UV-A/blue-light inactivation of the 'metal-free' hydrogenase (Hmd) from methanogenic archaea.

H2-forming methylenetetrahydromethanopterin dehydrogenase (Hmd) is an unusual hydrogenase present in many methanogenic archaea. The homodimeric enzyme dubbed 'metal-free' hydrogenase does not contain iron-sulfur clusters or nickel and thus differs from [Ni-Fe] and [Fe-Fe] hydrogenases, which are all iron-sulfur proteins. Hmd preparations were found to contain up to 1 mol iron per 40 kDa subunit, but the iron was considered to be a contaminant as none of the catalytic and spectroscopic properties of the enzyme indicated that it was an essential component. Hmd does, however, harbour a low molecular mass cofactor of yet unknown structure. We report here that the iron found in Hmd is most probably functional after all. Further investigation was initiated by the discovery that Hmd is inactivated upon exposure to UV-A (320-400 nm) or blue-light (400-500 nm). Enzyme purified in the dark exhibited an absorption spectrum with a maximum at approximately 360 nm and which mirrored its sensitivity towards light. In UV-A/blue-light the enzyme was bleached. The cofactor extracted from active Hmd was also light sensitive. It showed an UV/visible spectrum similar to that of the active enzyme and was bleached upon exposure to light. Photobleached cofactor no longer had the ability to reconstitute active Hmd from the apoenzyme. When purified in the dark, Hmd consistently contained per monomer about one Fe, which was tightly bound to the cofactor. The iron was released from the enzyme and from the cofactor upon light inactivation. Hmd activity was inhibited by high concentrations of CO and CO protected the enzyme from light inactivation indicating that the iron in Hmd is of functional importance. Therefore, reference to Hmd as 'metal-free' hydrogenase is no longer appropriate.     

The Na(+)-translocating methyltransferase complex from methanogenic archaea

Methanogenic archaea are dependent on sodium ions for methane formation. A sodium ion-dependent step has been shown to be methyl transfer from N(5)-methyltetrahydromethanopterin to coenzyme M. This exergonic reaction (DeltaG degrees '=-30 kJ/mol) is catalyzed by a Na(+)-translocating membrane-associated multienzyme complex composed of eight different subunits, MtrA-H. Subunit MtrA harbors a cob(I)amide prosthetic group which is methylated and demethylated in the catalytic cycle, demethylation being sodium ion-dependent. Based on the finding that in the cob(II)amide oxidation state the corrinoid is bound in a base-off/His-on configuration it is proposed that methyl transfer from MtrA to coenzyme M is associated with a conformational change of the protein and that this change drives the electrogenic translocation of the sodium ions.     

Heterodisulfide reductase from methanogenic archaea: a new catalytic role for an iron-sulfur cluster

Heterodisulfide reductase (HDR) from methanogenic archaea is an iron-sulfur protein that catalyzes reversible reduction of the heterodisulfide (CoM-S-S-CoB) of the methanogenic thiol-coenzymes, coenzyme M (CoM-SH) and coenzyme B (CoB-SH). Via the characterization of a paramagnetic reaction intermediate generated upon oxidation of the enzyme in the presence of coenzyme M, the enzyme was shown to contain a [4Fe-4S] cluster in its active site that catalyzes reduction of the disulfide substrate in two one-electron reduction steps. The formal thiyl radical generated by the initial one-electron reduction of the disulfide is stabilized via reduction and coordination of the resultant thiol to the [4Fe-4S] cluster.     

H2-forming methylenetetrahydromethanopterin dehydrogenase, a novel type of hydrogenase without iron-sulfur clusters in methanogenic archaea.

A novel hydrogenase has recently been found in methanogenic archaea. It catalyzes the reversible dehydrogenation of methylenetetrahydromethanopterin (CH2 = H4MPT) to methenyltetrahydromethanopterin (CH identical to H4MPT+) and H2 and was therefore named H2-forming methylenetetrahydromethanopterin dehydrogenase. The hydrogenase, which is composed of only one polypeptide with an apparent molecular mass of 43 kDa, does not mediate the reduction of viologen dyes with either H2 or CH2 = H4MPT. We report here that the purified enzyme from Methanobacterium thermoautotrophicum exhibits the following other unique properties: (a) the colorless protein with a specific activity of 2000 U/mg (Vmax) did not contain iron-sulfur clusters, nickel, or flavins; (b) the activity was not inhibited by carbon monoxide, acetylene, nitrite, cyanide, or azide; (c) the enzyme did not catalyze an isotopic exchange between 3H2 and 1H+; (d) the enzyme catalyzed the reduction of CH identical to H4MPT+ with 3H2 generating [methylene-3H]CH2 = H4MPT; and (e) the primary structure contained at most four conserved cysteines as revealed by a comparison of the DNA-deduced amino acid sequence of the proteins from M. thermoautotrophicum and Methanopyrus kandleri. None of the four cysteines were closely spaced as would be indicative for a (NiFe) hydrogenase or a ferredoxin-type iron-sulfur protein. Properties of the H2-forming methylenetetrahydromethanopterin dehydrogenase from Methanobacterium wolfei are also described indicating that the enzyme from this methanogenic archaeon is very similar to the enzyme from M. thermoautotrophicum with respect both to molecular and catalytic properties.     

Characterization of AMA, a new AAA protein from Archaeoglobus and methanogenic archaea

We have previously reported a new group of AAA proteins, which is only found in Archaeoglobus and methanogenic archaea (AMA). The proteins are phylogenetically basal to the metalloprotease clade and their N-terminal domain is homologous to the beta-clam part of the N-domain of CDC48-like proteins. Here we report the biochemical and biophysical characterization of Archaeoglobus fulgidus AMA, and of its isolated N-terminal (AMA-N) and ATPase (AMA-DeltaN) domains. AfAMA forms hexameric complexes, as does AMA-N, while AMA-DeltaN only forms dimers. The ability to hexamerize is dependent on the integrity of a GYPL motif in AMA-N, which resembles the pore motif of FtsH and HslU. While the physiological function of AMA is unknown, we show that it has ATP-dependent chaperone activity and can prevent the thermal aggregation of proteins in vitro. The ability to interact with non-native proteins resides in the N-domain and is energy-independent.     

Psychrotolerant methanogenic archaea: Diversity and cold adaptation mechanisms

Because of their diversity and abundance in a wide range of environments, particularly in cold regions, cold-adaptive archaea are expected to play a pivotal role in material recycling in cold environments. Methanogenic archaea are ubiquitous on earth and produce a large amount of methane (CH₄) as their main carbon metabolite. Methanogens are the most laboratory amendable archaea. The few psychrophilic archaea that have been cultured to date are mainly affiliated with methanogens, thus make them a good model for investigating mechanisms of archaeal cold adaptation. Studies of psychrotolerant methanogens have been ongoing since the 1990s. Using Methanococcoides burtonii, a methanogen isolated from Ace Lake in Antarctica, extensive studies on the genomic characteristics associated with cold adaptation have been carried out by the Cavicchioli laboratory. We recently analyzed the genome of another psychrophilic methanogen and identified the gene repertoire associated with cold adaptation. This review summarizes recent studies of psychroactive methanogens, particularly their diversity, the genomics and proteomics associated with their cold adaptation, and the cellular components and proteins likely involved in their cold protection.     

Molecular identification of methanogenic archaea from sheep in Queensland, Australia reveal more uncultured novel archaea

Molecular diversity of rumen methanogens in sheep in Queensland, Australia was investigated using 16S rRNA gene libraries prepared from pooled rumen contents from nine merino sheep. A total of 78 clones were identified revealing 26 different sequences. Of these 26 sequences, eight sequences (15 clones) were 95-100% similar to cultivated methanogens belonging to the orders Methanobacteriales and Methanomicrobiales, and the remaining 18 phylotypes (63 clones) were 72-75% similar to Thermoplasma acidophilum and Thermoplasma volcanium. These unique sequences clustered within a distinct and strongly supported (100% bootstrap support) phylogenetic group, exclusively composed of sequences from uncharacterized archaea from very diverse anaerobic environments. Members of this unique group that were previously considered atypical for the rumen environment were the predominant clones.     

Community analysis of methanogenic archaea within a riparian flooding gradient

Anoxic soils in river floodplains (or riparian soils) are a source of methane emission. However, little is known about the ecology and community structure of archaeal methanogenic microbes, which are a crucial component of methane flux in those habitats. We studied the archaeal community in the vertical profile of four different sites along the River Waal in the Netherlands. These sites differ in their annual flooding regime ranging from never or seldom to permanently flooded. The archaeal community structure has been characterized by terminal restriction fragment length polymorphism (T-RFLP) and comparative sequence analysis of the archaeal SSU rRNA gene and the mcrA gene. The latter gene codes for the alpha-subunit of methyl-coenzyme M reductase. Additionally, the potential methanogenic activity was determined by incubation of soil slurries under anoxic conditions. The community composition differed only slightly with the depth of the soil (0-20 cm). However, the diversity of archaeal SSU rRNA genes increased with the frequency of flooding. Terminal restriction fragment length polymorphism analysis of mcrA gene amplicons confirmed the results concerning methanogenic archaea. In the never and rarely flooded soils, crenarchaeotal sequences were the dominant group. In the frequently and permanently flooded soils, Methanomicrobiaceae, Methanobacteriaceae, Methanosarcinaceae and the uncultured Rice Clusters IV and VI (Crenarchaeota) were detectable independently from duration of anoxic conditions. Methanosaetaceae, on the other hand, were only found in the permanently and frequently flooded soils under conditions where concentrations of acetate were < 30 microM. The results indicate that methanogens as well as other archaea occupy characteristic niches according to the flooding conditions in the field. Methanosaetaceae, in particular, seem to be adapted (or proliferate at) to low acetate concentrations.     

The membraneless bioelectrochemical reactor stimulates hydrogen fermentation by inhibiting methanogenic archaea

The membraneless bioelectrochemical reactor (Ml-BER) is useful for dark hydrogen fermentation. The effect of the electrochemical reaction on microorganisms in the Ml-BER was investigated using glucose as the substrate and compared with organisms in a membraneless non-bioelectrochemical reactor (Ml-NBER) and bioelectrochemical reactor (BER) with a proton exchange membrane. The potentials on the working electrode of the Ml-BER and BER with membrane were regulated to ?0.9 V (versus Ag/AgCl) to avoid water electrolysis with a carbon electrode. The Ml-BER showed suppressed methane production (19.8?±?9.1 mg-C·L^?1·day^?1) and increased hydrogen production (12.6?±?3.1 mg-H·L^?1·day^?1) at pH_out 6.2?±?0.1, and the major intermediate was butyrate (24.9?±?2.4 mM), suggesting efficient hydrogen fermentation. In contrast, the Ml-NBER showed high methane production (239.3?±?17.9 mg-C·L^?1·day^?1) and low hydrogen production (0.2?±?0.0 mg-H·L^?1·day^?1) at pH_out 6.3?±?0.1. In the cathodic chamber of the BER with membrane, methane production was high (276.3?±?20.4 mg-C·L^?1·day^?1) (pH_out, 7.2?±?0.1). In the anodic chamber of the BER with membrane (anode-BER), gas production was low because of high lactate production (43.6?±?1.7 mM) at pH_out 5.0?±?0.1. Methanogenic archaea were not detected in the Ml-BER and anode-BER. However, Methanosarcina sp. and Methanobacterium sp. were found in Ml-NBER. Prokaryotic copy numbers in the Ml-BER and Ml-NBER were similar, as were the bacterial community structures. Thus, the electrochemical reaction in the Ml-BER affected hydrogenotrophic and acetoclastic methanogens, but not the bacterial community.     

Biomarker evidence for widespread anaerobic methane oxidation in Mediterranean sediments by a consortium of methanogenic archaea and bacteria. The Medinaut Shipboard Scientific Party

Although abundant geochemical data indicate that anaerobic methane oxidation occurs in marine sediments, the linkage to specific microorganisms remains unclear. In order to examine processes of methane consumption and oxidation, sediment samples from mud volcanoes at two distinct sites on the Mediterranean Ridge were collected via the submersible Nautile. Geochemical data strongly indicate that methane is oxidized under anaerobic conditions, and compound-specific carbon isotope analyses indicate that this reaction is facilitated by a consortium of archaea and bacteria. Specifically, these methane-rich sediments contain high abundances of methanogen-specific biomarkers that are significantly depleted in (13)C (delta(13)C values are as low as -95 per thousand). Biomarkers inferred to derive from sulfate-reducing bacteria and other heterotrophic bacteria are similarly depleted. Consistent with previous work, such depletion can be explained by consumption of (13)C-depleted methane by methanogens operating in reverse and as part a consortium of organisms in which sulfate serves as the terminal electron acceptor. Moreover, our results indicate that this process is widespread in Mediterranean mud volcanoes and in some localized settings is the predominant microbiological process.     

The energy conserving methyltetrahydromethanopterin:coenzyme M methyltransferase complex from methanogenic archaea: function of the subunit MtrH.

In methanogenic archaea the transfer of the methyl group of N5-methyltetrahydromethanopterin to coenzyme M is coupled with energy conservation. The reaction is catalyzed by a membrane associated multienzyme complex composed of eight different subunits MtrA-H. The 23 kDa subunit MtrA harbors a corrinoid prosthetic group which is methylated and demethylated in the catalytic cycle. We report here that the 34 kDa subunit MtrH catalyzes the methylation reaction. MtrH was purified and shown to exhibit methyltetrahydromethanopterin:cob(I)alamin methyltransferase activity. Sequence comparison revealed similarity of MtrH with MetH from Escherichia coli and AcsE from Clostridium thermoaceticum: both enzymes exhibit methyltetrahydrofolate:cob(I)alamin methyltransferase activity.     

Stress response of methanogenic archaea from Siberian permafrost compared with methanogens from nonpermafrost habitats

We examined the survival potential of methanogenic archaea exposed to different environmental stress conditions such as low temperature (down to −78.5°C), high salinity (up to 6 M NaCl), starvation (up to 3 months), long-term freezing (up to 2 years), desiccation (up to 25 days) and oxygen exposure (up to 72 h). The experiments were conducted with methanogenic archaea from Siberian permafrost and were complemented by experiments on well-studied methanogens from nonpermafrost habitats. Our results indicate a high survival potential of a methanogenic archaeon from Siberian permafrost when exposed to the extreme conditions tested. In contrast, these stress conditions were lethal for methanogenic archaea isolated from nonpermafrost habitats. A better adaptation to stress was observed at a low temperature (4°C) compared with a higher one (28°C). Given the unique metabolism of methanogenic archaea in general and the long-term survival and high tolerance to extreme conditions of the methanogens investigated in this study, methanogenic archaea from permafrost should be considered as primary candidates for possible subsurface Martian life.     

The tetrahydropyranopterin structure of the sulfur-free and metal-free molybdenum cofactor precursor

The molybdenum cofactor (Moco), a highly conserved pterin compound coordinating molybdenum (Mo), is required for the activity of all Mo-dependent enzymes with the exception of nitrogenase. Moco is synthesized by a unique and evolutionary old multi-step pathway with two intermediates identified so far, the sulfur-free and metal-free pterin derivative precursor Z and molybdopterin, a pterin with an enedithiolate function essential for Mo ligation. The latter pterin component is believed to form a tetrahydropyranopterin similar to the one found for Moco in the crystal structure of Mo as well as tungsten (W) enzymes. Here we report the spectroscopic characterization and structure elucidation of precursor Z purified from Escherichia coli overproducing MoaA and MoaC, two proteins essential for bacterial precursor Z synthesis. We have shown that purified precursor Z is as active as precursor Z present in E. coli cell extracts, demonstrating that no modifications during the purification procedure have occurred. High resolution electrospray ionization mass spectrometry afforded a [M + H]+ ion compatible with a molecular formula of C10H15N5O8P. Consequently 1H NMR spectroscopy not allowed structural characterization of the molecule but confirmed that this intermediate undergoes direct oxidation to the previously well characterized non-productive follow-up product compound Z. The 1H chemical shift and coupling constant data are incompatible with previous structural proposals and indicate that precursor Z already is a tetrahydropyranopterin system and carries a geminal diol function in the C1' position.     

Diversity and identification of methanogenic archaea and sulphate-reducing bacteria in sediments from a pristine tropical mangrove

Mangrove sediments are anaerobic ecosystems rich in organic matter. This environment is optimal for anaerobic microorganisms, such as sulphate-reducing bacteria and methanogenic archaea, which are responsible for nutrient cycling. In this study, the diversity of these two functional guilds was evaluated in a pristine mangrove forest using denaturing gradient gel electrophoresis (DGGE) and clone library sequencing in a 50 cm vertical profile sampled every 5.0 cm. DGGE profiles indicated that both groups presented higher richness in shallow samples (0–30 cm) with a steep decrease in richness beyond that depth. According to redundancy analysis, this alteration significantly correlated with a decrease in the amount of organic matter. Clone library sequencing indicated that depth had a strong effect on the selection of dissimilatory sulphate reductase (dsrB) operational taxonomic units (OTUs), as indicated by the small number of shared OTUs found in shallow (0.0 cm) and deep (40.0 cm) libraries. On the other hand, methyl coenzyme-M reductase (mcrA) libraries indicated that most of the OTUs found in the shallow library were present in the deep library. These results show that these two guilds co-exist in these mangrove sediments and indicate important roles for these organisms in nutrient cycling within this ecosystem.     

Assessment of active methanogenic archaea in a methanol-fed upflow anaerobic sludge blanket reactor

Methanogenic archaea enrichment of a granular sludge was undertaken in an upflow anaerobic sludge blanket (UASB) reactor fed with methanol in order to enrich methylotrophic and hydrogenotrophic methanogenic populations. A microbial community assessment, in terms of microbial composition and activity—throughout the different stages of the feeding process with methanol and acetate—was performed using specific methanogenic activity (SMA) assays, quantitative real-time polymerase chain reaction (qPCR), and high-throughput sequencing of 16S ribosomal RNA (rRNA) genes from DNA and complementary DNA (cDNA). Distinct methanogenic enrichment was revealed by qPCR of mcrA gene in the methanol-fed community, being two orders of magnitude higher with respect to the initial inoculum, achieving a final mcrA/16S rRNA ratio of 0.25. High-throughput sequencing analysis revealed that the resulting methanogenic population was mainly composed by methylotrophic archaea (Methanomethylovorans and Methanolobus genus), being also highly active according to the RNA-based assessment. SMA confirmed that the methylotrophic pathway, with a direct conversion of methanol to CH₄, was the main step of methanol degradation in the UASB. The biomass from the UASB, enriched in methanogenic archaea, may bear great potential as additional inoculum for bioreactors to carry out biogas production and other related processes.     

Structure of coenzyme F(420) dependent methylenetetrahydromethanopterin reductase from two methanogenic archaea

Coenzyme F(420)-dependent methylenetetrahydromethanopterin reductase (Mer) is an enzyme of the Cl metabolism in methanogenic and sulfate reducing archaea. It is composed of identical 35-40 kDa subunits and lacks a prosthetic group. The crystal structure of Mer from Methanopyrus kandleri (kMer) revealed in one crystal form a dimeric and in another a tetrameric oligomerisation state and that from Methanobacterium thermoautotrophicum (tMer) a dimeric state. Each monomer is primarily composed of a TIM-barrel fold enlarged by three insertion regions. Insertion regions 1 and 2 contribute to intersubunit interactions. Insertion regions 2 and 3 together with the C-terminal end of the TIM-barrel core form a cleft where the binding sites of coenzyme F(420) and methylene-tetrahydromethanopterin are postulated. Close to the coenzyme F(420)-binding site lies a rarely observed non-prolyl cis-peptide bond. It is surprising that Mer is structurally most similar to a bacterial FMN-dependent luciferase which contains a non-prolyl cis-peptide bond at the equivalent position. The structure of Mer is also related to that of NADP-dependent FAD-harbouring methylenetetrahydrofolate reductase (MetF). However, Mer and MetF do not show sequence similarities although they bind related substrates and catalyze an analogous reaction.