The continuous escalation in the global population is the biggest apprehension of the present era. To cope with the rising demand for food, the only solution is the enhancement of crop productivity, which is achievable by maintaining a healthy soil environment in agricultural lands and transforming degraded and marginal lands for cultivation. The worldwide estimate of marginal land is approx. 1.3 × 10⁹ ha, which can feed 1/3 of the global population (Kuang et al., 2022). India has 46.7 million ha of land affected by salinity and drought (Syed et al., 2021). Some arable soils become unfit for cultivation due to various abiotic stresses, including drought, salinity, temperature extremes, flooding, heavy metals, and organic contamination (Poria et al., 2022). The health of soil greatly depends on the type of microbiome (bacteria, fungi, etc.) it inhabits, as they are responsible for solubilizing and recycling essential nutrients in the soil, governing the biogeochemical cycles. Bacteria colonize the plant rhizosphere or the surrounding soil and deliver plenty of benefits to plants, such as nutrient acquisition, synthesis or modulation of plant hormones, and production of bioweapons against phytopathogens (Afridi et al., 2022; Sunar et al., 2023). They employ direct and indirect mechanisms to confer beneficial effects on plants (Ramakrishna et al., 2019). In direct mechanisms, essential phytohormones such as auxin, cytokinin, ethylene, and gibberellin are modulated and/or the supply of growth nutrients such as phosphate, nitrogen, potassium, iron, and zinc is enhanced. In indirect mechanism, they confer protection to plants against various abiotic stresses (salinity, drought etc.) by producing organic acids, polyamines, glycine, betaine, proline, butyric acid, trehalose, sucrose, mannitol, and glutamine (Kumawat et al., 2022) and phytopathogens through the production of lytic enzymes, antibiotics, siderophores, induction of systemic resistance, modulation in ethylene levels, and direct competition with phytopathogens (Akhilesh et al., 2018; Oleńska et al., 2020).

During stress regulation, bacteria have also been shown to be associated with the activation of plant antioxidant defense machinery by altering the activity of scavenging enzymes and regulating the production of reactive oxygen species (ROS), phytohormones, proteins, and polysaccharides (Sharma et al., 2021). Many rhizobacteria produce the 1-aminocyclopropane-1-carboxylate (ACC) deaminase enzyme, which initiates a cascade of physiological and biochemical changes in the plant, thus helping them withstand the effects of abiotic stresses (Forni et al., 2017). Efficient use of such beneficial microbes to alleviate abiotic stress effects in crops is the most convenient, inexpensive and environment-friendly approach for improving global food production.

Drought and salinity result in osmotic shock in plants leading to mortality and growth retardation, ultimately decreasing crop productivity (Gupta et al., 2022). The application of osmotolerant bacteria has the potential to reduce the symptoms induced by salinity or drought stress. The use of halo-tolerant bacteria increases the plant’s ability to adapt or tolerate salinity stress by improving osmoregulatory mechanisms and helping in plant survival (del Carmen Orozco-Mosqueda et al.,2020). However, limited reports are there showing the potential metabolite markers of bacteria that play a role in the alleviation of salinity or drought stress. The mechanism of secretion of various chemical stimulants synthesized by microbes under a stressful environment is largely unexplored. Also, there is a dearth of information about cell signaling networks and the involvement/role of bacteria in plant drought and salt adaptation.

Staphylococcus warneri is a facultative, anaerobic, gram-positive mesophilic bacterium that includes mostly non-pathogenic strains from extreme desert conditions, hypersaline environments, and oil-contaminated soils (Degtyareva et al., 2020; Shurigin et al., 2020; Caglayan, 2022), suggesting that it is a highly drought and salt-tolerant bacterium. It produces a variety of antibiotics and secondary metabolites and possesses hydrocarbon-oxidizing properties (Bastos et al., 2009). Some S. warneri strains have been shown to be part of human and mouse gut microbiota and have a positive effect on intestinal physiology (Louail et al., 2023). Staphylococcus warneri SG1 has been shown to tolerate up to 2.5% (vol/vol) butanol in rich liquid media, making it a candidate for biofuel production (Cheng et al., 2013). A range of genes, including heat shock proteins, transcription factors, and other stress-responsive proteins that contribute to tolerance against stressful environments, are upregulated in S. warneri. As it is a good multi-stress tolerant strain, it can be a good candidate for use in marginal lands as a microbial bio-inoculant for sustainable agriculture practices. Although salt tolerance has already been explored, drought tolerance is first reported in this study.

Despite similarities with other strains, the precise mechanism underlying S. warneri CPD1’s extreme stress adaptation remains unclear. Hence, this study hypothesizes that S. warneri CPD1 employs a unique and coordinated network of gene expression changes across the specific biochemical pathways to manage extreme osmotic stress, which could potentially enhance plant resilience in similar environmental conditions. By investigating the transcriptomic landscape of S. warneri CPD1 under salinity and drought stress, we aim to uncover the biochemical pathways and regulatory mechanisms facilitating its osmoadaptation and survival. Further, the present work seeks to elucidate how this stress-tolerant bacterial response may interact with the plants to manage drought and salinity. The findings aim to address the knowledge gap in understanding specific bacterial mechanisms to improve crop resilience in saline and drought-prone soils, a promising strategy for sustainable agriculture.

Several phosphate-solubilizing bacterial strains were isolated from a cotton field near Bathinda, Punjab, India. Screening for the presence of plant growth-promoting traits and stress attributes and 16S rRNA gene sequencing and characterization for selected candidates were performed previously (Rathore et al., 2021). For this study, we chose a single isolate with tolerance to PEG and NaCl, namely CPD1.

Sheep blood agar assay

S. warneri bacterial isolate CPD1 was revived from glycerol stock on NB agar plates. Colony was picked from an agar plate and inoculated in nutrient broth. Sheep blood agar plates (HiMedia) were streaked from nutrient broth inoculum and incubated at 37°C for 48 h.

Whole genome sequencing

DNA isolation was carried out using DNEasy Ultraclean Microbial kit (Qiagen Inc.). DNA was quantified using Nanodrop 1000. Libraries were constructed followed by quantification using Qubit 4.0 fluorometer (Thermofisher Inc.) using DNA HS assay kit (Thermofisher Inc.) following manufacturer’s protocols. The whole genome sequencing of CPD1 was performed on the NOVASEQ 6000 Illumina platform. The annotation of the draft genome was done using the Bakta tool using the following databases: AMRFinderPlus [release 2022-01-28], COG [release: 2020], DoriC [release: "10"], ISFinder [release: 2019-09-25], Mob-suite [release: 2.0], Pfam [release: 35], RefSeq [release: r210], Rfam [release: 14.7], UniProtKB/Swiss-Prot [release: 2021_04], and VFDB [release: 2022-01-28]. Functional annotation based on the protein sequences (obtained using Bakta) was done using PANNZER2 (Supplementary data Fig. S1). Genome-based classification and identification of prokaryotic strains and generation of the phylogenetic tree were performed using Type Strain Genome Server (TYGS) (https://tygs.dsmz.de/) and plotted using Interactive Tree Of Life (iTOL) (Letunic and Bork, 2019). The circular genome plot was plotted using Proksee/CGview (Grant and Stothard, 2008). The above process was performed by Neuberg Supratech, New Delhi, India.

Salt and drought tolerance assay

The salt tolerance of the strain was assessed by its ability to grow in LB medium with varying NaCl concentrations (1–10% w/v). To evaluate drought tolerance, LB medium supplemented with different concentrations, i.e., 10% to 40% of polyethylene glycol (PEG-6000), was prepared and inoculated with the bacterial culture followed by incubation on a shaker incubator (120 rpm) at 37°C. The cell growth was evaluated by measuring the absorbance at 600 nm every three hours for making a growth curve. The cells were pelleted after 24 h and sent to Neuberg Supratech (India) for transcriptomic analysis.

For transcriptomic studies, CPD1 was given both NaCl and polyethylene glycol (PEG-6000) treatments individually (CPD1S and CPD1PG, respectively) and combined treatment (CPD1PS). Thus, three treatments were used, i.e., 20% PEG, 10% NaCl, and both. Bacterial cultures were grown overnight at 37°C. The study was designed to gain insights into gene expression dynamics involved in osmo-adaptation, distinguish the genes between the two types of stress treatments, and find common among the other treatments, if any.

Transcriptomic analysis

Different sets of osmotic stress treatments were used for transcriptomic studies. The goal was to observe and analyze the differentially expressed genes (DEG) in individual sets and find the similarities and dissimilarities between different sets. RNA isolation was performed using Trizol reagent (Invitrogen). Extracted RNA was quantified on Qubit 4.0 fluorometer (Thermofisher) using RNA HS assay kit (Thermofisher) as per manufacturer’s protocol. Sequencing was performed on Illumina (NOVASEQ 6000). Raw fastq reads were subjected to quality assessment using FastQC v.0.11.9 (Brown et al., 2017). Fastp v.0.20.1 (parameters: phred 30; trim_front1 7; trim_front2 7; length_required 50; correction trim_poly_g) was used to preprocess the raw fastq reads (Chen et al., 2018), followed by quality re-assessment using FastQC (Supplementary data Fig. S2). The FastQC reports were further compiled into a single report using MultiQC (Ewels et al., 2016). The CPD1 draft genome was indexed using bowtie2 (Langmead and Salzberg, 2012). Processed samples were mapped to the indexed Staphylococcus warneri genome using bowtie2 (parameters: met-file’, un-conc-gz, al-conc-gz). Feature count v. 0.46 (Liao et al., 2014) (parameters: -g gene_id -F GTF -t CDS -p) was used to obtain gene counts from the aligned reads, which were used as inputs to DESeq2 (Love et al., 2014) for evaluating differential expression with threshold of statistical significance as alpha 0.05 with p-value adjustment method (http://www.bioconductor.org/packages/release/bioc/html/DESeq2.html). The above analysis was carried out by Neuberg Supratech, New Delhi, India. DEGs with a cut-off p-value ≤ 0.05 and an absolute log₂ ratio value ≥ 1 were chosen for further analysis. The association of DEGs with metabolic pathways was evaluated using Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis. The functional and pathway enrichment analysis revealed an upregulation of genes related to transport, metabolic processes, and stress-regulating enzyme activity.

Co-regulatory gene networks analysis

The genes with significant Log2-fold changes and their corresponding p-values under for CPD1PS were chosen for a comprehensive gene expression network analysis, aiming to explore the modular relationships among genes associated with stress tolerance. These selected genes were subsequently categorized into three distinct pathways: stress-related genes, metabolic genes, and molecular genes, based on the gene ontology annotations available in the KEGG database then mapped onto specific metabolic pathways using the KEGG mapper tool (Kanehisa et al., 2017).

STRING database (version 12.0) comprising experimental and predicted protein-protein interactions was used for analyzing interaction among proteins encoded by upregulated genes (Szklarczyk et al., 2023). Cytoscape 3.9.1 program was used to visualize and validate the biological networks (Saito et al., 2012). The CytoHubba plugin in the Cytoscape was used to identify hub genes in the network (Chin et al., 2014). To reduce the number of false positives, the threshold interaction score was set at 0.7 (high confidence). The thickness of the edge line indicates the degree of confidence in predicting the interaction based on the available interaction resources, including experimental and calculated data. Further, genes in this network were filtered based on the STRING database combined score > 0.7. The combined scores were calculated based on different types of evidence, including co-occurrence, neighbourhood, experimental evidence, and data from curated databases. The hub genes have a high degree of connectivity in the gene-gene network, which significantly influences the expression of other genes through activation, repression, or other regulatory roles identified using Cytoscape. In this study, we considered this network's top ten hub genes as the analysis captures the most influential nodes, which are more likely to yield biologically relevant insights.

Whole genome sequence analysis identifies features of S. warneri CPD1

The bacterial strain CPD1 was identified as Staphylococcus warneri based on the whole genome sequencing, with 14.73 million reads and a 78.11% overall alignment with the reference genome. The assembled genome of S. warneri comprised 2,273,098 base pairs, with 95.42% completeness and a GC content of 33%. A total of 2083 protein-coding sequences (CDS) were identified.

Comprehensive COG (Clusters of Orthologous Groups) analysis has categorized the identified proteins into 25 distinct functional groups (Fig. 1). The majority of annotated genes govern the synthesis of amino acids and their transport (189), carbohydrate transport and metabolism (129), nucleotide transport and metabolism (81), coenzyme (119), lipid metabolism (87), translation (205), transcription (107), DNA replication, recombination and repair (129), cell cycle and cell division (35), cell wall organization/membrane (104), and energy production and conversion (107). The full-genome sequencing and genome annotation of the Staphylococcus warneri strain identified several stress-responsive genes such as CsbA (general stress protein CsbA), UspA (nucleotide-binding universal stress protein), YzzA (general stress protein 26), etc.

Fig. 1. Genes of S. warneri CPD1 associated with general COG functional categories.

Further, genes related to osmoregulatory transporters such as OpuBA (ABC-type proline/glycine betaine transport system), ATPase component, ProX (ABC-type proline/glycine betaine transport system, periplasmic component), BetT (choline-glycine betaine transporter), PotE (serine transporter), etc., were identified under amino acid transport and metabolism.

The phylogenetic tree analysis revealed that CPD1 is closely related to Staphylococcus warneri NCTC11044 (Supplementary data Fig. S3). Furthermore, Staphylococcus pasteuri DSM10658 was found to be the most closely related species to CPD1. S. pasteuri is a non-pathogenic, coagulase-negative bacterium that serves as an endophyte, is known for plant growth promotion, and is commonly found in diverse environments, such as soil, water, humans, animals, and food (Alibrandi et al., 2018; Haidar et al., 2018; Jasim et al., 2014; Jayakumar et al., 2020). The sheep blood agar assay showed S. warneri CPD1 to be non-pathogenic as it did not show any zone around colonies demonstrating no hemolysis (Supplementary data Fig. S4).

Transcriptomic analysis identifies differentially expressed genes under osmotic stress

The bacterium exhibited a typical sigmoid growth curve when cultured in a nutrient-rich medium (Supplementary data Fig. S5). However, the growth rate declined upon adding 10% NaCl to the medium, although it still outperformed the growth observed in media supplemented with 20% PEG. Notably, a significantly slower growth pattern was observed when both 20% PEG and 10% NaCl were present in the medium simultaneously (Wang et al., 2021; Zhou et al., 2020). Overall, the bacterium demonstrated tolerance to PEG up to 40% and salt up to 10% when grown on nutrient agar plates.

The analysis of differentially expressed genes (DEGs), compared to the control (CPD1), revealed significant insights into the bacterium’s response to osmotic stress. When examining the individual treatments, CPD1PG and CPD1S, we observed a smaller number of genes exhibited up-regulation (>2-fold), with 107 genes in CPD1PG and 86 genes in CPD1S (p<0.05) (Fig. 2). However, the combined treatment of CPD1PS resulted in a substantial upregulation of 328 genes. Conversely, CPD1PG exhibited the highest number of downregulated genes (144 genes), followed by the combined treatment, with 104 downregulated genes, while CPD1S had only 62 downregulated genes. Notably, 29 genes were commonly downregulated in all three treatments. The comparative analysis revealed that 158 genes were commonly upregulated across CPD1PG and CPD1S, as well as CPD1 versus CPD1PS (Supplementary data Fig. S6). Additionally, we identified unique gene sets: 24 genes in CPD1PG, 26 in CPD1S, and 122 in CPD1 alone, emphasizing the distinct genetic responses to these specific treatments. In the case of downregulated genes, only a few were observed: 2 in CPD1PG, 12 in CPD1S and 27 in CPD1PS, with no common genes among the three treatments.

Fig. 2. Venn diagrams show overlapping DEGs in different sets of treatments in S. warneri. (A) Differentially expressed up-regulated genes in all three treatments vs. control. (B) Differentially expressed downregulated genes in all three treatments vs. control.

Upregulated genes

The transcriptomic analysis revealed significant upregulation of genes associated with critical metabolic pathways in response to osmotic stress. Notably, alterations in the tricarboxylic acid (TCA) cycle components and the electron transport chain (ETC) suggest a coordinated effect to enhance ATP synthesis, essential for energy-demanding stress responses. Under the combined stress of salt and water, key transporters and efflux pumps, including Na⁽⁺⁾/H⁽⁺⁾ antiporter (mnhF1), arginine/ornithine antiporter (ArcD), and glycine betaine transporter (OpuD) were significantly upregulated, indicating a strategic adaptation to maintain ion balance and osmotic homeostasis (Supplementary data Table S1). Remarkably, transcripts encoding components of the arginine deiminase system (ADS) showed an expression level increase up to 20-fold, indicating a substantial response to the osmotic stress conditions.

Additionally, genes involved in glycolysis/glucogenesis and fermentation pathways exhibited non-uniform yet significant changes in expression. Further, the upregulation of genes coding for cell division proteins and chaperones suggested an adaptive response that prioritizes cellular integrity and repair mechanisms during osmotic challenges. The activation of genes related to stress proteins further highlights the bacterium’s preparedness to cope with extreme environmental conditions. Notably, elevated levels of transcripts encoding enzymes involved in regulating oxidative stress, such as superoxide dismutase [Mn/Fe], quinol oxidase, catalase, NADH peroxidase, and alkyl hydroperoxide reductase indicate a robust antioxidant defense system, which is crucial for detoxifying reactive oxygen species generated under osmotic stress, further supporting cellular resilience.

Downregulated genes

Genes associated with transporter and efflux pumps, including the mnhF1 antiporter, (Na⁽⁺⁾/H⁽⁺⁾ antiporter subunit F1), fluoride ion transporter (CrcB), ancillary protein Kef, which may represent a strategic reallocation of cellular resources to enhance the efficiency of potassium ion homeostasis within bacterial cells (Supplementary data Table S2) (Checchetto et al., 2016; Feeney and Sleator, 2011; Gulati et al., 2023). The downregulation of these genes under osmotic stress may be an adaptive response that likely optimizes the bacterium's ability to manage intracellular ion concentrations, thereby improving stress tolerance.

Metabolic pathways regulated by osmotic stress in S. warneri CPD1

Under osmotic conditions, S. warneri CPD1 displayed significant regulation of genes linked to its core metabolic pathways that support cellular resilience. The DEGs were associated with 65 KEGG pathways that impact carbon metabolism, microbial metabolism in diverse environments, amino acid biosynthesis, oxidative phosphorylation, and secondary metabolites biosynthesis, which emerged as significant pathways critical for adapting to osmotic stress. DEGs to the control, CPD1PG vs. CPD1, CPD1S vs. CPD1, and CPD1PG vs. CPD1, were analyzed, and the top ten metabolic pathways were associated with several upregulated genes, which are shown in Table 1.

Table 1

Metabolic pathways regulated by osmotic stress in S. warneri CPD1

S. No.	Name of associated KEGG pathway	No. of genes upregulated
1	Biosynthesis of secondary metabolites	36
2	Microbial metabolism in diverse environments	27
3	Carbon metabolism	23
4	Biosynthesis of amino acids	19
5	Lipoic acid metabolism	7
6	Teichoic acid biosynthesis	3
7	Glycolysis/gluconeogenesis	9
8	TCA cycle	6
9	Oxidative phosphorylation	10
10	Nucleotide metabolism	4

The significant upregulation of enolase (Eno) (8.5-fold) and Phosphoglycerate kinase (Pgk) (6.1-fold) underscores an enhanced conversion of carbohydrates into energy, essential for growth and maintenance under stress. Further, genes associated with pyruvate metabolism, such as Phosphoglycerate kinase (Pgk) (6.1-fold), pyruvate dehydrogenase complex (PDHc), displayed substantial upregulation (PdhA, PdhB, and PdhC upregulated 11, 13, and 13.4-fold, respectively), highlighting the importance of connecting glycolysis to the citric acid cycle for energy production and redox homeostasis (Hirose et al., 2019; Krucinska et al., 2019; Moxley and Eiteman, 2021; Zhang et al., 2020) (Supplementary data Figs. S7 and S8). An upregulation of 7.5-fold was observed in the gene encoding glycerol kinase (glpK) which catalyzes the phosphorylation of glycerol to form glycerol 3-phosphate, an important intermediate in both glycolysis and gluconeogenesis (Tang et al., 2023). Further, the genes odhA and odhB (upregulated 13.4-fold) and the pdhD gene were also significantly upregulated by 13.4-fold and 30-fold, respectively. These genes encode the components of the 2-oxoglutarate dehydrogenase complex (ODHC), which is responsible for the oxidative decarboxylation of 2-oxoglutarate to form succinyl coenzyme A (succinyl-CoA) and subsequent degradation in the tricarboxylic acid (TCA) cycle (Supplementary data Fig. S9). These combined upregulations indicate a strategic shift towards efficient energy utilization in response to osmotic stress.

The regulation of amino acid metabolism emerged as a critical mechanism for stress resilience. The arginine deiminase (ADI) pathway genes, including arcABDC, showed a significant upregulation of 7.3-fold, enabling arginine as a source of energy for growth under anaerobic conditions (Favreau et al., 2023). In this study, we observed a significant increase in the expression of all the genes involved in the ADI pathway (ArcA, ArcB, ArcC, ArcD, and ArcR), which suggests the impact of salt and water stress on the arginine deiminase system (ADS) in S. warneri. In addition, the upregulation of the 1-pyrroline-5-carboxylate dehydrogenase (Pro/P5C dehydrogenase) gene by a 9.2-fold increase encodes its role in proline metabolism, which is crucial during osmotic stress adaption (Mansour and Salama, 2020). Further, significant increases in the expression of genes involved in the synthesis of compatible solutes, gluD (NAD-specific glutamate dehydrogenase, upregulated by 9.5-fold) and glnA (glutamine synthetase, upregulated by 12-fold), indicating the role of glutamate and glutamine as primary compatible solutes under saline conditions (Sleator and Hill, 2002; Vreeland, 1987). The serine hydroxymethyl transferase (GlyA) gene upregulated 10.9-fold, which catalyzes the synthesis of serine and tetrahydrofolate (THF) from glycine and the serine dehydratase gene (SdhA) (upregulated 6.9-fold) and SdhB gene (upregulated 10.4-fold) underscores the glycine metabolism, indicating a coordinated strategy to utilize glycine as a carbon and nitrogen source (Sarwar et al., 2016). The McsB gene, which encodes arginine kinase, was also upregulated 7.3-fold, which plays a critical role in regulating the bacterial stress response by controlling the expression of heat shock protein genes through regulating the activity of the central transcriptional repressor (CtsR) (Schmidt et al., 2014). The CysK gene, which encodes cysteine synthase, has an 8.8-fold increase in expression, catalyzing the conversion of O-acetyl serine (OAS) and sulfide ions into cysteine (Kushkevych et al., 2020). Further, the cysteine desulfurase encoding gene is upregulated 5.1-fold, facilitating sulfur mobilization from cysteine to various biomolecules and synthesizing sulfur-containing cofactors (Das et al., 2021). Cysteine is also required for methionine and glutathione synthesis and, therefore, is a central metabolite in antioxidant defense (Lithgow et al., 2004). These findings suggest that the amino acid metabolism, particularly involving Arginine, ornithine, Proline, Glutamate, Glycine, and cysteine, is significantly involved in the stress resistance mechanism of S. warneri (Fig. 3).

Fig. 3. Biosynthetic pathways for twenty amino acids of S. warneri. The green arrows indicate steps in a pathway where a differentially expressed gene enhances or stimulates the activity of another molecule or process. The molecule or product produced as a result of altered/upregulated genes is represented as purple circles.

Critical components of lipid metabolism, particularly those involved in lipoic acid biosynthesis, showed significant upregulation. Lipoyl synthase (LipA) and octanoyl-ACP: protein N-octanoyltransferase (LipB) are significantly upregulated by 6.4 and 7.3-fold, respectively. These enzymes are essential for lipoate biosynthesis, facilitating glycine detoxification, fatty acid biosynthesis, and redox homeostasis. The butA gene encoding diacetyl reductase shows a 12-fold increase in expression, which catalyzes the conversion of diacetyl (2,3-butanedione) to acetoin under aerobic conditions (Dias et al., 2018). Additionally, the acoA gene, which codes for acetoin:2,6-dichlorophenolindophenol oxidoreductase, is also upregulated, highlighting the importance of acetoin metabolism in S. warners’s osmotic tolerance (Supplementary data Fig. S10). Moreover, acetoin is a bacterial volatile compound that can activate the plant immune response and promote plant growth at the molecular level and in large-scale field applications (Rani et al., 2022; Silva Dias et al., 2021).

Stress response pathways were markedly upregulated, especially those managing oxidative stress. Key regulators, including superoxide dismutase (sodA), catalase (katA), alkyl hydroperoxide reductase (ahpC and ahpF), and NADH peroxidase, exhibited a notable increase in expression. The sodA, which mitigates free radical damage by converting hydrogen peroxide and oxygen, (Sharma et al., 2023) showed a 16.5-fold upregulation. AhpF (7.3-fold increase) and AhpC (13-fold increase) work together to detoxify peroxides and protect cells from DNA damage caused by alkyl hydroperoxides (Jiang et al., 2019; Zhang et al., 2019). Genes encoding subunits of the enzyme quinol oxidase (Oox), QoxA, OoxB, and OoxC were upregulated 13.2, 28.4, and 7.8-fold, respectively, under osmotic stress. This enzyme plays a critical role in respiration, reduction of oxygen to water, and aiding ATP generation, which is important for energy production under osmotic stress (Supplementary data Fig. S11). Further, stress response elements included general stress protein 13 (yugI_1) upregulated 9.4-fol, and Dps, a DNA binding protein that protects DNA from oxidative damage, was upregulated 6.2-fold (Orban and Finkel, 2022).

The upregulation of genes involved in cell wall synthesis highlights the role of Lipoteichoic acid (LTA) in the structural integrity under osmotic stress (Percy and Gründling, 2014). Lipoteichoic acid synthase (LtaS), upregulated 4.8-fold, polyglycerol phosphate to LTA and glycerol-3-phosphate cytidylyl transferase (TarD), upregulated to 7.5-fold, produces glycerol 3-phosphate for the synthesis of teichoic acid, thereby maintaining cell wall integrity under stress in S. warneri (Supplementary data Fig. S12).

Co-regulatory gene networks of S. warneri in response to salt and water stress

Network analysis of stress-related genes upregulated in S. warneri: The interaction network of nine stress-related genes with significant fold change showed 36 nodes and 91 edges, with an average node degree of 5.06. The top ten genes qoxB, atpB, atpC, atpD, sdhB, ctaB, ppaC, atpE, atpG, and atpH, with a high degree of connectivity as hub genes in the network are shown in Fig. 4. Interestingly, these hub genes (qoxB, atpC, atpD, sdhB, ppaC, atpG) show significant upregulation in our transcriptome data in the combined drought and saline treatment, indicating their contribution to drought and salinity tolerance of the bacterium. The significantly upregulated (4.8-fold) gene qoxB encoding quinol oxidase subunit 1 is a part of the cytochrome c oxidase, the terminal electron acceptor in the respiratory chain reaction. Further, the network analysis shows that qoxB is functionally associated with ctaB (protoheme IX farnesyltransferase), atpB (F₀F₁-ATP synthase subunit A), and sdhB (Fe-S protein subunit). Of these, ctaB catalyzes the conversion of protoheme IX to heme, whereas the product of sdhB, the subunit of the succinate dehydrogenase (SDH), is involved in the Fe-S exchange, and the hub gene atpB is involved in ATP hydrolysis. Further, the hub genes, ATP synthase β subunit (atpD), γ subunit (atpG), and ε subunit (atpC) showed significant Log2 fold changes in their expression (3.2, 2.9 and 2.9, respectively) indicating that the rising energy demand results in the upregulation of ATP biosynthesis, enabling the bacteria to survive under the stress conditions created by drought and salinity treatment (Suyal et al., 2017). This network also includes three genes, katA, trxB, and ypdA, involved in the reactive oxygen species metabolic process (GO: 0072593), two genes, trxB and ypdA, involved in the removal of superoxide radicals (GO: 0019430), five genes, katA, ahpC, trxB, ypdA, and A284_10370 involved in cellular oxidant detoxification (GO: 0098869) and response to oxidative stress (GO: 0006979). It is likely that that the upregulation of the above genes facilitates detoxification of the free radicals such as OH^- and O^2- produced in stress conditions (Chandra et al., 2021; Sudharsan et al., 2022).

Fig. 4. Interaction network of the proteins encoded by selected stress-related genes upregulated under osmotic stress in Staphylococcus warneri. (A) Interaction network was identified for six upregulated genes. String identified interactions for 6 genes out of an entry of 9 genes (katA, qoxA, qoxB, qoxC, sodA, yugI_1, dps, ahpF, and ahpC). The thickness of the edge line between each node indicates the degree of confidence in predicting the interaction between two genes. (B) Interaction network of top ten hub genes using degree of interaction. Hub genes have been selected using CytoHubba plug-in of Cytoscape. Node color reflects degree of connectivity. The pseudocolor scale from red (highest degree) to yellow (lowest degree) represents the top ten hub genes ranked from 1 to 10.

Network analysis of upregulated metabolic genes in S. warneri : The interaction network of selected metabolic genes, with a significant fold change, contained 55 nodes and 119 edges, with an average node degree of 4.33. This study identified ppaC, gcvH, atpA, atpB, atpC, atpD, atpE, atpF, atpG, and atpH as the top ten hub genes in the network (Fig. 5). ppaC is an inorganic soluble pyrophosphatase that catalyzes the hydrolysis of the inorganic pyrophosphate (PPi) (Chen et al., 1990) and maintains cellular homeostasis (Kukko-Kalske and Heinonen, 1985). Hence, the absence of pyrophosphatase leads to the accumulation of PPi, which can disrupt metabolic reactions and the normal functioning of biosynthetic processes. The gcvH gene encodes for glycine cleavage system protein in bacteria, which catalyzes the accumulated glycine degradation. Excess glycine can inhibit bacterial growth as it acts as a competitive inhibitor of proteases and transporters. Hence, the level of glycine is tightly regulated. Similar to the networks of stress genes, metabolic genes also showed the interaction pattern with the F₀F₁-ATP synthase subunits atpA (2.5-fold), atpC (2.9-fold), atpD (3.2-fold), and atpG (2.9-fold). In our expression data, the ppaC gene was upregulated (2.9-fold) in the combined treatment of drought and salt, which indicates that the expression of ppaC is needed for drought and saline tolerance in bacteria. The network analysis indicates that ppaC is interacting with all the subunits of the F₀F₁-ATP synthase system, including atpA, atpB, atpC, atpD, atpE, atpF, atpG, and atpH. This result shows that the inorganic pyrophosphatase, ppaC and the F₀F₁-ATP synthase system in the bacterial cell work hand in hand in the transfer of pyrophosphate, thereby regulating the production of ATP needed by the bacteria during stress conditions.

Fig. 5. Interaction network of the selected metabolic genes upregulated under osmotic stress and hub genes in Staphylococcus warneri. (A) Interaction network for thirty-six upregulated genes. String showed interactions for 36 genes out of an entry of 62 genes. The thickness of the edge line between each node indicates the degree of confidence in predicting the interaction between two genes. (B) Interaction network of top ten hub genes in the obtained network.

In the network, sixteen genes were identified to be involved in the ATP metabolic process (GO: 0046034), and seven genes (gcvH, gcvT, pruA, gcvPA, gcvPB, arcD, and A284_06575) in the cellular amino acid catabolic process (GO: 0009063), three genes (lipL, lipA, and gcvH) in protein lipoylation (GO: 0009249), four genes (gcvH, gcvT, gcvPA, gcvPB) in the glycine decarboxylation via glycine cleavage system (GO: 0019464), three genes (glpK, A284_07115, A284_10070) in the glycerol catabolic process (GO: 0019563), two genes (lipL and lipA) in the lipoate biosynthetic process (GO: 0009107), two genes (glpK and A284_07115) in the glycerol-3-phosphate metabolic process (GO: 0006072), and seven genes (arcD, gcvT, gcvH, gcvPB, gcvPA, pruA, and A284_06575) in the alpha amino acid catabolic process (GO: 1901606). The arcD gene (arginine/ornithine antiporter) coding for a membrane-bound amino acid transporter protein, which helps in the influx of ornithine and arginine efflux in an energy-independent manner (Ryan et al., 2009) is significantly upregulated (4.1-fold). The arcD gene knockout has been reported to reduce the growth rate in Listeria monocytogenes (NicAogáin and O’Byrne, 2016; Ryan et al., 2009). Experiments have validated the involvement of gcvT and gcvH in lipid transport and gcvP in the glycine cleavage system (Felix et al., 2021). These results agree with our network analysis results.

Network analysis of upregulated genes with molecular function in S. warneri

The interaction network of selected genes with molecular function showed 40 nodes and 371 edges, with an average node degree of 18.6. The average node degree is about 4-fold higher in the molecular genes network than in the stress and metabolic genes networks, indicating more functional and physical associations. Interestingly, this study identified the top ten hub genes, rplP, rpsE, rpsN, rplB, rplC, rplD, rpsK, rpoB, rpsJ, and rpsL (Supplementary data Fig. S13), which are constituents of prokaryotic ribosome (rplB, rplC, rplD, and rplP are 50S ribosomal proteins and rpsE, rpsN, rpsK, rpsJ, and rpsL are 30S ribosomal proteins). In addition to these hub genes, the upregulation of several other ribosomal proteins, including rplA (2.4-fold), rplY (3.1-fold), rplR (3.1-fold), rplV (3.2-fold), rplX (3.2-fold), rplN (3.2-fold), rplK (3.4-fold), rplF (3.6-fold), rpsH (3-fold), rpsG (3.7-fold), and rpsZ (4.3-fold) was observed in the combined drought and salinity stress.

The network also showed four genes (rpoA, rpoB, rpoC and sigB) involved in the DNA-template transcription process (GO: 0006351), twenty-three genes in the translation process (GO: 0006412), twenty-eight genes in the gene expression process (GO: 0010467), and thirty-two genes in the primary metabolic process (GO: 0044238). The expression of essential proteins such as stress response regulators, proteins involved in transcription and translation machinery and other cellular processes is essential for efficiently tackling stress (Ryan et al., 2009). Our combined transcriptome and network analysis clearly showed the upregulation of ribosomal genes to activate the functioning of ribosomal proteome components during bacterial abiotic stress.

Gaining insights into the osmo-adaptation mechanism in bacteria from soils holds significant importance in achieving the ultimate goal of enhancing plant-microbe interactions, thereby contributing to the improvement of crops cultivated in salt and drought-affected agricultural niche. Some of the Staphylococcus spp. have been investigated for their plant growth promoting characteristics (Mollety and Padal, 2021). There are a few reports of osmotolerance in Staphylococcus aureus. Seventy-eight genes of osmotic stress response systems including potassium, sodium and oligopeptide transport were identified in S. aureus ST772-MRSA-V (Casey and Sleator, 2021). The contamination of salted foods by S. aureus is a recurring issue that is attributed to its high osmotolerance (Feng et al., 2022). Improvement in cellular homeostasis is accomplished by the upregulation of aminoacyl-tRNA biosynthesis while protection of proteins and nucleic acids is achieved by upregulation of the betaine biosynthesis pathway. Further, L-proline levels are upregulated for osmotic stability. Osmotic stress induced the production of nukacin ISK-1, a lantibiotic in S. warneri ISK-1 by upregulating the transcription of the nukacin ISK-1 gene (Sashihara et al., 2001). S. warneri is one of the most salt tolerant among Staphylococcus species (Caglayan, 2022; Shurigin et al., 2020). The present study was conceived with the aim of delving into the mechanisms behind its exceptional osmotic resilience. The complete genetic blueprint of S. warneri CPD1 consists of protein-coding sequences GC content comparable to other sequenced S. warneri isolates, indicating a consistent pattern across this genus and species. This investigation holds the promise of unveiling valuable insights that could potentially position S. warneri as a promising microbe to mitigate the adverse impacts of abiotic stresses.

Bacteria trigger a cascade of gene expression changes in response to osmotic stress, which affect the expression of many genes involved in the maintenance of cell permeability and metabolic machinery. This study provides valuable transcriptomic insights into the osmotic stress response of S. warneri. Based on high number of genes (328) significantly upregulated during combined osmotic stress the existence of a complex regulatory network with common mechanisms governing both salt and drought tolerance can be envisaged. However, a higher number of genes were observed to be significantly down-regulated in PEG treatment (144) than in salt (62) and combined osmotic treatment (104) suggesting the existence of a lower number of common mechanisms employed by downregulated genes for combined osmotic stress tolerance.

The stress conditions triggered the expression of a suite of genes involved in the synthesis of stress proteins and osmolytes, effectively counteracting the detrimental effects of high osmolarity. The upregulation of McsB in the current study corroborates with improved stress tolerance attributed to the same gene in Staphylococcus aureus (Wozniak et al., 2012). Further, the accumulation of various amino acids and sugars was observed under osmotic stress to maintain cellular redox homeostasis and thus serve as osmolytes. The intrinsic resistance of staphylococci to high salt and water stress may be attributed to its tendency to sense alterations in the osmolarity of the external environment and adapt by accumulating solutes like glutamine, glycine, betaine, and proline in response to osmotic stress. These solutes act as osmoprotectants and help regulate the osmotic pressure within the bacterial cells, preventing dehydration and maintaining cellular functions. By exchanging extracellular protons for intracellular sodium ions, the antiporter could help maintain a more favorable intracellular pH, thereby enhancing stress tolerance (Fig. 6). Additionally, S. warneri may alter the expression of specific genes and proteins involved in stress response pathways, which help to maintain cell integrity and functionality. By coordinating these mechanisms, S. warneri demonstrates resilience in the face of osmotic stress, enhancing its survival and adaptation in diverse environments.

Fig. 6. Regulatory framework of different players responsible for osmotic resilience and adaptation. The Na⁽⁺⁾/H⁽⁺⁾ antiporter system, which includes the mnhC1 subunit, actively transports excess sodium ions out of the cell, preventing their toxic accumulation and maintaining ion balance. Superoxide dismutase, catalase, alkyl hydroperoxide reductase, and NADH peroxidase can help to protect the cell from oxidative damage. The QoxA-QoxB-QoxC- QoxD cytochrome aa3 oxidase complex, frdA, and ATP synthase subunits work together to generate ATP, which is important for energy production under osmotic stress. The transcriptional regulators ArcR and HrcA collaborate to control the expression of genes related to metabolism and stress response. Additionally, tarD and ltaS are essential components in the osmotic stress response involved in maintaining the cell wall integrity. Protein translocase subunit SecY is a component of the Sec translocation system, responsible for transporting proteins across the cytoplasmic membrane under osmotic stress to help maintain protein homeostasis and prevent misfolding of proteins.

In our study, through the transcriptomic and bioinformatic analysis, we identified genes involved in cellular homeostasis including the F₀F₁-ATP synthase system component genes which were found to be common hub genes in the stress and metabolic gene networks. This signifies the relevance of the genes associated with the production of ATP as energy source during osmotic stress. Overall, the genes encoding enzymes that constitute essential components of bacterial metabolism contribute to the regulation of energy production, carbon flux, osmoregulation, bacterial growth and survival amidst changing osmotic conditions. While some of these genes have primary functions unrelated to osmotic stress, they participate in metabolic and regulatory pathways influenced by osmolarity changes. Bacteria employ these genes to acclimatize to osmotic stress by fine-tuning their metabolic processes, preserving protein quality, and managing essential nutrient availability, such as nitrogen and sulfur. Understanding the molecular/biochemical mechanisms employed by bacteria for salt tolerance is crucial for their application in agriculture, particularly in saline areas worldwide. Employing salt tolerant bacteria such as S. warneri CPD1 to counteract decrease in crop yield is an indirect method. Future studies aimed at testing the bacteria on cereals such as wheat and rice for salinity tolerance and plant growth promotion would pave way for their application for sustainable agriculture.

Junior Research Fellowship and Senior Research Fellowship to PR and Junior Research Fellowship to PJ from Council of Scientific and Industrial Research (CSIR), New Delhi, India is acknowledged. Ruchi Sonowane and Rahul Beniwal are acknowledged for the blood agar assay. The authors acknowledge funding by DST-FIST (sanction order number SR/FST/LS-I/2018/125).

Parikshita Rathore performed experiments, analyzed data and wrote the initial draft. Priyanka Jeeth performed data analysis and wrote part of the initial draft. Saraboji Kadhirvel had a role in experimental design, data analysis and supervision. Wusirika Ramakrishna conceived the idea, designed experiments, supervised the work and prepared the final manuscript.