Cornelia Welte

Bioenergetics and anaerobic respiratory chains of aceticlastic methanogens

Methane-forming archaea are strictly anaerobic microbes and are essential for global carbon fluxes since they perform the terminal step in breakdown of organic matter in the absence of oxygen. Major part of methane produced in nature derives from the methyl group of acetate. Only members of the genera Methanosarcina and Methanosaeta are able to use this substrate for methane formation and growth. Since the free energy change coupled to methanogenesis from acetate is only -36kJ/mol CH4, aceticlastic methanogens developed efficient energy-conserving systems to handle this thermodynamic limitation. The membrane bound electron transport system of aceticlastic methanogens is a complex branched respiratory chain that can accept electrons from hydrogen, reduced coenzyme F420 or reduced ferredoxin. The terminal electron acceptor of this anaerobic respiration is a mixed disulfide composed of coenzyme M and coenzyme B. Reduced ferredoxin has an important function under aceticlastic growth conditions and novel and well-established membrane complexes oxidizing ferredoxin will be discussed in depth. Membrane bound electron transport is connected to energy conservation by proton or sodium ion translocating enzymes (F420H2 dehydrogenase, Rnf complex, Ech hydrogenase, methanophenazine-reducing hydrogenase and heterodisulfide reductase). The resulting electrochemical ion gradient constitutes the driving force for adenosine triphosphate synthesis. Methanogenesis, electron transport, and the structure of key enzymes are discussed in this review leading to a concept of how aceticlastic methanogens make a living. This article is part of a Special Issue entitled: 18th European Bioenergetic Conference.

A Metagenomics-Based Metabolic Model of Nitrate-Dependent Anaerobic Oxidation of Methane by Methanoperedens-Like Archaea

Methane oxidation is an important process to mitigate the emission of the greenhouse gas methane and further exacerbating of climate forcing. Both aerobic and anaerobic microorganisms have been reported to catalyze methane oxidation with only a few possible electron acceptors. Recently, new microorganisms were identified that could couple the oxidation of methane to nitrate or nitrite reduction. Here we investigated such an enrichment culture at the (meta) genomic level to establish a metabolic model of nitrate-driven anaerobic oxidation of methane (nitrate-AOM). Nitrate-AOM is catalyzed by an archaeon closely related to (reverse) methanogens that belongs to the ANME-2d clade, tentatively named Methanoperedens nitroreducens. Methane may be activated by methyl-CoM reductase and subsequently undergo full oxidation to carbon dioxide via reverse methanogenesis. All enzymes of this pathway were present and expressed in the investigated culture. The genome of the archaeal enrichment culture encoded a variety of enzymes involved in an electron transport chain similar to those found in Methanosarcina species with additional features not previously found in methane-converting archaea. Nitrate reduction to nitrite seems to be located in the pseudoperiplasm and may be catalyzed by an unusual Nar-like protein complex. A small part of the resulting nitrite is reduced to ammonium which may be catalyzed by a Nrf-type nitrite reductase. One of the key questions is how electrons from cytoplasmically located reverse methanogenesis reach the nitrate reductase in the pseudoperiplasm. Electron transport in M. nitroreducens probably involves cofactor F420 in the cytoplasm, quinones in the cytoplasmic membrane and cytochrome c in the pseudoperiplasm. The membrane-bound electron transport chain includes F420H2 dehydrogenase and an unusual Rieske/cytochrome b complex. Based on genome and transcriptome studies a tentative model of how central energy metabolism of nitrate-AOM could work is presented and discussed.

Electron transport during aceticlastic methanogenesis by Methanosarcina acetivorans involves a sodium-translocating Rnf complex

The anaerobic methanogenic archaeon Methanosarcina acetivorans lives under extreme energy limitation. Methanogenesis from acetate as carried out by M. acetivorans involves an anaerobic electron transport chain with ferredoxin as electron donor and heterodisulfide as electron acceptor, and so far only the heterodisulfide reductase has been shown to translocate H+. Here, we describe a second Na+‐translocating coupling site in this electron transport chain. Inside‐out membrane vesicles of M. acetivorans catalyzed Na+ transport coupled to an electron transport catalyzed by the ferredoxin:heterodisulfide oxidoreductase activity. Ionophore studies revealed that Na+ transport was primary and electrogenic. A ∆rnf mutant was unable to grow on acetate and the ferredoxin:heterodisulfide oxidoreductase‐coupled Na+ transport was abolished. These data are consistent with the hypothesis that the Rnf complex of M. acetivorans is an Na+‐translocating coupling site and the entry point of electrons derived from reduced ferredoxin into the electron transport chain leading to the heterodisulfide.

Involvement of Ech hydrogenase in energy conservation of Methanosarcina mazei

Methanosarcina mazei belongs to the group of aceticlastic methanogens and converts acetate into the potent greenhouse gases CO2 and CH4. The aceticlastic respiratory chain involved in methane formation comprises the three transmembrane proteins Ech hydrogenase, F420 nonreducing hydrogenase and heterodisulfide reductase. It has been shown that the latter two contribute to the proton motive force. The data presented here clearly demonstrate that Ech hydrogenase is also involved in energy conservation. ATP synthesis was observed in a cytoplasm‐free vesicular system of Ms. mazei that was dependent on the oxidation of reduced ferredoxin and the formation of molecular hydrogen (as catalysed by Ech hydrogenase). Such an ATP formation was not observed in a Δech mutant strain. The protonophore 3,5‐di‐tert‐butyl‐4‐hydroxybenzylidene‐malononitrile (SF6847) led to complete inhibition of ATP formation in the Ms. mazei wild‐type without inhibiting hydrogen production by Ech hydrogenase, whereas the sodium ion ionophore ETH157 did not affect ATP formation in this system. Thus, we conclude that Ech hydrogenase acts as primary proton pump in a ferredoxin‐dependent electron transport system.

Reverse Methanogenesis and Respiration in Methanotrophic Archaea

Anaerobic oxidation of methane (AOM) is catalyzed by anaerobic methane-oxidizing archaea (ANME) via a reverse and modified methanogenesis pathway. Methanogens can also reverse the methanogenesis pathway to oxidize methane, but only during net methane production (i.e., “trace methane oxidation”). In turn, ANME can produce methane, but only during net methane oxidation (i.e., enzymatic back flux). Net AOM is exergonic when coupled to an external electron acceptor such as sulfate (ANME-1, ANME-2abc, and ANME-3), nitrate (ANME-2d), or metal (oxides). In this review, the reversibility of the methanogenesis pathway and essential differences between ANME and methanogens are described by combining published information with domain based (meta)genome comparison of archaeal methanotrophs and selected archaea. These differences include abundances and special structure of methyl coenzyme M reductase and of multiheme cytochromes and the presence of menaquinones or methanophenazines. ANME-2a and ANME-2d can use electron acceptors other than sulfate or nitrate for AOM, respectively. Environmental studies suggest that ANME-2d are also involved in sulfate-dependent AOM. ANME-1 seem to use a different mechanism for disposal of electrons and possibly are less versatile in electron acceptors use than ANME-2. Future research will shed light on the molecular basis of reversal of the methanogenic pathway and electron transfer in different ANME types.

Interaction between Sox proteins of two physiologically distinct bacteria and a new protein involved in thiosulfate oxidation.

Organisms using the thiosulfate‐oxidizing Sox enzyme system fall into two groups: group 1 forms sulfur globules as intermediates (Allochromatium vinosum), group 2 does not (Paracoccus pantotrophus). While several components of their Sox systems are quite similar, i.e. the proteins SoxXA, SoxYZ and SoxB, they differ by Sox(CD)2 which is absent in sulfur globule‐forming organisms. Still, the respective enzymes are partly exchangeable in vitro: P. pantotrophus Sox enzymes work productively with A. vinosum SoxYZ whereas A. vinosum SoxB does not cooperate with the P. pantotrophus enzymes. Furthermore, A. vinosum SoxL, a rhodanese‐like protein encoded immediately downstream of soxXAK, appears to play an important role in recycling SoxYZ as it increases thiosulfate depletion velocity in vitro without increasing the electron yield.

Function of Ech Hydrogenase in Ferredoxin-Dependent, Membrane-Bound Electron Transport in Methanosarcina mazei

Reduced ferredoxin is an intermediate in the methylotrophic and aceticlastic pathway of methanogenesis and donates electrons to membrane-integral proteins, which transfer electrons to the heterodisulfide reductase. A ferredoxin interaction has been observed previously for the Ech hydrogenase. Here we present a detailed analysis of a Methanosarcina mazei Delta ech mutant which shows decreased ferredoxin-dependent membrane-bound electron transport activity, a lower growth rate, and faster substrate consumption. Evidence is presented that a second protein whose identity is unknown oxidizes reduced ferredoxin, indicating an involvement in methanogenesis from methylated C(1) compounds.

Membrane-Bound Electron Transport in Methanosaeta thermophila

The obligate aceticlastic methanogen Methanosaeta thermophila uses a membrane-bound ferredoxin:heterodisulfide oxidoreductase system for energy conservation. We propose that the system is composed of a truncated form of the F(420)H(2) dehydrogenase, methanophenazine, and the heterodisulfide reductase. Hence, the electron transport chain is distinct from those of well-studied Methanosarcina species.

Detoxifying symbionts in agriculturally important pest insects

Pest insects lead to excessive agricultural and therefore economical losses on crops worldwide. These insects have to withstand toxic molecules that are inherent to plant defences, as well as those that are produced and introduced by humans in the form of insecticides. In recent years, research on insect–microbe symbioses has recognized that microbial symbionts may play a role protecting against these toxins, leading to a form of defensive symbiosis between the pest insect and different types of microorganisms that we term detoxifying symbioses. In this minireview, we will highlight well‐characterized and emerging insect model systems of detoxifying symbioses and assess how the microorganisms influence the hosts success.

Novel phenazine crystals enable direct electron transfer to methanogens in anaerobic digestion by redox potential modulation

With one billion tons of methane produced annually by microorganisms, biogas production can be appreciated both for its role in global organic matter turnover and as an energy source for humankind. The importance of electron transfer from electrically conductive surfaces or from bacteria to methanogenic Archaea has been implicated in widespread commercial anaerobic digestion processes, though a mechanism for reception of electrons from conductive surfaces or pili by methanogens has never been demonstrated. Here we describe a novel crystalline form of the synthetic phenazine neutral red that harvests electrons from reduced inorganic and organic microbial sources in anaerobic environments and makes them available to methanogenic Archaea. The novel crystalline form is so effective at harvesting reducing equivalents because it displays a potential for reduction 444 mV higher than the soluble form (E′ = 70 mV). Neutral red molecules solubilised in the reduced state by protonation at the point of methanogen cell contact with the crystal surface deliver electrons to methanogens at a negative midpoint potential (E′ = −375 mV). We demonstrate that soluble neutral red delivers reducing equivalents directly to the membrane bound HdrED heterodisulfide reductase of Methanosarcina, replenishing the CoM-SH and CoB-SH pool for methanogenesis and generating proton motive force. An order of magnitude increase in methane production is recorded in pure acetate fed Methanosarcina and coal and food waste fed mixed cultures in the laboratory. The phenomenon is also demonstrated at field scale in a sub-bituminous coal seam 80 m below ground level.