area. As was observed in the growth curves, deletion of hvo_1885 in both the WT and the c-di-AMP-reduced strain did not strongly affect the cell shape and area of the cells, except when the deletion was made in the c-di-AMP-reduced strain and was grown on low sodium medium. Here, the cell shape and area more resembled the WT strain grown on normal conditions and significantly deviated from the c-di-AMP-reduced strain under these conditions. For the hvo_2211 deletion strain no changes in cell morphology or cell area were observed when compared to the WT strain, however, in the c-di-AMP-reduced background, deletion of hvo_2211 led to a reduction in cell size under normal and sodium-limiting conditions, while in contrast an increase in cell area was observed under potassium limitation.

Figure 6.

Representative images of H. volcanii strains with different gene knockouts in the WT and c-di-AMP-reduced (HTQ410) strain under different salt conditions. Phase-contrast images of wild-type (H26) and c-di-AMP–reduced (HTQ410) backgrounds with deletions of HVO_1055, HVO_1058, HVO_1885, and HVO_2211 after 50 h of cultivation. Cells were grown under three conditions: normal salt (2.5 M NaCl, 55 mM KCl), low potassium (2.5 M NaCl, 5 mM KCl), and low sodium (1.8 M NaCl, 55 mM KCl). Representative fields from each strain and condition are shown at 1000X magnification. Scale bar: 5 µm.

Deletion of hvo_1055 strongly affects survival rates under potassium-limiting conditions

To evaluate the functional relevance of selected c-di-AMP–associated genes under enhanced osmotic stress, we conducted spot-dilution assays across a range of extracellular potassium and sodium concentrations (Fig. 7A and B). Growth was assessed based on the last dilution showing visible colony formation and semiquantitative scores were assigned, and scores are presented as log₁₀-transformed values and used for comparative analysis. Under potassium-limiting conditions (0–1 mM potassium), deletion of hvo_1055 abolished growth in both the wild-type and c-di-AMP-reduced strains, highlighting its essential role in potassium acquisition or homeostasis. In contrast, deletion of hvo_1058, hvo_1885, or hvo_2211 had no discernible impact on growth under the same conditions, suggesting that these genes are either dispensable or functionally redundant in potassium-limited environments. Under varying sodium concentrations, the c-di-AMP-reduced strain exhibited reduced viability compared to the wild type across all tested conditions. However, no significant differences in survival were observed among the deletion strains in either the wild-type or c-di-AMP-reduced backgrounds.

Figure 7.

Colony forming units after serial dilution of the WT and c-di-AMP-reduced strains containing the different Trk domain deletions on plates with different potassium and sodium concentrations. Spot-dilution assays were performed to assess the growth capacity of wild-type (H. volcanii H26), individual gene deletion strains (ΔHVO_1055, ΔHVO_1058, ΔHVO_1885, and ΔHVO_2211), and c-di-AMP-reduced strain HTQ410 with or without additional gene deletions, under different concentrations of potassium (top) and sodium (bottom). Growth was determined based on colony dilution series and are plotted on a log₁₀ scale (y-axis). The x-axis indicates the salt concentration in the plate (A, potassium: 0–50 mM; B, sodium: 1.6–2.5 M). Solid symbols represent single deletion strains in the WT background, while open symbols indicate deletions in the HTQ410 (low c-di-AMP) background.

HVO_1055 and HVO_1058 bind c-di-AMP

To determine whether c-di-AMP directly interacts with TrkA-like RCK domain proteins in H. volcanii, we performed DRaCALA using radiolabelled c-di-AMP and purified recombinant proteins (Fig. 8). HVO_1055, HVO_1058, HVO_1885, and HVO_2211 were successfully expressed in E. coli and confirmed by SDS-PAGE (Fig. 8, left panel), indicating sufficient yield and purity for binding analysis. In the absence of a competitor, strong c-di-AMP binding signals were observed for HVO_1055 and HVO_1058. These signals were progressively reduced by the addition of unlabelled c-di-AMP at concentrations of 1 µM and 1 mM, demonstrating specific, dose-dependent binding. In contrast, the addition of 1 mM ATP did not affect c-di-AMP binding, indicating highly specific c-di-AMP binding. By comparison, HVO_1885 and HVO_2211 exhibited only weak or negligible binding under the same assay conditions. These findings identify HVO_1055 and HVO_1058, both cotranscribed with TrkH-like transporter genes, as specific c-di-AMP–binding proteins in H. volcanii. The ability of c-di-AMP to bind HVO_1055 and HVO_1058 supports a model in which c-di-AMP serves as an allosteric regulator of RCK-containing potassium transport complexes, consistent with its proposed role in modulating TrkH-type transporter activity to maintain ion homeostasis. These findings also demonstrate that both single TrkA-like RCK domain proteins HVO_1885 and HVO_2211 do not bind c-di-AMP and mediate osmotic adaptation through alternative, c-di-AMP independent mechanisms.

Figure 8.

DRaCALA performed with different Trk domain proteins overexpressed in E. coli. (A) SDS-PAGE analysis confirming expression of His-tagged candidate TrkA-like proteins (HVO_1885, HVO_2211, HVO_1058, and HVO_1055) and control lysate. As is often observed for proteins of H. volcanii,, the acidic proteins were detected at higher apparent molecular weights then their predicted masses of 24, 25, 48, and 25 kDa. (B) Image of a DRaCALA using radiolabelled c-di-AMP to assess direct ligand binding. Competition with 1 mM cold ATP and with 1 µM and 1 mM nonradioactive c-di-AMP.

Discussion

To assess how osmotic stress and reduced c-di-AMP levels affect gene expression in H. volcanii, we analysed transcriptomic differences between the wild-type and a c-di-AMP-reduced strain under both standard and low-salt conditions. The results revealed that although both factors significantly shape global transcriptional responses, osmotic stress impacted a broader range of genes than c-di-AMP depletion alone. In this study, we specifically concentrated on the transcriptional changes associated with reduced c-di-AMP levels, and identified several genes that were highly up- or downregulated.

Two highly expressed genes, ftsZ2 and cdrS, were upregulated in the c-di-AMP-reduced strain. FtsZ is a tubulin-like GTPase that polymerizes into a Z-ring at the division site, playing a central role in bacterial and archaeal cytokinesis. Haloferax volcanii encodes two distinct FtsZ homologs: FtsZ1, which localizes early to the division site and likely recruits other division proteins, and FtsZ2, which contributes to constriction and stabilizes the divisome (Liao et al. 2021a). SepF, CdpB1, CdpB2, and CdpA are additional key components of the archaeal divisome in H. volcanii, coordinating membrane anchoring and spatial organization of the FtsZ2 ring during cell division. SepF acts as a membrane anchor and interacts with CdpB1. The CdpB1–CdpB2 heterodimers further polymerize in a head-to-tail fashion, possibly creating a filament-like structure that resembles a chain aligned along the inner membrane at the division site (Nußbaum et al. 2021, Pende et al. 2021, 2024, Zhao et al. 2024). CdpA was shown to interact with FtsZ2 to ensure proper ring positioning (Liao et al. 2025). Together, these proteins form a complex network essential for the accurate assembly and functioning of the cell division machinery.

CdrS is a transcriptional regulator encoded by a small ORF (open reading frame) within the ftsZ2 operon and appears to act as a key intermediate linking c-di-AMP signalling to cell division control (Liao et al. 2021b, Darnell et al. 2020). In H. volcanii, CdrS positively regulates several division-related genes, including ftsZ1, ftsZ2, sepF, cdpB1, and cdpA, and it also binds to the promoter of dacZ. CdrS regulates its own expression, suggesting an autoregulatory or feedback loop. It has been proposed that the CdrS–FtsZ2 circuit coordinates the timing of cell division with growth conditions in archaea (Darnell et al. 2020). In c-di-AMP-reduced cells, expression of cdrS and several cdrS-regulated genes (ftsZ2, sepF, cdpB1, and cdpA) is upregulated. This suggests that CdrS not only regulates dacZ expression but is itself regulated by c-di-AMP levels, implying that c-di-AMP indirectly regulates cell division proteins like FtsZ2 through CdrS.

Hvo_2919, which encodes a dihydrofolate reductase, is also upregulated in strains with reduced c-di-AMP levels. This observation aligns with previous suggestions that folate metabolism and c-di-AMP signalling are functionally connected. In Firmicutes, perturbations in folate/thymidine pathways can feed back into c-di-AMP production: treatment of Staphylococcus aureus or Listeria with antifolate drugs (e.g. DHFR inhibitors such as trimethoprim) elevates c-di-AMP levels (Tang et al. 2022). Similarly, thymidine auxotrophic small-colony variants of S. aureus, which carry folate cycle disruptions, also overproduce c-di-AMP (Tang et al. 2022). A block in folate-dependent thymidine synthesis is sensed as a metabolic stress, triggering elevated c-di-AMP accumulation (Tang et al. 2022). By analogy, the inverse scenario may also occur: if c-di-AMP levels decrease, cells could experience a metabolic imbalance that prompts compensatory adjustments in folate metabolism. In H. volcanii, which uses tetrahydrofolate as its one-carbon carrier (in contrast to methanopterin in some archaea) (Pfeiffer and Dyall-Smith 2021), a disturbance in c-di-AMP signalling may disrupt nucleotide pool balance or replication processes, thereby triggering folate-related metabolic responses and resulting in the upregulation of hvo_2919.

Hvo_2091, which most likely encodes a putative pyridoxal phosphate-dependent aminotransferase capable of generating glutamate from aspartate using α-ketoglutarate, was strongly downregulated in the c-di-AMP-reduced strain. Glutamate is a well-known compatible solute and serves as the primary counter-ion for potassium, helping to maintain electroneutrality. Indeed, potassium and glutamate are the most abundant intracellular cation and anion, respectively, and their concentrations must be tightly balanced (Gundlach et al. 2018). In B. subtilis, depletion of c-di-AMP leads to glutamate toxicity (Krüger et al. 2021). This toxicity arises from glutamate’s role in stimulating potassium uptake via channels, such as KtrCD, thereby exacerbating the stress associated with dysregulated potassium homeostasis in the absence of c-di-AMP (Krüger et al. 2021). These findings highlight the need to restrict intracellular glutamate levels to preserve cellular homeostasis. Thus, the reduced expression of hvo_2091 in c-di-AMP–reduced strains may serve to prevent excessive accumulation of glutamate and potassium.

In this study, we further investigated the effects of reduced extracellular sodium and potassium concentrations on the growth and morphology of wild-type and c-di-AMP-reduced strains of H. volcanii. Consistent with previous findings, the c-di-AMP–reduced strain exhibited a marked increase in cell area under standard conditions, attributed to excessive intracellular accumulation of potassium due to deregulated uptake (Braun et al. 2019). This phenotype was partially suppressed under low-potassium conditions, supporting the role of c-di-AMP in limiting potassium influx. However, under low-sodium conditions, growth and morphological defects were significantly more pronounced, particularly in the c-di-AMP-reduced background. The hypoosmotic stress induced by reduced external sodium led to increased water influx and cell swelling. While wild-type cells mitigated this through effective regulation of ion homeostasis, most likely through inhibition of sodium uptake and activation of efflux pathways, cells with lowered c-di-AMP levels failed in this adaptive response, resulting in uncontrolled potassium accumulation and severe osmotic imbalance. These findings underscore the critical role of c-di-AMP in orchestrating ion fluxes during osmotic stress, especially under sodium-limiting conditions. Moreover, since sodium gradients contribute to membrane potential and transport dynamics, their disruption further exacerbates the cellular inability to maintain homeostasis in the absence of proper regulatory control. Collectively, our results demonstrate that sodium limitation poses an even greater challenge to cellular osmoadaptation than potassium limitation.

To further elucidate the roles of RCK domain-containing proteins in ion homeostasis, the phenotypes of four genes, hvo_1055, hvo_1058, hvo_1885, and hvo_2211, were analysed by generating deletion mutants in both wild-type and c-di-AMP–reduced backgrounds. The resulting phenotypes were interpreted in the context of established mechanisms of c-di-AMP-mediated regulation and osmoadaptation.

HVO_1055

Hvo_1055 encodes a single RCK domain and lies in an operon with a gene encoding the potassium transporter domain protein HVO_1056. The binding of c-di-AMP to HVO_1055 could be shown in DRaCALA assays. Its deletion had a dramatic impact on growth, especially under ionic stress. In wild-type cells, the deletion mutant grew poorly when potassium was limited and even worse when sodium was low, which is consistent with a loss of a major potassium importer needed for adaptation to low potassium levels or osmotic down shock. At 0–1 mM external potassium, the mutant essentially could not grow on plates, indicating that the HVO_1055/HVO_1056 transporter is critical for importing potassium at low concentrations. This is analogous to losing a Kdp/Ktr system in bacteria, which causes severe potassium starvation under potassium limitation (Cereija et al. 2021). Under normal salt conditions, the hvo_1055 deletion strain grew normal, but under low salt stress, the strain showed a growth defect and most likely had problems to maintain potassium homeostasis. In the c-di-AMP-reduced background, a reversal was seen: the hvo_1055 deletion strain showed improved growth under standard conditions compared to the c-di-AMP mutant alone. However, growth was again reduced under low potassium and even more severely under low sodium conditions. This suggests that when c-di-AMP levels are low, the presence of HVO_1055 becomes detrimental, likely because an unchecked HVO_1056 transporter would allow excessive potassium influx. In the strain with a reduced c-di-AMP level, potassium importers are hyperactive (since c-di-AMP is normally needed to restrain them), so these cells tend to accumulate too much potassium, leading to internal ionic imbalance and enlarged cells (Braun et al. 2019). Removing the high-affinity potassium uptake system reduces the import of potassium and the internal potassium levels, so that it grew better and had a more normal cell size under optimal salt conditions. However, the hvo_1055 deletion strain in the low-c-di-AMP background again showed reduced growth and an increase in cell volume under low sodium under stress, indicating that under these conditions the transporter is still required. In summary, HVO_1055/1056 likely constitutes the primary potassium uptake system (similar to a Trk/Ktr high-affinity transporter). It is normally regulated (inhibited) by c-di-AMP binding to HVO_1055. When deleted, cells cannot acquire enough potassium in stressful conditions causing poor growth in low potassium, and they mismanage volume under hyposaline conditions, causing extreme cell enlargement as water influx is not countered by proper ion adjustment. The severity of the effects of the hvo_1055 deletion underscores its key role in potassium homeostasis and osmoregulation.

HVO_1058

Hvo_1058 encodes a protein with two RCK domains and lies in an operon with the gene encoding the potassium transporter domain protein HVO_1057. Binding of c-di-AMP to HVO_1058 was demonstrated in DRaCALA assays. The phenotype of the hvo_1058 deletion strain was similar to the phenotype of the hvo_1055 deletion strain but milder. In the WT background, deletion of hvo_1058 caused only a slight growth defect in normal medium, but a larger defect was observed under low potassium and especially low sodium conditions. This indicates that the HVO_1057/1058 transporter is a secondary potassium uptake system, which is not as critical as the HVO_1055/1056 transporter but still important when cells are grown under low salt conditions. Perhaps it provides additional potassium uptake capacity or fine-tuning under suboptimal conditions. Notably, the hvo_1058 deletion strain did not abolish growth at low potassium as was observed for the hvo_1055 deletion strain, implying functional redundancy. Under low sodium conditions however, even this secondary system becomes important, as indicated by the substantial growth impairment of the hvo1058 deletion strain under these conditions. This fits a model where multiple potassium uptake systems are recruited when the cell is coping with osmotic stress. In the c-di-AMP–reduced background, the hvo_1058 deletion again resembled the hvo_1055 deletion but to a lesser extent: early in growth, the OD (optical density) of the hvo_1058 deletion strain increased faster than the c-di-AMP-reduced parental strain, possibly because removing a potassium importer slightly relieved the excessive potassium influx, but final growth yields were similar. At low potassium concentrations, the hvo_1058 deletion in the c-di-AMP-reduced strain showed a minor additional growth defect, and under low sodium a strong defect was observed, confirming that in a cell already reduced in c-di-AMP, losing any potassium transporter severely harms the cells capability to respond to osmotic stress. The observed cell shapes mirrored these results: The hvo_1058 deletion strain had normal-sized cells in standard and low potassium medium, but under low sodium it produced many enlarged cells. Interestingly, introducing the hvo_1058 deletion into the c-di-AMP–reduced strain did not dramatically worsen the cell-size phenotype beyond what low c-di-AMP alone caused. This further suggests that the HVO_1057/1058 transporter is less central to osmoregulation than the HVO_1055/1056 transporter, or that in the absence of c-di-AMP the primary transporter’s effect dominates. In summary, HVO_1058 appears to regulate a potassium uptake system that is auxiliary: its loss is tolerable in normal conditions but becomes deleterious as external conditions become more challenging, especially under low sodium conditions. Like HVO_1055, it is presumably a c-di-AMP target that normally helps adjust the potassium flux. The presence of two RCK domains in HVO_1058 in comparison with only one RCK domain in HVO_1055 might allow to integrate multiple signals like energy charge via ATP and c-di-AMP levels to modulate HVO_1057 activity, ensuring potassium uptake responds appropriately to both cellular energy status and osmotic state.

HVO_1885

Hvo_1885 encodes a stand-alone RCK domain protein that is not linked to any identifiable potassium transporter and shows no impact on growth or morphology when deleted under standard or potassium-limiting conditions. This suggests that HVO_1885 is not essential for potassium homeostasis under normal conditions and may serve a redundant or condition-specific role. Its expression is upregulated under low-salt conditions, even in wild-type cells, pointing to a potential function in osmoadaptation. In a c-di-AMP-reduced background, where cells typically exhibit large swelling under sodium-limiting conditions due to the excessive accumulation of potassium, deletion of hvo_1885 suppressed the cell enlargement phenotype. This initially suggested that HVO_1885 might act as a c-di-AMP ‘sink’, binding and sequestering c-di-AMP and thereby exacerbating dysregulation. However, in our DRaCALAs HVO_1885 did not bind c-di-AMP, ruling out this mechanism. This suggests HVO_1885 modulates osmotic balance through a c-di-AMP–independent mechanism, potentially through physical interactions with transporter complexes or other regulatory proteins, rather than by sequestering c-di-AMP. One possibility is that HVO_1885 influences the gating or activity of potassium channels via RCK–RCK domain interactions, thereby affecting potassium flux during osmotic stress. The alleviation of the low-sodium phenotype in c-di-AMP-reduced cells lacking hvo_1885 may reflect an indirect effect on transporter dynamics or signalling pathways that compensate for the absence of c-di-AMP regulation. Overall, HVO_1885 appears to play a modulatory, c-di-AMP-independent role in osmotic adaptation that becomes evident only when primary regulatory systems are impaired. Its lack of phenotype in wild-type cells, contrasted with its suppressive effect in the c-di-AMP-reduced strain, highlights its potential function as a conditional regulator of ionic balance during hypoosmotic stress.

HVO_2211

Hvo_2211 encodes a solitary RCK domain-containing protein with no associated membrane transporter and is upregulated under low-salt conditions. This suggests a role in the osmotic stress response of H. volcanii. Deletion of hvo_2211 in a wild-type background caused a severe growth defect specifically under sodium-limiting conditions, indicating hypersensitivity to hypoosmotic stress. In contrast, no growth impairment was observed under standard salinity or potassium limitation, suggesting that HVO_2211 is dispensable under nonstress conditions but becomes essential during osmotic downshift. Although many RCK proteins function as c-di-AMP effectors, our biochemical analysis showed that HVO_2211, like HVO_1885 does not bind c-di-AMP, and implies that its role in osmo-adaptation is c-di-AMP–independent. The pronounced phenotype under sodium limitation suggests that HVO_2211 may function as a regulatory factor in a parallel or auxiliary pathway, potentially interacting with ion transporters or signalling proteins to promote potassium export or limit intracellular osmolyte accumulation. Its synteny with a putative transcriptional regulator gene (hvo_2212), which is also conserved among other halobacteria, suggests that HVO_2211 may participate in a stress-responsive regulatory module activated under salt stress. Interestingly, deletion of hvo_2211 in the c-di-AMP-reduced background produced mixed phenotypes: under sodium limitation, cell swelling was partially alleviated, whereas under potassium limitation, cell enlargement increased. These contrasting effects point to a complex and context-dependent role for HVO_2211 in managing ionic balance during stress, possibly by coordinating compensatory mechanisms when c-di-AMP-mediated regulation is impaired. Taken together, our results indicate that HVO_2211 is a critical, c-di-AMP-independent factor required for osmotic resilience under sodium stress, acting through an as-yet undefined mechanism to maintain ion homeostasis and cell integrity.

In summary, each of the four RCK domain proteins plays a distinct role: HVO_1055 and HVO_1058 are integral to potassium import systems, with HVO_1055/1056 being the primary high-affinity uptake for potassium and HVO_1057/1058 a secondary system, whereas HVO_1885 and HVO_2211 are regulatory/accessory RCK proteins that come into play mainly during osmotic stress. The stand-alone RCK proteins HVO_1885 and HVO_2211 appear to fine-tune the c-di-AMP regulatory network: HVO_1885 may buffer excessive signalling, whereas HVO_2211 could facilitate appropriate responses. Deletion of either gene results in subtle phenotypes that manifest only under stress or in combination with altered c-di-AMP levels. Together, our findings suggest that haloarchaea have evolved a robust, multifaceted osmotic stress response integrating c-di-AMP monophosphate signalling and RCK domain regulators. This expands the paradigm of second messenger signalling by demonstrating that even in highly halophilic archaea, c-di-AMP coordinates ion homeostasis and cell volume control, much like in bacteria, highlighting a similar strategy for environmental stress adaptation.

Conflict of interest

The authors declare no conflicts of interest.

Funding

This work was supported by a grant to S.V.A from the Deutsch Forschungsgemeinschaft (DFG) within the scope of SPP1879: Nucleotide Second Messenger Signaling in Bacteria. S.V.A. also received funding from the DFG under Germany’s Excellence Strategy (CIBSS-Exc_2189-Project ID 390939984). D.G. gratefully acknowledges financial support by the Deutsche Forschungsgemeinschaft (SFB960-TP7).

Data availability

RNA-seq data are available at the European Nucleotide Archive (ENA, https://www.ebi.ac.uk/ena) under project accession number PRJEB94923.