Dwi Susanti

Genomic Characterization of Methanomicrobiales Reveals Three Classes of Methanogens

Background Methanomicrobiales is the least studied order of methanogens. While these organisms appear to be more closely related to the Methanosarcinales in ribosomal-based phylogenetic analyses, they are metabolically more similar to Class I methanogens. Methodology/Principal Findings In order to improve our understanding of this lineage, we have completely sequenced the genomes of two members of this order, Methanocorpusculum labreanum Z and Methanoculleus marisnigri JR1, and compared them with the genome of a third, Methanospirillum hungatei JF-1. Similar to Class I methanogens, Methanomicrobiales use a partial reductive citric acid cycle for 2-oxoglutarate biosynthesis, and they have the Eha energy-converting hydrogenase. In common with Methanosarcinales, Methanomicrobiales possess the Ech hydrogenase and at least some of them may couple formylmethanofuran formation and heterodisulfide reduction to transmembrane ion gradients. Uniquely, M. labreanum and M. hungatei contain hydrogenases similar to the Pyrococcus furiosus Mbh hydrogenase, and all three Methanomicrobiales have anti-sigma factor and anti-anti-sigma factor regulatory proteins not found in other methanogens. Phylogenetic analysis based on seven core proteins of methanogenesis and cofactor biosynthesis places the Methanomicrobiales equidistant from Class I methanogens and Methanosarcinales. Conclusions/Significance Our results indicate that Methanomicrobiales, rather than being similar to Class I methanogens or Methanomicrobiales, share some features of both and have some unique properties. We find that there are three distinct classes of methanogens: the Class I methanogens, the Methanomicrobiales (Class II), and the Methanosarcinales (Class III).

Ribosomal protein L7Ae is a subunit of archaeal RNase P

To the mounting evidence of nonribosomal functions for ribosomal proteins, we now add L7Ae as a subunit of archaeal RNase P, a ribonucleoprotein (RNP) that catalyzes 5′-maturation of precursor tRNAs (pre-tRNAs). We first demonstrate that L7Ae coelutes with partially purified Methanococcus maripaludis (Mma) RNase P activity. After establishing in vitro reconstitution of the single RNA with four previously known protein subunits (POP5, RPP21, RPP29, and RPP30), we show that addition of L7Ae to this RNase P complex increases the optimal reaction temperature and kcat/Km (by ∼360-fold) for pre-tRNA cleavage to those observed with partially purified native Mma RNase P. We identify in the Mma RNase P RNA a putative kink-turn (K-turn), the structural motif recognized by L7Ae. The large stimulatory effect of Mma L7Ae on RNase P activity decreases to ≤ 4% of wild type upon mutating either the conserved nucleotides in this K-turn or amino acids in L7Ae shown to be essential for K-turn binding. The critical, multifunctional role of archaeal L7Ae in RNPs acting in tRNA processing (RNase P), RNA modification (H/ACA, C/D snoRNPs), and translation (ribosomes), especially by employing the same RNA-recognition surface, suggests coevolution of various translation-related functions, presumably to facilitate their coordinate regulation.

Genome Sequence of Thermofilum pendens Reveals an Exceptional Loss of Biosynthetic Pathways without Genome Reduction

We report the complete genome of Thermofilum pendens, a deeply branching, hyperthermophilic member of the order Thermoproteales in the archaeal kingdom Crenarchaeota. T. pendens is a sulfur-dependent, anaerobic heterotroph isolated from a solfatara in Iceland. It is an extracellular commensal, requiring an extract of Thermoproteus tenax for growth, and the genome sequence reveals that biosynthetic pathways for purines, most amino acids, and most cofactors are absent. In fact, T. pendens has fewer biosynthetic enzymes than obligate intracellular parasites, although it does not display other features that are common among obligate parasites and thus does not appear to be in the process of becoming a parasite. It appears that T. pendens has adapted to life in an environment rich in nutrients. T. pendens was known previously to utilize peptides as an energy source, but the genome revealed a substantial ability to grow on carbohydrates. T. pendens is the first crenarchaeote and only the second archaeon found to have a transporter of the phosphotransferase system. In addition to fermentation, T. pendens may obtain energy from sulfur reduction with hydrogen and formate as electron donors. It may also be capable of sulfur-independent growth on formate with formate hydrogen lyase. Additional novel features are the presence of a monomethylamine:corrinoid methyltransferase, the first time that this enzyme has been found outside the Methanosarcinales, and the presence of a presenilin-related protein. The predicted highly expressed proteins do not include proteins encoded by housekeeping genes and instead include ABC transporters for carbohydrates and peptides and clustered regularly interspaced short palindromic repeat-associated proteins.

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. 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.

An Intertwined Evolutionary History of Methanogenic Archaea and Sulfate Reduction

Hydrogenotrophic methanogenesis and dissimilatory sulfate reduction, two of the oldest energy conserving respiratory systems on Earth, apparently could not have evolved in the same host, as sulfite, an intermediate of sulfate reduction, inhibits methanogenesis. However, certain methanogenic archaea metabolize sulfite employing a deazaflavin cofactor (F420)-dependent sulfite reductase (Fsr) where N- and C-terminal halves (Fsr-N and Fsr-C) are homologs of F420H2 dehydrogenase and dissimilatory sulfite reductase (Dsr), respectively. From genome analysis we found that Fsr was likely assembled from freestanding Fsr-N homologs and Dsr-like proteins (Dsr-LP), both being abundant in methanogens. Dsr-LPs fell into two groups defined by following sequence features: Group I (simplest), carrying a coupled siroheme-[Fe4-S4] cluster and sulfite-binding Arg/Lys residues; Group III (most complex), with group I features, a Dsr-type peripheral [Fe4-S4] cluster and an additional [Fe4-S4] cluster. Group II Dsr-LPs with group I features and a Dsr-type peripheral [Fe4-S4] cluster were proposed as evolutionary intermediates. Group III is the precursor of Fsr-C. The freestanding Fsr-N homologs serve as F420H2 dehydrogenase unit of a putative novel glutamate synthase, previously described membrane-bound electron transport system in methanogens and of assimilatory type sulfite reductases in certain haloarchaea. Among archaea, only methanogens carried Dsr-LPs. They also possessed homologs of sulfate activation and reduction enzymes. This suggested a shared evolutionary history for methanogenesis and sulfate reduction, and Dsr-LPs could have been the source of the oldest (3.47-Gyr ago) biologically produced sulfide deposit.

Complete genome sequence of Desulfurococcus fermentans, a hyperthermophilic cellulolytic crenarchaeon isolated from a freshwater hot spring in Kamchatka, Russia.

Desulfurococcus fermentans is the first known cellulolytic archaeon. This hyperthermophilic and strictly anaerobic crenarchaeon produces hydrogen from fermentation of various carbohydrates and peptides without inhibition by accumulating hydrogen. The complete genome sequence reported here suggested that D. fermentans employs membrane-bound hydrogenases and novel glycohydrolases for hydrogen production from cellulose.

A novel F420-dependent Thioredoxin Reductase Gated by Low Potential FAD: A Tool for Redox Regulation in an Anaerobe.

A recent report suggested that the thioredoxin-dependent metabolic regulation, which is widespread in all domains of life, existed in methanogenic archaea about 3.5 billion years ago. We now show that the respective electron delivery enzyme (thioredoxin reductase, TrxR), although structurally similar to flavin-containing NADPH-dependent TrxRs (NTR), lacked an NADPH-binding site and was dependent on reduced coenzyme F420 (F420H2), a stronger reductant with a mid-point redox potential (E′0) of −360 mV; E′0 of NAD(P)H is −320 mV. Because F420 is a deazaflavin, this enzyme was named deazaflavin-dependent flavin-containing thioredoxin reductase (DFTR). It transferred electrons from F420H2 to thioredoxin via protein-bound flavin; Km values for thioredoxin and F420H2 were 6.3 and 28.6 μm, respectively. The E′0 of DFTR-bound flavin was approximately −389 mV, making electron transfer from NAD(P)H or F420H2 to flavin endergonic. However, under high partial pressures of hydrogen prevailing on early Earth and present day deep-sea volcanoes, the potential for the F420/F420H2 pair could be as low as −425 mV, making DFTR efficient. The presence of DFTR exclusively in ancient methanogens and mostly in the early Earth environment of deep-sea volcanoes and DFTRs characteristics suggest that the enzyme developed on early Earth and gave rise to NTR. A phylogenetic analysis revealed six more novel-type TrxR groups and suggested that the broader flavin-containing disulfide oxidoreductase family is more diverse than previously considered. The unprecedented structural similarities between an F420-dependent enzyme (DFTR) and an NADPH-dependent enzyme (NTR) brought new thoughts to investigations on F420 systems involved in microbial pathogenesis and antibiotic production.

Permanent Draft Genome Sequence of Desulfurococcus amylolyticus Strain Z-533 T , a Peptide and Starch Degrader Isolated from Thermal Springs in the Kamchatka Peninsula and Kunashir Island, Russia

ABSTRACT Desulfurococcus amylolyticus Z-533T, a hyperthermophilic crenarcheon, ferments peptide and starch, generating acetate, isobutyrate, isovalerate, CO2, and hydrogen. Unlike D. amylolyticus Z-1312, it cannot use cellulose and is inhibited by hydrogen. The reported draft genome sequence of D. amylolyticus Z-533T will help to understand the molecular basis for these differences.

Permanent draft genome sequence of Desulfurococcus mobilis type strain DSM 2161, a thermoacidophilic sulfur-reducing crenarchaeon isolated from acidic hot springs of Hveravellir, Iceland

This report presents the permanent draft genome sequence of Desulfurococcus mobilis type strain DSM 2161, an obligate anaerobic hyperthermophilic crenarchaeon that was isolated from acidic hot springs in Hveravellir, Iceland. D. mobilis utilizes peptides as carbon and energy sources and reduces elemental sulfur to H2S. A metabolic construction derived from the draft genome identified putative pathways for peptide degradation and sulfur respiration in this archaeon. Existence of several hydrogenase genes in the genome supported previous findings that H2 is produced during the growth of D. mobilis in the absence of sulfur. Interestingly, genes encoding glucose transport and utilization systems also exist in the D. mobilis genome though this archaeon does not utilize carbohydrate for growth. The draft genome of D. mobilis provides an additional mean for comparative genomic analysis of desulfurococci. In addition, our analysis on the Average Nucleotide Identity between D. mobilis and Desulfurococcus mucosus suggested that these two desulfurococci are two different strains of the same species.

A Reexamination of Thioredoxin Reductase from Thermoplasma acidophilum, a Thermoacidophilic Euryarchaeon, Identifies It as an NADH-Dependent Enzyme

Flavin-containing Trx reductase (TrxR) of Thermoplasma acidophilum (Ta), a thermoacidophilic facultative anaerobic archaeon, lacks the structural features for the binding of 2′-phosphate of nicotinamide adenine dinucleotide phosphate (NADPH), and this feature has justified the observed lack of activity with NADPH; NADH has also been reported to be ineffective. Our recent phylogenetic analysis identified Ta-TrxR as closely related to the NADH-dependent enzymes of Thermotoga maritima and Desulfovibrio vulgaris, both being anaerobic bacteria. This observation instigated a reexamination of the activity of the enzyme, which showed that Ta-TrxR is NADH dependent; the apparent Km for NADH was 3.1 μM, a physiologically relevant value. This finding is consistent with the observation that NADH:TrxR has thus far been found primarily in anaerobic bacteria and archaea.