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Methanothermobacter Met2 is a metagenome-assembled genome (MAG) that encodes a putative mixotrophic methanogen constituting the major populations in thermophilic fixed-bed anaerobic digesters. In order to characterize its physiology, the present work isolated an archaeon (strain Met2-1) that represents Met2-type methanogens by using a combination of enrichments under a nitrogen atmosphere, colony formation on solid media and limiting dilution under high partial pressures of hydrogen. Strain Met2-1 utilizes hydrogen and carbon dioxide for methanogenesis, while the growth is observed only when culture media are additionally supplemented with acetate. It does not grow on acetate in the absence of hydrogen. The results demonstrate that Methanothermobacter sp. strain Met2-1 is a novel methanogen that exhibits obligate mixotrophy.

The acetyl-CoA “Wood–Ljungdahl” pathway couples the folate-mediated one-carbon (C1) metabolism to either CO ₂ reduction or acetate oxidation via acetyl-CoA. This pathway is distributed in diverse anaerobes and is used for both energy conservation and assimilation of C1 compounds. Genome annotations for all sequenced strains of Dehalococcoides mccartyi , an important bacterium involved in the bioremediation of chlorinated solvents, reveal homologous genes encoding an incomplete Wood–Ljungdahl pathway. Because this pathway lacks key enzymes for both C1 metabolism and CO ₂ reduction, its cellular functions remain elusive. Here we used D. mccartyi strain 195 as a model organism to investigate the metabolic function of this pathway and its impacts on the growth of strain 195. Surprisingly, this pathway cleaves acetyl-CoA to donate a methyl group for production of methyl-tetrahydrofolate (CH ₃-THF) for methionine biosynthesis, representing an unconventional strategy for generating CH ₃-THF in organisms without methylene-tetrahydrofolate reductase. Carbon monoxide (CO) was found to accumulate as an obligate by-product from the acetyl-CoA cleavage because of the lack of a CO dehydrogenase in strain 195. CO accumulation inhibits the sustainable growth and dechlorination of strain 195 maintained in pure cultures, but can be prevented by CO-metabolizing anaerobes that coexist with D. mccartyi , resulting in an unusual syntrophic association. We also found that this pathway incorporates exogenous formate to support serine biosynthesis. This study of the incomplete Wood–Ljungdahl pathway in D. mccartyi indicates a unique bacterial C1 metabolism that is critical for D. mccartyi growth and interactions in dechlorinating communities and may play a role in other anaerobic communities.

We present a Nip site model of acetyl coenzyme-A synthase (ACS) within a de novo-designed trimer peptide that self-assembles to produce a homoleptic Ni(Cys)3 binding motif. Spectroscopic and kinetic studies of ligand binding demonstrate that Ni binding stabilizes the peptide assembly and produces a terminal NiI-CO complex. When the CO-bound state is reacted with a methyl donor, a new species is quickly produced with new spectral features. While the metal-bound CO is albeit unactivated, the presence of the methyl donor produces an activated metal-CO complex. Selective outer sphere steric modifications demonstrate that the physical properties of the ligand-bound states are altered differently depending on the location of the steric modification above or below the Ni site.

In a recent study, it has been shown that biosynthesis of triacylglycerol (TAG) in the oleaginous green alga Chlorella desiccata is preceded by a large increase in acetyl-coenzyme A (Ac-CoA) levels and by upregulation of plastidic pyruvate dehydrogenase (ptPDH). It was proposed that the capacity to accumulate high TAG critically depends on enhanced production of Ac-CoA. In this study, two alternative Ac-CoA producers—plastidic Ac-CoA synthase (ptACS) and ATP citrate lyase (ACL)—are shown to be upregulated prior to TAG accumulation under nitrogen deprivation in the oleaginous species C. desiccata, but not in the moderate TAG accumulators Dunaliella tertiolecta and Chlamydomonas reinhardtii. Measurements of endogenous acetate production and of radiolabelled acetate incorporation into lipids are consistent with the upregulation of ptACS, but suggest that its contribution to the overall TAG biosynthesis is negligible. Induction of ACS and production of endogenous acetate are correlated with activation of alcohol dehydrogenase, suggesting that the upregulation of ptACS is associated with activation of PDH-bypass in C. desiccata. It is proposed that activation of the PDH-bypass in C. desiccata is needed to enable a high rate of lipid biosynthesis under nitrogen deprivation by controlling the level of pyruvate reaching ptPHD and/or mtPDH. This may be an important parameter for massive TAG accumulation in microalgae.

Thermoanaerobacter kivui is a thermophilic acetogen that can grow on carbon monoxide as sole carbon and energy source. To identify the gene(s) involved in CO oxidation, the genome sequence was analyzed. Two genes potentially encoding CO dehydrogenases were identified. One, cooS, potentially encodes a monofunctional CO dehydrogenase, whereas another, acsA, potentially encodes the CODH component of the CODH/ACS complex. Both genes were cloned, a His-tag encoding sequence was added, and the proteins were produced from a plasmid in T. kivui. His-AcsA copurified by affinity chromatography with AcsB, the acetyl-CoA synthase of the CO dehydrogenase/acetyl CoA synthase complex. His-CooS copurified with CooF1, a small iron-sulfur center containing protein likely involved in electron transport. Both protein complexes had CO:ferredoxin oxidoreductase as well as CO:methyl viologen oxidoreductase activity, but the activity of CooSF1 was 15-times and 231-times lower, respectively. To underline the importance of CooS, the gene was deleted in the CO-adapted strain. Interestingly, the ∆cooS deletion mutant did not grow on CO anymore. These experiments clearly demonstrated that CooS is essential for growth of T. kivui on CO. This is in line with the hypothesis that CooS is the CO-oxidizing enzyme in cells growing on CO.

ATG5-induced autophagy is triggered in the early stages after SAH, which plays a vital role in subarachnoid hemorrhage (SAH). Acyl-CoA synthetase short-chain family 2 (ACSS2) is not just involved in energy metabolism but also binds to TEFB to form a complex translocated to related autophagy genes to regulate the expression of autophagy-related genes. However, the contribution of ACSS2 to the activation of autophagy in early brain injury (EBI) after SAH has barely been discussed. The purpose of this study was to investigate the alterations of ACSS2 and its neuroprotective effects following SAH. We first evaluated the expression of ACSS2 at different time points (6, 12, 24, and 72 h after SAH) in vivo and primary cortical neurons stimulated by oxyhemoglobin (OxyHb). Subsequently, adeno-associated virus and lentivirus were used to regulate ACSS2 expression to investigate the effect of ACSS2 after SAH. The results showed that the ACSS2 level decreased significantly in the early stages of SAH and was minimized at 24 h post-SAH. After artificial intervention to overexpress ACSS2, ATG5-induced autophagy was further enhanced in EBI after SAH, and neuronal apoptosis was alleviated to protect brain injury. In addition, brain edema and neurological function scores were improved. These results suggest that ACSS2 plays an important role in the neuroprotection against EBI after SAH by increasing ATG5-induce autophagy and inhibiting apoptosis.

Lignocellulosic wastes (LCW) have enormous potential to be recycled for bioethanol production. Although yeasts ( Saccharomyces sp.) are commonly used bio-agents for fermentation, their efficiency is inhibited in cellulosic feedstocks. This study isolated novel ethanologenic bacteria from vermicomposting systems for bioenergy generation from fruit waste without pre-treatment. Initially, six strains out of 22, showing remarkable ethanol production ability, were characterized via 16S rRNA sequencing. Specifically, two strains ( Bacillus alcalophilus C5 and Rhizobium spp. S10) produced more ethanol (5.5 and 15.7 g L −1 ) than the yeast (5 g L −1 ) from banana epicarps. These strains' dramatically high sedimentation rate and ethanol tolerance strongly justified their industrial applicability. Significant upregulation of alcohol dehydrogenase and acetyl CoA synthase endowed greater ethanol-producing capacity in C5 and S10 than in S. cerevisiae . The flow cytometry and confocal microscopy evidenced that ethanologenic bacteria uniquely defend the reactor-induced sugar and ethanol stresses through reverse/delayed apoptosis and robust membrane integrity. The waste-to-wealth conversion efficiency and cost–benefit analyses estimated that bacteria-mediated LCW-to-bioethanol conversion was a more profitable venture than vermicomposting or composting. Overall, this research demonstrated that the C5 and S10 isolates were more effective than widely used commercial yeast strains for bioethanol generation from LCW. Graphical Abstract

Lateral gene transfer (LGT) is an important mechanism of evolution for protists adapting to oxygen-poor environments. Specifically, modifications of energy metabolism in anaerobic forms of mitochondria (e.g., hydrogenosomes) are likely to have been associated with gene transfer from prokaryotes. An interesting question is whether the products of transferred genes were directly targeted into the ancestral organelle or initially operated in the cytosol and subsequently acquired organelle-targeting sequences. Here, we identified key enzymes of hydrogenosomal metabolism in the free-living anaerobic amoebozoan Mastigamoeba balamuthi and analyzed their cellular localizations, enzymatic activities, and evolutionary histories. Additionally, we characterized 1) several canonical mitochondrial components including respiratory complex II and the glycine cleavage system, 2) enzymes associated with anaerobic energy metabolism, including an unusual D-lactate dehydrogenase and acetyl CoA synthase, and 3) a sulfate activation pathway. Intriguingly, components of anaerobic energy metabolism are present in at least two gene copies. For each component, one copy possesses an mitochondrial targeting sequence (MTS), whereas the other lacks an MTS, yielding parallel cytosolic and hydrogenosomal extended glycolysis pathways. Experimentally, we confirmed that the organelle targeting of several proteins is fully dependent on the MTS. Phylogenetic analysis of all extended glycolysis components suggested that these components were acquired by LGT. We propose that the transformation from an ancestral organelle to a hydrogenosome in the M. balamuthi lineage involved the lateral acquisition of genes encoding extended glycolysis enzymes that initially operated in the cytosol and that established a parallel hydrogenosomal pathway after gene duplication and MTS acquisition.

Despite its toxicity for the majority of living matter on our planet, numerous microorganisms, both aerobic and anaerobic, can use carbon monoxide (CO) as a source of carbon and/or energy for growth. The capacity to employ carboxidotrophic energy metabolism anaerobically is found in phylogenetically diverse members of the Bacteria and the Archaea. The oxidation of CO is coupled to numerous respiratory processes, such as desulfurication, hydrogenogenesis, acetogenesis, and methanogenesis. Although as diverse as the organisms capable of it, any CO-dependent energy metabolism known depends on the presence of carbon monoxide dehydrogenase. This review summarizes recent insights into the CO-dependent physiology of anaerobic microorganisms with a focus on methanogenic archaea. Carboxidotrophic growth of Methanosarcina acetivorans, thought to strictly rely on the process of methanogenesis, also involves formation of methylated thiols, formate, and even acetogenesis, and, thus, exemplifies how the beneficial redox properties of CO can be exploited in unexpected ways by anaerobic microorganisms.

This review focuses on how microbes live on CO as a sole source of carbon and energy and with CO by generating carbon monoxide as a metabolic intermediate. The use of CO is a property of organisms that use the Wood-L jungdahl pathway of autotrophic growth. The review discusses when CO metabolism originated, when and how it was discovered, and what properties of CO are ideal for microbial growth. How CO sensing by a heme-containing transcriptional regulatory protein activates the expression of CO metabolism-linked genes is described. Two metalloenzymes are the cornerstones of growth with CO: CO dehydrogenase (CODH) and acetyl-CoA synthase (ACS). CODH oxidizes CO to CO2, providing low-potential electrons for the cell, or alternatively reduces CO2 to CO. The latter reaction, when coupled to ACS, forms a machine for generating acetyl-CoA from CO2 for cell carbon synthesis. The recently solved crystal structures of CODH and ACS along with spectroscopic measurements and computational studies provide insights into novel bio-organometallic catalytic mechanisms and into the nature of a 140 A gas channel that coordinates the generation and utilization of CO. The enzymes that are coupled to CODH/ACS are also described, with a focus on a corrinoid protein, a methyltransferase, and pyruvate ferredoxin oxidoreductase.