Methanosarcina Acetivorans C2A Research Articles

Chemical Dynamics of Selenium Nanoparticles in Archaeal Systems.

Methanogenic archaea, characterized by their cell membrane lipid molecules consisting of isoprenoid chains linked to glycerol-1-phosphate via ether bonds, exhibit exceptional adaptability to extreme environments. However, this distinct lipid architecture also complicates the interactions between methanogenic archaea and nanoparticles. This study addresses this challenge by exploring the interaction and transformation of selenium nanoparticles (SeNPs) within archaeal Methanosarcina acetivorans C2A. We demonstrated that the effects of SeNPs are highly concentration-dependent, with chemical stimulation of cellular processes at lower SeNPs concentrations as well as oxidative stress and metabolic disruption at higher concentrations. Notably, we observed the formation of a protein corona on SeNPs, characterized by the selective adsorption of enzymes critical for methylotrophic methanogenesis and those involved in selenium methylation, suggesting potential alterations in protein function and metabolic pathways. Furthermore, the intracellular transformation of SeNPs into both inorganic and organic selenium species highlighted their bioavailability and dynamic transformation within archaea. These findings provide vital insights into the nano-bio interface in archaeal systems, contributing to our understanding of archaeal catalysis and its broader applications.

A hybrid photocatalytic system enables direct glucose utilization for methanogenesis

Integration of methanogenic archaea with photocatalysts presents a sustainable solution for solar-driven methanogenesis. However, maximizing CH4 conversion efficiency remains challenging due to the intrinsic energy conservation and strictly restricted substrates of methanogenic archaea. Here, we report a solar-driven biotic-abiotic hybrid (biohybrid) system by incorporating cadmium sulfide (CdS) nanoparticles with a rationally designed methanogenic archaeon Methanosarcina acetivorans C2A, in which the glucose synergist protein and glucose kinase, an energy-efficient route for glucose transport and phosphorylation from Zymomonas mobilis, were implemented to facilitate nonnative substrate glucose for methanogenesis. We demonstrate that the photo-excited electrons facilitate membrane-bound electron transport chain, thereby augmenting the Na+ and H+ ion gradients across membrane to enhance adenosine triphosphate (ATP) synthesis. Additionally, this biohybrid system promotes the metabolism of pyruvate to acetyl coenzyme A (AcCoA) and inhibits the flow of AcCoA to the tricarboxylic acid (TCA) cycle, resulting in a 1.26-fold augmentation in CH4 production from glucose-derived carbon. Our results provide a unique strategy for enhancing methanogenesis through rational biohybrid design and reprogramming, which gives a promising avenue for sustainably manufacturing value-added chemicals.

In Situ Probing of the Intrinsic Adhesion Strength of Single Anaerobic Microbial Cells.

Probing the single-cell mechanobiology in situ is imperative for microbial processes in the medical, industrial, and agricultural realms, but it remains a challenge. Herein, we present a single-cell force microscopy method that can be used to measure microbial adhesion strength under anaerobic conditions in situ. This method integrates atomic force microscopy with an anaerobic liquid cell and inverted fluorescence microscopy. We obtained the nanomechanical measurements of the single anaerobic bacterium Ethanoligenens harbinense YUAN-3 and the methanogenic archaeon Methanosarcina acetivorans C2A and their nanoscale adhesion forces in the presence of sulfoxaflor, a successor of neonicotinoid pesticides. This study presents a new tool for in situ single-cell force measurements of various anoxic and anaerobic species and provides new perspectives for evaluating the potential environmental risk of neonicotinoid applications in ecosystems.

Single bubble probe atomic force microscope and impinging-jet technique unravel the interfacial interactions controlled by long chain fatty acid in anaerobic digestion

Anaerobic digestion of lipid-rich wastewater generally suffers from foaming induced by long chain fatty acid (LCFA). However, a systematic understanding of LCFA inhibition, especially the physical inhibition on interfacial interaction still remains unclear. Here, we combined bubble probe atomic force microscope and impinging-jet technique to unravel the interfacial interactions controlled by long chain fatty acids in anaerobic digestion. We showed that LCFA had a significant inhibition on methane production in anaerobic reactors for the inhibition of the conversion of VFAs to methane. By measuring the LCFA influence on methanogenic archaea Methanosarcina acetivorans C2A, the results demonstrated that methanogenesis was limited for substrates utilization but not metabolic pathways. The impinging-jet technique results indicated that LCFA enhanced bubble separation from anaerobic granules and reduced the bubble-bubble coalescence probability. In addition, the bubble probe atomic force microscope (AFM) revealed that LCFA enhanced the adhesion force between bubbles by enhancing electrical double layer (EDL) repulsion and decreasing hydrophobic interactions. Overall, these results complement framework of LCFA inhibition in anerobic digestion and provide a nanomechanical insight into the fundamental interfacial interactions related to bubbles in anaerobic reactors.

The surface groups of polystyrene nanoparticles control their interaction with the methanogenic archaeon Methanosarcina acetivorans

A better understanding of the interaction between nanoplastics and archaea is crucial to fill the knowledge gaps regarding the ecological safety of nanoplastics. As a vital source for global methane emissions, methanogenic archaea have unique cell membranes that are distinctly different from those in all other forms of life, little is known about their interaction with nanoplastics. Here, we show that polystyrene nanoparticles functionalized with sulfonic acid (PS-SO3H) and amino (PS-NH2) interact with this methanogenic archaeon in distinct ways. Although both of them have no significant phenotype effects on Methanosarcina acetivorans C2A, these nanoparticles could affect DNA-mediated transposition of this methanogenic archaeon, and PS-SO3H also downregulated nitrogen fixation, nitrogen cycle metabolic process, oxidoreductase activity, etc. In addition, both nanoplastics decreased the protein contents in the extracellular polymer substances (EPS), with distinct binding sequences to the functional groups of the EPS. The single particle atomic force microscopy revealed that the force between the amino group and the M. acetivorans C2A was greater than that of sulfonic acid group. Our results exhibit that the surface groups of polystyrene nanoparticles control their risk on the methanogenic archaea, and these effects might influence their contribution on global methane emission.

Effect of different concentrations of sodium selenite on anaerobic digestion of waste sewage sludge

The effect of sodium selenite on anaerobic digestion of waste sewage sludge (WSS) was studied. Interestingly, methane production was enhanced at 2 mg/L and inhibited at 100 mg/L of sodium selenite during the anaerobic digestion of WSS. Hydrolytic activity increased in correlation with increased concentrations of sodium selenite (0 to 100 mg/L). At the acidogenesis stage, acetic acid accumulated at the concentrations more than 25 mg/L of sodium selenite whereas it was consumed for 10 days at the concentration of 2 mg/L of sodium selenite. Also, the concentration of propionic acid decreased and that of butyric acid increased at 100 mg/L of sodium selenite. Although methanogenic activities were totally inhibited at 100 mg/L of sodium selenite, a high hydrogenotrophic methanogenic activity was observed at 2 mg/L of sodium selenite. The abundance of hydrogenotrophic methanogens ( Methanospirillum and Methanocorpusculum ) increased in the WSS at 2 mg/L of sodium selenite. Moreover, the results of pure-culture experiments using Methanosarcina acetivorans C2A show that the growth promotion or inhibition was observed at 2 mg/L or at 100 mg/L of sodium selenite. In conclusion, sodium selenite has the double-sided effect during the anaerobic digestion of WSS as shown by the results that methane production was promoted at a low concentration and inhibited at a high concentration. • Sodium selenite affects methane production through the anaerobic digestion of sewage sludge. • Methanogenic activities were totally inhibited at 100 mg/L of sodium selenite. • Sodium selenite at 2 mg/L enhanced the activity of hydrogenotrophic methanogenesis. • The growth of Methanosarcina acetivorans C2A was promoted at 2 mg/L and inhibited at 100 mg/L of sodium selenite.

Bioaugmentation with methanogens cultured in a micro-aerobic microbial community for overloaded anaerobic digestion recovery

Anaerobic digestion (AD) is widely used for conversion of waste materials into biogas, but inhibition of methane production caused by overloading can be a major problem. The micro-aerobic microbial community MC1 was used to successfully culture methanogens, Methanosarcina acetivorans C2A and Methanosaeta thermophila NBRC 101360. The maximum 16S rRNA gene concentrations of Methanosarcina acetivorans C2A and Methanosaeta thermophila NBRC101360 were 1.06 × 106 and 1.35 × 103 copies/mL, respectively. The five key bacteria in MC1 were quantified to assess the effect of inoculation on the abundances of the bacteria in the mixed culture. The original MC1 total 16S rRNA gene concentration was 1.93 × 108 copies/mL, and the total 16S rRNA gene concentration had increased to 4.79 × 109 copies/mL on day 9 (p < 0.05). The proportions of the key strains in MC1+MST had changed by day 9. Cells were harvested and used to bioaugment and increase the pH values of the high- and medium-temperature anaerobic systems. After bioaugmentation, thermophilic AD recovered well. The cumulative amounts of gas produced were 44.78% and 28.28% higher in the MC1+MST and MC1 groups, respectively, than the sterilized control. The MC1+MST group gave better results than the chemical addition control group (CaCO3). There was no clear effect of bioaugmentation in mesophilic AD. When compared with traditional pure culture of methanogens as inoculants, methanogen cultivation in MC1 was simple and there was no need to separate and purify the target strains. This simplified methanogenic bioaugmentation agent was useful to study the mechanism of bioaugmentation for the recovery from low pH inhibition, showing the potential for practical application.

Impact of 5-fluorouracil on anaerobic digestion using sewage sludge

The role of bacterial interaction is vital to control bacterial functions; however, it has not been fully understood in microbial consortia (including anaerobic digestion). In this study, fluorouracil (FU), which is an anticancer agent and a quorum sensing (QS) inhibitor to some of the Gram-negative bacteria was found to inhibit methane production from sewage sludge under anaerobic conditions, as shown in a result where methane production in the presence of FU was eight times lower than the control (sewage sludge without FU). Whereas FU did not influence the hydrolysis process, in the acidogenesis/acetogenesis process, butyrate, and acetate accumulated in samples with FU. Also, in the methanogenesis process, FU remarkably inhibited methane production by acetoclastic methanogens rather than by the hydrogenotrophic ones. This result agreed with the result that growth and methane production of Methanosarcina acetivorans C2A was inhibited in the presence of FU. However, the inhibitory effect of FU was high in the condition that both bacteria and archaea were active. It indicates that FU influences methanogens and bacteria in the process of methane fermentation. The analyses of microbial communities (bacteria and archaea) showed that the abundance ratio of the Firmicutes phyla is high, and hydrogenotrophic methanogens become dominant in the presence of FU. Conversely, the abundance of Spirochaetes significantly decreased under FU. The inhibition of methane production by FU was due to the growth inhibition of methanogenic archaea and the changes in the composition of the bacterial population.

Effect of sodium tungstate on anaerobic digestion of waste sewage sludge: Enhanced methane production via increased acetoclastic methanogens

In this study, the effect of sodium tungstate on anaerobic digestion of waste sewage sludge (WSS) was investigated. Methane production was enhanced by sodium tungstate in a dose-dependent manner. Sodium tungstate did not affect the hydrolysis stage as the same level of protease activity was detected in the WSS sample with or without sodium tungstate. At the acidogenesis stage, the concentration of acetate decreased during the anaerobic digestion of WSS supplemented with sodium tungstate whereas that of isobutyrate increased. The pH of WSS increased in the presence of sodium tungstate, correlating with the result of acetate consumption with time during the anaerobic digestion of WSS. The analyses of methanogenesis via acetoclastic and hydrogenotrophic methanogens indicate that methane production from acetate was high in the WSS with sodium tungstate. In addition, the addition of sodium tungstate to the initial fresh WSS did not enhance methane production from acetate; therefore, sodium tungstate may increase the number of acetoclastic methanogens rather than promote the catalytic reaction related to the methane production. The analyses of archaeal population supported that acetoclastic methanogens (mostly Methanosaeta) increased with time in the WSS with sodium tungstate. In detail, when compared to the control WSS, the percentage of Methanosaeta significantly increased from 3.02% to 31.20% in the presence of sodium tungstate. Furthermore, the addition of sodium tungstate enhanced the growth of Methanosarcina acetivorans C2A. Taken together, sodium tungstate increases methane production via the anaerobic digestion of WSS by increasing the activity of acetoclastic methanogenesis.

Insights on the inhibition of anaerobic digestion performances under short-term exposure of metal-doped nanoplastics via Methanosarcina acetivorans.

Anaerobic digestion is an attractive waste treatment technology, achieving both pollution control and energy recovery. Though the inhibition of polystyrene nanoplastics in anaerobic granular sludge is well studied, no direct evidence has been found on the interaction of methanogens and nanoplastics. In this study, to characterize the location of nanoplastics, Pd-doped polystyrene nanoplastics (Pd-PS) were used to explore the inhibition mechanism of anaerobic sludge through short-term exposure to Methanosarcina acetivorans C2A. The results showed that Pd-PS inhibited the methanogenesis of the anaerobic sludge, and the methane production decreased as the Pd-PS increased, with a 14.29% reduction at the Pd-PS concentration of 2.36×1010 particles/mL. Also, Pd-PS interacted with the protein in the extracellular polymeric substances (EPS). Furthermore, Pd-PS inhibited the methanogenesis of M.acetivorans C2A without exhibiting an evident reduction in the growth. The inhibition of Pd-PS on methane was due to the inhibition of methane production related genes, MtaA and mcrA. These results provide potential explication for the inhibition of nanoplastics on the methanogens, which will fulfill the knowledge on the stability of methanogens under the short-term exposure of nanoplastics.

Kinetic modeling of Stickland reactions-coupled methanogenesis for a methanogenic culture

Studying amino acid catabolism-coupled methanogenesis is the important standpoints to decipher the metabolic behavior of a methanogenic culture. l-Glycine and l-alanine are acted as sole carbon and nitrogen sources for acidogenic bacteria. One amino acid is oxidized and another one is reduced for acetate production via pyruvate by oxidative deamination process in the Stickland reactions. Herein, we have developed a kinetic model for the Stickland reactions-coupled methanogenesis (SRCM) and simulated objectively to maximize the rate of methane production. We collected the metabolic information from enzyme kinetic parameters for amino acid catabolism of Clostridium acetobutylicum ATCC 824 and methanogenesis of Methanosarcina acetivorans C2A. The SRCM model of this study consisted of 18 reactions and 61 metabolites with enzyme kinetic parameters derived experimental data. The internal or external metabolic flux rate of this system found to control the acidogenesis and methanogenesis in a methanogenic culture. Using the SRCM model, flux distributions were calculated for each reaction and metabolite in order to maximize the methane production rate from the glycine–alanine pair. Results of this study, we demonstrated the metabolic behavior, metabolite pairing while mutually interact, and advantages of syntrophic metabolism of amino acid-directed methane production in a methanogenic starter culture.

Fine-Tuned Protein Production in Methanosarcina acetivorans C2A.

Methanogenic archaea can be integrated into a sustainable, carbon-neutral cycle for producing organic chemicals from C1 compounds if the rate, yield, and titer of product synthesis can be improved using metabolic engineering. However, metabolic engineering techniques are limited in methanogens by insufficient methods for controlling cellular protein levels. We conducted a systematic approach to tune protein levels in Methanosarcina acetivorans C2A, a model methanogen, by regulating transcription and translation initiation. Rationally designed core promoter and ribosome binding site mutations in M.acetivorans C2A resulted in a predicable change in protein levels over a 60 fold range. The overall range of protein levels was increased an additional 3 fold by introducing the 5' untranslated region of the mcrB transcript. This work demonstrates a wide range of precisely controlled protein levels in M.acetivorans C2A, which will help facilitate systematic metabolic engineering efforts in methanogens.

Genetic techniques for studies of methyl-coenzyme M reductase from Methanosarcina acetivorans C2A.

Methanogenic archaea generate methane as a by-product of anaerobic respiration using CO2, C1 compounds (like methanol or methylated amines), or acetate as terminal electron acceptors. Methanogens are an untapped resource for biotechnological advances related to methane production as well as methane consumption. However, key biological features of these organisms remain poorly understood. One such feature is the enzyme methyl-coenzyme M reductase (referred to as MCR), which catalyzes the last step in the methanogenic pathway and results in methane formation. Gene essentiality has limited genetic analyses of MCR thus far. Therefore, studies of this important enzyme have been limited to biochemical and biophysical techniques that are especially laborious and often reliant on sophisticated instrumentation that is not commonly available. In this chapter, we outline our recently developed CRISPR-Cas9-based genome editing tools and describe how these tools have been used for the introduction of a tandem affinity purification tag at the chromosomal mcr locus in the model methanogen, Methanosarcina acetivorans C2A. We also report a protocol for rapid affinity purification of MCR from M. acetivorans C2A that will enable high-throughput studies of this enzyme in the future.

The biosynthetic pathway of coenzyme F430 in methanogenic and methanotrophic archaea.

Methyl-coenzyme M reductase (MCR) is the key enzyme of methanogenesis and anaerobic methane oxidation. The activity of MCR is dependent on the unique nickel-containing tetrapyrrole known as coenzyme F430. We used comparative genomics to identify the coenzyme F430 biosynthesis (cfb) genes and characterized the encoded enzymes from Methanosarcina acetivorans C2A. The pathway involves nickelochelation by a nickel-specific chelatase, followed by amidation to form Ni-sirohydrochlorin a,c-diamide. Next, a primitive homolog of nitrogenase mediates a six-electron reduction and γ-lactamization reaction before a Mur ligase homolog forms the six-membered carbocyclic ring in the final step of the pathway. These data show that coenzyme F430 can be synthesized from sirohydrochlorin using Cfb enzymes produced heterologously in a nonmethanogen host and identify several targets for inhibitors of biological methane formation.

Identification and characterization of arsenite methyltransferase from an archaeon, Methanosarcina acetivorans C2A.

Arsenic is a ubiquitous toxic contaminant in the environment. The methylation of arsenic can affect its toxicity and is primarily mediated by biological processes. Few studies have focused on the mechanism of arsenic methylation in archaea although archaea are widespread in the environment. Here, an arsenite [As(III)] methyltransferase (ArsM) was identified and characterized from an archaeon Methanosarcina acetivorans C2A. Heterologous expression of MaarsM was shown to confer As(III) resistance to an arsenic-sensitive strain of E. coli through arsenic methylation and subsequent volatilization. Purified MaArsM protein was further identified the function in catalyzing the formation of various methylated products from As(III) in vitro. Methylation of As(III) by MaArsM is highly dependent on the characteristics of the thiol cofactors used, with some of them (coenzyme M, homocysteine, and dithiothreitol) more efficient than GSH. Site-directed mutagenesis demonstrated that three conserved cysteine (Cys) residues (Cys62, Cys150, and Cys200) in MaArsM were necessary for As(III) methylation, of which only Cys150 and Cys200 were required for the methylation of monomethylarsenic. These results present a molecular pathway for arsenic methylation in archaea and provide some insight into the role of archaea in As biogeochemistry.

Structure and Haem-Distal Site Plasticity in Methanosarcina acetivorans Protoglobin

Protoglobin from Methanosarcina acetivorans C2A (MaPgb), a strictly anaerobic methanogenic Archaea, is a dimeric haem-protein whose biological role is still unknown. As other globins, protoglobin can bind O2, CO and NO reversibly in vitro, but it displays specific functional and structural properties within members of the hemoglobin superfamily. CO binding to and dissociation from the haem occurs through biphasic kinetics, which arise from binding to (and dissociation from) two distinct tertiary states in a ligation-dependent equilibrium. From the structural viewpoint, protoglobin-specific loops and a N-terminal extension of 20 residues completely bury the haem within the protein matrix. Thus, access of small ligand molecules to the haem is granted by two apolar tunnels, not common to other globins, which reach the haem distal site from locations at the B/G and B/E helix interfaces. Here, the roles played by residues Trp(60)B9, Tyr(61)B10 and Phe(93)E11 in ligand recognition and stabilization are analyzed, through crystallographic investigations on the ferric protein and on selected mutants. Specifically, protein structures are reported for protoglobin complexes with cyanide, with azide (also in the presence of Xenon), and with more bulky ligands, such as imidazole and nicotinamide. Values of the rate constant for cyanide dissociation from ferric MaPgb-cyanide complexes have been correlated to hydrogen bonds provided by Trp(60)B9 and Tyr(61)B10 that stabilize the haem-Fe(III)-bound cyanide. We show that protoglobin can strikingly reshape, in a ligand-dependent way, the haem distal site, where Phe(93)E11 acts as ligand sensor and controls accessibility to the haem through the tunnel system by modifying the conformation of Trp(60)B9.

Function and Regulation of Isoforms of Carbon Monoxide Dehydrogenase/Acetyl Coenzyme A Synthase in Methanosarcina acetivorans

Conversion of acetate to methane (aceticlastic methanogenesis) is an ecologically important process carried out exclusively by methanogenic archaea. An important enzyme for this process as well as for methanogenic growth on carbon monoxide is the five-subunit archaeal CO dehydrogenase/acetyl coenzyme A (CoA) synthase multienzyme complex (CODH/ACS) catalyzing both CO oxidation/CO(2) reduction and cleavage/synthesis of acetyl-CoA. Methanosarcina acetivorans C2A contains two very similar copies of a six-gene operon (cdh genes) encoding two isoforms of CODH/ACS (Cdh1 and Cdh2) and a single CdhA subunit, CdhA3. To address the role of the CODH/ACS system in M. acetivorans, mutational as well as promoter/reporter gene fusion analyses were conducted. Phenotypic characterization of cdh disruption mutants (three single and double mutants, as well as the triple mutant) revealed a strict requirement of either Cdh1 or Cdh2 for acetotrophic or carboxidotrophic growth, as well as for autotrophy, which demonstrated that both isoforms are bona fide CODH/ACS. While expression of the Cdh2-encoding genes was generally higher than that of genes encoding Cdh1, both appeared to be regulated differentially in response to growth phase and to changing substrate conditions. While dispensable for growth, CdhA3 clearly affected expression of cdh1, suggesting that it functions in signal perception and transduction rather than in catabolism. The data obtained argue for a functional hierarchy and regulatory cross talk of the CODH/ACS isoforms.

Structural heterogeneity and ligand gating in ferric methanosarcina acetivorans protoglobin mutants

AbstractProtoglobin from Methanosarcina acetivorans C2A (MaPgb), a strictly anaerobic methanogenic Archaea, displays peculiar structural and functional properties within members of the hemoglobin superfamily. In fact, MaPgb‐specific loops and a N‐terminal extension (20 amino acid residues) completely bury the heme within the protein matrix. Therefore, the access of diatomic gaseous molecules to the heme is granted by two apolar tunnels reaching the heme distal site from locations at the B/G and B/E helix interfaces. The presence of two tunnels within the protein matrix could be partly responsible for the slightly biphasic ligand binding behavior. Unusually, MaPgb oxygenation is favored with respect to carbonylation. Here, the crucial role of Tyr(B10)61 and Ile(G11)149 residues, located in the heme distal site and lining the protein matrix tunnels 1 and 2, respectively, on ligand binding to the heme‐Fe‐atom and on distal site structural organization is reported. In particular, tunnel 1 accessibility is modulated by a complex reorganization of the Trp(B9)60 and Phe(E11)93 side‐chains, triggered by mutations of the Tyr(B10)61 and Ile(G11)149 residues, and affected by the presence and type of the distal heme‐bound ligand.

Structural heterogeneity and ligand gating in ferric methanosarcina acetivorans protoglobin mutants

Protoglobin from Methanosarcina acetivorans C2A (MaPgb), a strictly anaerobic methanogenic Archaea, displays peculiar structural and functional properties within members of the hemoglobin superfamily. In fact, MaPgb-specific loops and a N-terminal extension (20 amino acid residues) completely bury the heme within the protein matrix. Therefore, the access of diatomic gaseous molecules to the heme is granted by two apolar tunnels reaching the heme distal site from locations at the B/G and B/E helix interfaces. The presence of two tunnels within the protein matrix could be partly responsible for the slightly biphasic ligand binding behavior. Unusually, MaPgb oxygenation is favored with respect to carbonylation. Here, the crucial role of Tyr(B10)61 and Ile(G11)149 residues, located in the heme distal site and lining the protein matrix tunnels 1 and 2, respectively, on ligand binding to the heme-Fe-atom and on distal site structural organization is reported. In particular, tunnel 1 accessibility is modulated by a complex reorganization of the Trp(B9)60 and Phe(E11)93 side-chains, triggered by mutations of the Tyr(B10)61 and Ile(G11)149 residues, and affected by the presence and type of the distal heme-bound ligand.

Redundant Synthesis of Cysteinyl-tRNACys in Methanosarcina mazei

A subset of methanogenic archaea synthesize the cysteinyl-tRNA(Cys) (Cys-tRNA(Cys)) needed for protein synthesis using both a canonical cysteinyl-tRNA synthetase (CysRS) as well as a set of two enzymes that operate via a separate indirect pathway. In the indirect route, phosphoseryl-tRNA(Cys) (Sep-tRNA(Cys)) is first synthesized by phosphoseryl-tRNA synthetase (SepRS), and this misacylated intermediate is then converted to Cys-tRNA(Cys) by Sep-tRNA:Cys-tRNA synthase (SepCysS) via a pyridoxal phosphate-dependent mechanism. Here, we explore the function of all three enzymes in the mesophilic methanogen Methanosarcina mazei. The genome of M. mazei also features three distinct tRNA(Cys) isoacceptors, further indicating the unusual and complex nature of Cys-tRNA(Cys) synthesis in this organism. Comparative aminoacylation kinetics by M. mazei CysRS and SepRS reveals that each enzyme prefers a distinct tRNA(Cys) isoacceptor or pair of isoacceptors. Recognition determinants distinguishing the tRNAs are shown to reside in the globular core of the molecule. Both enzymes also require the S-adenosylmethione-dependent formation of (m1)G37 in the anticodon loop for efficient aminoacylation. We further report a new, highly sensitive assay to measure the activity of SepCysS under anaerobic conditions. With this approach, we demonstrate that SepCysS functions as a multiple-turnover catalyst with kinetic behavior similar to bacterial selenocysteine synthase and the archaeal/eukaryotic SepSecS enzyme. Together, these data suggest that both metabolic routes and all three tRNA(Cys) species in M. mazei play important roles in the cellular physiology of the organism.