Thioredoxin targets fundamental processes in a methane-producing archaeon, Methanocaldococcus jannaschii

Significance

This study extends thioredoxin (Trx)-based oxidative redox regulation to the archaea, the third domain of life. Our study suggests that Trx is nearly ubiquitous in anaerobic methanogens, enabling them to recover from oxidative stress and synchronize cellular processes, including methane biogenesis, with the availability of reductants. As methane is a valuable fuel, an end product of anaerobic biodegradation and a potent greenhouse gas, Trx may now be considered a critical participant in the global carbon cycle, climate change, and bioenergy production. Because methanogenesis developed before the oxygenation of the earth, our work raises the possibility that Trx functioned in a complex redox regulatory network in anaerobic prokaryotes at least 2.5 billion years ago.

Abstract

Thioredoxin (Trx), a small redox protein, controls multiple processes in eukaryotes and bacteria by changing the thiol redox status of selected proteins. The function of Trx in archaea is, however, unexplored. To help fill this gap, we have investigated this aspect in methanarchaea—strict anaerobes that produce methane, a fuel and greenhouse gas. Bioinformatic analyses suggested that Trx is nearly universal in methanogens. Ancient methanogens that produce methane almost exclusively from H₂ plus CO₂ carried approximately two Trx homologs, whereas nutritionally versatile members possessed four to eight. Due to its simplicity, we studied the Trx system of Methanocaldococcus jannaschii—a deeply rooted hyperthermophilic methanogen growing only on H₂ plus CO₂. The organism carried two Trx homologs, canonical Trx1 that reduced insulin and accepted electrons from Escherichia coli thioredoxin reductase and atypical Trx2. Proteomic analyses with air-oxidized extracts treated with reduced Trx1 revealed 152 potential targets representing a range of processes—including methanogenesis, biosynthesis, transcription, translation, and oxidative response. In enzyme assays, Trx1 activated two selected targets following partial deactivation by O₂, validating proteomics observations: methylenetetrahydromethanopterin dehydrogenase, a methanogenesis enzyme, and sulfite reductase, a detoxification enzyme. The results suggest that Trx assists methanogens in combating oxidative stress and synchronizing metabolic activities with availability of reductant, making it a critical factor in the global carbon cycle and methane emission. Because methanogenesis developed before the oxygenation of Earth, it seems possible that Trx functioned originally in metabolic regulation independently of O₂, thus raising the question whether a complex biological system of this type evolved at least 2.5 billion years ago.

Thioredoxins (Trxs) are small (∼12-kDa) redox proteins typically bearing a characteristic Cys-Gly-Pro-Cys motif that reduce specific disulfide bonds of selected proteins (1). Reduction alters the biochemical properties of the proteins targeted—e.g., by increasing their activity or solubility (1). Trxs are found in the three domains of life: bacteria, eukarya, and archaea (2). In eukarya and bacteria, the regulatory role of Trx has been shown to span the major aspects of metabolism, including photosynthesis, biosynthesis, replication, transcription, translation, and stress response (1). Trx also acts as an electron donor for enzymes, notably ribonucleotide reductase, phosphoadenosinephosphosulfate reductase, methionine sulfoxide reductase, and peroxiredoxins (1). However, in contrast to the wealth of information for bacteria and eukaryotes, our understanding of archaeal Trx is limited to its biochemical and structural properties (3–9). Its physiological role remains a mystery.

To help fill this gap, we have investigated the role of Trx in a group of archaea known as methanogens or methanarchaea—strict anaerobes that produce methane, a prominent greenhouse gas and important fuel. We have focused on Methanocaldococcus jannaschii—a deeply rooted, hyperthermophilic methanogen living in deep-sea hydrothermal vents (10) where conditions mimic those of early Earth. M. jannaschii produces methane exclusively from H₂ and CO₂ via a process believed to represent an ancient form of respiration (11). M. jannaschii thus presents an opportunity to explore the role of Trx in an archaeon and, at the same time, gain insight into the evolutionary history of redox regulation. Our results suggest that Trx alleviates oxidative stress in methanogens via a thiol-based mechanism that could also regulate fundamental processes by redox transitions in the absence of O₂. The role formulated for this anaerobic archaeon confirms and extends that established for aerobic forms of life.

Results

Thioredoxin Homologs of Methanarchaea.

Iterative BLAST searches (12) using Escherichia coli and M. jannaschii Trxs as queries and screening output for hits with the C-X-X-C motif and appropriate sizes of 70- to 110-aa residues (13) showed that Trx homologs exist in almost all methanogen genomes represented in the National Center for Biotechnology Information (NCBI) database (Fig. 1 and Table S1). Methanopyrus kandleri AV19, a hydrothermal vent-associated hyperthermophilic methanogen (optimum growth temperature, 98 °C), was apparently the only exception in lacking a recognizable homolog of Trx (14).

Fig. 1.

Distribution of thioredoxin homologs in methanogens. A 16S-ribosomal RNA gene-based maximum-likelihood phylogenetic tree constructed as described previously (18) provides a platform for this presentation. Black dots at the branches, confidence values ≥700 (out of 1,000 replicates). Scale bar, number of base substitution per site. The 16S-rRNA gene of Desulfurococcus fermentans (not shown) was used as outgroup. *Abbreviations: IPA and IBA, isopropanol and isobutanol; Me, methanol and mono-, di-, and trimethylamines; Me-H, methanol + H₂; Me-S, dimethylsulfide, and methanethiol. ^†Not detected via BLAST searches.

Methanococci and Methanobacteria carried an average of two Trx homologs, with their numbers ranging from one to four, whereas Methanomicrobia possessed two to eight Trx homologs, with an average of four. Methanocorpuscullum labreanum, a member of the latter class, was an exception in possessing two Trx homologs.

Trxs of M. jannaschii.

M. jannaschii (Mj) carries two Trx homologs, Mj_0307 and Mj_0581 (9, 15), here called Trx1 and Trx2, respectively. The sequence identity and similarity between Trx1 and Trx2 are 23% and 49%, respectively. Both proteins have homologs in Methanothermobacter thermautotrophicus ΔH (7, 8), where Trx1 is closely related to MTH807 (identity, 51%; similarity, 67%) and Trx2 corresponds to MTH895 (identity, 37%; similarity, 54%). Purified recombinant Trx1 and Trx2 were reduced by dithiothreitol (DTT) (Fig. S1A). However, the proteins were distinct in two well-characterized activities in which Trx1 exhibited a closer resemblance to E. coli Trx, a standard in the field. First, in the insulin reduction assay, Trx1 showed 80-fold higher activity than Trx2, and Trx2 exhibited a longer lag, 35 vs. 10 min for Trx1 (Fig. S1B). Second, Trx1 but not Trx2 was reduced by E. coli nicotinamide adenine dinucleotide phosphate (NADP)-thioredoxin reductase (Ec-NTR) with NADPH. It is noteworthy that the homolog of Trx2, M. thermautotrophicus ΔH (MTH895), unlike the M. jannaschii protein, accepts electrons from E. coli NTR (8).

Identification of Trx1 Targets.

A fluorescent gel/proteomics approach that proved successful in several plant investigations (1, 16) was used to identify the M. jannaschii proteins reduced by Trx1 (Trx1 targets). Briefly, in this procedure, M. jannaschii cell extracts were oxidized by aerobic dialysis, and the remaining free sulfhydryl groups of the air-exposed proteins were blocked by alkylation. The extract was then treated with Trx1 using either DTT or NADPH (plus E. coli NTR) as reductant, anticipating that Trx1 would reduce the regulatory disulfide (S–S) groups formed in aerobic dialysis. The newly available free –SH groups were derivatized with the fluorescent probe monobromobimane (mBBr), and the labeled proteins were resolved in 2D gels (Fig. S2 A and B). The fluorescent spots, which were either absent or less intense in control gels, were analyzed by mass spectrometry (17). The experiment with DTT was performed in triplicate and that with Ec-NTR+NAPDH was performed once. From these experiments, we identified a total of 152 potential Trx1 targets (Table 1 and Table S2). Of these, 19 proteins were identified in all four experiments, and 18, 38, and 77 were detected in three, two, and one of the experiments, respectively.

Table 1.

Potential M. jannaschii Trx1 targets

Metabolic function or structural unit	Potential targets
ATP synthesis	V-type ATP synthase subunit A*, V-type ATP synthase subunit B*
Biosynthesis	Capsular polysaccharide biosynthesis protein, GMP synthase II, inosine-5′-monophosphate dehydrogenase I, orotate phosphoribosyltransferase-like protein, phosphoribosylaminoimidazole synthetase, phosphoribosylaminoimidazole-succinocarboxamide synthase, phosphoribosylformylglycinamidine synthase II, pyridoxal biosynthesis lyase PdxS, ribose-phosphate pyrophosphokinase, spermidine synthase*, uridylate kinase*
Coenzyme M biosynthesis	Phosphosulfolactate synthase
Defense against foreign DNA	Csm3 family CRISPR-associated RAMP protein
Hypothetical protein	Hypothetical protein MJ_0164, hypothetical protein MJ_0308, hypothetical protein MJ_1099
Metabolism	2-Oxoglutarate ferredoxin oxidoreductase subunit γ, acetyl-CoA decarbonylase/synthase complex subunit γ, fructose-bisphosphate aldolase*, fructose-1,6-bisphosphatase*, phosphoenolpyruvate synthase, phosphopyruvate hydratase (enolase)*, putative transaldolase*, pyruvate carboxylase subunit B, pyruvate ferredoxin oxidoreductase subunit α PorA*, UDP-glucose dehydrogenase*
Methanogenesis (energy generation)	F420-dependent methylenetetrahydromethanopterin dehydrogenase, formylmethanofuran–tetrahydromethanopterin formyltransferase, H2-dependent methylenetetrahydromethanopterin dehydrogenase, H2-dependent methylenetetrahydromethanopterin dehydrogenase-like protein I, H+-transporting ATP synthase subunit E AtpE, methyl coenzyme M reductase I subunit McrA, methyl coenzyme M reductase I subunit McrB, methylenetetrahydromethanopterin reductase, methylviologen-reducing hydrogenase subunit α, N5,N10-methenyltetrahydromethanopterin cyclohydrolase
Miscellaneous	AMMECR 1 domain protein, methanogenesis marker protein 17, methyltransferase, iron-sulfur flavoprotein
Nitrogen and amino acid metabolism	(R)-2-Hydroxyglutaryl-CoA dehydratase activator, 2-hydroxyglutaryl-CoA dehydratase, 3-dehydroquinate synthase, acetolactate synthase catalytic subunit*, anthranilate synthase component II TrpD, argininosuccinate synthase*, aspartate aminotransferase*, aspartate-semialdehyde dehydrogenase*, branched-chain amino acid aminotransferase, d-3-phosphoglycerate dehydrogenase*, dihydrodipicolinate reductase, dihydrodipicolinate synthase, dihydroxy-acid dehydratase*, ketol-acid reductoisomerase*, phosphoribosylformimino-5-aminoimidazole carboxamide ribotide, -isomerase HisA1, S-adenosylmethionine synthetase*
Oxidative stress response	Flavoprotein FpaA, NADH oxidase, peroxiredoxin*
Replication, transcription, and translation	30S ribosomal protein S7, 50S ribosomal protein L6, acidic ribosomal protein P0, arginyl-tRNA synthetase, cell division protein CDC48*, cell division protein FtsZ I*, elongation factor 1-α*, elongation factor EF-2, thermosome
Structural proteins	Flagella-like protein E, S-layer protein
Sulfite detoxification	F420-dependent sulfite reductase†
Transport proteins	High-affinity branched-chain amino acid transport protein BraC

Targets that were identified in at least two independent experiments are reported here. A more extensive list appears in Table S2.

^*Previously identified as Trx target in eukaryotic and bacterial systems.

^†A non–F₄₂₀-dependent sulfite reductase has been identified as a Trx target in certain bacteria and eukaryotes.

Effect of Reduction by Trx1 on the Activity of Selected M. jannaschii Enzymes.

F₄₂₀-dependent sulfite reductase.

An air-exposed 7,8-didemethyl-8-hydroxy-5-deazaflavin-5′-phosphoryllactyl glutamate [coenzyme F₄₂₀ (F₄₂₀)]-dependent sulfite reductase (Fsr) preparation showed two-thirds the activity observed with the corresponding anaerobic preparation, the respective values being 0.132 and 0.200 U/mg. Assay of oxidized Fsr with Trx1 (20 µM) at 65 °C in the presence of 1 mM DTT increased the activity of the enzyme by 2.9-fold (Fig. 2); with twice as much Trx1, activation was 4.6-fold. DTT alone (1 mM) inhibited the enzyme, and Trx1 alone activated Fsr 1.5-fold, likely due to a protein concentration effect.

Fig. 2.

Activation of F₄₂₀-dependent sulfite reductase or Fsr and F₄₂₀-dependent methylenetetrahydromethanopterin dehydrogenase or Mtd by Trx1. Fsr and Mtd were preincubated with Trx1, DTT, or both at 65 °C for 5 min followed by an additional incubation at 25 °C for 20 min and then assayed for activity. Enzyme without a treatment was used as the control. Solid bar, an average of values from replicates (three independent experiments for Fsr and two for Mtd). Error bar, SD. Number on a solid bar, fold of activation. Label below a bar, reagents used for treatment.

F₄₂₀-dependent methylenetetrahydromethanopterin dehydrogenase.

Nonreducing SDS/PAGE developed with a monobromobimane (mBBr)-treated preparation revealed that purified recombinant F₄₂₀-dependent methylenetetrahydromethanopterin dehydrogenase (Mtd) was recovered mostly in reduced form. To generate potential redox active cystine disulfides, the enzyme was treated with several oxidants, H₂O₂, CuCl₂, and Aldrithiol-2. Based on mBBr gel analysis, Aldrithiol-2 proved most effective in oxidizing Mtd. This oxidation deactivated the enzyme by 53%. A treatment of the deactivated enzyme with fivefold molar excess Trx1 and 0.05 mM DTT yielded a 4.4-fold increase in activity vs. a 1.4-fold enhancement seen with DTT alone (Fig. 2).

Discussion

Distribution of Trx Homologs in Methanogens.

The evidence presented above suggests that Trx homologs are nearly universal in methanogenic archaea. M. kandleri, the most deeply rooted and the most thermophilic methanogen known (growth occurs at 84–110 °C), was the only exception. This organism, which is solely dependent on H₂ and CO₂ for methanogenesis (14), lacked a recognizable homolog of Trx. In the other methanogens, the distribution of Trx homologs followed a pattern (Fig. 1 and Table S1). Phylogenetically deeply rooted representatives belonging to the classes of Methanococci and Methanobacteria carried a limited number of Trx homologs (two to four; two on average). These organisms have relatively smaller genomes (1.24–2.94 Mbp; NCBI data), include almost all hyperthermophilic or thermophilic methanogens (19), and are mostly restricted to H₂-dependent methanogenesis (19). By contrast, the late-evolving Methanomicrobia with larger genomes (1.8–5.75 Mbp; NCBI data) and more complex metabolism carried up to eight Trxs (on average, four). The methylotrophic Methanomicrobia use methanol and methylamines, and some of these perform methanogenesis from acetate as well as H₂ and CO₂ (19, 20); most are mesophiles and relatively O₂ tolerant.

It is possible that the Trx system came into play in the deeply rooted methanogenic archaea as these organisms faced a more oxidizing environment brought about by H₂ limitation or O₂ exposure. It remains to be seen whether the larger number of Trxs in late-evolving methanogens is a result of horizontal gene transfer or gene duplication coupled with subsequent diversification. We note that these organisms’ ability to use a range of methanogenic substrates is thought to be due to a large number of genes acquired from the Clostridia and other anaerobes (21).

M. jannaschii Trxs.

Trx1 and Trx2 were distinct in terms of amino acid sequence, reactivity with insulin, and activity with E. coli NTR. These features are possibly related to the nature of the putative redox active site motif C-X-X-C as the two internal residues (X’s) influence the redox properties of the protein (22). In Trx1 and Trx2, this motif is, respectively, C-P-H-C and C-P-K-C, which differ from each other and from the classical C-G-P-C (21). It is thus not surprising that Trx1 and Trx2 showed different specificities. Trx1 was typical—i.e., similar to its E. coli counterpart in primary structure, robust insulin reduction activity, and reduction by E. coli NTR. This protein was, therefore, chosen for identifying candidate Trx target proteins in M. jannaschii.

Targets of Trx1.

Proteomics and enzyme activity measurements suggested that Trx1 influences multiple processes in M. jannaschii, including methanogenesis—the hallmark of the methanogens. Our proteomics analysis revealed a total of 152 M. jannaschii polypeptides as potential Trx1 targets, representing ∼10% of the total ORFs in the organism’s genome (Table S2). Of these, 75 targets were detected in at least two of four independent experiments (Table 1) and more than one-half were observed only once. As shown in Table S2, most of the targets contain at least two Cys residues, indicative of Trx-reducible intramolecular or intermolecular Cys disulfide bonds (Table S2). Curiously, a few of the targets have only one Cys, raising the possibility that in these instances Trx reduces intermolecular disulfides as described for yeast 1-Cys peroxiredoxin (23). The putative peroxiredoxin of M. jannaschii (MJ_0736), however, contains five Cys.

The Trx1 target proteins participate in multiple processes in addition to methanogenesis: biosynthesis, information processing, cell division, sulfite detoxification, oxidative response, and resistance to phages and invasion by foreign DNA. Structural proteins were also identified as Trx1 targets. The results reveal the obvious vulnerability of an ancient methanogen cell to oxidative stress and the suitability of Trx for repairing the resulting damage.

To confirm and extend the proteomics results, we tested the effect of reduced Trx1 on the in vitro activity of two candidate target enzymes: Mtd, a core enzyme of the methanogenesis pathway (24), and Fsr (25), an enzyme that enables certain methanogens to tolerate and use sulfite as a source of sulfur. Oxidized forms of both enzymes were activated by Trx1, giving further credence to the fluorescent/gel approach of target identification. Mtd and Fsr were selected based on two criteria: being specific to methanarchaea and the availability of assay tools in our laboratories.

M. jannaschii Systems Targeted by Trx1.

Methanogenesis.

It is significant that many of the enzymes identified as Trx targets function in the reduction of CO₂ to CH₄ (Fig. 3, Table 1, and Table S2). Based on the nature of our experiments, this observation suggests that, reminiscent of plants, Trx activates enzymes of M. jannaschii that have been deactivated following O₂ exposure (1, 26). The archaeon may use a similar mode of action of Trx to regulate the activity of selected enzymes, thereby synchronizing metabolism with the availability of reductant such as H₂ under normal anaerobic conditions. In the deep-sea hydrothermal vents M. jannaschii inhabits, changes in partial pressure of H₂ can be extreme (4 Pa to 200 kPa) (19, 25, 27, 28) and exposure to O₂ may occur following the entry of aerobic seawater (27, 28).

Fig. 3.

Select reactions and pathways of M. jannaschii targeted by Trx1 (Mj_0307). The methanogenesis pathway was redrawn from ref. 23. Color codes: red and green, enzymes identified as Trx1 targets in two or more and one experiment(s), respectively; blue, not targeted by Trx1. The dashed arrows show extended biosynthetic routes. 1,3-BPG, 1,3-bisphosphoglycerate; [CO], enzyme-bound carbon monoxide (CO); CoB, coenzyme B; CoM, coenzyme M; DHAP, dihydroxyacetone phosphate; Ech, energy-converting hydrogenase; F₄₂₀, coenzyme F₄₂₀; FBP aldolase, fructose bisphosphate aldolase; FBPase, fructose bisphosphatase; *Fd, specific ferredoxin; Frd, fumarate reductase; Ftr, formylmethanofuran-H₄MPT formyltransferase; Fwd and Fmd, tungsten- and molybdenum-dependent formylmethanofuran dehydrogenase; Gapdh, glyceraldehyde-3-phosphate dehydrogenase; GD3P, glyceraldehyde-3-phosphate; H₄MPT, tetrahydromethanopterin; Hdr-H₂ase, electron-bifurcating hydrogenase-heterodisulfidereductase complex; HS-CoA, CoA; α-Kgfor and Pfor, α-ketoglutarate- and pyruvate-ferredoxin oxidoreductase; Mch, methenyl-H₄MPT cyclohydrolase; Mcr, methyl-coenzyme M reductase; Mdh, malate dehydrogenase; Mer, methylene-H₄MPT reductase; MF, methanofuran; Mtd and Hmd, F₄₂₀- and H₂-dependent methylene-H₄MPT dehydrogenase; Mtr, methyl-H₄MPT-coenzyme M methyltransferase; Δμ Na⁺, electrochemical sodium ion potential; PEP, phosphoenolpyruvate; 3-PG and 2-PG, 3- and 2-phosphoglycerate; Pgi, phosphoglycerate isomerase; Pgk, phosphoglycerate kinase; 2-Pgm, 2-phosphoglycerate mutase; Pps, phospoenolpyruvate synthase; Pyc, pyruvate carboxylase; Sdh, succinate dehydrogenase; Tpi, triose phosphate isomerase.

The expression of several methanogenesis-related genes in M. jannaschii and other H₂-oxidizing methanogens is transcriptionally regulated by H₂ availability, and our observations suggest the presence of parallel posttranslational control effected by Trx (Table 1 and Table S2; Fig. 2). This possibility is also consistent with the proposal that Mtd is the primary enzyme for the reduction of methylenetetrahydromethanopterin under H₂ limitation (29, 30) and with our finding of a direct effect of Trx on the activity of the enzyme. Significantly, the alternate H₂-dependent enzyme [H₂-dependent methylenetetrahydromethanopterin dehydrogenase (Hmd)], also a potential Trx1 target, is active under high H₂ partial pressure (31). In view of these results, it is timely to determine whether Trx regulates the activity of Hmd and other methanogenesis enzymes identified as targets (Table 1 and Table S2) as well as counterparts in terrestrial systems where O₂ exposure and changes in reductant supply are common (19, 20). The ability to down-regulate methanogenesis, the sole avenue for energy generation, via the oxidation of sulfhydryl groups would enable methanogens to attain a dormant-type state. With the return of favorable environmental conditions, Trx could reactivate target methanogenesis enzymes via disulfide reduction and thereby restore growth and other vital cell processes. This situation resembles the role of Trx in initiating processes associated with seed germination (32). Modification of F₄₂₀ via adenylation and guanylation provides an alternate avenue for enabling a methanogen to shut down energy production and achieve a dormant state (33).

Sulfite detoxification.

Identified as a target in proteomics studies (Table S2), Fsr was deactivated upon exposure to O₂, and the altered enzyme was partially reactivated by reduced Trx1 (Fig. 2). These observations make physiological sense. Sulfite inhibits methyl-coenzyme M reductase and, thereby, impedes methanogenesis (25). In the habitat of M. jannaschii, sulfite is formed when O₂-containing cold seawater mixes with the hot sulfide-rich vent fluid (25). Fsr detoxifies the newly formed sulfite by reducing it to sulfide, an essential nutrient for methanogens (25). Because sulfite can oxidize protein sulfhydryl (SH) groups to the disulfide (S–S) level (34), it is not surprising that oxidatively deactivated Fsr can be reductively activated by Trx1. It is possible that activation by Trx is a general feature of sulfite reductases as the enzyme of wheat starchy endosperm appears also to be a Trx target (35).

Biosynthesis.

The de novo synthesis of acetate and pyruvate from CO₂ are key initial anabolic steps for an autotroph such as M. jannaschii (10). It is significant that the two enzymes of this process, acetyl-CoA decarbonylase/synthase (ACDS) and pyruvate:ferredoxin oxidoreductase (PFOR), were both identified as Trx1 targets (Table 1 and Fig. 3). Trx is known to revive oxidatively damaged PFOR in Desulfovibrio africanus, an anaerobic sulfate reducing bacterium (36, 37). However, the situation may be different in M. jannaschii as PFOR and ACDS of methanogens have been reported to be irreversibly inactivated by O₂ exposure in vitro (38, 39). In methanogens, the role of Trx could lie in recovery from less severe O₂ exposure or in redox regulation of activity. Methanogens could invoke the latter in response to a drop in H₂ partial pressure—a fact of life in most, if not all, of their natural habitats (19, 20, 25, 27, 28). In these cases, the prevailing midpoint potential values of H⁺/H₂ redox couple would make the formation of protein disulfides thermodynamically feasible. Because the level of coenzyme F₄₂₀ in a methanogen cell is in equilibrium with environmental H₂ partial pressure (40, 41), this mechanism of sulfhydryl oxidation could be performed by an F₄₂₀-dependent enzyme acting directly or via an intermediary.

The synthesis of sugars via gluconeogenesis is an energy-intensive pathway that requires a reductant. In eukarya, many of the associated enzymes are linked to Trx (26). Significantly, several of their M. jannaschii counterparts were also identified as potential Trx targets (Fig. 3; Table 1 and Table S2), suggesting that gluconeogenesis could also be redox regulated in this organism. Following this same theme, several enzymes of amino acid biosynthesis reported to be Trx targets in plants (26)—notably, glutamine synthetase, threonine synthase, and aspartate semialdehyde dehydrogenase were reduced by Trx1 in M. jannaschii extracts. Phosphosulfolactate synthase, an enzyme needed for the biosynthesis of coenzyme M—a requirement for methane formation with all substrates (42), also appeared to be linked to Trx1 (42). A role in coenzyme M synthesis falls within the broader function of Trx in the repair and regulation of the methanogenesis process.

Transcription, translation, and cell division.

Transcription and translation have previously been linked to Trx-based regulation in Bacteria and Eukarya (1). Modification of the RNA polymerase ω subunit and elongation factors in E. coli (43) and several chloroplast ribosomal proteins fall in this category (26). Similar controls likely exist in M. jannaschii where a ribosomal protein S7 and several tRNA synthetases were identified as Trx1 targets (Table 1 and Table S2).

Like its counterparts in chloroplasts and E. coli (16, 43), FtsZ, a cytoskeletal protein similar to tubulin in eukaryotes (44), was reduced by Trx1 in M. jannaschii (Table 1 and Table S2). Thus, as with E. coli, Trx acting through FtsZ could contribute to the regulation of cell division in this organism.

Structural proteins.

One of the two S-layer proteins, which are major cell envelope components in methanogens (45), was reduced by Trx1. Interestingly, the level of S-layer protein decreases under H₂ limitation in Methanococcus maripaludis, a close relative of M. jannaschii (46). Considering that Trx is a posttranslational modifier, it is possible that both the generation and assembly of the S-layer is redox controlled in methanogens.

Defense against reactive oxygen species and foreign DNA.

Similar to chloroplasts (16), a peroxiredoxin was identified as a Trx target in M. jannaschii (Table 1 and Table S2). Peroxiredoxins are critical antioxidant enzymes catalyzing the reduction of hydroperoxides and alkyl hydroperoxides to water and respective alcohols (1). Three clustered regularly interspaced short palindromic repeats (CRISPR)-associated proteins, namely Csm 2-, 3-, and 5-family proteins, were also targeted by Trx1 (Table 1 and Table S2). CRISPR elements and associated proteins provide defense against invasion by external DNA materials such as plasmids and phage in archaea as well as bacteria (47), raising the possibility that this process is regulated by Trx in M. jannaschii.

Concluding Remarks

The present work extends Trx-based redox transition to the third domain of life. The methanarchaeon M. jannaschii was found to use this protein to protect a range of cellular processes against oxidative damage. Interestingly, many of the Trx targets identified have counterparts in plants where an oxidative type of regulation is known to occur (16, 48).

The present findings have far-reaching implications to our understanding of the evolution of redox regulation as well as to areas of current societal interest. Because M. jannaschii performs hydrogenotrophic methanogenesis, a process that developed before the appearance of O₂ (11, 49), Trx may have originally functioned in an anaerobic regulatory capacity in this ancient organism. Its participation in protecting cells against O₂ would have developed later. Accordingly, the redox network created by Trx, and the attendant cellular complexity, would have developed in prokaryotes at least 2.5 billion years ago. Future research will be directed toward this question. On the pragmatic side, due to the role of methanogens in producing methane and the attendant changes in the biosphere, Trx emerges as a key participant in the global carbon cycle, climate change, and bioenergy production.

Materials and Methods

Purified Preparations of M. jannaschii Trxs, Mtd, and Fsr, and Methanogen Cofactors, and Insulin Reduction Assay.

Previously described methods were used for generating homogeneous preparations of recombinant His-tagged Trx and Mtd (50) and F₄₂₀ (51), partial purification of Fsr from M. jannaschii cell extracts (25), and the insulin assay for Trx (52).

Trx-Mediated Reduction of M. jannaschii Cell Extract Proteins, 2D Gel Electrophoresis, and Mass-Spectrometric Analysis.

Cell-free extracts of M. jannaschii (25) were oxidized and treated with thiol reagents as described in SI Materials and Methods. Methods for reducing this preparation with Trx, fluorescent labeling, and identifying the potential Trx targets are also given in SI Materials and Methods.

Activation and Activity Assay for Mtd and Fsr.

Fsr was oxidized by aerobic dialysis and Mtd via a reaction with an oxidant, H₂O₂, CuCl₂, or Aldrithiol-2. Oxidized preparations were activated by anaerobic incubation in the following mixtures: Fsr: 14 μg of partially purified enzyme in a 200-µL solution containing 50 mM potassium phosphate buffer (pH 7.0), 100 mM KCl, 20 μM Trx1, and 1 mM DTT; Mtd: homogenous enzyme (1 μM) in a solution containing 100 mM potassium phosphate buffer (pH 7.0), 0.5 M KCl, 5 μM Trx1, and 0.05 mM DTT. Fsr activity was assayed as previously (25), except 100 mM KCl was added to the assay mixture. For Mtd, a previously described assay (53) was modified by replacing tetrahydromethanopterin with tetrahydrosarcinapterin (a gift from Dr. D. Grahame, Uniformed Services University of the Health Sciences, Bethesda, MD), changing the phosphate buffer concentration (to 100 mM, pH 7.0) and including KCl (0.5 M). KCl enhanced the activities of Fsr and Mtd.