Process of energy conservation in the extremely haloalkaliphilic methyl-reducing methanogen Methanonatronarchaeum thermophilum.

The recently isolated methanogen Methanonatronarchaeum thermophilum is an extremely haloalkaliphilic and moderately thermophilic archaeon and belongs to the novel class Methanonatronarchaeia in the phylum Halobacteriota. The knowledge about the physiology and biochemistry of members of the class Methanonatronarchaeia is still limited. It is known that M. thermophilum performs hydrogen or formate-dependent methyl-reducing methanogenesis. Here, we show that the organism was able to grow on all tested C1 -methylated substrates (methanol, trimethylamine, dimethylamine, monomethylamine) in combination with formate or molecular hydrogen. A temporary accumulation of intermediates (dimethylamine or/and monomethylamine) in the medium occurred during the consumption of trimethylamine or dimethylamine. The energy conservation of M. thermophilum was dependent on a respiratory chain consisting of a hydrogenase (VhoGAC), a formate dehydrogenase (FdhGHI), and a heterodisulfide reductase (HdrDE) that were well adapted to the harsh physicochemical conditions in the natural habitat. The experiments revealed the presence of two variants of energy-conserving oxidoreductase systems in the membrane. These included the H2 : heterodisulfide oxidoreductase system, which has already been described in Methanosarcina species, as well as the novel formate: heterodisulfide oxidoreductase system. The latter electron transport chain, which was experimentally proven for the first time, distinguishes the organism from all other known methanogenic archaea and represents a unique feature of the class Methanonatronarchaeia. Experiments with 2-hydroxyphenazine and the inhibitor diphenyleneiodonium chloride indicated that a methanophenazine-like cofactor might function as an electron carrier between the hydrogenase/ formate dehydrogenase and the heterodisulfide reductase.

Energy conservation in the gut microbe Methanomassiliicoccus luminyensis is based on membrane-bound ferredoxin oxidation coupled to heterodisulfide reduction.

Methanomassiliicoccus luminyensis was originally isolated from human feces and belongs to the seventh order of methanogens, the Methanomassiliicoccales, which are only distantly related to other methanogenic archaea. The organism forms methane from the reduction of methylamines or methanol using molecular hydrogen as reductant. The energy-conserving system in M. luminyensis is unique and the enzymes involved in this process are not found in this combination in members of the other methanogenic orders. In this context our central question was how the organism is able to generate ATP. Energy transduction was dependent on a membrane-bound ferredoxin: heterodisulfide oxidoreductase composed of reduced ferredoxin as an electron donor, at least one protein in the membrane fraction and the heterodisulfide reductase HdrD, which reduced the electron acceptor CoM-S-S-CoB. Electron transfer of this respiratory chain proceeded with a rate of 145 nmol reduced heterodisulfide min-1 ·mg-1 membrane protein. Methanomassiliicoccus luminyensis is the first example of a methanogenic archaeon that does not require Na+ ions for energy conservation. Only protons were used as coupling ions for the generation of the electrochemical ion gradient. The membrane-bound F420 H2 :phenazine oxidoreductase complex (without the electron input module FpoF) probably catalyzed the oxidation of reduced ferredoxin and potentially acted as primary proton pump in this electron transport system. In summary, the energy-conserving system of M. luminyensis possesses features found in the pathways of hydrogenotrophic and methylotrophic/aceticlastic methanogenesis. Consequently, the composition of the enzymes involved in ion translocation across the cytoplasmic membrane is different from all other methanogenic archaea.