Functional redundancy of ubiquitin-like sulfur-carrier proteins facilitates flexible, efficient sulfur utilization in the primordial archaeon Thermococcus kodakarensis

ABSTRACT Ubiquitin-like proteins (Ubls) in eukaryotes and bacteria mediate sulfur transfer for the biosynthesis of sulfur-containing biomolecules and form conjugates with specific protein targets to regulate their functions. Here, we investigated the functions and physiological importance of Ubls in a hyperthermophilic archaeon by constructing a series of deletion mutants. We found that the Ubls (TK1065, TK1093, and TK2118) in Thermococcus kodakarensis are conjugated to their specific target proteins, and all three are involved in varying degrees in the biosynthesis of sulfur-containing biomolecules such as tungsten cofactor (Wco) and tRNA thiouridines. TK2118 (named UblB) is involved in the biosynthesis of Wco in a glyceraldehyde 3-phosphate:ferredoxin oxidoreductase, which is required for glycolytic growth, whereas TK1093 (named UblA) plays a key role in the efficient thiolation of tRNAs, which contributes to cellular thermotolerance. Intriguingly, in the presence of elemental sulfur (S0) in the culture medium, defective synthesis of these sulfur-containing molecules in Ubl mutants was restored, indicating that T. kodakarensis can use S0 as an alternative sulfur source without Ubls. Our analysis indicates that the Ubl-mediated sulfur-transfer system in T. kodakarensis is important for efficient sulfur assimilation, especially under low S0 conditions, which may allow this organism to survive in a low sulfur environment. IMPORTANCE Sulfur is a crucial element in living organisms, occurring in various sulfur-containing biomolecules including iron-sulfur clusters, vitamins, and RNA thionucleosides, as well as the amino acids cysteine and methionine. In archaea, the biosynthesis routes and sulfur donors of sulfur-containing biomolecules are largely unknown. Here, we explored the functions of Ubls in the deep-blanched hyperthermophilic archaeon, Thermococcus kodakarensis. We demonstrated functional redundancy of these proteins in the biosynthesis of tungsten cofactor and tRNA thiouridines and the significance of these sulfur-carrier functions, especially in low sulfur environments. We propose that acquisition of a Ubl sulfur-transfer system, in addition to an ancient inorganic sulfur assimilation pathway, enabled the primordial archaeon to advance into lower-sulfur environments and expand their habitable zone.

form an enzyme-bound persulfide, without releasing harmful reactive sulfur species in solution.The terminal sulfur of the persulfide in CD is subsequently transferred to sulfur-carrier proteins.There exist two types of sulfur carrier: (1) protein persulfide (R-SSH) exemplified by CD and rhodanese homology domain-containing proteins with a conserved cysteine residue; and (2) protein thiocarboxylate (R-COSH) formed at conserved C-terminal di-glycine residues of Ubls (8).Finally, the sulfur atoms of sulfur- carrier proteins are incorporated into target molecules by specific sulfurtransferases in each biosynthesis pathway.
Although these sulfur-transfer systems are widely conserved in all domains of life, many hyperthermophilic archaea do not possess CD orthologs (Fig. S1a) (9).Many live under inorganic sulfur compound-rich conditions and are assumed to take environmen tal inorganic sulfur into their cytoplasm and utilize it as a sulfur source directly for final sulfurtransferases in biosynthesis pathways of sulfur-containing biomolecules (9).However, the facultatively sulfur-reducing hyperthermophilic archaeon Thermococcus kodakarensis does possess a CD ortholog (Fig. S1a).CD is dispensable for iron-sulfur cluster biosynthesis and cell growth in the presence of elemental sulfur (S 0 , cyclo-octasul fur) (5), suggesting that T. kodakarensis can use both L-cysteine and environmental S 0 as sulfur sources.However, unlike CDs, sulfur-carrier protein Ubls are highly conserved in archaea (10), although their functions remain largely unknown.
Here, we characterized the molecular functions and biological importance of three Ubls (TK1065, TK1093, and TK2118) of T. kodakarensis and found that these proteins act both as sulfur-carrier proteins and protein modifiers.TK2118 (named UblB) is required for growth with maltodextrin (Mdx) as a sole carbon source, suggesting that it mediates incorporation of tungsten cofactor (Wco) into the key enzyme, glyceraldehyde-3-phos phate:ferredoxin oxidoreductase (GOR), essential for glycolysis in the Mdx medium.TK1093 (UblA) mediates sulfur-transfer for the synthesis of tRNA thionucleosides and contributes to cell growth at high temperature.Moreover, we revealed that Ubls were partially dispensable for the synthesis of these sulfur biomolecules under S 0 -rich conditions.These results clearly indicate the presence of an inorganic sulfur assimilation pathway in T. kodakarensis, which may reflect the metabolic heritage of protobionts.The findings also suggest that acquisition of CDs and Ubls enables efficient synthesis of sulfur-containing biomolecules under low concentrations of sulfur donors, thereby allowing these organisms to advance into lower-sulfur environments and expand their habitable zone.

Three Ubls contribute differently to the growth of T. kodakarensis, depending on temperature and sulfur availability
To uncover the biological functions of the three Ubl orthologs in T. kodakarensis, we constructed deletion mutants for each Ubl gene and a triple Ubl deletion mutant (∆ubls) from the parent strain DAD (∆pyrF and ∆pdaD; Fig. S3).The growth phenotypes of these strains on artificial seawater (ASW)-YT-S 0 medium (S 0 -containing medium) at optimal (85°C) and high (93°C or 90°C) temperatures were examined (Fig. 2a).T. kodakarensis grows at temperatures ranging from 60°C to 95°C, with an optimum growth temperature of 85°C.Since wild-type strains grow slowly at 93°C on Pyr medium, the "high" tempera ture was set to 90°C when Pyr medium was used.At the optimal temperature, these strains showed almost the same growth rates.However, the ∆ublA strain showed growth retardation at 93°C compared with the parent strain and other Ubl-deficient strains.The ∆ubls strain also showed growth phenotypes similar to ∆ublA.In ASW-YT-Pyr medium (Pyr medium, no S 0 ; Fig. 2b), ∆ublA and ∆ublB strains displayed severe growth retardation at optimal (85°C) and high (90°C) temperatures.The ∆ubls strain also exhibited similar growth defects to ∆ublA and ∆ublB strains.These results suggest that UblA and UblB are required for cell growth especially at higher temperature in T. kodakarensis in the absence of environmental S 0 , but their functions could be partially compensated for in the presence of environmental S 0 .

The roles of Ubls in MPT biosynthesis
MPT consists of a pyranopterin, a complex heterocycle featuring a pyran fused to a pterin ring, and the pyran ring contains two thiolates that serve as ligands for a molybdenum or a tungsten atom to form Moco or Wco, respectively (Fig. 1a).Moco and Wco are organometallic cofactors, which are essential for the activities of various oxidoreductases (19)(20)(21)(22)(23)(24).GOR, a glyceraldehyde 3-phosphate:ferredoxin oxidoreductase mediating an essential step in maltose fermentation, requires Wco as a cofactor (24)(25)(26).To investigate the requirement of each Ubl for the synthesis of GOR-incorporated Wco, the strains were cultured in ASW-YT-based medium supplemented with maltodextrin (Mdx medium, no S 0 ).In Mdx medium, the ∆ublB strain was unable to grow, while the ∆ublA strain showed severe growth retardation (Fig. 2c), as observed in Pyr medium (Fig. 2b).The growth defect of the ∆ublB strain was restored by ectopic expression of the wild-type ublB gene but not the ublB gene lacking the C-terminal two glycine residues (Fig. 2c), suggesting that UblB and its C-terminus are essential for MPT synthesis in GOR.
We next measured the quantity of MPT in the cell.MPT in the cell extract from each strain was oxidized to FormA-phospho by iodine under acidic conditions (Fig. 3a) (5,27,28) then subjected to liquid chromatography with mass spectrometry (LC-MS) analysis.As shown in Fig. 3b, the proton adduct ion identical to FormA-phospho (m/z 328.1) was detected at ~6.7 min and further confirmed by co-injection analysis with FormAphospho prepared from bovine xanthine oxidase (29) (Fig. 3b).Next, the quantity of FormA-phospho in each Ubl-deficient strain and the ∆ubls strain was quantified (Fig. 3c).When cultivated in Pyr medium, approximately half the amount of FormA-phospho was detected in these mutants compared with the parent strain.However, in S 0 medium, the levels of FormA-phospho in each Ubl-deficient strain were completely restored (Fig. 3c).These results indicate that all Ubls are involved in Wco biosynthesis under sulfur-poor conditions but are dispensable for MPT biosynthesis under sulfur-rich conditions.
Furthermore, we measured the activities of Wco-containing oxidoreductases AOR, formate:ferredoxin oxidoreductase (FOR), and GOR in the cell lysates of Ubl-deficient strains grown in three different media (S 0 medium, Pyr medium, and Mdx medium; Fig. 4a through c).No differences were observed in the activities of the three Wco-containing enzymes in parent and ∆ubls strains grown in S 0 medium (Fig. 4a).These results were consistent with the result that the levels of MPT derivatives in the ∆ubls strain and each Ubl-deficient strain were comparable to that of the parent strain (Fig. 3).In Pyr medium, the AOR activities of the ∆ublA, ∆ublB, and ∆ubls were less than half of those of the parent strain.Also, the AOR activity of ∆ublC was ~54% of that of the parental strain.The FOR activity of ∆ublA was less than half of that of the parent strain, and no FOR activity was detected for ∆ublC, ∆ublB, and ∆ubls.GOR activity could not be determined because the activity was below the detection limit (Fig. 4b).By contrast, GOR activity was enhanced in Mdx medium (25), and the GOR activities of ∆ublC and ∆ublA strains were almost the same as that of the parent strain, suggesting that UblC and UblA are not involved in the maturation of active GOR (Fig. 4c).As a control, the activity of ferredoxin:NADP + oxidoreductase (FNOR), which does not contain Wco and instead contains an iron-sulfur cluster (30) formed without Ubls, was also measured.The FNOR activity of each Ubl-deficient strain was comparable to that of the parent strain under all culture conditions (Fig. 4a through c).These results suggest that Wco in GOR is biosynthesized only by a specific Ubl (UblB) under S 0 -poor conditions.

UblA is required for efficient s 2 U formation in tRNAs
Ubl, belonging to the ThiS family, is involved in the formation of 2-thiolated uridine derivatives at positions 34 and 54 of tRNAs (31)(32)(33).In T. kodakarensis tRNAs, m 5 s 2 U is located at position 54 (34), but the chemical structure of the 2-thiolated uridine derivative at position 34 is still unclear.Therefore, we investigated the 2-thiolated uridine derivative in T. kodakarensis before evaluating the involvement of Ubls in 2-thiolated uridine formation.The tRNA fraction prepared from the parent strain of T. kodakarensis was digested into nucleosides and subjected to liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis.The proton adduct ion of 5-methyl-2-thiouridine (m 5 s 2 U, m/z 275.1) was observed at ~10.3 min (Fig. S4a), consistent with a previous report showing m 5 s 2 U located at position 54 in tRNA of T. kodakarensis (34).In addition, we successfully detected 5-cyanomethyl-2-thiouridine (cnm 5 s 2 U, m/z 300.1 at ~9.7 min) and its precursor 5-cyanomethyluridine (cnm 5 U, m/z 284.1 at ~13.2 min) with confirmation by MS 2 analysis and co-injection analysis with digested tRNA of Methanocaldococcus jannaschii, in which cnm 5 s 2 U is attached to position 34 of tRNA(35) (Fig. S4a through c).
Next, we investigated the involvement of the T. kodakarensis Ubls in the biosynthesis of the two thionucleosides.Total tRNA fractions prepared from Ubl-deficient strains and the ∆ubls strain cultivated at 85°C in Pyr medium were subjected to LC-MS/MS analysis.In the parent strain, m 5 s 2 U and cnm 5 s 2 U were clearly detected (Fig. 5a through  c).However, the levels of these thionucleosides in the ∆ublA strain were drastically reduced to ~14% and ~8%, respectively, with the elevation of their precursors, m 5 U and cnm 5 U.The levels of m 5 s 2 U and cnm 5 s 2 U were not altered in ∆ublC and ∆ublB strains (Fig. 5a through c).In the ∆ubls strain, levels of these were also decreased, as observed in the ∆ublA strain (Fig. 5a through c).Conversely, in S 0 -containing ASW-YT-S 0 medium, the thiolation levels of m 5 s 2 U and cnm 5 s 2 U in ∆ublA and ∆ubls strains were partially but significantly recovered (Fig. 5d through f).In addition to cnm 5 s 2 U and m 5 s 2 U, four thiolated nucleosides (4-thiouridine, s 4 U; 2-methylthio-N 6 -hydroxy-norvalyl carbamoyladenosine, ms 2 hn 6 A; 2-thiocytidine, s 2 C; and 2-methylthio-N 6 -threonylcarba moyladenosine, ms 2 t 6 A) were also identified based on mass spectrum data and retention time (35).The levels of these four thionucleosides were comparable between parent and ∆ubl strains (Fig. S5).These results clearly indicate that UblA is the only sulfur-carrier Ubl responsible for s 2 U formation in T. kodakarensis tRNA and is required for efficient 2-thiolation of m 5 U and cnm 5 U under sulfur-poor conditions, although UblA is partially dispensable for modifications under sulfur-rich conditions.

Three Ubls conjugate to specific proteins in T. kodakarensis
We next explored whether the three Ubls are able to form protein conjugates, as observed in other organisms (8,18,36), in addition to their sulfur-carrier roles.Similar to eukaryotic ubiquitin, Ubls in bacteria also covalently attach to the Lys residues of target proteins via their C-terminal Gly residues.To this end, whole soluble proteins from cells grown in S 0 -containing medium were separated by sodium dodecyl-sulfate polyacry lamide gel electrophoresis (SDS-PAGE) and subjected to Western blotting with each Ubl-specific antibody (Fig. S6).In the parent strain, many signals indicating Ubl-conju gating proteins were observed, along with signals corresponding to monomeric Ubls at ~10 kDa [theoretical mass: UblC (TK1065), 9.1 kDa; UblA (TK1093), 7.2 kDa; and UblB (TK2118), 10.1 kDa], whereas these bands were absent in the deletion mutants.Moreover, the major bands observed were also detected in lysates prepared from Ubl-deficient strains cultured in the absence of S 0 , suggesting that Ubls are conjuga ted to specific proteins, irrespective of the presence or absence of S 0 .In addition, the amount of each Ubl-conjugation band tended to be greater in the Pyr medium than in the S 0 -containing medium.Because there are different gene expression patterns in the presence or absence of S 0 (17), the Ubl-conjugation pattern may also differ.

DISCUSSION
Here, we investigated the cellular functions of three Ubls in T. kodakarensis and revealed the functional versatility and essentiality of these ancient Ubls in the biosynthesis of MPT/Wco and tRNA thiouridine (outlined in Fig. 6), as well as their roles in post-transla tional modification.Wco in GOR is specifically synthesized via UblB (TK2118), although all three Ubls are redundantly involved in Wco biosynthesis in several oxidoreductases except GOR.For tRNA thiouridine synthesis, only one Ubl (UblA, TK1093) serves as sulfur carrier for both cnm 5 s 2 U and m 5 s 2 U.
In general, different Ubls function strictly in their cognate pathways for sulfur-contain ing biomolecules (1,8).Ubls belonging to the ThiS family are relatively shorter in length (~70 amino acid residues) than those in the MoaD family (~80 amino acid residues, Fig. 1d) and have a simpler topology with fewer secondary structural elements (Fig. 1e).Ubls belonging to MoaD and ThiS families in bacteria are essential for sulfur transfer in MPT synthesis (37)(38)(39) and tRNA thiolation (12,18), respectively.In eukaryotes, MOCS2A and Urm1 are required for the biosynthesis of Moco (40,41) and tRNA thiouridine (42,43), respectively.Herein, we showed that the ThiS family protein UblA is involved in Moco biosynthesis, in addition to thiouridine synthesis and protein conjugation, implying functional redundancy of an ancient-type Ubl and modest substrate specif icity between Ubls and their interaction partners.Related functional redundancy of sulfur-carrier proteins has been reported for the biosynthesis of 2-thiosugar-containing antibiotic secondary metabolites in Actinobacteria; the reaction of 2-thioglucose-6-phos phate synthase BexX of Amycolatopsis orientali borrows sulfur-carrier proteins for primary metabolites including MPT (44).
Interestingly, our study clearly revealed flexible utilization of sulfur sources in T. kodakarensis; in the presence of environmental S 0 , all Ubls were partially dispensable for the syntheses of these sulfur-containing biomolecules.We previously reported that impairment of iron-sulfur cluster biosynthesis due to the loss of CD was restored in the presence of environmental S 0 (5).Therefore, we propose that sulfur atoms for sulfurcontaining biomolecules in T. kodakarensis are supplied from a sulfur-transfer system involving Ubl and CD or from environmental S 0 .Intriguingly, our study also revealed another layer of complexity for the sulfur donor in T. kodakarensis; almost 50% of MPT derivative in cells (Fig. 3c) and ~10% of m 5 s 2 U (Fig. 5c) in tRNA were estimated to be biosynthesized through a Ubl-independent sulfur-transfer pathway, even in the absence of S 0 , suggesting the existence of unknown sulfur sources such as S 2− equivalents.We speculated sulfur-transfer pathways for the biosynthesis of MPT/Wco and tRNA thionucleosides in T. kodakarensis based on homology searches of the enzymes in these pathways identified in other model organisms (Fig. S7).There is only one E1/MoeB/ThiF family adenylyltransferase homolog (TK2117) in the T. kodakarensis genome, and TK2117 presumably activates the glycine residue at the C-terminus of each Ubl in the Wco and thiouridine biosynthesis pathways.There are several reports that one E1/MoeB/ThiF family adenylyltransferase can activate multiple Ubls (4,18,40,41).Our current results showed a decrease in MPT-derivative levels in each Ubl-deficient strain and therefore low or no AOR/FOR activity (Fig. 3, 4b and c).On the other hand, the ∆ublB strain showed growth impairment in Mdx medium (Fig. 2c), where GOR activity is required for growth.Therefore, UblB seems to be a major sulfur-carrier protein for Wco biosynthesis in GOR, implying the existence of an unknown mechanism for specific incorporation of Wco into certain target protein(s) with cognate Ubl(s).Molybdopterin might receive its sulfur atoms after apo-cofactor has bound to the target protein.UblB is the only Ubl that can activate GOR, while all three Ubls can activate FOR and AOR.In plants, Cnx6 (MPT synthase) and Cnx1 (MPT:Mo transferase), which are responsible for two successive steps in Moco biosynthesis, reportedly form a multiprotein complex (45).Such a complex may contribute to secure and specific targeting of the cofactor to the corresponding apo-enzymes, although the mechanism that links specific Ubls and their corresponding apo-enzymes is not known.
For tRNA thiolation, the sulfur atom of UblA thiocarboxylate is likely transferred to tRNAs by sulfurtransferases for m 5 s 2 U54 and cnm 5 s 2 U34 syntheses (Fig. S7).T. kodakar ensis has two possible 2-thiolase-encoding genes, ttuA (tk1556) and ncs6/ncsA (tk1821) (46), suggesting that their encoded enzymes recognize and catalyze the modification at positions 34 and 54, respectively, with only one Ubl, UblA (Fig. S7b).We previously showed that TtuB, a homolog of UblA in Thermus thermophilus, is a sulfur-carrier Ubl responsible for 2-thiolation at position 54 of tRNA (12).Meanwhile, others reported that SAMP2, a homolog of UblA in Haloferax volcanii, is a sulfur-carrier Ubl responsible for 2-thiolation at position 34 of tRNA Lys (33).By contrast, our current study demonstrated for the first time that one sulfur-transfer pathway has two target sites on tRNA, and in vitro characterization of the molecular basis of this unique pathway, including putative RNA sulfurtransferases, is under way.We also found that the ∆ublA strain showed growth retardation in Pyr or Mdx media at both optimal and higher temperatures.Since 2-thiolation at positions 34 and 54 of tRNA contribute crucially to decoding accuracy in translation and structural stability of tRNA, respectively (47), significant reductions in the frequencies of these modifications in ∆ublA (Fig. 5a through c) should induce abnormal translation and instability of tRNA, thus resulting in growth retardation and temperature sensitivity.Especially in thermophiles, tRNAs are excessively decorated with various modifications to stabilize the characteristic L-shaped structure (48,49).Of note, some tRNA modifications, including 2-thiolation of U54, are introduced in a temperaturedependent manner to adapt the structural rigidity and flexibility of tRNA for appropriate protein synthesis at a given temperature (12,50).Therefore, direct use of sulfide by 2-thiolase would enable faster adaption to a sudden temperature change without a sulfur-transfer system.The direct utilization of sulfide by sulfurtransferases may just be a remnant of protobionts, but for T. kodakarensis, which lives in solfatara where acute changes in sulfur concentration and temperature occur, being able to respond to sudden changes in environmental conditions is a great advantage (see below).
Organisms that inhabit inorganic sulfur compound-rich environments are the most likely to have S 0 reducibility and/or sulfite reductase orthologs and do not have CD orthologs; hence, they probably utilize sulfide (5,51).For example, the methanogenic archaeon Methanococcus maripaludis, which grows in sulfide-rich environments (52), uses sulfide directly for tRNA thiolation and iron-sulfur cluster biosynthesis without CD and Ubls (9,53).Iron-sulfur cluster synthesis can proceed in vitro on an apoprotein in the presence of iron salts and sulfide at millimolar concentrations under anaerobic conditions (54,55).T. thermophilus TtuA can use sulfide as sulfur donor for in vitro s 2 U formation (31) without in vivo sulfur donor TtuB (12).The C-terminus of Escherichia coli MoaD could be thiocarboxylated by sulfide at millimolar concentration, in which case sulfide may be able to support MPT formation in vitro (56,57).A Ubl-mediated sulfur-transfer pathway would compensate the disadvantage of S 0 -dependency, which is susceptible to fluctuation of environmental S 0 , providing an opportunity to survive in a low or no sulfur environment.CD in T. kodakarensis likely serves as sulfur donor for Ubls.Our analysis also revealed that all Ubls in T. kodakarensis form protein conjugates with specific targets (Fig. S6).It is possible that these post-translational modifications could regulate both MPT and tRNA thiouridine biosynthesis, although further analysis including the identification of target proteins is required.In H. volcanii, the Ubls SAMP1 and SAMP2 are essential for Moco biosynthesis and tRNA thiolation, respectively, and these are attached through isopeptide bonds to lysine residues on each protein target, including MoaE and MoeB in Moco biosynthesis (4,10).Furthermore, in T. thermophilus, TtuA and TtuC enzymes for thiouridine synthesis are post-translationally modified by the cognate Ubl TtuB (36).
T. kodakarensis probably modulates sulfur-transfer pathways by sensing the fluctuation of S 0 concentration in the environment, as demonstrated in the present study, and T. kodakarensis is also known to switch respiration mode in response to S 0 availability (58)(59)(60).It is believed that protobionts evolved under thermal and sulfur-rich conditions similar to the environments of present hydrothermal vents and volcanic marine sediments (61,62).Protobionts likely rely on direct utilization of inorganic sulfurs in the biosynthesis of sulfur-containing molecules; therefore, extant hyperthermophiles presumably possess a similar primitive sulfur assimilation mechanism that is dependent on inorganic sulfur.Our characterization of a sulfur-transfer system involving Ubls in T. kodakarensis provides insight into an evolutional snapshot of a sulfur assimilation pathway which might have allowed organisms that lived only in solfataric environments to advance into lower-sulfur environments.

Complementation of the tk2118 deletion mutant
Primers Tk2118-Fw3 and Tk2118-Rv3 were used to clone tk2118.PCR-amplified DNA fragments of the tk2118 gene were ligated to the pCSG (67) fragment, which was PCR-amplified using primer set pCSG-Fw/pCSG-Rv, yielding the pCSG-TK2118 plasmid.The QuickChange site-directed mutagenesis method (Qiagen) was used to generate Tk2118 mutants with primer set Tk2118∆GG-Fw/Tk2118∆GG-Rv. The pCSG-TK2118 and derivative constructs were individually introduced into the T. kodakarensis ∆ublB strain (ΔpdaD, ΔpyrF, and Δtk2118), and transformants were selected by growth in the absence of agmatine.Cells were maintained in ASW-YT-based medium supplemented with 2 g/L Mdx.

TK2117 enzyme assay
ATP:TK2118 adenylyltransferase reaction was performed in a reaction mixture containing 50 mM Tris-HCl buffer (pH 8.0), 10 mM ATP, 5 mM MgCl 2 , and TK2117 (4.0 µM) in the presence or absence of TK2118 (10.0 µM) at 80°C.Enzyme activity was measured by quantifying the phosphate concentration converted from the pyrophosphate product in the reaction mixture.The amount of phosphate was determined using a Biomol Green phosphate assay reagent kit (Enzo Life Sciences) following addition of 1-2 units of inorganic pyrophosphatase (Sigma-Aldrich) to the reaction mix.One unit was defined as the amount of enzyme that produced 1 µmol phosphate/min.

Extraction and mass spectrometry analysis of FormA-phospho from T. kodakarensis
Wco and MPT in T. kodakarensis cell was extracted (5,27,28) and quantified by LC-MS as follows.T. kodakarensis cells were cultivated to logarithmic phase in 100 mL of Pyr medium or S 0 medium.Cell pellets were washed twice with 30 mL of 1× ASW, resus pended in 300 µL of 10 mM Tris-HCl (pH 8), sonicated, and centrifuged.The protein concentration in the supernatant was determined by the Bradford method (68).The supernatant was adjusted to pH 2.3 by adding 6 M HCl, 200 µL of a solution containing 1 wt/vol % iodine and 2 wt/vol % potassium iodide was added to the cell lysate, and the lysate was heated at 100°C for 30 min to obtain the MPT fluorescent derivative FormA-phospho.Excess iodine was removed by addition of 50 µL of 100 mM ascorbic acid, and the sample was adjusted to pH 8.0 by adding 6 M NaOH.MPT derivative was also extracted from xanthine oxidase in bovine milk (Sigma-Aldrich) for comparison.The LC-MS system consisted of a Nexera X2 high-performance liquid chromatography system and an LCMS-8060NX triple quadrupole mass spectrometer (Shimadzu).Samples were chromatographed using a Kinetex EVO C18 column (100 × 4.6 mm, GL Science) at a flow rate of 0.5 mL/min with a solvent consisting of 50 mM ammonium acetate (pH 6.8) and 7 vol/vol % methanol, and the eluent was sprayed directly into the mass spectrometer.Ions were scanned in positive polarity mode over an m/z range of 80-350 throughout the separation.The amount of FormA-phospho in each sample was quantified from the MS peak area, and the value was normalized against the total protein amount in each sample.

Oxidoreductase enzyme assays
T. kodakarensis cells were cultivated to logarithmic phase in the presence of S 0 (0.2 wt/vol %), then collected and disrupted anaerobically by incubating with a lytic solution containing 2 mM dithiothreitol and 2-mM dithionite in 50 mM Tris-HCl (pH 8) supple mented with 5 U/mL DNase I (Takara Bio).Substrate-dependent reduction of methyl viologen in the cell-free extract was assayed as a change in A 603 nm to determine tungstopterin enzyme activities, crotonaldehyde for AOR (22), formaldehyde for FOR (23), and glyceroaldehyde-3-phosphate for GOR (24) at 80°C under anaerobic conditions (O 2 <1 ppm).NADH-dependent reduction of methyl viologen (Sigma-Aldrich) was measured to detect FNOR activity (30).Protein concentrations were determined by the Bradford method with bovine serum albumin as a standard (68).One unit was defined as the amount of enzyme that produced 1 µmol of product/min.

Preparation and mass spectrometry analysis of total tRNAs from T. kodakar ensis
T. kodakarensis cells were cultivated to logarithmic phase in the presence or absence of S 0 .Total RNA was extracted from harvested cells using the acid guanidinium thiocya nate-phenol-chloroform method (34).After isopropanol precipitation, the RNA solution was mixed with 2-butoxyethanol and purified by ethanol precipitation.Total RNAs were separated by 10 wt/vol % denaturing PAGE containing 7 M urea, and bands correspond ing to total tRNAs were excised from the gel stained with 0.05 wt/vol % toluidine blue (Wako).Total tRNAs were eluted from the gel using elution buffer consisting of 0.3 M sodium acetate (pH 5.3), 1 mM EDTA-NaOH (pH 8.0), and 0.1 wt/vol % SDS, and filtered (Ultrafree-MC, HV, 0.45 µm; Merck Millipore).Total tRNAs were recovered by ethanol precipitation and desalted by drop dialysis on a nitrocellulose membrane (MFMillipore, Merck Millipore) against ultrapure water for 2 h.The principles underlying mass spectrometry analysis of nucleosides have been described previously (34,48).tRNA mixtures were digested with nuclease P1 (Wako) and bacterial alkaline phosphatase (BAP.C75, Takara Bio).The digest dissolved in acetonitrile was applied to a nanoflow high-performance LC-MS/MS system.The solvent system consisted of 5 mM ammonium acetate, pH 5.3 (solvent A) and acetonitrile (solvent B).Nucleotides were chromato graphed by a ZIC-cHILIC column (2.1 × 150 mm, Merck Millipore) with a multistep linear gradient of 90%-50% B from 0 to 30 min, 50% B for 10 min, and 50%-90% B from 40 to 45 min, then initialized to 90% B at a flow rate of 100 µL/min.The chromatographic eluent was sprayed directly into the ESI source of a Q Exactive Hybrid Quadrupole-Orbi trap Mass Spectrometer (Thermo Fisher Scientific).Ions were scanned in positive polarity mode over an m/z range of 110-900 throughout the separation.

Expression and purification of Ubls and TK2117 proteins
Plasmids used to overexpress the tk1065, tk1093, tk2118, and tk2117 genes in E. coli were constructed.Briefly, PCR-amplified DNA fragments encoding tk1065, tk1093, tk2118, and tk2117 genes generated using primer set Tk1065-pET-Fw/Tk1065-pET-Rv, Tk1093-pET-Fw/Tk1093-pET-Rv, Tk2118-pET-Fw/Tk2118-pET-Rv, and Tk2117-pET-Fw/Tk2117-pET-Rv were separately cloned into the NdeI/BamHI sites of the pET28a plasmid (Merck Millipore).E. coli BL21-CodonPlus (DE3)-RIL cells were used for protein expression.The recombinant E. coli cells were grown in 100 mL of Luria-Bertani medium containing 100 µg/mL of ampicillin at 37°C until the OD 600 reached 0.6.After induction with 1-mM isopropyl-β-D-thiogalactopyranoside for 4 h, cells were harvested by centrifugation, resuspended in buffer A containing Tris-HCl (pH 8) and 150 mM NaCl, and disrupted by sonication.The crude extract was incubated at 85°C for 20 min and centrifuged, and recombinant protein was purified from the supernatant to homogeneity (Fig. S8).The supernatant was applied to 5 mL Ni Sepharose high-performance resin (Cytiva) charged with nickel.The column was washed with buffer A containing 10 mM imidazole, and recombinant proteins were eluted with buffer A containing 200 mM imidazole.Imidazole was removed from the protein solution by a PD-10 desalting column (Cytiva).Protein concentrations were determined by the Bradford method with bovine serum albumin as a standard (68).

Immunoblotting
Extracts (30 µg) prepared from cells grown in S 0 medium were separated by SDS-PAGE and transferred to polyvinylidene difluoride membranes (ATTO).Immunodetection was performed with antibodies raised against purified TK1065, TK1093, and TK2118 in rabbit and goat anti-rabbit IgG-AP Conjugate (BioRad), and the signal was visualized using an ImageQuant LAS4000 instrument (Cytiva).

FIG 3
FIG 3 Quantification of the amount of molybdopterin in cells.(a) Molybdopterin was oxidized by iodine, and the derivatized FormA-phospho was detected and quantified by LC-MS.(b) Mass chromatograms of monovalent positive ions of FormA-phospho (m/z 328.1) extracted from T. kodakarensis KU216 cells grown in Pyr medium (upper) and extracted from xanthine oxidase for reference (middle).Co-injection analyses of the above (lower).FormA-phospho was eluted around 6.7 min and is indicated by an arrowhead.(c) Normalized amounts of FormA-phospho in each sample are shown.The mass spectrometry peak area values for FormA-phospho were normalized against their corresponding protein amounts in cell extracts.The value of the KU216 strain in Pyr medium was set to 100%.Means ± standard deviations of three independent measurements are shown.Statistical significance among the relative abundances of KU216 and the Ubl-deficient strains was determined using the Dunnett test.**P < 0.01.

FIG 4
FIG 4 Activities of Wco-containing enzymes and FNOR in Ubl-deficient strains of T. kodakarensis.The activities of Wco-containing enzymes and FNOR in cell extracts prepared from cells grown in S 0 (a), Pyr (b), and Mdx (c) media were measured at 85°C.Values are averages of triplicate experiments, and their statistical significance among the relative abundances of KU216 and the Ubl-deficient strains was determined using the Dunnett test.**P < 0.01.FNOR, ferredoxin:NADP + oxidoreductase; FOR, formate:ferredoxin oxidoreductase; N.D., not detected.

FIG 5
FIG 5 LC-MS/MS analysis of thiolated nucleosides in tRNA.The mass chromatograms show monovalent positive ions of the modified nucleosides of m 5 s 2 U (m/z 275.1) and m 5 U (m/z 259.1), cnm 5 s 2 U (m/z 300.1), and cnm 5 U (m/z 284.1) in total tRNAs prepared from cells of the T. kodakarensis KU216 strain, Ubl-deficient strains, and the ∆ubls strain grown in the absence (a and b) or presence (d and e) of S 0 .Relative abundance represents the relative intensity of each peak normalized by the intensity of 1-methylinosine.The values of m 5 s 2 U and cnm 5 s 2 U in KU216 strain were set to 100%, respectively.For m 5 U and cnm 5 U, the values in ∆ublA strain in Pyr medium was set to 100%.The results of LC-MS/MS analyses (a, b, d, and e) are displayed as bar graphs (c and f).Relative abundances of m 5 s 2 U and cnm 5 s 2 U in KU216 (gray), ∆ublC (cyan), ∆ublA (red), ∆ublB (blue), and ∆ubls (black) strains grown in the absence (c) or presence (f) of S 0 are shown.Means ± standard deviations of three independent measurements are shown.Statistical significance among the relative abundances of KU216 and the Ubl-deficient strains was determined using two-sided Student's t-test.*P < 0.05, **P < 0.01.

FIG 6
FIG 6 Proposed sulfur-transfer scheme for Wco and tRNA thionucleosides in T. kodakarensis.Arrows indicate sulfur flow to the target proteins or tRNA nucleosides.T. kodakarensis utilizes two alternative mechanisms, depending on environmental conditions, such as the absence or presence of S 0 .