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Table of Contents
Abstract
Results
Discussion
Materials And Methods
RNA-Seq
Proteome
LC-MS/MS Analysis
Metabolome Analyses
Molecular Cloning
Complementation Studies
Analytical Assays
Bioinformatic Analyses
Statistics And Reproducibility
Reporting Summary
Data Availability
Acknowledgements
Author Contributions
Funding
Competing Interests
Additional Information
Abstract
Glycerol is highly abundant in natural ecosystems and serves as both an important carbon source for microorganisms as well as a promising feedstock for industrial applications. However, the pathways involved in glycerol degradation in Archaea remain unclear. Here, we show that the thermoacidophilic Crenarchaeon Sulfolobus acidocaldarius can grow with glycerol as its sole carbon source and characterize the mechanisms involved in glycerol utilization. We show that after uptake involving facilitated diffusion, glycerol is phosphorylated to glycerol-3-phosphate by glycerol kinase (GK), followed by oxidation to dihydroxyacetone phosphate catalyzed by an unusual glycerol-3-phosphate dehydrogenase (G3PDH) with a previously undescribed type of membrane anchoring via a CoxG-like protein. Furthermore, we show that while S. acidocaldarius has two paralogous GK/G3PDH copies (saci_1117-1119, saci_2031-2033) with similar biochemical activity, only saci_2031-2033 is highly upregulated and essential on glycerol, suggesting that distinct enzyme pairs may be regulated by different environmental conditions. Finally, we explore the diversity of glycerol metabolism enzymes across theArchaea domain, revealing a high versatility ofG3PDHswith respect to interacting proteins, electron transfer mechanisms, and modes of membrane anchoring. Our findings help to elucidate the mechanisms involved in glycerol utilization in Archaea, highlighting unique evolutionary strategies that likely enabled adaptation to different lifestyles.
communications biology Article A Nature Portfolio journal https://doi.org/10.1038/s42003-025-07953-9 An unusual glycerol-3-phosphate dehydrogenase in Sulfolobus acidocaldarius elucidates the diversity of glycerol metabolism across Archaea Check for updates Christian Schmerling1,9, Carsten Schroeder1,9, Xiaoxiao Zhou1,9, Jan Bost2, Bianca Waßmer2, Sabrina Ninck 3, Tobias Busche4, Lidia Montero5,6,8, Farnusch Kaschani3,7, Oliver J. Schmitz5,6, Jörn Kalinowski4, Markus Kaiser 3, Sonja-Verena Albers 2, Christopher Bräsen 1 & Bettina Siebers 1 Glycerol is highly abundant in natural ecosystems and serves as both an important carbon source for microorganisms as well as a promising feedstock for industrial applications. However, the pathways involved in glycerol degradation in Archaea remain unclear. Here, we show that the thermoacidophilic Crenarchaeon Sulfolobus acidocaldarius can grow with glycerol as its sole carbon source and characterize the mechanisms involved in glycerol utilization. We show that after uptake involving facilitated diffusion, glycerol is phosphorylated to glycerol-3-phosphate by glycerol kinase (GK), followed by oxidation to dihydroxyacetone phosphate catalyzed by an unusual glycerol-3-phosphate dehydrogenase (G3PDH) with a previously undescribed type of membrane anchoring via a CoxG-like protein. Furthermore, we show that while S. acidocaldarius has two paralogous GK/G3PDH copies (saci_1117-1119, saci_2031-2033) with similar biochemical activity, only saci_2031-2033 is highly upregulated and essential on glycerol, suggesting that distinct enzyme pairs may be regulated by different environmental conditions. Finally, we explore the diversity of glycerol metabolism enzymes across theArchaea domain, revealing a high versatility ofG3PDHswith respect to interacting proteins, electron transfer mechanisms, and modes of membrane anchoring. Our findings help to elucidate the mechanisms involved in glycerol utilization in Archaea, highlighting unique evolutionary strategies that likely enabled adaptation to different lifestyles. Glycerol (C₃H₈O₃) is a simple organic compound that is an integral constituent of membrane phospholipids and storage lipids like triglycerides, and thus is highly abundant in plant, animal and microbial cells. Accordingly,many organisms, fromBacteria andArchaea to complexEukarya, can utilize glycerol as a carbon and energy source. Furthermore, glycerol has important physiological and ecological roles, such as serving as an osmolyte or regulating cross-species interactions between photosynthetic algae and archaea in hypersaline environments1. Glycerol also has many biotechnological uses, from the food and pharmaceutical industries to being increasingly regarded as an attractive feedstock to support the production of 1Molecular Enzyme Technology and Biochemistry (MEB), Environmental Microbiology and Biotechnology (EMB), Centre for Water and Environmental Research (CWE), Faculty of Chemistry, University of Duisburg-Essen, Essen, Germany. 2Molecular Biology of Archaea, Institute of Biology II—Microbiology, University of Freiburg, Freiburg, Germany. 3Chemical Biology, Center of Medical Biotechnology, Faculty of Biology, University of Duisburg-Essen, Essen, Germany. 4Center for Biotechnology (CeBiTec), BielefeldUniversity, Bielefeld, Germany. 5Applied Analytical Chemistry (AAC), University of Duisburg-Essen, Essen,Germany. 6Teaching andResearch Center for Separation (TRC), University of Duisburg-Essen, Essen, Germany. 7Analytics Core Facility Essen (ACE), Center ofMedical Biotechnology, University of Duisburg-Essen, Essen, Germany. 8Present address: Laboratory of Foodomics, Institute of Food Science Research, CIAL, CSIC, Madrid, Spain. 9These authors contributed equally: Christian Schmerling, Carsten Schroeder, Xiaoxiao Zhou. e-mail: christopher.braesen@uni-due.de; bettina.siebers@uni-due.de Communications Biology | (2025) 8:539 1 12 34 56 78 90 (): ,; 12 34 56 78 90 (): ,; value-added molecules by recombinant microbes2. Therefore, elucidating themechanisms involved in glycerolmetabolism is critical to not only better understand the physiological and ecological roles mediated by glycerol but also to support improved use of this substrate in biotechnological applications. Despite the importance of glycerol across all domains of life, the detailed pathways involved in glycerol utilizationhave only been explored in a fewspecies, particularly inBacteria andEukarya.The initial step inglycerol utilization is its uptake across the cytoplasmic membrane, which often involves facilitated diffusion via glycerol-uptake facilitators (GUF), including aqua(glycerol)porins such as GlpF3 (Fig. 1). Additionally, glycerol can also enter the cell via passive diffusion4 or through alternative transporters5–9. Then, glycerol metabolism can follow two routes, which are differentially distributed across organisms (Fig. 1). The most prevalent pathway, predominantly employed by respiring organisms9–11, converts glycerol into sn-glycerol-3-phosphate (G3P) via anATP-dependent glycerol kinase (GK) (encoded by glpK)12. Then, G3P is oxidized to dihydroxyacetone phosphate (DHAP) by one of two membrane-bound glycerol-3phosphate dehydrogenases (G3PDH), designated as GlpD (encoded by glpD) andGlpABC (encoded by the glpABC operon), with the simultaneous reduction of a non-covalently bound flavin adenine dinucleotide (FAD) to FADH2. From FADH2 the electrons are transferred to the quinone pool of the respiratory chain13–16. GlpD is active under aerobic conditions, e.g. in E. coli11,14–16, transferring electrons via ubiquinone to oxygen or nitrate, and is also the G3P oxidizing enzyme in the mitochondria of Eukarya15. Ubiquinone is the prevalent quinone of the respiratory chain under aerobic conditions in Bacteria and mitochondria. The GlpABC respiratory complex that is mainly known from Bacteria, is induced under anaerobic conditions and reducesmenaquinonewith the final acceptors nitrate or fumarate (inE. coli)11,17. Menaquinone is commonly used by anaerobic bacteria, and facultative anaerobes including E. coli change their quinone pool from ubiquinone under aerobic to menaquinone under anaerobic conditions. The A and B subunits of the complex form a soluble and active dimer18 which is likely anchored to themembrane via the C subunit17,19. In addition to these twomembrane-bound G3PDH enzymes, G3P oxidation to DHAP canalso be performedby theG3PoxidaseGlpO (encoded by the glpO gene), a cytosolic, soluble FAD-dependent enzyme directly reducing oxygen to H2O2, which is detoxified via peroxidase or catalase. GlpO ismainly known from aerotolerant/microaerophilic lactic acid bacteria and Mycoplasma spp20–25. The second route of glycerol processing is less abundant and restricted to fermentatively growing organisms including E. coli, some additional Enterobacteriaceae and a few other bacterial species26,27. In these organisms, glycerol is first oxidized to dihydroxyacetone (DHA) in an NAD+-dependent manner by the glycerol dehydrogenase GldA (encoded by the gldA gene). Then, DHA is phosphorylated to DHAP in a phosphoenolpyruvate (PEP)-dependentmannerbyDhaK (encodedby the dhaK gene) or in an ATP-dependent manner by GlpK (encoded by the gene glpK in Klebsiella pneumoniae)11,26. The NADH derived from glycerol oxidation is reoxidizedwith anothermolecule of glycerol, and finally converted to 1,3- propandiol or 1,2-propanediol26. Both of these metabolic routes result in DHAP production, which is then channelled into the central metabolism and further metabolized via the lower common shunt of the Entner–Doudoroff (ED) and the Embden-Meyerhof-Parnas (EMP) pathway or utilized for gluconeogenesis. G3P serves as a building block for phospholipid and membrane synthesis in Bacteria and Eukarya. While different routes of glycerol metabolism have been characterized in Bacteria and Eukarya, comparatively little is known in Archaea, with studies focusing primarily onHaloferax volcanii.H. volcanii utilizes glycerol via homologues of the bacterial GlpK and GlpABC, while GlpD is absent1,28–30. Althoughgenes encodingputativeGKsandG3PDHshavebeen described in representatives of other taxa31, and some enzymes have been characterized29,32–36, no other Archaea have so far been shown to grow with glycerol as sole carbon and energy source. Here, we expand the characterization of glycerol metabolism in Archaea by delineating the mechanisms involved in glycerol utilization in the thermoacidophilic Crenarchaeon Sulfolobus acidocaldarius. S. acidocaldarius grows heterotrophically at temperatures around 75 °C and a pH of 2.0–3.0 with a variety of carbon sources, although like other Sulfolobales, S. acidocaldarius has been reported to not grow on glycerol37. S. acidocaldarius is regarded as a promising chassis for biotechnological applications, due to its ability to grow in extreme environments, availability of its genome sequence and genetic tools38–40 and because some of its metabolic pathways, particularly those involved in carbohydrate metabolism, have been characterized in detail38,41. With regards to enzymes involved in glycerolmetabolism, onlyGKhomologues have been identified, whileG3PDH homologues appear to be absent31. However, S. acidocaldarius has recently been shown to cleave triacylglycerides by esterases and to grow with fatty acids42. As cleavage of triacylglycerides results in the production of glycerol, here we reexamined the growth of S. acidocaldarius on this substrate. Using a combination of growth studies, genetic mutants, and biochemical and multi-omics analyses, we demonstrate that S. acidocaldarius grows using glycerol as its sole carbon source. Furthermore, we show that glycerol utilization uses a conserved ‘classical’ GK, which is homologous to GlpK, for glycerol phosphorylation, followed by G3P oxidation, which is catalysed by an unusual G3PDH enzyme, resembling a truncated version of GlpA in Communications Biology | (2025) 8:539 2 which membrane association is facilitated by a small carbon monoxide dehydrogenase subunit G (CoxG)-like protein. Finally, we reveal a diverse repertoire of G3PDHs across Archaea, featuring various interacting proteins, electron transfer pathways, and potential modes of membrane anchoring. Our results expand our knowledge on the diversity of mechanisms used for glycerol metabolism in Archaea and are likely to support future biotechnological uses of S. acidocaldarius.
Results
S. acidocaldarius glycerol catabolism involves GK and G3PDH Following a period of adaption, S. acidocaldarius MW001 exhibited exponential growth when supplied with glycerol (10mM, 20mM, and 40mM) as sole carbon and energy source. Notably, similar growth rates of approximately 0.0287 ± 0.0005 h−1 were observed across all glycerol concentration, and cell densities up to an OD600 of 4 (at 40mM glycerol) were achieved, coinciding with complete consumption of glycerol (Fig. 2a–c, Supplementary Table 1). The adapted strain was named S. acidocaldarius MW00G. For comparison, S. acidocaldariusMW00G grew on 0.2% (w/v) D-xylose, a commonly used carbon source, to a much lower final OD600 of 0.8 and with a slower growth rate of 0.0195 ± 0.0005 h−1 (Fig. 2d). Having shown that S. acidocaldarius can grow faster and to higher biomass yields (corresponding to increased yield coefficients) (Supplementary Table 1) on glycerol than on D-xylose, we then compared the cellular responses to these carbon sources using transcriptomics (RNA-Seq) and proteomics (LC-MS-MS). On glycerol, a total of 39 transcripts/proteins were significantly upregulated while 14 transcripts/proteins were downregulated with at least a log2-fold change of 2 (Supplementary Tables 2 and 3). Downregulation was observed for the Weimberg pathway for pentose degradation (saci_1938, α-ketoglutarate semialdehyde dehydrogenase; saci_1939, 2-dehydro-3-deoxy-D-arabinonate dehydratase) and for the sugar binding subunit of the xylose/arabinose transporter (saci_2122). This coincided with previous findings demonstrating this pathway to be up-regulated by D-xylose43. Other central carbohydrate metabolic pathways (e.g. branched ED pathway, tricarboxylic acid (TCA) cycle, and EMP)were unaffected. Notably, we found that glycerol induced a significant upregulation of the gene cluster saci_2031-2034 (Fig. 3a) and its encodedproteins, including a putativeG3PDH (Saci_2032, GlpA-like) with a downstream encoded protein annotated as CoxG (Saci_2031), as well as a putative GK (Saci_2033) and a putative glycerol uptake facilitator (GUF; Saci_2034). Both, saci_2032-2031 and saci_2033-2034 formoperons and are divergently oriented (Fig. 3b). In accordance with the regulation pattern of saci_2031-2034, we also observed the induction of enzymatic activities in soluble crude extracts of cells grown in glycerol compared to cells grown on D-xylose: GK activity increased from 0.06 ± 0.002Umg−1 to 0.89 ± 0.048Umg−1, whereas G3PDH activity increased from nonmeasurable to 0.12 ± 0.006 Umg−1 activity (with DCPIP as electron acceptor) (Fig. 3c). Neither NAD+-dependent G3P oxidation nor DCPIPor NAD+-dependent glycerol oxidation could be observed, indicating that no alternative pathway for glycerol dissimilation is present. Notably, the DCPIP-dependent G3PDH activity was not only located in the soluble fraction (crude extract) but also in the resuspendedmembrane fractionwith 0.13 ± 0.018Umg−1 (Fig. 3c), whereas GK activity was exclusively found in the soluble fraction. In addition to saci_2031-34, we identified a second gene cluster (saci_1117-1119) encoding isoenzymes for GK (saci_1117), G3PDH (saci_1118) and the CoxG-like protein (saci_1119). However, in contrast to Saci_2031-2034, a homologue encoding a GUF (or other transporter) is missing from this cluster (Fig. 3b). Furthermore, this second cluster saci_1117-1119 was only slightly upregulated in response to glycerol G ly ce ro l [ m M ] 0 2 4 6 8 10 Time [hours] O D 60 0 1.6 1.4 1.2 1 0.8 0.6 0.4 0.2 0 0 20 40 60 80 100 120 140 160 180 2.5 2 1.5 1 0.5 0 Time [hours] G ly ce ro l [ m M ] O D 60 0 a b 50 40 30 20 10 0 0 50 100 150 200 250 5 4 3 2 1 0 Time [hours] G ly ce ro l [ m M ] O D 60 0 O D 60 0 c d Time [hours] Xy lo se [m M ] 0 20 40 60 80 100 120 140 160 180 0 0.1 0.2 0.3 0.4 0.5 0.6 0 3 6 9 12 15 0 20 40 60 80 100 120 160 180140 20 15 10 5 0 Fig. 2 | Growth and substrate consumption of S. acidocaldarius MW00G. Cells were grown onBrock’s basalmedium containing 10 mM (a), 20 mM(b), and 40 mM (c) glycerol as sole carbon and energy source under aerobic conditions. For comparison the growth with 14 mMD-xylose (d) was studied. Growth wasmonitored as increase in OD600. Substrate concentrations in the cell free supernatant were determined enzymatically as described in the materials and methods part. Experiments were performed in triplicate (n = 3, biological replicates) and error bars indicate the standard deviation (SD) of the mean and individual data points are shown as grey dots. Communications Biology | (2025) 8:539 3 compared to D-xylose, at both the transcript and protein level (Fig. 3a).We also used targeted LC(HILIC)-MS/MS-based metabolome analyses to further characterize glycerol metabolism in S. acidocaldarius. Focusing on central metabolic intermediates, especially those involved in glycerol catabolism, we found minimal levels of free glycerol in glycerol grown cells, while the concentrations of G3P and DHAP were substantially increased, compared to cells grown on D-xylose (Fig. 3d). These multi-omics analyses demonstrate that S. acidocaldarius effectively utilizes glycerol as its sole carbon and energy source. Our data support a metabolic route for glycerol utilization in which following transport into the cell, likely involving the GUF (Saci_2034), glycerol is phosphorylated to G3P via GK (GlpK, Saci_2033), and further oxidized to DHAP by G3PDH (GlpA-like, Saci_2032). DHAP is then channelled either into the common lower shuntof theEDandEMPpathway for glycolysis or theupperEMP for gluconeogenesis. Purification and characterization of GK and G3PDH isoenzymes To further characterize the enzymes involved in glycerol metabolism in S. acidocaldarius, the two putative GKs Saci_1117 and Saci_2033 were homologously produced in S. acidocaldariusMW001 as C-terminally Hisand Twin-Strep-tagged proteins, respectively, and purified. Saci_1117 and Saci_2033 comprise 498 and 497 amino acids with calculated molecular masses of 55.6 and 55.3 kDa, respectively, coinciding well with the approximately 55 kDa determined by SDS-PAGE (Supplementary Fig. 1). The native molecular weight of ~110 kDa for both proteins determined by SEC indicated a homodimeric structure (α2) (Supplementary Fig. 2). Both isoenzymes showed GK activity in vitro, which followedMichaelis-Menten kinetics. For Saci_1117, we determined Vmax values of 45.7 ± 0.25 Umg −1 andKM values of 0.024 ± 0.001mM(glycerol) and0.205 ± 0.024mM(ATP) at 50 °C (Fig. 4, Table 1). The kinetic properties of Saci_2033, with a Vmax value of 88.2 ± 2.5 Umg−1 and KM values of 0.024 ± 0.004mM (glycerol) and 0.174 ± 0.019mM(ATP), revealed a two-fold higher catalytic efficiency compared to Saci_1117 (Table 1). For additional information on substrate specificity, pH- and temperature optima, thermal stability, substrate and phosphoryl donor specificity and effect of fructose-1,6-bisphosphate see Supplementary Information ‘In-depth comparison of structural, enzymatic, and regulatory properties of glycerol kinases across the three domains of life’ (Supplementary Figs. 3 and 4, Supplementary Table 3). We also characterized the in vitro activity of the two GlpA-like G3PDHs, Saci_1118 and Saci_2032. Heterologous expression in E. coli and purification of both N-terminally His-tagged G3PDHs, with calculated molecular mass of 46.9 kDa for Saci_1118 and 47.6 kDa for Saci_2032, yielded soluble, yellow proteins with subunit sizes of around 50 kDa (SDSPAGE) and nativemolecularmasses of 95 kDa (Supplementary Fig. 5), thus a b c d 1117 1118 1119 GK G3PDH CoxG 2031 2032 2033 2034 GUFGKG3PDHCoxG 0 3 20 25 30 35 40 45 Pe ak a re a [1 07 m AU m in -1 ] 15 Glycerol 10 20 40 X 10 20 40 X 10 20 40 X G3P DHAP 10 mM Glycerol 20 mM Glycerol 40 mM Glycerol 0.2% (w/v) D-xylose uptake facilitator (GUF). cGKandG3PDH activity in the soluble (S) andmembrane fraction (M) (after cell lysis) of S. acidocaldariusMW00G grown on 40 mM glycerol in comparison to 0.2% (w/v) D-xylose. d Glycerol, G3P and DHAP determined by targetedmetabolomics in S. acidocaldariusMW00Ggrown on glycerol (10 mM(10), 20 mM (20) and 40 mM (40) glycerol) compared to D-xylose (0.2% (w/v) (X)). All values represent the average of three (crude extract activities) or eight (metabolic analyses) independent measurements (biological replicates). Error bars represent the SD of the mean and individual data points are shown as dots. Communications Biology | (2025) 8:539 4 representinghomodimers. The yellow colour indicated thepresenceof FAD cofactor, and a FAD content of two per homodimer was determined for both enzymes (Fig. 5a). After reduction with G3P, without adding an artificial electron acceptor, the FAD cofactor remained stable in its colourless, reduced state. To exclude G3P oxidase (GlpO) activity, the direct electron transfer from FAD to oxygen forming H2O2 was excluded using the 2,2’- azinobis-(3-ethylbenzothiazoline-6-sulfonate) (ABTS) assay (25, data not shown). Saci_1118 displayed KM and Vmax values of 0.019 ± 0.003mM (G3P) and 19.7 ± 0.73 U mg−1, respectively, while Saci_2032 exhibited values of 0.055 ± 0.009mM (G3P) and 44.5 ± 2.47 Umg−1, with DCPIP serving as the artificial electron acceptor (Fig. 5b, Table 1). Also, ubiquinone-Q1 was confirmed as the electron acceptor, as evidenced by G3P-dependent decrease of absorption at 280 nm (Fig. 5c) and kinetic characterization (Fig. 5d, Table 1). Ubiquinone-Q1 is a water-soluble analogue of ubiquinones which are found in bacterial and mitochondrial respiratory chains. Its short side chain of one isoprenoid unit renders it water-soluble and thus suitable for aqueous biochemical assays, unlike the longer-chain ubiquinones (up to ten isoprenoid units comprising side chains) found in bacteria and eukaryotes, which are water-insoluble and unsuitable for such assays. For additional information on (co)substrate specificity, pH- and temperatureoptima, thermal stability, andeffect of nonionic detergents and phospholipids see Supplementary Information ‘Indepth comparison of structural, enzymatic, and regulatory properties of glycerol-3-phosphate dehydrogenases across the three domains of life’ (Supplementary Figs. 6 and 7, Supplementary Table 4). Collectively, these analyses demonstrate that the GK and G3PDH enzymes of S. acidocaldarius are functional in vitro and reveal minor differences in catalytic activity between isoenzymes that may be relevant for glycerol metabolism under different conditions. CoxG homologues serve as membrane anchor for S. acidocaldarius G3PDHs The ability of both S. acidocaldarius G3PDHs to reduce quinones suggests that these enzymes are associated with the cellular membrane. However, while the archaeal G3PDH enzymes share similarities with the GlpA subunit from the bacterial GlpABC complex, S. acidocaldarius does not encode a GlpC subunit, which mediates membrane anchoring in Bacteria. Notably, both genes encoding G3PDH (saci_1118 and saci_2032) form operons with a CoxG homologue (saci_1119 and saci_2031, respectively), suggesting a functional association. Furthermore, CoxG proteins have been shown to mediate membrane associations in Bacteria, although this function has never been demonstrated in archaeal cells or in connection to glycerol metabolism. However, both G3PDHs were active in vitro in the absence of their corresponding CoxG homologue, indicating that CoxG is not essential for G3PDH activity or electron transfer to quinones. To elucidate a potential role of the CoxG homologues Saci_1119 and Saci_2031 as membrane anchors for G3PDHs, we heterologously cooverexpressed saci_1118/saci_1119 and saci_2032/saci_2031 from the pETDuet-1-vector in E. coli, which resulted in an enrichment of the respective G3PDH in the membrane fraction as monitored via immunodetection using anti-His antibodies. By contrast, expression of saci_1118 and saci_2032 alonewithout theCoxGproteins resulted inG3PDHproteins being exclusively localized in the cytoplasmic fraction (Figs. 6a, b). Upon homologous production of Saci_1119 and Saci_2031 CoxGs in S. acidocaldarius MW00G, both HA-tagged proteins also predominantly localized in the membrane fraction. Furthermore, they could be solubilized usingDDM, suggesting that bothCoxGhomologues are indeedmembraneassociated in S. acidocaldarius in vivo (Fig. 6c). Co-immunoprecipitation experiments utilizing magnetic beads coupled with Anti-HA antibodies further validated the interaction between C-terminally HA-tagged CoxGs Saci_1119 and Saci_2031 and their G3PDHs Saci_1118 and Saci_2032. MS analysis of interacting proteins unveiled a specific interaction of Saci_1119 with Saci_1118 and Saci_2031 with Saci_2032 (Supplementary Table 5). These findings indicate that the membrane interaction of G3PDHs is mediated by the CoxG homologues, which represents an additional function of CoxG proteins in Archaea and an uncommon mechanism of membrane anchoring of G3PDHs in S. acidocaldarius.
Deletionofonlyoneof twoGK-encodinggenesaffectsgrowthon glycerol
To elucidate the importance of individual GK paralogues in glycerol conversion, single deletion mutants Δsaci_1117 and Δsaci_2033, as well as the double-deletion mutant Δsaci_1117Δsaci_2033, were constructed in the parental strain MW00G (Fig. 7). The Δsaci_1117 mutant exhibited a reduced growth rate on glycerol compared to the MW00G strain (Fig. 7a), along with a slightly delayed glycerol consumption (Fig. 7b), and only a minor decrease in GK activity in crude extracts (Supplementary Fig. 8a). By contrast, growth of both the single Δsaci_2033 and double Δsaci_1117Δsaci_2033 mutant was completely abolished (Fig. 7a) and accordingly glycerol consumption was entirely blocked (Fig. 7b), highlighting the essential role of Saci_2033 for growth on glycerol. Since the single Δsaci_2033 and double Δsaci_1117Δsaci_2033 mutant did not exhibit any growth on glycerol, the effect on GK crude extract activity of the three mutants was determined in cells grown on N-Z-amine as alternative carbon source (Supplementary Fig. 8b). In Δsaci_1117, GK activity remained unaffected (115 ± 16mUmg−1), while onlyminor (20 ± 5mUmg−1) or noGK activity could be detected in Δsaci_2033 and Δsaci_1117Δsaci_2033, respectively. This further confirms the significant contribution of Saci_2033 to the total GK activity. Both single deletion strains, Δsaci_1117 and Δsaci_2033, were complemented in trans by ectopic integrationof thewildtype gene saci_1117 and Fig. 4 | Kinetic characterization of the purified recombinant GK isoenzymes Saci_1117 and Saci_2033. The kinetic properties of Saci_1117 (red circles) and Saci_2033 (blue squares) with ATP (a) and glycerol (b) were analysed using a continuous assay at 50 °C. The glycerol-dependent conversion of ATP to ADPwas coupled to NADH oxidation via pyruvate kinase (PK) and lactate dehydrogenase (LDH), with the decrease in absorbance at 340 nm used to monitor the reaction. All experiments were performed in triplicate (n = 3, technical replicates), and error bars represent the SD of the mean and individual data points are shown as grey dots. S pe ci fic A ct iv ity [U /m g] 0 20 40 60 80 100 Glycerol [mM] 0 0,5 1 b Sp ec ifi c Ac tiv ity [U /m g] 0 20 40 60 80 100 0 0,5 ATP [mM] a 1 1,5 2 Communications Biology | (2025) 8:539 5 a b c d 0.00 0.05 0.10 0.15 0.20 0.25 0.30 0 10 20 30 40 G3P [mM] Sp ec ifi c ac tiv ity [U m g- 1 ] 0.0 0.01 0.02 0.03 0.04 0.05 Ubiquinone-Q1 [mM] Sp ec ifi c ac tiv ity [U m g- 1 ] 0 5 10 15 20 25 artificial redox active dye DCPIP following the decrease of absorbance at 600 nm. cAbsorption spectra of ubiquinone-Q1 at 70 °C in the absence (solid line) and in the presence of Saci_1118 (red dotted line) or Saci_2032 (blue dashed line) and 200 µM of G3P after 60 seconds. Loss of absorption at 280 nm indicates reduction of ubiquinone-Q1 by G3PDH. d The kinetic properties of Saci_1118 (red circle) and Saci_2032 (blue square) with respect to ubiquinone-Q1 were determined at 70 °C in a continuous assay following the G3P-dependent reduction of ubiquinone-Q1 to ubiquinol-Q1 at 280 nm. Experiments were performed in triplicate (n = 3, technical replicates) and error bars indicate the SD of the mean and individual data points are shown as grey dots. Table 1 | Kinetic characterization of the recombinant GK and G3PDH isoenzymes from S. acidocaldarius Enzyme Substrate KM [mM] Vmax [U mg−1] kcat/KM [mM−1 s−1] Calculated mass [kDa] Native mass [kDa] Oligomeric state Temperature optimum pH optimum Saci_1117a (GK) Glycerol 0.024 ± 0.001 45.7 ± 0.25 (170 ± 9)b 6397 ± 507.4 55.6 105 Homodimer 75 °Ca 7 ATP 0.205 ± 0.024 50.8 ± 2 229 ± 28.4 Saci_2033a (GK) Glycerol 0.024 ± 0.004 88.2 ± 2.5 (339.7 ± 7.3)b 13129 ± 2205 55.3 110 Homodimer 75 °Ca 6.5 ATP 0.174 ± 0.019 91 ± 4 485 ± 57.1 Saci_1118c (G3PDH) G3P 0.019 ± 0.003 19.65 ± 0.73 808 ± 131.4 46.9 95 Homodimer 70 °C 6.5 Ubiquinone-Q1 0.086 ± 0.019 18.66 ±1.41 170 ± 39.7 Saci_2032c (G3PDH) G3P 0.055 ±0.009 44.5 ±2.47 642 ± 110.9 47.6 94 Homodimer 70 °C 6.5 Ubiquinone-Q1 0.179 ± 0.075 21.1 ± 5.25 128 ± 58.9 Parameter values are given ± standard deviation. a Kinetic constants for the Saci_1117 and Saci_2033 GKs were determined at 50 °C. b Specific activities were determined under Vmax conditions at 75 °C. c Kinetic constants for the Saci_1118 and Saci_2032 G3PDH were determined at 70 °C. For comparison to other characterized GKs and G3PDHs see Supplementary Tables 3 and 4. Communications Biology | (2025) 8:539 6 the saci_2033/34 operon, respectively, into the upsE gene locus (saci_1494), under control of their respective native promoters. For bothΔsaci_1117 and Δsaci_2033 mutants, complementation significantly restored growth (Fig. 7c), glycerol consumption (Fig. 7d) and crude extract activities to wild type levels (Supplementary Fig. 8a). Together, these results indicate that Saci_2033 is the primarily expressed and essential GK during growth on glycerol, whereas Saci_1117 plays a minor role under the conditions tested. Sequence and structural comparisons of G3PDHs To further characterize the S. acidocaldarius G3PDH enzymes, we performed sequence analysis and structural comparison with other G3PDHs. As exemplified by Saci_2032 (Supplementary Figs. 9 and 10), these comparisons revealed homology between S. acidocaldarius and other archaeal G3PDHs in their N-terminal region of approximately 370 amino acids, with GlpDs, GlpAs and GlpOs, all exhibiting the D-amino acid oxidase (DAAO) superfamily fold (pfam01266), which includes for instance the glycine oxidase from Bacillus subtilis lacking any C-terminal extension (1ng3)24,44. These proteins consist of a ‘glutathione-reductase-2’ type FAD-binding domain and an antiparallel β-sheet-based substratebinding domain24. Notably, both the FAD and the G3P binding site are conserved across these proteins (Supplementary Fig. 9). However, the comparison revealed substantial differences in the C-terminus of the various proteins. While glycine oxidase consists solely of the DAAO fold without any C-terminal extensions, the proteins GlpD, GlpO, GlpA as well as the archaeal homologues differ in length and domain organization of the C-terminus (Supplementary Figs. 10–12, for detailed discussion see Supplementary Information ‘Extended analysis of sequence and structural comparisons of G3PDHs’). For example, the four highly conserved cysteine residues for FeS cluster binding, present in the C-terminus of GlpA sequences, are absent in GlpDs, GlpOs and the S. acidocaldarius G3PDHs (Supplementary Fig. 9). These structural differences in the C-terminal domains of the various proteins are likely correlated with their distinct functions. In GlpD, the C-terminus was shown to play a role in dimer formation, whereas in GlpOs the C-terminus is not involved in dimerization15,20. Though no crystal structure is available for GlpA, the presence of a FeS cluster in its C-terminus suggests its involvement in electron transfer to GlpB, supported by studies demonstrating that GlpAB forms a catalytically active unit. Our structural predictions of the Saci_G3PDH complex, coupled with the finding that Saci_2032 alone forms a soluble dimer (Supplementary Fig. 13), suggest that the C-terminus might also contribute to dimer formation. However, the predicted structure shows a different spatial orientation of the DAAO domains compared to GlpD, which combined with the interaction between Saci_G3PDH and CoxG, may facilitate optimal electron transfer to the quinone pool in the cytoplasmic membrane in S. acidocaldarius. Distribution, sequence similarities and phylogenetic affiliation of GK and G3PDH isoenzymes from S. acidocaldarius The identification of unusual features in the G3PDH enzymes of S. acidocaldarius, including a distinct C-terminus and a previously undescribed association with CoxG, prompted us to examine the distribution and composition of GK and G3PDH enzymes across Archaea more generally. Notably, previous studies have analyzed the distribution of glycerol degrading genes/enzymes in Archaea, describing enzyme sets comprising GlpK/GlpA in several euryarchaeal orders including Thermococcales, Halobacteriales, Archaeoglobales and Thermoplasmatales, as well as in the crenarchaeal lineages such as Thermoproteales, Thermofilales, Desulfurococcales, and Sulfolobales31,45. However, GK/G3PDHhomologues are only found in some representatives of these lineages, rather than being universally present or predominant across all Archaea, resulting in a patchy distribution. The overall sequence identity among archaeal GKs (40-70% identity) is higher compared to archaeal G3PDHs (20-50% identity). Furthermore, the closest homologues to S. acidocaldarius GKs were found in Thermococcales (61-68% identity), while the closest homologues to S. acidocaldarius G3PDHs were found in Thermoproteales and had lower sequence similarity (45-48% identity). The S. acidocaldarius GKs, show a high degree of sequence conservation not only to other archaeal homologues but also to bacterial ( > 50% sequence identity) and eukaryotic (~44%) enzymes, whereas archaeal G3PDHs are more diverse. These findings support that GKs and G3PDHs experienced distinct evolutionary histories and suggest that while GKs have a conserved function across organisms, G3PDHs have greater functional variability concerning interaction partners, membrane anchoring, and electron acceptors. To better explore the differences between G3PDH enzymes, we used phylogenetic analyses of representative FAD-dependentG3PDH sequences across all domains of life, including Archaea, Bacteria and Eukarya. While our analyses yielded tree topologies consistent with previous reports1,30,45, they also offer novel insights into the evolution and potential function of these enzymes (Fig. 8). For example,whilewe found that allG3PDHs cluster together within theDAAO superfamily, these enzymes could be subdivided based on their distinct C-terminal extensions: GlpAs and GlpDs form separate subgroups, with GlpOs exhibiting closer relation to GlpDs (falling ca b Saci_2032 (His-G3PDH) + + + + - -+ + Cy to pla sm Me mb ran e Saci_2032 (His-G3PDH) Saci_2031 (CoxG) + + + + - -+ + Cy to pla sm Me mb ran e Saci_1118 (His-G3PDH) Saci_1119 (CoxG) Saci_1118 (His-G3PDH) Saci_1119 (CoxG-HA) Saci_2031 (CoxG-HA) Fig. 6 | CoxG homologues Saci_1119 and Saci_2031 facilitatemembrane binding of their respective G3PDH isoenzymes Saci_1118 and Saci_2032 in E. coli and S. acidocaldarius. The effect of heterologous co-expression of CoxGs on the localization of His-tagged G3PDHs in the cytoplasmic and membrane fractions of E. coli was analysed viawesternblotting, followedby immunodetection using an anti-His antibody. Only upon co-expression of CoxG (Saci_1119) and G3PDH (Saci_1118) (a) as well as CoxG (Saci_2031) and G3PDH (Saci_2032) (b) the respective G3PDH is found in the membrane fraction. c Isolatedmembrane fractions from S. acidocaldarius cells following homologous overexpression of HA-tagged CoxG homologues Saci_1119 (left lane) and Saci_2031 (right lane), utilizing the pSVAaraFX-HA vector, were analyzed. Western blotting and immunodetection with an anti-HA antibody clearly demonstrated the membrane localization of both CoxG homologues. Communications Biology | (2025) 8:539 7 within the GlpD cluster) than to GlpAs. Furthermore, we found that G3PDHs from Sulfolobales, Thermoplasmatales,Caldivirgamaquilingensis and Vulcanisaeta moutnovskia, as well as from Thermofilales form distinct subclusters within the GlpA cluster, distinct from canonical bacterial and haloarchaeal sequences. Notably, sequences from Thermococcales constitute a separate family within the DAAO superfamily, here designated as GlpTk, which also includes sequences from anaerobic Crenarchaea (Desulfurococcales), anaerobic Bacteria (e.g. Thermotoga maritima and Clostridiumperfringens), and amitochondriate protists (Giardia intestinalis, Entamoeba histolytica, and Spironucleus salmonicida). The observed phylogenetic clusters also coincide with conserved downstream gene synteny (Fig. 9): (i) Canonical glpA genes consistently co-occur with glpB and C genes; (ii) in the subcluster of Thermofilumrelated species, glpA is followed by genes encoding putative counterparts of succinate dehydrogenase b, c, and d subunits, which are involved in membrane anchoring and electron transfer46; (iii) the glpA genes in the subcluster that includes S. acidocaldarius are associated with coxG; (iv) the genes in the glpD cluster lack obvious operon-like structures; (v) Thermococcus-related glpTk genes cluster with two genes encoding a NADH oxidase (NOX) and a molybdopterin oxidoreductase (MOX), and protein complex formation has been shown in vitro35. Notably, these three genes are fused in protists47. Collectively, the phylogenetic and gene synteny analyses suggest that G3PDHs form distinct complexes with a variety of proteins encoded downstream of the respective G3PDHs, which mediate membrane association and electron transfer. These observations suggest that the acquisition of glycerol utilization necessitated the concurrent evolution of tailored membrane anchoring and/or electron transfer systems to suit a variety of metabolisms and terminal electron acceptors, which likely reflect distinct lifestyles across a range of organisms.
Discussion
Glycerol is an abundant and important carbon source for many microbes and a highly regarded substrate for industrial applications.However, studies elucidating the pathways involved in glycerol degradation in Archaea have so far been mainly restricted to H. volcanii1,28–30. Here, we show that S. acidocaldarius grows on glycerol and demonstrate that glycerol involves a classical GK and an unusual G3PDHwith a previously undescribed type of membrane anchoring via a CoxG-like protein. S. acidocaldariuswas previously reported not to grow on glycerol37 and not to possess canonical G3PDH homologues31. However, it has been demonstrated that S. acidocaldarius can cleave short-chained triacylglycerols using esterases and can utilize short-chain fatty acids as carbon and energy sources for growth42,48. This metabolic capability leaves glycerol as a valuable byproduct, prompting us to investigate the capacity of S. acidocaldarius to utilize this substrate.We demonstrate that, after an adaptation period, S. acidocaldarius utilizes glycerol as sole carbon and energy source, even outperforming sugars such as D-xylose, as observed inH. volcanii30,49. Notably, a similar adaptation phasewas needed to enablePseudomonas spp. to grow on glycerol, which was attributed to transcriptional regulation induced by G3P11. In S. acidocaldarius, the higher growth yield on glycerol likely reflects its higher reduction state, suggesting amore efficient energetic couplingduringglycerolutilization.This coincideswith theup-regulationof the SoxEFGHIM gene cluster (saci_2258-saci_2263), one of three terminal oxidases with a higher H+/e− ratio compared to the other two50–53 (Supplementary Table 2). Fig. 7 | Comparison of growth, glycerol consumption and GK activities in S. acidocaldarius parental (MW00G), GK deletion and complementation strains. a Growth comparison of the parental strain MW00G with the single deletion strains Δsaci_1117 and Δsaci_2033, and the double deletion strain Δsaci_1117Δsaci_2033. Cells were cultivated in Brock’s basal medium containing 10 mM glycerol as the sole carbon and energy source. b Glycerol consumption of the different strains. c Growth and (d) glycerol consumption of the single deletion strains Δsaci_1117 and Δsaci_2033, complemented in trans by ectopic integration of the wildtype genes saci_1117 and the saci_2033-34 operon, respectively, into the upsE gene locus (saci_1494) under control of their native promoters. For comparison, the growth of the single deletion strains and the parental strain as in (a) is included. GK activity of the parental MW00G, deletion mutants, and complementation strains was measured and is presented in Supplementary Fig. 8. The lower panel presents the colour code and symbols used throughout the figure. Experiments were conducted in triplicate (n = 3, biological replicates) and error bars indicate the SD of the mean and individual data points are shown as grey dots. b d 0 50 100 150 200 O D 60 0 1,2 1 0,8 0,6 0,4 0,2 0 Time [hours] 12 G ly ce ro l c on ce nt ra tio n [m M ] 10 8 6 4 2 0 Time [hours] 0 50 100 150 200 0 50 100 150 200 Time [hours] O D 60 0 1,2 1 0,8 0,6 0,4 0,2 0 12 10 8 6 4 2 0G ly ce ro l c on ce nt ra tio n [m M ] Time [hours] 0 50 100 150 200 a c MW00G WT MW00G ∆saci_1117 MW00G ∆saci_2033 MW00G ∆saci_1117 ∆upsE:P1117 saci_1117 MW00G ∆saci_2033 ∆upsE:P2033 saci_2033-2034 MW00G ∆saci_1117∆saci_2033 Communications Biology | (2025) 8:539 8 Our multi-omics analyses revealed that glycerol uptake in S. acidocaldarius involves a GUF homologous to GlpF, suggesting a mechanism similar to that observed in other organisms3. Additionally, simple diffusion may also contribute to glycerol uptake, as observed in Bacteria4. Notably, GlpF homologues are less abundant in Archaea than GKs and G3PDHs1,30, suggesting that some organisms utilize alternative transporters, which is supported by the gene neighbourhoods of the GKs and G3PDHs often including genes encoding for putative MFS (major facilitator superfamily) transporters. In S. acidocaldarius, alongside the glpF gene, we observed that glycerol induced the up-regulation of a putative ABC transporter (saci_1762-1765), suggesting its potential involvement in glycerol transport. Fig. 8 | Phylogenetic affiliation of the S. acidocaldarius G3PDH isoenzymes Saci_2032 and Saci_1118 with selected GlpDs, GlpOs, and GlpAs from other Archaea, Bacteria and Eukarya. GlpA homologues are shaded green, with the canonical bacterial and haloarchaeal GlpAs shown in dark green, the Thermophilum homologues in green and the S. acidocaldarius homologues in light green. The GlpD cluster shaded blue comprises the GlpDs and GlpOs highlighted in light and dark blue, respectively. The Thermococcus-like G3PDHs, designated as GlpTk are shown in orange. Organism names and uniprot accession numbers are given. The evolutionary history was inferred by using the Maximum Likelihood method and Le/Gascuel_2008 model82. Themodel was selected based on the lowest Bayesian information criterion value using the ‘Find best proteinmodel’ option implemented in theMEGA11 package. The tree with the highest log likelihood (−18468.08) is shown. The percentage of trees in which the associated taxa clustered together is shown next to the branches. Initial tree(s) for the heuristic search were obtained automatically by applying Neighbor-Join and BioNJ algorithms to a matrix of pairwise distances estimated using the JTT model, and then selecting the topologywith superior log likelihood value. A discrete Gamma distribution was used to model evolutionary rate differences among sites (5 categories (+G, parameter = 1.8751)). The rate variation model allowed for some sites to be evolutionarily invariable ([+I], 2.12% sites). The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. This analysis involved 55 amino acid sequences. All positions containing gaps and missing data were eliminated (complete deletion option). There were a total of 262 positions in the final dataset. Evolutionary analyses were conducted in MEGA1180. P0A9C2 Shigella flexneri GlpA P0A9C0 Escherichia coli GlpA P43799 Haemophilus influenzae GlpA Q5V4I2 Haloarcula marismortui C7NYT5 Halomicrobium mukohataei Q9HNS4 Halobacterium salinarum Q18JE7 Haloquadratum walsbyi L9UU94 Halogeometricum borinquense D4GYI2 Haloferax volcanii GlpA1 M0MF86 Halococcus saccharolyticus A0A1I6RV48 Halostagnicola kamekurae A0A1I6UVM2 Halostagnicola kamekurae D4GQU6 Haloferax volcanii GlpA2 Q18GV9 Haloquadratum walsbyi D3SR72 Natrialba magadii E4NLP9 Halogeometricum borinquense C7NYY9 Halomicrobium mukohataei Q5V0Y0 Haloarcula marismortui A1RZ95 Thermofilum pendens A0A3G1A4N2 Thermofilum adornatum A0A7L9FIM8 Infirmifilum lucidum Q6KYY1 Picrophilus torridus Q9HKG6 Thermoplasma acidophilum A8MAZ1 Caldivirga maquilingensis F0QSI0 Vulcanisaeta moutnovskia A0A348B1W5 Sulfodiicoccus acidiphilus Q4J9Q8 Sulfolobus acidocaldarius Saci 1118 Q4J7A2 Sulfolobus acidocaldarius Saci 2032 Q97VT4 Saccharolobus solfataricus P2 P9WN81 Mycobacterium tuberculosis GlpD P13035 Escherichia coli GlpD P52111 Pseudomonas aeruginosa GlpD P74257 Synechocystis PCC 6803 GlpD P35571 Rattus norvegicus P43304 Homo sapiens Q9SS48 Arabidopsis thaliana Q06B39 Dunaliella salina P32191 Saccharomyces cerevisiae Q99UH2 Staphylococcus aureus GlpD P18158 Bacillus subtilis GlpD C9A595 Enterococcus casseliflavus D0VWP7 Streptococcus sp. P75063 Mycoplasma pneumoniae B0EN20 Entamoeba histolytica V6LVD7 Spironucleus salmonicida A8BBJ1 Giardia intestinalis A0A0X1KM21 Thermococcus guaymasensis Q8XHD4 Clostridium perfringens Q9X1E6 Thermotoga maritima I3XS74 Desulfurococcus amylolyticus A3DL61 Staphylothermus marinus Q5JGZ7 Thermococcus kodakarensis Q9V205 Pyrococcus abyssi Q8TZI7 Pyrococcus furiosus O31616 Bacillus subtilis 100 53 100 100 100 100 100 92 87 100 100 98 100 66 93 89 100 61 43 70 64 54 100 89 99 64 100 100 28 73 99 100 75 47 100 47 88 99 87 100 41 30 100 100 81 82 92 100 63 97 0.50 GlpA GlpD GlpTK Communications Biology | (2025) 8:539 9 We found that in S. acidocaldarius, glycerol degradation follows the GK/G3PDH pathway, leading to the production of DHAP, which is then channelled into central metabolism (Fig. 10). While this pathway is similar to that used inH. volcanii29, these organisms used distinct G3PDH enzymes for G3P oxidation. In H. volcanii, FAD-dependent G3P oxidation with electron transfer to the quinone occurs via a ‘classical’ bacterial-like ‘anaerobic’ GlpABC, whereas in S. acidocaldarius this process is catalysed by a structurally unusual G3PDH, in which membrane association is mediated by aCoxG-like subunit, which represents a previously undescribed function for CoxG proteins in Archaea and glycerol metabolism in general.Whereas in anaerobic bacteria and aerobic halophilic archaea, menaquinone derivatives serve as electron acceptors, in Sulfolobales, including S. acidocaldarius, the caldariellaquinone54 is the primary quinone and thus likely functions as an electron acceptor for G3P oxidation in vivo (Fig. 10). Fig. 9 | Comparison of different G3PDHs from Bacteria and Archaea. The respective genome neighbourhood, the derived reconstructed protein complexes with potential interaction partners, membrane or cytoplasmic localization and cofactor content, as well as the catalytic oxidoreductasemonomer 3D-structure of the G3PDHs GlpABC (E. coli (a), GlpA-SdhBCD (T. pendens) (b), GlpACoxG (S. acidocaldarius) (c), GlpD (E. coli) (d)GlpO (Streptocoocus) (e) and GlpTk-NOX-MOX (T. kodakarensis) (f) are shown. For colour code see Fig. 8. For all 3D structures the principal FADbinding G3P-oxidoreductase domain is coloured in grey while the highly divergent C-terminus is coloured according to Fig. 8. GlpD GlpD FAD FAD G3P DHAP Quinone (ox) Quinol (red) GlpA GlpB FAD G3P DHAP Quinone (ox) Fe-S GlpC Quinol (red) SdhC G3P DHAP Quinone (ox) Quinol (red) GlpA FAD SdhB SdhD ? ? Fe-S ? CoxG ? G3P DHAP Quinone (ox) Quinol (red) GlpA FAD GlpA FAD CoxG ? G3P DHAP GlpA FAD GlpA FAD NOX NOX FAD FAD MOX Fe-S NADH NAD+ Acceptor (ox) Acceptor (red) ? GlpO GlpO FAD FAD G3P DHAP O2 H2O2 coxGglpAglpA glpB glpC glpA sdhB sdhC sdhD glpD glpO glpA NOX 1667 a b c d e f E. coli GlpA T. pendens GlpA S. acidocaldarius GlpA E. coli GlpD Streptococcus GlpO T. kodakarensis GlpTK Communications Biology | (2025) 8:539 10 Ourfindings also show that S. acidocaldarius encodes twogene clusters for glycerol metabolism, saci_1117-1119 and saci_2031-2034, which exhibit a similar organization (Fig. 3). However, these clusters differ in the presence of theGUF encoding gene saci_2034. Furthermore, only the saci_2031-2034 gene cluster was substantially up-regulated on glycerol, and only the GK Saci_2033 was essential for growth on glycerol, indicating that the saci_2031-2034 gene cluster is primarily involved in glycerol degradation under the tested conditions. The presence of twoGK/G3PDHpairs is rare in both Archaea and Bacteria, although some species, including some Haloarchaea, encode two G3PDHs and a single GK1,10,11,30. In H. volcanii, only one of two G3PDH isoenzymes (GlpABC) was essential and upregulated by glycerol30,55. High constitutive levels of G3PDH activity have been reported, with approximately a two-fold upregulation in the presence of glycerol. This is consistent with the finding that glycerol serves as the primary carbon source in halophilic environments29,32. In E. coli, the two G3PDHs, GlpD and GlpABC, are known to be primarily expressed under different environmental conditions10,15,17. Similarly, studies in species with two GK paralogues suggest functional specialization1,55,56. In S. acidocaldarius, the two G3PDH and GK paralogues exhibit very similar functional parameters and might thus be responsible for glycerol degradation under different growth conditions. In contrast to the saci_2031-2033 GK/ G3PDH/CoxG encoding genes co-occurring with the GlpF glycerol transporter, the genomic context of saci_1117-1119 comprisesgeneswhichmight be involved in lipid hydrolysis and fatty acid β oxidation42. On solid media containing triglycerides like tributyrine, S. acidocaldarius was shown to be active and able to cleave these lipids by means of esterases encoded in the saci_1103-1126 gene neighbourhood, i.e. Saci_1105 and Saci_111648. Thus, this gene cluster might be involved in the glycerol utilization during lipid breakdown, but further analyses are needed to elucidate the specific functions of each glycerol gene cluster. Our analyses suggest that S. acidocaldarius GKs have a homodimeric structure, which is consistent with some other GKs from Bacteria and Eukarya. Similarly, the S. acidocaldariusG3PDHs seem to form catalytically active homodimers, and directly reduce the ubiquinone Q1, resembling bacterial and eukaryotic GlpDs. Additionally, their FAD content of one per monomer aligns more closely with GlpDs15,57,58. GlpDs are monotopic membrane proteins that transfer electrons to the quinone pool, relying on membrane interaction, phospholipids, or non-denaturing detergents for conformational integrity and thus full activity15. By contrast, S. acidocaldarius G3PDHs alone are soluble and active without membrane association or detergent addition. We show that these G3PDHs interact with CoxG-like proteins, and that the G3PDHs only associate with the membrane when co-expressed with the CoxG-like proteins. The quinone reactivity of both G3PDH dimers in the absence of CoxG suggests that CoxG is not involved in electron transfer, but rather anchors the protein to the membrane. Our findings are consistent with the membrane-anchoring ability of CoxG homologues, including that from Oligotropha carboxydovorans, which has been shown to recruit the carbon monoxide dehydrogenase from the aerobic xanthin oxidase type to the membrane without enzymatic implications59. A similar function for a CoxG homologue in membrane anchoring of 3-hydroxypyridine dehydrogenase of Ensifer adhaerens HP1 has also been proposed60. The identification of unusual G3PDH isoenzymes in S. acidocaldarius led us to investigate the distribution, sequence similarities, and phylogenetic affiliation of GK and G3PDH enzymes across Archaea. Enzyme sets comprising GlpK/GlpA for glycerol degradation are widespread among various euryarchaeal orders, such as Thermococcales, Halobacteriales, Archaeoglobales, and Thermoplasmatales, as well as crenarchaeal lineages like Thermoproteales, Thermofilales, Desulfurococcales, and Sulfolobales. However, the evolutionary history of GKs andG3PDHs inArchaea appears divergent. In contrast to GK, which seems more widespread and conserved across archaeal lineages, the distribution of G3PDH homologues across these lineages appears patchy, suggesting a complex evolutionary history. While GK likely originated from horizontal gene transfer from Bacteria, G3PDHmayhavebeenpresent in theuniversal commonancestor, aswell as in bacterial and archaeal common ancestors45. Our sequence analysis revealed high conservation among S. acidocaldarius GKs, belonging to the FGGY family within the sugar kinase/HSP70/actin superfamily, with significant similarity to both archaeal and bacterial enzymes, as well as eukaryotic counterparts. Our structural and sequence analyses of G3PDHs also highlighted strong homology in the N-terminal region among S. acidocaldarius and other archaeal G3PDHs (GlpAs) with canonical bacterial and haloarchaeal GlpAs (GlpABC complex), GlpDs, and GlpOs, all exhibiting the D-amino acid oxidase (DAAO) superfamily fold. However, despite conservation in the FAD and G3P binding sites, significant variability exists in the C-terminal domains of archaeal G3PDHs, likely correlating with their distinct functions. For example, the C-terminus of the canonical GlpA contains a bfd-like domain with an FeS cluster, suggesting involvement in electron transfer to GlpB, while GlpD and GlpO lack such extensions, consistent with their roles as homodimers. Our additional phylogenetic and gene synteny analyses provide additional support for these structural differences among different G3PDH subgroups. However, the extent of diversity observed in Archaea is unexpected, especially when contrasted with the pattern observed in Bacteria. In Bacteria, canonical glpA genes consistently co-occur with glpB and glpC, mirroring the pattern found in Haloarchaea. By contrast, among various other archaeal lineages, distinct patterns of gene co-occurrence emerge. For instance, in Thermofilum-related species, glpA genes are associated with genes encoding succinate dehydrogenase subunits. InThermococcus-related Communications Biology | (2025) 8:539 11 species, glpA genes cluster with genes encoding NADH oxidase and molybdopterin oxidoreductase. Lastly, in the S. acidocaldarius subgroup, glpA genes are consistently linkedwith coxG. These findings suggest that the acquisition of glycerol utilization required the simultaneous evolution of specialized membrane anchoring and/or electron transfer systems to align with the organism’s lifestyle, metabolism, and terminal electron acceptors. Our findings will also likely have important implications for future biotechnological applications. The utilization of extremophiles such as S. acidocaldarius offers several benefits for industrial biotechnology, including increased reaction rates and improved substrate solubility that promote higher biomass conversion, and a reduction of microbial contamination at higher temperatures and low pH, making antibiotics obsolete38. S. acidocaldarius iswell established as an industrial chassis due to the availability of its genome sequence and an extensive genetic toolbox, detailed metabolic characterization, and existing bioprocesses. Importantly, glycerol is generated during biodiesel production, and the rapid growth of biofuels has led to a significant oversupplyof glycerol anddecliningprices, rendering it a highly regarded wasteful by-product that can support the production of a range of value-added products. Therefore, our demonstration that S. acidocaldarius grows efficiently on glycerol, coupled with our detailed characterization of themetabolic pathways involved in glycerol utilization, are likely to facilitate the development of engineered strains that can support the production of a range of valuable products from this cheap source material. In summary, we show that glycerol degradation in S. acidocaldarius proceeds via the GK/G3PDH pathway involving ‘classical’ bacterial-like GKs andG3PDHs homologous to GlpA that are remarkably different from their bacterial counterparts. The G3PDHs from S. acidocaldarius lack the B and C subunits present in bacterial GlpABC complexes, differ in the C-terminal domain of the catalytic subunit, and are anchored to the membrane viaCoxGhomologues.Of twoparalogousGK/G3PDHcopies in the S. acidocaldarius genome, only saci_2031-2033 is highly upregulatedand essential during glycerol degradation. Furthermore, sequence analyses suggest that glycerol metabolism in Archaea is much more versatile with respect to the G3PDHs, their interaction partners, modes of membrane association, and electron transfer mechanisms, underscoring the intricate adaptation of glycerol metabolism to diverse environmental niches and metabolic pathways.
Materials and methods
Cultivation of S. acidocaldarius strains S. acidocaldariusMW001 (uracil auxotroph mutant)61 was adapted to glycerol utilization. The adapted strain designated S. acidocaldarius MW00G was routinely grown at 76 °C under constant shaking at 120 rpm (New Brunswick Innova 44127 incubator, Eppendorf, Hamburg, Germany) in Brock’s basal salt medium62, supplemented with 10 µgmL−1 of uracil and 10mM, 20mM, or 40mM glycerol, or for comparison with 0.2% (w/v) D-xylose as sole carbon and energy source. Growth was monitored as increase in OD600 over time. S. acidocaldarius MW00G deletion mutant strains were pre-grown in Brock medium with 0.1% (w/v) N-Z-amine and 0.2% (w/v) dextrin and then subcultured in Brock’s basal salt medium containing 10 µgmL−1 of uracil and 10mM glycerol. The glycerol concentration in the medium was determined enzymatically using the recombinant glycerol kinase (Saci_2033, see below), pyruvate kinase (PK) and lactate dehydrogenase (LDH) (both from rabbit muscle; Merck, Darmstadt, Germany). For each mole of glycerol, one mole of NADH is oxidized, with the reaction monitored at 340 nm in 96-well plates (BRANDplates®, BRAND, Wertheim, Germany). A NADH calibration curve (0–0.7 mM NADH) was used for quantification, and measurements were conductedusing aTecan InfiniteM200plate reader (TecanGroupAG, Männedorf, Switzerland). The reaction was performed in 200 µL total volume at 42 °C in 0.1M MOPS-KOH buffer pH 6.5, 0.6mM NADH, 1mM PEP, 5mM ATP, 5mM MgCl2, 5.5 U LDH, 2.8 U PK, 0.4 U Saci_2033, using 20 µL (diluted) samples with a glycerol concentration of at most 0.5 mM.Formulti-omics analyses, S. acidocaldariusMW00Gwaspregrown on minimal medium with the indicated carbon source (10mM, 20mM, 40mM glycerol, 0.2% (w/v) D-xylose) or 0.1% (w/v) N-Z-amine (control) as described above.With these precultures, 400mLmain cultures (four replicates) were inoculated to an initial OD600 of 0.05 and grown to exponential phase (OD600 of 0.8). Culture samples were cooled down with liquid nitrogen and cells were harvested by centrifugation at 9000 × g, 15min, 4 °C and stored at −70 °C for transcriptomic, proteomic and metabolomic analyses.
RNA-seq
RNA isolation, library preparation, and next-generation cDNA sequencing - RNA was isolated using Zymo Direct-zol RNAMiniprep kit following the manufactures instructions. TheRNAqualitywas checked byTrineanXpose (Gentbrugge, Belgium) and the Agilent RNA Nano 6000 kit using an Agilent 2100 Bioanalyzer (Agilent Technologies, Böblingen, Germany). PanArchaea riboPOOL kit from siTOOLs Biotech was used to remove the rRNA.TruSeq StrandedmRNALibrary PrepKit from Illuminawas applied toprepare the cDNAlibraries. The cDNAswere sequencedpaired endonan IlluminaNextSeq 500 system(SanDiego, CA,USA) using 74 bp read length mid output. Bioinformatics data analysis, read mapping and analysis of differential gene expression—The paired-end cDNA reads were mapped to the S. acidocaldarius DSM 639/MW001 genome sequence (accession number CP000077.1) using bowtie2 v2.2.7. with default settings for paired-end read mapping. All mapped sequence data were converted from SAM to BAM format with SAMtools v1.363 and imported to the software ReadXplorer v.2.264. Differential gene expression analysis of four replicates including normalization was performed using Bioconductor package DESeq265 included in the ReadXplorer v2.2 software64. The signal intensity value (A-value) was calculated by the log2 mean of normalized read counts and the signal intensity ratio (M-value) by the log2 fold change. The evaluation of the differential datawasperformedusing an adjustedP-value cutoff of P ≤ 0.01 and a signal intensity ratio (M-value) cutoff of ≥2 or ≤−2. Genes with an M-value/log2 fold change outside this range and adjusted P ≤ 0.01 were considered as differentially up- or downregulated.
Proteome
Samples for proteomic analysis were prepared using the single-pot, solidphase-enhanced sample-preparation (SP3) strategy66. All buffers and solutions were prepared with mass spectrometry (MS)-grade water (Avantor, Radnor, PA, USA). Cell pellets were taken up in 200 µL 1× sample buffer (50mM HEPES pH 8.0, 1% (w/v) SDS, 1% (v/v) NP-40, 10mM TCEP, 40mM chloroacetamide) and the samples were heated for 5min at 95 °C prior to sonication with a Bioruptor UCD-200 (Diagenode, Seraing, Belgium) device for ten cycles of 1min pulse and 30 sec pause at high power. The protein extracts were centrifuged (20,000 × g, room temperature (RT), 20min) and the protein concentration of the cleared lysateswas determined using the Pierce 660 nmProtein Assay Reagent (#22660; Thermo Scientific, Waltham, MA, USA) with the Ionic Detergent Compatibility Reagent (#22663; Thermo Scientific, Waltham, MA, USA) according to the manufacturers’ instructions. Next, 15 µg of total protein in a volume of 47 µL 1× sample buffer was treated with 7 U of benzonase (#70746; MerckMillipore, Burlington, MA, USA) in dilution buffer (20mM HEPES pH 8.0, 2mM MgCl2; 37 °C, 30min, 1500 rpm) followed by the addition of iodoacetamide to afinal concentrationof 10mMto complete alkylationof cysteine residues (RT, 30min, 1500 rpm in the dark), resulting a sample volume of 50 µL. Then, 3 µL of a 50 µg µL−1 1:1 mixture of hydrophilic (#45152105050250) and hydrophobic (#65152105050250) carboxylate modified Sera-Mag™ SpeedBeads (Cytiva, Marlborough, MA, USA) that were washed twice with MS-gradewater were added to the samples. Protein bindingwas induced by the addition of an equal sample volume of pure ethanol (24 °C, 20min, 1500 rpm), the beads were collected using a magnetic stand. Beads were allowed to bind for at least 5 min before the supernatant was removed. The beads were washed thrice with 180 µL 80% (v/v) ethanol prior to the addition of the digestion enzyme mix (0.6 μg of trypsin (V5111; Promega, Communications Biology | (2025) 8:539 12 Madison, WI, USA) and 0.6 µg LysC (125-05061; FUJIFILM Wako Pure Chemical, Osaka, Japan) in 25mM ammonium bicarbonate) and incubation of the samples at 37 °C (16 h, 1500 rpm). Next day, the samples were briefly centrifuged (10 s, 2000 × g, RT) and placed on a magnet for 5min. The clear solution containing the tryptic peptides was transferred to a new Eppendorf tube. The beads were taken up in 47 µL 25mM ammonium bicarbonate (RT, 10min, 1500 rpm). After incubation, the tubes were placed oncemore on amagnetic stand and after 5min the clear supernatant was again collected and combined with the recovered peptidemix, followed by the additionof trifluoroacetic acid (TFA; 2% (w/v)final concentration) to the samples. Prior to LC-MS/MS analysis, peptides were desalted as described in ref. 67 with the only modification of not re-applying the flowthrough to the C18StageTips again.
LC-MS/MS analysis
of peptide samples was performed on an Orbitrap Fusion Lumosmass spectrometer (Thermo Scientific,Waltham,MA,USA) coupled to an EASY-nLC 1200 liquid chromatography (LC) system (Thermo Scientific, Waltham, MA, USA) that was operated in the onecolumn mode. The samples were separated on a self-packed analytical column (see supplementary File 1) filled with Reprosil-Pur 120 C18-AQ 1.9 μm (Dr. Maisch, Ammerbuch-Entringen, Germany) that was encased by a PRSO-V2 column oven (Sonation, Biberach an der Riß, Germany) and attached to a nanospray flex ion source (Thermo Scientific, Waltham, MA, USA). During data acquisition, the column oven temperature was adjusted to 50 °C.TheLCwas equippedwith solventA (0.1% (w/v) formic acid (FA), in water) and solvent B (0.1% FA (w/v), in 80% acetonitrile (ACN)) prepared from UHPLC (ultra-high-performance liquid chromatography)grade solvents (Honeywell, Charlotte, NC, USA) asmobile phases. Peptides were directly loaded onto the analytical column with a maximum flow rate so that the set pressure limit of 980 bar would not be exceed (usually around 0.5–0.8 μl min−1) and separated by running gradients with different length and composition (for details see supplementary File 1). The mass spectrometer was operated in the positive ion mode using Xcalibur software (v4.3.7.3.11). Precursor ions (MS1) were scanned in the Orbitrap analyzer (FTMS; Fourier TransformMass Spectrometry) with the internal lock mass option switched on (lock mass was 445.120025m/z, polysiloxane68). Data-dependent product ion spectra (MS2) were recorded in the ion trap. All relevant MS settings (resolution, scan range, AGC, ion acquisition time, charge states, isolation window, fragmentation type and details, cycle time, number of scans performed, and various other settings) can be found in supplementary File 1. Peptide and protein identification using MaxQuant and perseus Recorded RAW data was analyzed with MaxQuant (v. 1.6.17.0 or v.2.0.1.0) using the default settings69 with the Label-free quantification (LFQ)70 and match between runs options activated. MS/MS spectra were searched against the UniProt S. acidocaldarius (DSM639) (UP000001018_330779.fasta; 2222 entries; downloaded on 2020-08-02) database. A search against a contaminants database as implemented in MaxQuant (contains known MS contaminants; 246 sequences) was included to estimate the level of contamination. For further data analysis and filtering of the MaxQuant output, LFQ intensities were loaded from the proteinGroups.txt file into Perseus (v. 1.6.14.0 or v.1.6.15.0)71. Contaminants as well as hits only identified based on peptides with a modification site and hits from the reverse database were removed. To allow comparison of the different treatment groups, biological replicates were combined into categorical groups and the data was transformed to the log2(x) scale. For the full proteome analysis, only protein groups (PGs) with a valid LFQ intensity in at least three out of four replicates in a minimum of one categorical group were kept for further analysis. For the identification of interaction partners of the CoxG homologues Saci_2031 and Saci_1119, the data was separately filtered to only keep hits with a valid LFQ intensity in at least two out of three replicates for samples containing the respective HA-tagged protein. The log2-fold change in normalized protein group quantities between the different categorical groups was determined based on the mean LFQ intensities of replicate samples (relative quantification). To enable quantification, missing LFQ intensitieswere imputed fromanormal distribution (full proteome analysis: width 0.3, down shift 1.8; identification of interaction partners: width 0.3, down shift 2.0). The statistical significance of the difference in LFQ intensity was determined via a two-sided Student’s t-test. Full MS data for the comparative full proteome analysis and the identification of interaction partners of Saci_2031 and Saci_1119 can be found in Supplementary Files 2 and 3. Genes that were up- or downregulatedwith a log2-fold change ≥2 in response to growth on glycerol and are shared between the transcriptomics andproteomics analyses are reported inSupplementaryTable 2. Proteins enriched by co-IPwith a log2-fold change ≥2, excluding ribosomal proteins, are reported in Supplementary Table 5.
Metabolome analyses
Metabolite extraction. The metabolite extraction was based on72 with slight modifications. Cells were disrupted by resuspension in 500 µL of prechilled (−80 °C) methanol and the addition of 20 µL of internal standards (fructose 6-phosphate, arginine 13C6, and succinic acid d4), followed by vortexing and sonication both for 2 min. Sampleswere frozen at −80 °C for 5 min and the vortex and sonication steps were repeated. The methanol was evaporated by vacuum concentration (Concentrator plus, Eppendorf, Hamburg, Germany).When the extract was completely dry, 250 µL of water were added and the process was repeated. During the whole process the cells were kept on ice. After drying the water, extracts were resuspended in 100 µL ACN/water (85:15, v/v), sonicated 2 min, and vortexed 2 min. The mixture was centrifugated at 12,000 × g for 2 min (MiniSpin centrifuge, Eppendorf, Hamburg, Germany). The supernatant was transferred to a LC vial. A quality control (QC) sample was prepared by pooling a same-volume aliquot from each sample to evaluate the performance and quality of the analytical instrumentation during the batch analysis. The samples were analyzed in a randomized order. LC-ESI-QTOF method for the targeted metabolomics analysis. The samples were analyzed by a 1290 Infinity II LC instrument coupled to a 6546 LC/Q-TOF high-resolution mass spectrometer, and the ionization was performed using a Dual Jet Stream source in negative mode (Agilent Technologies, Waldbronn, Germany). For the LC separation, a iHILIC(P)Classic (150 × 2.1 mm, 5 µm) (Hilicon, Umeå, Sweden) was used. The mobile phases consisted of 5 mM ammonium acetate, pH 5 (A) and ACN/5 mM ammonium acetate, 85:15 (v/v), pH 5 (B). The gradient elution was stablished as follows: 0 min, 90% B; 22 min, 80% B, 30 min, 65% B; 35 min, 65% B. The flow rate was set at 0.2 mLmin−1 and the column temperature was kept at 40 °C. The electrospray ion source parameters were: gas temperature, 200 °C; dry gas, 10 mLmin−1; nebulizer, 40 psi; sheath gas temperature, 300 °C; sheath gas flow, 12 Lmin−1; fragmentor, 125 V; skimmer, 65 V; capillary voltage, 3000 V. Full scandata-dependent acquisition was used to perform the tandem MS experiments. The retention time, the MS and the MS/MS spectra of the targeted metabolites were compared with commercial standards. The acquired data was processed by MS-Dial and Skyline software. Crude extract enzyme measurements For the preparation of crude extracts, 50mL cultures of S. acidocaldarius MW00G were grown using 40mM glycerol or 0.2% (w/v) D-xylose as the sole carbon source until reaching an OD600 of 0.6. The S. acidocaldarius MW00G deletion mutants were cultured in 50mLmedia containing either 10mM glycerol or 0.2% (w/v) N-Z-amine as the carbon source and grown to an OD600 between 0.8 and 1. Cells were collected by centrifugation (4300 × g, 15min, 4 °C), resuspended in 5mLof 50mMMES-KOHpH6.5, 20mM KCl, lysed by sonication (3 × 8min, 60%, 0.5 s−1, on ice) (UP200S, Hielscher Ultrasonics, Brandenburg, Germany), and cell debris was removed by centrifugation (4300 × g, 30min, 4 °C). The supernatant was used as crude extract for enzyme activity measurements of GK. To separate Communications Biology | (2025) 8:539 13 the membrane fraction, the supernatant was further centrifuged at 150,000 × g, 60min at 4 °C. The resulting soluble supernatant (crude extract) was used to determine G3PDH activity. For preparation of the membrane fraction the pellet was washed in 5mL of 50mM MES-KOH, 20mMKCl, pH 6.5, centrifuged (150,000 × g, 60min at 4 °C), resuspended in 500 µL of the same buffer and finally sonicated (20%, 0.5 s−1, on ice) until membranes were fully suspended. This membrane fraction was then used for GK and G3PDH enzyme measurements. Enzyme activities in soluble and membrane fraction were assayed spectrophotometrically with protein amounts of 50 µg (crude extract) and 40 µg (membrane fraction) in a total volume of 500 µL (Specord UV/visible-light (Vis) spectrophotometer; Analytic Jena, Jena, Germany). Assay mixtures were preincubated at the respective temperatures before starting the reaction with substrate (glycerol or G3P). G3PDH activity was determined at 70 °C as G3P-dependent reduction of DCIPIP at 600 nm (extinction coefficient 20.7 mM−1 cm−1) in 50mMMES-KOH pH 6.5, 20mM KCl supplemented with 50 µM DCPIP and 200 µM G3P. GK activity was assayed at 50 °C in 100mM TRIS-HCl pH7, 1mMMgCl2, 1mMATP, 5mMglycerol, 0.2 mMNADH, and 2mM PEP by coupling the glycerol-dependent formation of ADP from ATP to NADH oxidation via PK (7 U) and LDH (14 U) at 340 nm (extinction coefficient 6.22mM−1 cm−1). It was ensured that the auxiliary enzymeswere not rate limiting. One unit (1 U) of enzyme activity is defined as 1 µmol substrate consumed or product formed per minute.
Molecular cloning
E. coli DH5α, used for plasmid construction and propagation, was cultivated at 37 °C with shaking at 180 rpm (Unitron, INFORS HT, Bottmingen, Switzerland) in Lysogeny Broth (LB) supplemented with the appropriate antibiotics (150 μgmL−1 ampicillin, 50 μg mL−1 kanamycin, or 25 μg mL−1 chloramphenicol). For heterologous overexpression in E. coli either the pET15b (saci_2032, saci_1118) or the pETDuet-1 vector (co-expresssion of saci_2032/saci_2031 and saci_1118/1119, expression of saci_2032 or saci_1118 alone) and for homologous overexpression in S. acidocaldarius MW001 the pSVAmalFX-SH10 vector (saci_1117), pBS-Ara-albaUTR-FX-vector (saci_2033)40, and the pSVAAraFX-HA vector (saci_2032, saci_1119)73 were used. Plasmids and strains are listed in Supplementary Table 6. Open reading frames (ORFs) were amplified from genomic DNA of S. acidocaldarius DSM 639 and cloned into the respective vectors using the primers and restriction sites/endonucleases listed in Supplementary Table 7. For construction of pETDuet-1 coexpression vectors the gene pairs saci_2032 and saci_2031 or saci_1118 and saci_1119 were sequentially ligated into the multiple cloning site 1 (saci_2032/saci_1118) and 2 (saci_2031/saci_1119). Successful cloning was confirmed by sequencing. Construction of markerless single and double glycerol kinase deletion mutants Δsaci_1117, Δsaci_2033 and Δsaci_1117 Δsaci_2033 To obtain the markerless GK deletion mutants of saci_1117 and saci_2033, the plasmids pSVA12818 and pSVA12822 were constructed. Briefly, 500 bp of the upstream and downstream region of each gene were amplified by PCR and the respective PCR products were annealed via overlap extension PCR. The resulting products were then cloned into pSVA407 or pSVA431 (for plasmids and strains, see Supplementary Table 6). The resulting plasmids were then methylated by transformation in E. coli ER1821 to prevent plasmid degradation in the recipient strain. Methylated plasmid was then used to transform MW00G as described previously for MW00161. To prepare competent MW00G, a pre-culture was grown in Brock media supplemented with 0.1% (w/v) N-Z-amine and 10mM glycerol and 20 µgmL−1 uracil to an OD600 of 0.5–0.7 and used to inoculate 50mL Brock media supplemented with 0.1% (w/v) NZ-amine and 10mM glycerol. Notably, competence could only be obtained after addition of glycerol to the culture medium. The culture was harvested at an OD600 of 0.2–0.3 and further prepared as described previously61.
Complementation studies
For the complementation of the GKdeletionmutants,MW1257Δ1117 and MW1258 Δ2033, saci_1117 and the operon saci_2033-2034 with 200 bp of the respective native promoter region and 500 bp of the upsE (saci_1494) upstreamanddownstream regionwere amplified using the primers listed in the Supplementary Table 7. The resulting PCR products, either Psaci_1117 saci_1117 or Psaci_2033 saci_2033-2034 combined with the upsE upstream and downstream PCR products, were then assembled in an ApaI and SacII digested pSVA407 vector via Gibson Assembly (New England Biolabs, Frankfurt a.M., Germany) following the manufacturer’s instructions. The respective deletion mutants were then transformed with the resulting knock-in plasmids pSVA407-ΔupsE:P1117 saci_1117 CtSS and pSVA407ΔupsE:P2033 saci_2033-2034. Correct integration was confirmed by colony PCR and sequencing. Complementationwas assessed by comparing growth and crude extract activities in Brock medium supplemented with 10mM glycerol or 0.2% (w/v) of N-Z-amine, as described previously. Homologous overexpression and affinity purification of glycerol kinase For homologous GK expression, the vectors pSVAmalFX-saci_1117SH10 and pBS-Ara-albaUTR-FX-saci_2033-CtSS were methylated by transforming them into E. coli ER1821, thereby preventing degradation in the recipient strain. The methylated plasmids were transformed into S. acidocaldarius MW001 via electroporation using a Gene Pulser Xcell (BioRad, Munich, Germany) with a constant time protocol (input parameters 2 kV, 25 μF, 600Ω in 0.5 mm cuvettes). Cells were incubated for 45min at 75 °C in recovery solution61, and then plated on Brock’s basal salt mediumwith0.1% (w/v)N-Z-amine, 0.2% (w/v) dextrin solidifiedby gelrite without uracil. Successful transformation was confirmed via colony-PCR. Clones were precultured in 50mL liquid Brock’s basal salt medium supplementedwith 0.1% (w/v)N-Z-amine, 0.2% (w/v) dextrin and then used to inoculate 2 L liquidBrock’s basal saltmedium (OD600 0.05) containing 0.1% (w/v) N-Z-amine and 0.2% (w/v) D-xylose for induction. Cells were grown for two days at 76 °C under constant shaking at 120 rpm, harvested by centrifugation at 7000 × g and 4 °C for 15min and stored at −80 °C for further use. For protein purification, the cells were resuspended in 50mM TRIS-HCl buffer (pH 8.0), 250mMNaCl at a ratio of 3mL buffer per 1 g of wet cell weight. The cells were then disrupted by sonication (3 × 8min, 60% amplitude, 0.5 s−1, on ice). Cell debris was removed by centrifugation at 30,000 × g and 4 °C for 45min and the resulting crude extract was applied onto a Strep-Tactin®XT 4Flow® column (IBA Lifesciences, Göttingen, Germany) and purified following the manufacturers’ instructions. HeterologousoverexpressionandaffinitypurificationofG3PDHs For the expression of G3PDHs, E. coliRosetta (DE3) cells transformedwith the vectors pET15b-saci_2032 and pET15b-saci_1118, respectively, were cultured in terrific broth (TB) supplemented with 150 µgmL−1 ampicillin and 25 µgmL−1 chloramphenicol at 37 °C. When the culture reached an OD₆₀₀ of 0.8, expression was induced with 1mM IPTG, followed by further growth at 20 °C for 17 hours. Cells were harvested by centrifugation (6000 × g, 20 min, and 4 °C) and stored at−80 °C for further use. Cells were resuspended in 10mL of buffer (50mM TRIS-HCl pH 7.8, 10mM imidazole, 150mM NaCl, and 1mM FAD) per gram of cells (wet weight) and disrupted by sonication (3 × 8min, 50% amplitude, 0.5 s−1, on ice). The cell debris was removed by centrifugation at 16,000 × g for 45min at 4 °C. After filtration (0.45 µm polyvinylidene fluoride membrane; Starlab, Hamburg, Germany), the supernatant was applied onto a 1mL Nickel-IDA column (Cytiva,Marlborough,MA,USA) equilibrated in 50mMTRIS-HCl pH7.8, 10mM imidazole, 150mM NaCl and after washing (20 column volumes) proteins were eluted with a linear gradient from 10–300mM imidazole in equilibration buffer using an ÄktaPurifier system (Cytiva, Marlborough, MA, USA). In G3PDH containing fractions imidazole was removed using a centrifugal concentrator (10 kDa cutoff, Sartorius AG, Göttingen, Germany). Protein aliquots were frozen in liquid nitrogen and stored at−80 °C for further use. Communications Biology | (2025) 8:539 14 Determination of the native molecular mass of purified proteins Size exclusion chromatography (SEC) was used to determine the native molecularmass of all purified enzymes. To this end, pooled enzyme samples after affinity chromatography were concentrated using centrifugal concentrators (10 kDa cutoff) and applied to a Superose 6 Increase 10/300 GL column (Cytiva, Marlborough, MA, USA). For both GKs, Saci_1117 and Saci_2033, 50 µg protein in 200 µL were applied. For G3PDHs, 19 mg Saci_2032 or 6 mg Saci_1118 each in 500 µL were loaded to the column. 50mM TRIS-HCl pH 7.5, 250mM NaCl was employed as running buffer. For calibration, the LMWandHMWGel FiltrationCalibration Kits (Cytiva, Marlborough, MA, USA) were used. Localization of G3PDH upon co-expression with CoxG in E. coli To investigate the role of CoxG (Saci_2031 and Saci_1119) for membrane anchoring of the G3PDHs (Saci_2032 and Saci_1118) in S. acidocaldarius, co-expression studies in E. coli were performed. Therefore, saci_2032 and saci_2031 or saci_1118 and saci_1119 were co-expressed from pETDuet-1 expression vectors in E. coli Rosetta (DE3) as described above. As a control, saci_2032 or saci_1118 were expressed alone from the same vector without saci_2031 or saci_1119. After expression, cells were disrupted as described above and the resulting supernatant was centrifuged at 150,000 × g at 4 °C for 120min. The soluble fraction (supernatant) was separated from the membrane fraction,whichwaswashed twicewith50mMTRIS-HCl, pH7.0 and centrifuged (150,000 × g, 4 °C, 120min). Finally, the membrane fraction and soluble fraction were aliquoted, flash frozen in liquid nitrogen, and stored at−80 °C. Isolatedmembranes were resuspended in 500 µL of buffer (50mMTRIS-HCl, pH7.0) and sonicatedat lowamplitude (20%, 0.5 s−1, on ice) until fully homogenized. 50 µg of the membrane or soluble protein fractions were subjected to SDS-PAGE and transferred to a PVDF membrane (Roth, Karlsruhe, Germany). Immunodetection was performed with horseradish peroxidase (HRP) conjugated anti-His antibody (1:10,000 dilution; Abcam, Cambridge, United Kingdom) and the ClarityTM Western ECL substrate (BioRad, Munich, Germany) using a VersaDoc (BioRad) or Fusion FX (Vilber Lourmat, Marne-la-Vallée, France) imaging system. Localization of CoxG homologues in vivo and coimmunoprecipitation (co-IP) assays with anti-HAmagnetic beads Saci_2031 or saci_1119 were homologously expressed from the pSVAaraFX-HA vector construct transformed into S. acidocaldarius MW00G to yield a C-terminally HA-tagged recombinant protein. Expression cultures were grown in Brock’s basal saltmediumwith 10mMglycerol as carbon source for 72 h to an OD600 0.6–0.7 and expression was induced by the addition of 0.2% (w/v) L-arabinose. After 4 h of additional growth (OD₆₀₀ 0.8–0.9), the cells were cooled and harvested by centrifugation at 5000 × g for 20min at 4 °C. For localization of the CoxG homologues, cells were disrupted and soluble and membrane fractions were separated as described above for co-expression studies in E. coli. HA-tagged CoxG homologues in the separated fractionsweredetectedbywesternblotting and immunodetection using anti-HA and horse radish peroxidase-coupled secondary antibodies (Invitrogen, Thermo Fisher Scientific, Waltham, Massachusetts, United States). For Co-IP/pull-down experiments, pellets obtained from 500mL expression cultures were resuspended to a calculated OD600 of 40 in 25mM TRIS-HCl pH 7, 150mM KCl, 5% (v/v) glycerol, 10mMEDTA, and 2% (w/v) n-dodecyl β-maltoside (DDM) and incubated for 1 h at 37 °Cwhile rotating. Sampleswere centrifuged (30min, 15,000 × g, room temperature (RT)) to remove cell debris and the supernatantwas used for co-immunoprecipitation. Therefore, 35 µL of Pierce™ anti-HAMagnetic Beads (PierceTM HA-tag IP/co-IP kit (Pierce, Thermo Fisher Scientific)) were added to 10mL of the cell lysate and incubated for 1 h at room temperature while rotating. The beads were collected on a magnetic stand and washed twice with 25mMTRIS-HCl pH 7 containing 150mMKCl, 5% (v/ v) glycerol, 10mMEDTAandfinally oncewithLC-MSgradewater (Merck, Darmstadt, Germany). After washing, 35 µL of LC-MS grade water was added and 25 µL bead suspension was used for on-bead digestion as described previously67. Characterization of purified enzymes Enzyme assays were performed spectrophotometrically using a Specord UV/visible-light (Vis) spectrophotometer in a total volume of 500 µL (unless stated otherwise). Assay mixtures were pre-warmed to the designated assay temperature, and the reaction was typically initiated by the addition of the substrate. Experimental data were fitted, and kinetic constants were determined using the OriginPro 2022 software package (OriginLab). Glycerol kinase. GK activity was determined as described above for the crude extract measurements with the following modifications: For the continuous PK-LDH assay at 50 °C, the assays were performed for Saci_1117 in 0.1 MTRIS-HCl buffer (pH 7 at 50 °C), 1 mMMgCl2, 1 mM ATP, 0.2 mM NADH, 2 mM PEP, 14 U LDH, 7 U PK using 0.5 µg of purified Saci_1117. For Saci_2033 the assay was conducted in 0.1 M MOPS-KOH buffer (pH 6.5 at 50 °C), 5 mMMgCl2, 5 mMATP, 0.2 mM NADH, 2 mM PEP, 14 U LDH, 7 U PK using 0.25 µg of purified Saci_2033. To determine the kinetic properties varying concentrations of glycerol (0–2 mM), DHA (0–30 mM), DL-glyceraldehyde (0–20 mM) or ATP (0-5 mM) were used at a constant concentration of 1 mM (Saci_1117) and 5 mM (Saci_2033) ATP, 1 mM (Saci_1117) and 5 mM (Saci_2033) MgCl2 or 2 mM glycerol, respectively. One unit (1 U) of enzyme activity is defined as 1 µmol of product (ADP) formed per minute. Substrate specificity was tested using the continuous PK-LDH assay at 50 °C with 5 mM of various substrates, including DLglyceraldehyde (GA), dihydroxyacetone (DHA), DL-glyceric acid, Dxylose, D-glucose, meso-erythritol, D-sorbitol, or xylitol, in place of glycerol. Nucleotide specificity was assessed using the G3PDH-coupled assay, as described below, by substituting 5 mM GTP, CTP, or phosphoenolpyruvate (PEP) for ATP at 75 °C. The pH optimumwas determined using the PK-LDH assay at 50 °C in a mixed buffer system ranging from pH 5.0 to pH 8.0 containing 50mM MES, 50mM HEPES and 50mM TRIS-HCl. The temperature optimum was determined between 60 °C to 80 °Cusing the continuousG3PDHassay. Therefore, the glycerol kinase (GK)-mediated glycerol-3-phosphate (G3P) formation was coupled to DCPIP reduction, monitored at 600 nm, using purified recombinant G3PDH (0.25 U) from Sulfolobus acidocaldarius (Saci_2032). The assay for Saci_1117 was conducted in 0.1M TRIS-HCl buffer (pH 7, temperature adjusted), containing 1mMATP, 1mMMgCl2, 0.1mM DCPIP, and 2mM glycerol. For Saci_2033, the assay was performed in 0.1M MOPS-KOH buffer (pH 6.5, temperature adjusted), with 5mMATP, 5mMMgCl2, 0.1mMDCPIP, and2mMglycerol. To study the thermal stability, theGKs Saci_1117 and Saci_2033were incubated at 70 °C, 80 °C, and 90 °C in 150 µL (total volume) 100mM TRIS-HCl (pH 7) or MOPS-KOH (pH 6.5) (pH temperature adjusted), respectively, at a protein concentration of 0.05mgmL−1. At regular time intervals (1 h, 3 h, 6 h, and 24 h), samples (10 µL for Saci_1117 and 5 µL for Saci_2033)were taken, and the residual enzyme activity was measured using the continuous PK-LDH assay at 50 °C, as previously described. The effect of fructose 1,6-bisphosphate (F1,6BP)onGKactivitywas also evaluated using the continuous PKLDH assay (described above) with F1,6BP concentrations of up to 1mM, as indicated. Glycerol-3-phosphate dehydrogenase. The determination of the reduction of enzyme-bound FAD was performed at 70 °C (200 µL total volume) in 50 mM HEPES-KOH pH 6.5 (temperature adjusted), 100 mM KCl, 50 µM of G3P and 70 µg of Saci_2032 or Saci_1118, corresponding to a final concentration of 5 µM. The reaction was started by addition of G3P and after 5 min absorption spectra were measured between 400 and 500 nm in 96 well plates in a Tecan Infinite M200 plate reader. Loss of absorption at 450 nm indicates the reduction of bound FAD. To determine the FAD concentration of the purified G3PDH, the Communications Biology | (2025) 8:539 15 proteinwas denatured in 50 mMHEPES-KOHpH6.5, 100 mMKClwith 0.5% (w/v) SDS (500 µL total volume) at room temperature and FAD absorbance spectra were determined from 300 nm to 600 nm (Specord UV/visible-light (Vis) spectrophotometer). The FAD content was calculated using the extinction coefficient of 11.300 mM−1 cm−1 at 450 nm. To test the quinone reactivity, 0.6 µg of Saci_2032 or 1.2 µg of Saci_1118 were preincubated with 30 µM of water-soluble ubiquinoneQ1 (500 µL total volume) in 50mM HEPES-KOH pH 6.5, 100 mM KCl. Native ubiquinones used in Bacteria and eukaryotes are water-insoluble due to side chains comprising up to ten isoprenoid units. Afterwards 100 µM of G3P was added to the samples and reduction of ubiquinoneQ1 was followed in 15 s intervals by recording the absorption spectra between 260 and 330 nm using a Specord UV/visible-light (Vis) spectrophotometer. Loss of absorption at 280 nm indicates the reduction of the ubiquinone-Q1. For enzyme characterization continuous enzyme assays were conducted at 70 °C (500 µL total volume) by monitoring the decrease in absorbance during the reduction of the artificial electron acceptorDCPIP at 600 nm or ubiquinone-Q1 at 280 nm. Kinetic parameters for G3P were determined in 50mM HEPES-KOH pH 6.5, 100mM KCl, 0.06mM DCPIP, with 0.35 µg of Saci_2032 or 0.5 ug of Saci_1118 at 70 °C and varying concentrations of G3P (0–0.3mM). To determine the kinetic constants for ubiquinone-Q1, DCPIPwas omitted and the concentration of thequinonewas variedbetween0mMand0.06mMwith a concentrationof 0.6 µg for either Saci_2032 or Saci_1118, while maintaining a fixed G3P concentration of 0.4 mM. One unit (1 U) of enzyme activity is defined as 1 µmol of product (DCPIPred or ubiquinone-Q1red) formed per minute. Substrate specificity was evaluated by replacing glycerol with 0.3mM D-glycerol 1-phosphate, D-glycerol, DL-glyceric acid, D-glyceraldehyde 3- phosphate, and D-phosphoglyceric acid. Both, the pH and the temperature optimum were determined using 0.4mM G3P and 0.06mM DCPIP. For the pH optimum a mixed buffer systemcontaining 50mMMES, 50mMHEPES, and 50mMTRISwas used in the range of pH 5.0 to pH 8.0. The temperature optimum between 50 °C and 80 °C was determined in 50mM MES-KOH pH 6.5 adjusted at the respective temperature. Glycerol oxidase activity of both G3PDHs was tested as G3P-dependent hydrogen peroxide formation coupled to the oxidation of 2,2’-azinobis-(3-ethylbenzothiazoline-6-sulfonate) (ABTS) via horseradish peroxidase (HPR, Merck, Darmstadt, Germany) measured as increase in absorbance at 420 nm (extinction coefficient 42.3mM−1cm−1)25. The assays were performed in 100mM MES-KOH pH 6.5, 1mM ABTS, and 0.2 U of HRP with 0.045 µg of protein. The thermostability of the Saci_2032 and Saci_1118 G3PDHs was analyzed by incubating the enzyme at 70 °C, 80 °C, and 90 °C in 50mM MES-KOH, pH 6.5 (temperature adjusted, 400 µL total volume) at a protein concentration of 0.18 µg µL−1. After regular time intervals (1, 3, and 6 h) 10 µL aliquots were removed and the residual activity of the enzyme was determined at 70 °C with G3P and DCPIP as described above. The influence of detergents and membrane lipids on Saci_2032 and Saci_1118 activity was analysed with G3P and DCPIP at 50 °C in the presence of either 0.5% (w/v) DDM, 0.5% (v/v) of triton X-100, 50 µg of phosphatidylcholine (Merck, Darmstadt, Germany) or 50 µg of isolated S. acidocaldarius MW00G membrane fractions (prepared as described above).
Analytical assays
If not stated otherwise, protein purity was analyzed by SDS-PAGE and protein concentrationwas determined by amodified Bradford assay74 using bovine serum albumin (Carl Roth, Germany) as standard.
Bioinformatic analyses
Structural models were retrieved from the AlphaFold Protein Structure database75,76 or predicted using the ColabFold software77. Structural analyses, comparisons, and visualizations were done using UCSF Chimera package from the Resource for Biocomputing, Visualization, and Informatics at theUniversity ofCalifornia, SanFrancisco (supportedbyNIHP41 RR-01081)78. For phylogenetic analyses sequences were aligned with clustal omega using the EMBL server79. The clustal omega alignment was used in the MEGA11 software package for phylogenetic tree constructions80 (for further details see legend to Fig. 8).
Statistics and Reproducibility
All data was generated in three independent experiments (n = 3), with the exception of the metabolite analysis that was preformed using eight replicates (n = 8). Sample sizes were chosen based on standard sample sizes in literature.All graphswere createdusingOriginPro2024.All statistical details are described in the figure legends or in the methods section of the corresponding experiment.
Reporting summary
Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.
Data availability
The mass spectrometry proteomics data for the on-bead digestions have been deposited to the ProteomeXchange Consortium via the PRIDE81 partner repository (https://www.ebi.ac.uk/pride/archive/) with the dataset identifier PXD050086. All other data generated during the current study are available from the corresponding author upon request. RNA-seq data were deposited in in the ArrayExpress database (www.ebi.ac.uk/arrayexpress) under accession number E-MTAB-14293. Numerical source data for all graphs can be found in Supplementary File 4 and unedited versions of gel pictures and western blots are shown in Supplementary Fig. 14. Received: 29 November 2024; Accepted: 18 March 2025;
Acknowledgements
This researchwas fundedby theFederalMinistryofEducationandResearch (BMBF)andby theVolkswagenFoundation.B.S.,M.K.,O.J.S. (031B0848A), J.K. (031B0848B) andS-V.A. (031B0848C)wish toexpress their gratitude for the financial support received for the HotAcidFACTORY project, titled ‘Sulfolobus acidocaldarius as a novel thermoacidophilic bio-factory’, under the BMBF National Bioeconomy Strategy (Microbial Biofactories for Industrial Bioeconomy—Novel Platform Organisms for Innovative Products andSustainableBioprocesses).B.S. andC.B.wish toexpress their gratitude for the financial support received for the Lipid║Divide—‘Resolving the ‘lipid divide’by unravelling the evolution and role of fatty acidmetabolic pathways inArchaea’projectwithin the ‘Life?’—A freshscientificapproach to thebasic principlesof life initiative (VolkswagenFoundation, grant number96725).We acknowledge support by the Open Access Publication Fund of the University of Duisburg-Essen.
Author contributions
Ch.S., X.Z. andCa.S. conducted growth studies, crude extractmeasurements, cloning and expression, as well as enzyme purification and characterization. T.B. performed RNA sequencing and related data analysis. S.N. and F.K. executed proteomics and corresponding data analysis. L.M. carried out metabolomics and associated data analyses. J.B. and B.W. constructed the GK deletion mutants, cloned and homologously expressed CoxG, and conducted co-immunoprecipitation experiments. C.B. handled sequence, structural, and phylogenetic analyses. The study was conceived and designed byS-V.A., J.K.,M.K., O.J.S., C.B. andB.S. All authors contributed towriting the initial draft, while Ch.S., Ca.S., C.B. andB.S. reviewed and edited the final draft. Project administration was managed by B.S., and funding acquisition was secured by J.K., S-V.A., O.J.S., M.K., C.B. and B.S. All authors contributed to the article and approved the final submitted version. Communications Biology | (2025) 8:539 18
Funding
Open Access funding enabled and organized by Projekt DEAL.
Competing interests
The authors declare no competing interests.
Additional information
Supplementary information The online version contains supplementary material available at https://doi.org/10.1038/s42003-025-07953-9. Correspondence and requests for materials should be addressed to Christopher Bräsen or Bettina Siebers. Peer review information Communications Biology thanks the anonymous reviewers for their contribution to the peer review of this work. Primary Handling Editors: Kaliya Georgieva. A peer review file is available. Reprints and permissions information is available at http://www.nature.com/reprints Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in anymedium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. © The Author(s) 2025 Communications Biology | (2025) 8:539 19
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