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Proteome Cold-Shock Response in the Extremely Acidophilic Archaeon, Cuniculiplasma divulgatum

The archaeon Cuniculiplasma divulgatum is ubiquitous in acidic environments with low-to-moderate temperatures. However, molecular mechanisms underlying its ability to thrive at lower temperatures remain unexplored. Using mass spectrometry (MS)-based proteomics, we analysed the effect of short-term (3 h) exposure to cold. The C. divulgatum genome encodes 2016 protein-coding genes, from which 819 proteins were identified in the cells grown under optimal conditions. In line with the peptidolytic lifestyle of C. divulgatum, its intracellular proteome revealed the abundance of proteases, ABC transporters and cytochrome C oxidase. From 747 quantifiable polypeptides, the levels of 582 proteins showed no change after the cold shock, whereas 104 proteins were upregulated suggesting that they might be contributing to cold adaptation. The highest increase in expression appeared in low-abundance (0.001–0.005 fmol%) proteins for polypeptides’ hydrolysis (metal-dependent hydrolase), oxidation of amino acids (FAD-dependent oxidoreductase), pyrimidine biosynthesis (aspartate carbamoyltransferase regulatory chain proteins), citrate cycle (2-oxoacid ferredoxin oxidoreductase) and ATP production (V type ATP synthase). Importantly, the cold shock induced a substantial increase (6% and 9%) in expression of the most-abundant proteins, thermosome beta subunit and glutamate dehydrogenase. This study has outlined potential mechanisms of environmental fitness of Cuniculiplasma spp. allowing them to colonise acidic settings at low/moderate temperatures.


Introduction
The recently isolated archaea Cuniculiplasma divulgatum belong to the order Thermoplasmatales (together with Thermoplasma, Picrophilus, Ferroplasma, Thermogymnomonas and Acidiplasma) [1]. C. divulgatum and related organisms were before their isolation designated as "G-plasma" and found in metagenomic sequences from acidic environments worldwide [2][3][4]. Among them, acid mine drainage (AMD) systems generating high level of pollution via production of highly acidic and heavy metal-containing waters accommodate C. divulgatum and other members of the family Cuniculiplasmataceae [4]. Metagenomic and metaproteomic studies have previously showed high relative abundances of these microorganisms in mature biofilms in AMD systems and sulfide-rich caves [5,6]. Mechanisms underlying their successful colonisation of acidic environments and their interactions

Culture Conditions
Triplicate cultures (200 mL) of C. divulgatum S5 (=JCM 30642 T =VKM B-2941 T ) were grown under optimal conditions (37 • C, modified medium 88 DSMZ, on an orbital shaker at 100 rpm) for 90 h to reach the exponential growth phase [1]. After that, half of each culture (100 mL) was transferred into other Erlenmeyer flasks and subjected to the cold shock introduced by placing of cultures in an ice-water bath (0 • C) for 3 h. During that incubation time, the remaining halves of the cultures were maintained under optimal conditions, as above. All variants were harvested by centrifugation (8000× g for 15 min) and washed twice in the same sterile growth medium without beef extract.

Protein Extraction
Protein extraction and digestion were carried out as previously described [17]. In brief, proteins were extracted by resuspending the biomass in 300-500 µL of breakage buffer containing one tablet of Roche complete-mini protease inhibitors (without EDTA) (Roche Diagnostics Ltd., West Sussex, UK) in 7 mL of 25 mM NH 4 HCO 3 (Sigma-Aldrich, Dorset, UK).
Acid washed glass beads (150-212 µm; 70-100 US sieve) (Sigma-Aldrich, Dorset, UK) were added to the cell pellet (200 µL). The pellet was subjected to repeated rounds of bead-beating (15 bursts of 30 s with 1 min cool-down periods on wet ice in between individual bursts). The biomass was centrifuged (10 min at 4 • C, 12,000 rcf) and the supernatant was transferred to a new Eppendorf tube (Eppendorf UK, Stevenage, UK). 250 µL of fresh breakage buffer were added to the pellet, which was then resuspended by vortexing. The bottom of the extraction vial was pierced with a heated syringe needle (BD Microlance, gauge 21G or narrower) and placed on top of a fresh Eppendorf tube. The device was inserted into a 50-mL Falcon tube (Sigma-Aldrich, Dorset, UK), and the supernatant spun through at low speed (4000 rcf) for 5 min at 4 • C to separate the beads from the resulting supernatant. The protein content of the combined supernatant and flow-through was assessed using a standard Bradford assay (Sigma-Aldrich, Dorset, UK) [18]. The protein extracts were stored at −80 • C prior to subsequent digestion.

Protein Digest
100 µg of protein aliquots were transferred into a 0.5-mL LoBind Eppendorf tube (Eppendorf UK, Stevenage, UK), and the volume adjusted to a total of 160 µL with fresh NH 4 HCO 3 . Proteins were denatured by adding 10 µL of 1% (w/v) of RapiGest™ (Waters UK Ltd., Wilmslow, Cheshire, UK) in 25 mM NH 4 HCO 3 , followed by a 10-min incubation (80 • C; 400 rpm). The samples were reduced by adding 10 uL of freshly prepared 60-mM Dithiothreitol (DTT) (Sigma-Aldrich, Dorset, UK) in 25-mM NH 4 HCO 3 , followed by incubation at 60 • C (10 min; 400 rpm). The samples were cooled to room temperature before alkylation was initiated by adding 10 µL of freshly prepared 180mM iodoacetamide (IAM) (Sigma-Aldrich, Dorset, UK). Samples were incubated for 30 min at room temperature in the dark.
After the addition of 4.5 µL fresh NH 4 HCO 3 to each tube, mass spectrometry grade trypsin (Promega UK, Southampton, UK) was reconstituted in 50mM acetic acid (Sigma-Aldrich, Dorset, UK). 10 µL of a 0.2-mg/mL trypsin solution was added to the reaction. Samples were incubated for 4.5 h (37 • C; 400 rpm). Another 10 µL aliquot of trypsin was added to each digestion tube, and complete digestion was carried out overnight (37 • C; 400 rpm).
Then, 5.5 µL of acetonitrile (Sigma-Aldrich, Dorset, UK) was added to each tube and RapiGest™ was precipitated by adding of 1.5 µL of trifluoroacetic acid (TFA) (Sigma-Aldrich, Dorset, UK) and incubated for a minimum 45 min (37 • C; 400 rpm). The digest was then incubated at 4 • C for a minimum of 2 h prior to 15 min of centrifugation (14,000 rpm at 4 • C). The final volume of the digest was 221.5 µL. 180 µL of the resulting supernatant was transferred into a fresh LoBind Eppendorf tube.
Digests were diluted 1:1 with glycophosphorylase B (Uniprot Accession P00489) MassPREP™ digestion-standard (Waters UK, Wilmslow, Cheshire, UK) to give a final concentration 50 fmol/uL glycophosphorylase B. 1 µL per injection of the prepared sample was used for analysis.

Analytical Instrumentation and Data Acquisition
The resulting spiked digests were analysed by nanoLC-HDMS e using an M-Class nano-Acquity system (Waters UK, Wilmslow, Cheshire, UK) with a trap valve manager, coupled to a Synapt G2-Si mass spectrometer (Waters UK, Wilmslow, Cheshire, UK).
The sample (1.0 µL) was loaded onto the trapping column (Waters UK, Wilmslow, Cheshire, UK; NanoEase™ M/Z Symmetry C18 100Å, 5 um, 180 um × 20 mm trap column), using partial loop injection with a sample loading time of 3 min at a flow rate of 0.3 µL/min.
The sample was resolved on an analytical column (nanoEase™ M/Z Peptide BEH C18 130Å, 1.7 µm, 75 µm × 100 mm column) using a gradient of 99% A (Optima ® Water with 0.1% formic acid) 1% B (Optima ® ACN with 0.1% formic acid) (Sigma-Aldrich, Dorset, UK) to 60% A, 40% B over 90 min at a flow rate of 0.3 µL/min and then to 15% A, 85% B over 90 min. At 95 min, the conditions returned to the initial state and held for 15 min in preparation for the next injection.
The mass spectrometry (MS) data was acquired in HDMS e mode, e.g., ion mobility was enabled and all ions within a specified m/z range were observed and fragmented. In essence, four signals are available from an HDMS e acquisition: firstly, a low energy MS, secondly, a high energy MS/MS, the third signal contains the lockmass data for mass axis correction and the fourth relates to the ion mobility aspect of HDMS e .
A mass range of 50 to 2000 Da was selected in resolution mode (ToF W mode) using a positive polarity continuum acquisition. The scan time was set at 0.5 s and a ramp transfer collision energy of 15-45 eV was used for the high energy signal. The time-of-flight analyser was calibrated before running batches against the fragment ions of glufibrinopeptide and throughout the analytical run at 6 min intervals using the Waters ZSpray™ NanoLockSpray™ source with leucine enkephaline.

Peptide Identification and Quantification
HDMS e data was processed with Progenesis QI for proteomics version 4.1 (Nonlinear Dynamics, Newcastle upon Tyne, UK). Label-free analysis and quantification was performed using an integrated Hi 3 workflow [19], applying standard settings for HDMS e peptide/protein identification.
The Progenesis Ion Accounting workflow was used, and the data was searched against the C. divulgatum, strain S5 T proteome (Uniprot proteome ID: UP000195607) including the protein sequence of glycophosphorylase B (P00489).
Peptides were identified assuming standard trypsin degradation rules with one missed cleavage allowed. The following modifications were also included: fixed carbamidomethyl (C), and variable oxidation (M), deamidation (Q), deamidation (N). The peptide mass tolerance and fragment mass tolerance were set to auto with a 4% FDR, and ion matching requirements were set to: 3, 7, 1 for fragments/peptide, fragments/protein, and peptides/protein, respectively.
Fold-change data and quantities were determined using the absolute quantification workflow within the proteomics software Progenesis QIP (Nonlinear Dynamics, Newcastle upon Tyne, UK), which is based on the Hi 3 algorithm. Amounts of proteins quantified in this manner are expressed in "fmol" amounts and refer to the total amount quantified in 1 µL of digest.

Bioinformatics Analysis
Sequences for all identified proteins were fetched from Uniprot database using the url-based API. Functional annotation and classification have been developed against eggNOG Archaea database using emapper v1.0.3, arCOG database using Blastp from BLAST 2.2.31+ tools and KEGG database using BlastKOALA. All figures have been developed using R programming language.
The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE [20][21][22] partner repository with the dataset identifier PXD017828 and 10.6019/PXD017828.

Analysis of the Total Intracellular Proteome of C. divulgatum Cells
Based on the complete sequence of the C. divulgatum genome, it encodes 2016 protein-coding genes. MS analysis of the total intracellular proteome of C. divulgatum revealed the presence of 819 different proteins, from which 747 proteins were found to be quantifiable (e.g., fmol amounts and ratios could be calculated).
Comparison of protein abundances in C. divulgatum cells at control conditions and after cold shock demonstrated that the levels of 582 proteins were similar in both types of cells suggesting that they represent the "core" proteins. One hundred and four proteins were upregulated (in comparison to control conditions) under cold shock (p < 0.05).
All proteomic data, including polypeptides' locus tags, accession numbers, annotations, quantity, statistical metrics and functional categories, are presented in Supplementary Tables.
Interestingly, proteins from the categories 'Post translational modification, protein turnover and chaperones' were expressed under standard conditions in higher numbers compared to stressed cells. These include the proteasome 20S, alpha subunit (CSP5_0278), predicted ATP-dependent serine protease (CSP5_1287), and DnaJ-class molecular chaperone with C-terminal Zn finger domain (CSP5_1163) which were revealed in proteomic pool under standard conditions from this category.
Analysis of data presented in Volcano Plot ( Figure 1) revealed that under optimal growth conditions the highest expression level was shown for STO (stomatin regulator of protease activity, CSP5_0371). This protein shared high sequence similarity with related peptidases from Acidiplasma aeolicum (77.6% seq. identity), Thermoplasma volcanium (70.7%), T. acidophilum (69.1%) and other archaea from the order Thermoplasmatales without taxonomic status (e.g., Thermoplasmatales "E-" and "A-plasma"). These proteolytic membrane-associated proteins are also known to occur in diverse archaea [23]. However, it must be noted that this protein has one of the lowest abundances in the proteome, only 0.001% in the cold and 0.015% under optimal growth conditions. Another minor (0.001% of the total 'cold' proteome) protein was expressed, the ATP-dependent protease LonB, (CSP5_1287), related with ATP-dependent proteases from Thermoplasmatales: Thermoplasma acidophilum, T. volcanium and Picrophilus oshimae with amino acid identity levels 67.61 69.93, and 70.29%, respectively. A number of archaeal LonB homologs have been characterised, including LonB from Thermoplasma acidophilum, which exhibited ATPase and proteolytic activity [24,25]. In Haloverax volcanii, LonB protease was found to control membrane lipids composition and was essential for viability [26]. Another expressed protein was PsmA, a subunit of proteasome, a large protease complex involved in protein degradation (CSP5_0278). This protein was homologous to proteasome alpha subunits from close phylogenetic relatives of C. divulgatum, Thermoplasma species: T. volcanium, and T. acidophilum with 76.39% and 74.25% AA sequence identity. Proteasome subunit alpha functional activity was studied in considerable details in T. acidophilum [27]. According to the UniProtKB, the protein belongs to the peptidase T1A family (arCOG00971) with endopeptidase activity. The expression of STO, LonB and PsmA proteins is consistent with peptidolytic lifestyle of C. divulgatum.
Furthermore, FeCT family ABC transporter substrate-binding component, Fect (CSP5_1116) with the closest identical protein from "Ferroplasma acidarmanus", 47.66% and PanK, type I (CSP5_1916), are shown on Volcano plot as significantly expressed at optimal growth conditions. This protein, apart from a 62.5% sequence identity with the type I pantothenate kinase from "E-plasma", also exhibited 53% sequence identity with bacterial type I pantothenate kinases from acidophilic bacteria Alicyclobacillus acidocaldarius and to a lesser extent with similar proteins from other bacilli.
Another minor protein, which was strongly expressed, FAAH fumarylacetoacetate hydrolase family protein of the MhpD superfamily (CSP5_1964), increased from 0.001 in the cold to 0.005 fmol% ( Figure 1, Table S1).

Proteins Overexpressed under Cold Shock Conditions
The cold shock proteome of C. divulgatum showed a number of proteins expressed at higher levels under cold conditions (Table S1). Metal-dependent hydrolase of the beta-lactamase superfamily II (CSP5_0007, 3.06-fold) showed relatively low identity to MBL fold metallo-hydrolases proteins from Thermoplasmatales archaea (highest, 39.15% identity to Thermoplasma acidophilum). KorB; 2-oxoglutarate/2-oxoacid ferredoxin oxidoreductase subunit beta (CSP5_0285), involved in central metabolism, i.e., tricarboxylic acid cycle via the CoA-dependent oxidation of pyruvate and 2-oxoglutarate [28][29][30] (Table S1). Moreover, the FADox protein, a FAD-dependent oxidoreductase of the DAO superfamily (CSP5_1916, 2-fold increase) with high sequence identity with DadA glycine/D-amino acid oxidase (deaminating)/Amino acid transport and metabolism category, showed the highest (52.35%) identity with the predicted beta subunit of SoxB-like protein (sarcosine oxidase) from uncultured "A-plasma". Furthermore, histidine phosphatase PhoE domain protein (CSP5_0806) was 3-fold overexpressed, yet its production was at the maximal level of only 0.003% of the total proteome ( Figure 1, Table S1). ADH-acryloyl coenzyme A reductase (CSP5_0778, 25% up) and D-arabinose 1-dehydrogenase from the Zn-dependent alcohol dehydrogenase family (30% up) were shown to be overexpressed. V-type ATP synthase (CSP5_0042), subunit I with 44.24% sequence identity to similar proteins from Thermoplasmatales archaea, showed a 27% increase in abundance. Since this enzyme complex is generating ATP, it would make perfect sense for it to be upregulated in light of increased ATP requirement for de novo protein synthesis and transport events required to meet the challenge to adapt to a new environment. The expression of this energy conservation enzyme was reported to be important for survival under stress conditions [31]. PyrL (CSP5_1108), the aspartate carbamoyltransferase regulatory subunit (Nucleotide transport and metabolism category) was detected as 50% overexpressed. The protein could be involved in allosteric regulation of aspartate carbamoyltransferase and showed 51.25% identity to similar proteins from "Candidatus Bathyarchaeota" archaea, from Thermoplasma species and Thermoplasmatales "E-" and "A-plasma" variants. PyrG protein (CSP5_0024), a CTP synthase (glutamine hydrolysing) (with high levels of sequence identity to similar proteins from Thermoplasmatales archaea) involved in pyrimidine ribonucleotide/ribonucleoside metabolism was 20% overexpressed.

Differential Expression under Optimal Growth and Cold Shock Conditions of Functional Categories of Proteins
Our study has observed significant changes in expression of functional categories of proteins COG and eggnog expressed under cold shock conditions.
These observations imply the importance of proteins involved in amino acids metabolism, translation, and ribosomal biogenesis for cold shock resistance of C. divulgatum cells. The shock response to the longer (up to 5 h) exposure to the cold led to an increase in the biosynthesis of certain amino acids, as previously observed in cells of Pyrococcus furiosus [33]. In general, genes encoding proteins engaged in translation, transport of solutes, amino acids biosynthesis, carbon and tungsten metabolism together with some hypothetical proteins were mentioned as upregulated after early, late shock and adapted response conditions [33].
Carbohydrate transport and metabolism category-related proteins that were overexpressed in the proteome of cold shock-imposed cells of C. divulgatum included acryloyl-coenzyme A reductase alcohol dehydrogenase (CSP5_0778, 1.52-fold), glyceraldehyde-3-phosphate dehydrogenase (CSP5_0332, 1.21), ribokinase (PfkB family carbohydrate kinase) (CSP5_1289, 1.13), and short-chain dehydrogenase/reductase (CSP5_1330, 1.13). The numbers of proteins of this category that were upregulated in cold-stressed proteome exceeded those expressed under optimal growth conditions ( Figure 2). All this suggests that the cold shock has induced defined metabolic changes reflected in intracellular carbohydrate metabolism. Further experimental work is required to reveal more details of this metabolic shift.
Lipid transport and metabolism category included proteins of mevalonate pathway that were expressed in C. divulgatum (Figure 4). In particular, mevalonate 3,5-biphosphate decarboxylase, (CSP5_0962, 1.25-fold) and acetyl-CoA acetyltransferase, (CSP5_0855, 1.24-fold), were upregulated under cold shock. HMG (hydroxymethylglutaryl)-CoA synthase (CSP5_0230, 5% higher in the cold), HMG-CoA reductase (CSP5_1096, 5% lower in cold), mevalonate 3-kinase (CSP5_0479, 2.5% down in the cold) and IPP isomerase (CSP5_0048, 20% down in the cold) were found in proteomic data but were not overexpressed. However, IPP kinase (and mevalonate 3-phosphate 5-kinase) could not be identified in this dataset. Isoprenoid lipid biosynthesis from mevalonate was considered early to be associated with cold adaption in Methanocaldococcus jannaschii [34]. Cell membranes are known to play a vital role in stress sensing and signalling, and changes in the lipid composition of membranes have been reported in a variety of species from higher plants to archaea [35][36][37]. We detected an overexpression of two proteins involved in mevalonate pathway under cold shock conditions: acetyl-CoA-acetyltransferase and mevalonate-3,5-bisphosphate decarboxylase, the latter being important for biosynthesis of isopentenyl diphosphate, a fundamental precursor for isoprenoids [38]. We believe that efficiency of biosynthesis of isoprenoids that are the principal component of membrane tetraether lipids is important for survival strategy of C. divulgatum. This statement would be more solid after experimental characterisation of lipid content of cold-stressed C. divulgatum. . Metabolic reconstruction of membrane phospholipids biosynthesis through mevalonate pathway. Two enzymes, Acetyl-CoA acetyltransferase and Mevalonate 3,5-bisphosphate are overexpressed (blue arrows) under cold conditions, catalysing key reactions for the synthesis of mevalonate and isopentenyl bisphosphate (IPP), respectively, inside the mevalonate pathway for the synthesis of isoprenoids. These molecules are closely related with the membrane phospholipids biosynthesis, which some enzymes have been also identified, although not overexpressed, in this proteomics dataset as long as other enzymes are involved in mevalonate pathway (black arrows). Absent reactions, catalysed by enzymes not detected in this dataset, are coloured in grey.
Signal transduction mechanisms category showed the presence of expressed CDC48 family ATPase of the AAA+ class (CSP5_1374, 1.21). The protein of the CDC48 family ATPase of the AAA+ class cloned from Thermoplasma acidophilum showed the complex appearance resembling 20S proteasome and Hsp60/GroEL [39].
Inorganic transport and metabolism category proteins downregulated in cold included DrsE family protein (CSP5_0480, 1.38). FeCT family ABC transporter substrate-binding component (CSP5_1116) and ATPase subunits (CSP5_1114) were detected at exactly same levels in the proteome under both . Metabolic reconstruction of membrane phospholipids biosynthesis through mevalonate pathway. Two enzymes, Acetyl-CoA acetyltransferase and Mevalonate 3,5-bisphosphate are overexpressed (blue arrows) under cold conditions, catalysing key reactions for the synthesis of mevalonate and isopentenyl bisphosphate (IPP), respectively, inside the mevalonate pathway for the synthesis of isoprenoids. These molecules are closely related with the membrane phospholipids biosynthesis, which some enzymes have been also identified, although not overexpressed, in this proteomics dataset as long as other enzymes are involved in mevalonate pathway (black arrows). Absent reactions, catalysed by enzymes not detected in this dataset, are coloured in grey.
Signal transduction mechanisms category showed the presence of expressed CDC48 family ATPase of the AAA+ class (CSP5_1374, 1.21). The protein of the CDC48 family ATPase of the AAA+ class cloned from Thermoplasma acidophilum showed the complex appearance resembling 20S proteasome and Hsp60/GroEL [39].
Inorganic transport and metabolism category proteins downregulated in cold included DrsE family protein (CSP5_0480, 1.38). FeCT family ABC transporter substrate-binding component (CSP5_1116) and ATPase subunits (CSP5_1114) were detected at exactly same levels in the proteome under both conditions. However, cold shock cells of C. divulgatum showed the certain overexpression of rhodanese-related sulfurtransferase (CSP5_1158, 1.15-fold). Altogether, the genome of C. divulgatum S5 encodes four rhodanese-related sulfurtransferases and two rhodanese domains fused to Zn-dependent hydrolase of glyoxylase family. The overexpressed protein (CSP5_1158) with conserved domains on RHOD superfamily showed relatively low (36.65%) identity to metallo-beta-lactamase superfamily protein from Thermoplasmatales uncultured archaea of "E-plasma" and 34.67-29.15% identity to sulfurtransferases from Sulfobacillus species. Interestingly, other rhodanese-related sulfurtransferases, represented in C. divulgatum S5 genome exhibited, along with sulfurtransferases from "A-" and "E-plasma") highest sequence identities with bacterial sulfurtransferases (C. divulgatum proteins CSP5_854, CSP5_1347, CSP5_1466, with Acetoanaerobacterium spp. (42.55%), and Sulfobacillus spp. (40.71% and 31.33%), correspondingly)). Noteworthy, CSP5_1465 and CSP5_1975 with rhodanese domain fused to Zn-dependent hydrolase of glyoxylase family were 31.3% identical to bacterial proteins MBL fold metallo-hydrolase from Sulfobacillus spp., from Crenarchaeota (45.31% identical to those from Sulfurisphaera tokodaii and other archaea) and from some bacteria. Consequently, we hypothesize that Cuniculiplasma might have experienced horizontal gene transfer of sulfurtransferases from archaeal or bacterial community members. Proteins with rhodanese-like domains were considered associated with stress in bacteria previously [40]. Functions of those proteins may include (i) chaperone activity to provide assembly of iron-sulfur complexes, (ii) detoxification of some toxic compounds (arsenate and cyanide), (iii) maintenance of redox homeostasis and (iv) biosynthesis of cofactors, enzymes and vitamins [40][41][42][43].
Altogether, the relative number from categories 'Energy production' and 'Conversion and amino acid transport and metabolism' was higher under cold conditions in comparison to optimally grown cells. In addition, 'Nucleotide transport and metabolism', 'Coenzyme transport and metabolism', 'Lipid transport and metabolism', 'Inorganic ion transport and metabolism', and proteins belonging to unknown function were overexpressed in the cold shock proteome ( Figure 2, Table S1).
Comparison of our data with those in archaeon Pyrococcus furiosus showed certain similarities [33]. For example, the early (1-2 h) shock response in P. furiosus caused upregulation of proteins (17 of 55) involved in metabolism of amino acids and primary carbohydrates, in the translation and oxidoreductase-type processes and also in transport of solutes [33]. In the cold, four-fold upregulation was detected for S-adenosylmethionine synthetase in P. furiosus, whereas in C. divulgatum there was a 20% increase in this protein biosynthesis. Additionally, upregulation of oxidoreductases was detected in C. divulgatum with a FADox protein, FAD-dependent oxidoreductase of DAO superfamily (CSP5_1916, 2-fold increase). The cold-shock response in P. furiosus showed upregulation of biosynthesis of branched-chain amino acids and methionine. Our data revealed the upregulation of methionine, as well [33]. However, solute-binding proteins CipA and CipB identified as two major membrane glycoproteins in P. furiosus, representing archaeal type of bacterial cold shock protein (Csp) family, are non-existent in C. divulgatum.

Most Abundant Proteins
Combined, alpha and beta subunits of thermosome were the most significant proteins, making up to 3.18% and 3.29% of the total identified proteins in both, exponentially-grown and cold-imposed C. divulgatum, which, unlike Sulfolobales [44], does not encode in its genome a gamma-subunit to form α, β, γ heterooligomeric complex in response to the cold. Further, the most-abundant proteins (Table S2) were glutamate dehydrogenase (CSP5_1203, 2.0 and 2.19%) peroxiredoxin (CSP5_0886, 1% of the sum of identified proteins under both conditions), rubrerythrin (CSP5_0891, ca. 0.9% under both conditions), malate dehydrogenase (CSP5_0838, ca. 0.87% and 0.83%), and 2-oxoacid ferredoxin oxidoreductase (CSP5_0284, ca. 0.72% and 0.79%) and S-adenosylmethionine (SAM) synthetase (CSP5_0006, 0.8% and 0.97%, i.e., 20% increase in the cold). Glutamate dehydrogenases (GDH) are linked in humans to many cellular processes-including acid-base homeostasis, redox homeostasis, ammonia metabolism and lipid biosynthesis [45]. In bacteria and archaea, GDH are known to be important for carbon and nitrogen metabolism and responsible for oxidative deamination of glutamate and for reductive inclusion of ammonium into 2-oxoglutarate [46]. For Thermococcus profundus grown on peptides, GDH function was suggested for deamination of glutamate, accompanied with formation of NAD(P)H+(reduced) and 2-oxoglutarate, which is channelled into tricarboxylic acids cycle and used as an energy source [47]. We consider a similar role of GDH in deamination of glutamate originated from polypeptides used for cultivation of C. divulgatum. Other proteins associated with oxidative stress were also found synthesised at a high level, such as vitamin B 12 -dependent ribonucleotide reductase (CSP5_2009, ca. 0.48% under both conditions), and lactaldehyde dehydrogenase (CSP5_1304, ca. 0.51% under both conditions). The abundance of glutamate dehydrogenase, which tops the list of most-produced cellular proteins with an enzymatic function, does reflect the lifestyle of Cuniculiplasma as polypeptide-scavenging organism, and which enables the conversion of glutamate from externally acquired glutamate-containing proteinaceous substrates into α-ketoglutarate that is then funnelled into the tricarboxylic acid cycle. Interestingly, SAM synthetase is one of the most abundant proteins in the cold proteome (5th ranked, 0.97%), which provides the substrate for SAM-dependent methyltransferases. SAM synthetase generates the methyl group donor involved into polyamine synthesis, methylation of DNA and other organic molecules [48] that may contribute to the cold stress response in C. divulgatum. While the Cuniculiplasma genome encodes 12 SAM-dependent methyltransferase homologues (including one pseudogene), only three of them were detected in this proteome dataset: (i) CSP5_0484 (low-identity to isoaspartyl methylransferase from Archaeoglobus and 6% downregulated in the cold), (ii) CSP5_0705 (50% AA similarity with bacterial ubiquinone/menaquinone biosynthesis C-methyltransferases and arsenite methyltransferase in Methanosarcina, with exactly the same expression levels under both conditions) and (iii) CSP5_1828 (48% AA similarity with bacterial rRNA guanine or uracil methyltransferases, 4% up in the cold). Their expression levels (ranging from 0.1% to 0.15% fmol) did show less than 6% variations under both conditions. FKBP-type peptidyl-prolyl cis-trans isomerase was another abundant protein (CSP5_1482, 0.67% of total proteome, which increased its numbers in the cold (5% up)). These proteins, apart from PPIase activity, also demonstrate a chaperone-like activity [49], supporting their importance of protein folding under stress conditions. Protein folding is a critical factor, and chaperones, which are well-known for being involved in the general stress response, exhibited a lower expression in the cold. DnaK (CSP5_1162) was 2.4% down, Hsp20 (CSP5_0928 and CSP5_1387)was 17% and 4% down, respectively, DnaJ (CSP5_1163) 30% down, and GrpE (CSP5_1161), 15% down), although they were expressed at very low levels (0.1-0.3% fmol) (Table S2). Iron-sulfur cluster assembly protein (CSP5_1945, ca. 0.125 vs. 0.131%) and peptide methionine sulfoxide reductase (CSP5_1232, 0.088 vs. 0.087%) were found to be expressed at significant low levels (ca. 0.125 vs. 0.131%); these are redox enzymes commonly over-synthesised under oxidative stress periods by acidophilic bacteria [50].

Conclusions
Under cold-shock conditions, the majority of the top 10 most-abundant proteins (0.73-2 fmol% of the quantifiable proteins in this dataset) of C. divulgatum S5 T were marginally, yet statistically reliably, overexpressed. These included proteins involved in folding (thermosome subunits and FKBP-type peptidyl-prolyl cis-trans isomerase), glutamate deamination (glutamate dehydrogenase, the most abundant protein with enzymatic function), methylation (SAM synthetase), citric acid cycle (malate dehydrogenase and 2-oxoacid ferredoxin oxidoreductase) and in signal transduction pathways. Growth-associated elongation factor 1-alpha (down 1.4%), rubrerythrin and all four peroxiredoxins present in this dataset (equal levels under both conditions) were the only exceptions.
Low-abundance proteins (0.001-0.005 fmol%) that showed the greatest fold-changes in the proteome of actively metabolising C. divulgatum cells were the enzymes mainly involved in proteolytic machinery. Stomatin-a regulator of protease activity, ATP-dependent LonB protease, alongside ABC transporters and a cytochrome C oxidase of the heme copper oxidase 1 superfamily-were shown to be among the most highly expressed proteins during active growth. Cold-shock proteome analysis has pinpointed proteins that exhibited overexpression from functional categories 'Amino acid transport and metabolism', 'Energy production and conversion', 'Coenzyme transport and metabolism' and proteins belonging to unknown function groups. Overall, this study provided first insights into the intracellular proteome of C. divulgatum and suggested several potential mechanisms underlying its physiological adaptation to a short-term cold shock: overexpression in pathways for aspartate, methionine and cysteine synthesis and also in central metabolic pathways for carbohydrates transformation, pyrimidine biosynthesis and coenzyme transport. Since C. divulgatum is a ubiquitous acidophilic microorganism, these adaptive mechanisms could contribute to their broad distribution across a variety of acidic environments with different temperatures.