Heterodisulfide reductases (Hdr) of the HdrABC class are ancient enzymes and a component of the anaerobic core belonging to the prokaryotic common ancestor. The ancient origin is consistent with the widespread occurrence of genes encoding putative HdrABC homologs in metabolically diverse prokaryotes predicting diverse physiological functions; however, only one HdrABC has been characterized and that was from a narrow metabolic group of obligate CO ₂ -reducing methanogenic anaerobes (methanogens) from the domain Archaea . Here we report the biochemical characterization of an HdrABC homolog (HdrA2B2C2) from the acetate-utilizing methanogen Methanosarcina acetivorans with unusual properties structurally and functionally distinct from the only other HdrABC characterized. Homologs of the HdrA2B2C2 archetype are present in phylogenetically and metabolically diverse species from the domains Bacteria and Archaea . The expression of the individual HdrA2, HdrB2, and HdrB2C2 enzymes in Escherichia coli , and reconstitution of an active HdrA2B2C2 complex, revealed an intersubunit electron transport pathway dependent on ferredoxin or coenzyme F ₄₂₀ (F ₄₂₀ H ₂ ) as an electron donor. Remarkably, HdrA2B2C2 couples the previously unknown endergonic oxidation of F ₄₂₀ H ₂ and reduction of ferredoxin with the exergonic oxidation of F ₄₂₀ H ₂ and reduction of the heterodisulfide of coenzyme M and coenzyme B (CoMS-SCoB). The unique electron bifurcation predicts a role for HdrA2B2C2 in Fe(III)-dependent anaerobic methane oxidation (ANME) by M. acetivorans and uncultured species from ANME environments. HdrA2B2C2, ubiquitous in acetotrophic methanogens, was shown to participate in electron transfer during acetotrophic growth of M. acetivorans and proposed to be essential for growth in the environment when acetate is limiting. IMPORTANCE Discovery of the archetype HdrA2B2C2 heterodisulfide reductase with categorically unique properties extends the understanding of this ancient family beyond CO ₂ -reducing methanogens to include diverse prokaryotes from the domains Bacteria and Archaea . The unprecedented coenzyme F ₄₂₀ -dependent electron bifurcation, an emerging fundamental principle of energy conservation, predicts a role for HdrA2B2C2 in diverse metabolisms, including anaerobic CH ₄ -oxidizing pathways. The results document an electron transport role for HdrA2B2C2 in acetate-utilizing methanogens responsible for at least two-thirds of the methane produced in Earth’s biosphere. The previously unavailable heterologous production of individual subunits and the reconstitution of HdrA2B2C2 with activity have provided an understanding of intersubunit electron transfer in the HdrABC class and a platform for investigating the principles of electron bifurcation.

The mitogen-activated protein kinase (MAPK) pathways are important signal transduction pathways conserved in essentially all eukaryotes, but haven't been subjected to functional studies in the most important cellulase-producing filamentous fungus Trichoderma reesei. Previous reports suggested the presence of three MAPKs in T. reesei: Tmk1, Tmk2, and Tmk3. By exploring the phenotypic features of T. reesei Δtmk3, we first showed elevated NaCl sensitivity and repressed transcription of genes involved in glycerol/trehalose biosynthesis under higher osmolarity, suggesting Tmk3 participates in high osmolarity resistance via derepression of genes involved in osmotic stabilizer biosynthesis. We also showed significant downregulation of genes encoding chitin synthases and a β-1,3-glucan synthase, decreased chitin content, ‘budded’ hyphal appearance typical to cell wall defective strains, and increased sensitivity to calcofluor white/Congo red in the tmk3 deficient strain, suggesting Tmk3 is involved in cell wall integrity maintenance in T. reesei. We further observed the decrease of cellulase transcription and production in T. reesei Δtmk3 during submerged cultivation, as well as the presence of MAPK phosphorylation sites on known transcription factors involved in cellulase regulation, suggesting Tmk3 is also involved in the regulation of cellulase production. Finally, the expression of cell wall integrity related genes, the expression of cellulase coding genes, cellulase production and biomass accumulation were compared between T. reesei Δtmk3 grown in solid state media and submerged media, showing a strong restoration effect in solid state media from defects resulted from tmk3 deletion. These results showed novel physiological processes that fungal Hog1-type MAPKs are involved in, and present the first experimental investigation of MAPK signaling pathways in T. reesei. Our observations on the restoration effect during solid state cultivation suggest that T. reesei is evolved to favor solid state growth, bringing up the proposal that the submerged condition normally used during investigations on fungal physiology might be misleading.

Background: Acetate is the major source of methane in nature. The majority of investigations have focused on acetotrophic methanogens for which energy-conserving electron transport is dependent on the production and consumption of H₂ as an intermediate, although the great majority of acetotrophs are unable to metabolize H₂. The presence of cytochrome c and a complex (Ma-Rnf) homologous to the Rnf ( R hodobacter n itrogen f ixation) complexes distributed in the domain Bacteria distinguishes non-H₂-utilizing Methanosarcina acetivorans from H₂-utilizing species suggesting fundamentally different electron transport pathways. Thus, the membrane-bound electron transport chain of acetate-grown M. acetivorans was investigated to advance a more complete understanding of acetotrophic methanogens. Results: A component of the CO dehydrogenase/acetyl-CoA synthase (CdhAE) was partially purified and shown to reduce a ferredoxin purified using an assay coupling reduction of the ferredoxin to oxidation of CdhAE. Mass spectrometry analysis of the ferredoxin identified the encoding gene among annotations for nine ferredoxins encoded in the genome. Reduction of purified membranes from acetate-grown cells with ferredoxin lead to reduction of membrane-associated multi-heme cytochrome c that was re-oxidized by the addition of either the heterodisulfide of coenzyme M and coenzyme B (CoM-S-S-CoB) or 2-hydoxyphenazine, the soluble analog of methanophenazine (MP). Reduced 2-hydoxyphenazine was re-oxidized by membranes that was dependent on addition of CoM-S-S-CoB. A genomic analysis of Methanosarcina thermophila, a non-H₂-utilizing acetotrophic methanogen, identified genes homologous to cytochrome c and the Ma-Rnf complex of M. acetivorans. Conclusions: The results support roles for ferredoxin, cytochrome c and MP in the energy-conserving electron transport pathway of non-H₂-utilizing acetotrophic methanogens. This is the first report of involvement of a cytochrome c in acetotrophic methanogenesis. The results suggest that diverse acetotrophic Methanosarcina species have evolved diverse membrane-bound electron transport pathways leading from ferredoxin and culminating with MP donating electrons to the heterodisulfide reductase (HdrDE) for reduction of CoM-S-S-CoB.

A methanogenic organism from the domain Archaea (SD1T) was isolated from saline water released from a coal seam located 926 m below the surface via a methane-producing well near Monroe, Louisiana, USA. Growth and methanogenesis were supported with methanol, monomethylamine, dimethylamine or trimethylamine, but not with dimethylsulfide, formate, acetate or H2/CO2. Cells grew in high-salt minimal medium but growth was stimulated with yeast extract or tryptone. Cells were single, non-motile, irregular coccoids 0.5–1.0 μm in diameter and the cell wall contained protein. Conditions for the maximum rate of growth were 40–50 °C, 0.2–0.6 M NaCl, 100–≥200 mM MgCl2, and pH 7.0–8.0. The G+C content of the genomic DNA was 42±1mol %. A comparison of 16S rRNA gene sequences indicated that strain SD1T was most closely related to Methanolobus oregonensis DSM 5435T with 96 % gene sequence similarity. It is proposed that strain SD1T represents a novel species, Methanolobus zinderi sp. nov. The type strain is SD1T (=ATCC BAA-1601T=DSM 21339T).

China is the largest sweet potato producer and exporter in the world. Sweet potato residues (SPRs) separated after extracting starch account for more than 10 % of the total dry matter of sweet potatoes. In China, more than 2 million tons of SPRs cannot be utilized, and the unutilized SPRs are perishable and result in environmental pollution. Thus, an environmentally friendly and highly efficient process for bioethanol production from SPRs should be developed. The swelling behaviour of cellulose causes high-gravity sweet potato residues to be recalcitrant to enzymatic hydrolysis. Cellulase plays a major role in viscosity reduction and glucose production. In contrast, pectinase has a minor role in viscosity reduction but acts as a “helper protein” to assist cellulase in liberating glucose, especially at low cellulase activity levels. In total, 153.46 and 168.13 g/L glucose were produced from high-gravity SPRs with cellulase and a mixture of cellulase and pectinase, respectively. These hydrolysates were fermented to form 73.37 and 79.00 g/L ethanol, respectively. Each kilogram of dry SPR was converted to form 209.62 and 225.71 g of ethanol, respectively. The processes described in this study have an enormous potential for industrial production of bioethanol because they are environmentally friendly, highly productive, economic with low cost, and can be easily manipulated.