Metagenomic and metatranscriptomic characterization of a microbial community that catalyzes both energy-generating and energy-storing electrode reactions

Electroactive bacteria are living catalysts, mediating energy-generating reactions at anodes or energy storage reactions at cathodes via extracellular electron transfer (EET). The Cathode-ANode (CANode) biofilm community was recently shown to facilitate both reactions, however, the identity of the primary constituents and underlying molecular mechanisms remain unknown. Here, we used metagenomics and metatranscriptomics to characterize the CANode biofilm. We show that a previously uncharacterized member of the family Desulfobulbaceae, Desulfobulbaceae -2, which had <1% relative abundance, had the highest relative gene expression and accounted for over 60% of all differentially expressed genes. At the anode potential, differential expression of genes for a conserved flavin oxidoreductase (Flx) and heterodisulfide reductase (Hdr) known to be involved in ethanol oxidation suggests a source of electrons for the energy-generating reaction. Genes for sulfate and carbon dioxide reduction pathways were expressed by Desulfobulbaceae -2 at both potentials and are the proposed energy storage reactions. Reduction reactions may be mediated by direct electron uptake from the electrode, or from hydrogen generated at the cathode potential. The Desulfobulbaceae -2 genome is predicted to encode at least 85 multi-heme (≥3 hemes) c -type cytochromes, some with as many as 26 heme-binding domains, that could facilitate reversible electron transfer with the electrode. Gene expression in other CANode biofilm species was also affected by the electrode potential, although to a lesser extent, and we cannot rule out their contribution to observed current. Results provide evidence of gene expression linked to energy storage and energy-generating reactions and will enable development of the CANode biofilm as a microbially-driven rechargeable battery. IMPORTANCE Microbial electrochemical technologies (METs) rely on electroactive bacteria to catalyze energy-generating and energy storage reactions at electrodes. Known electroactive bacteria are not equally capable of both reactions and METs are typically configured to be unidirectional. Here we report on genomic and transcriptomic characterization of a recently described microbial electrode community called the Cathode-ANode (CANode). The CANode community is able to generate or store electrical current based on the electrode potential. During periods where energy is not needed, electrons generated from a renewable source, such as solar power, could be converted into energy storage compounds to later be reversibly oxidized by the same microbial catalyst. Thus, the CANode system can be thought of as a living “rechargeable battery”. Results show that a single organism may be responsible for both reactions demonstrating a new paradigm for electroactive bacteria.

The Synechocystis ORF slr0201 product is involved in succinate dehydrogenase-mediated cyclic electron transfer around PSI

The Synechocystis sp. PCC 6803 open reading frame (ORF) slr0201 was originally annotated as heterodisulfide reductase B subunit (HdrB). The slr0201 encodes a 301-amino acid hypothetical protein with the predicted amino acid sequence significantly homologous to not only the HdrB from methanogenic bacteria, but also some novel succinate dehydrogenase C subunit (SdhC) found in Archaea and Campylobacter. Genetic manipulation via knocking-out approach created a Δslr0201 mutant showing a ΔsdhB-like phenotype that was characterized by impaired succinate-dependent DCPIP reduction activities, reduced SDH-mediated respiratory electron transports, lower cellular contents of succinate and fumarate, slower KCN-induced increases in Chl fluorescence yield in the dark, and weak state 2/strong state 1 transitions, being indicative of a more oxidized PQ pool. In addition, slower re-reductions of the photosystem (PS) I reaction center P700 upon light-off were also monitored in the Δslr0201, indicating functional involvements of Slr0201 in cyclic electron transfer around PSI. Both photoautptrophical and photomixotrophical growth rates of the Δslr0201 strain resembled to those of the wild type, but substantial growth deteriorations occurred when arginine (~25 mM) or other two urea-cycle relevant amino acids (citrulline and ornithine) were added, which were attributed to generations and accumulations of certain hazardous metabolites. Based on the ΔsdhB-resembling phenotype, in conjunction with its high sequence similarities to some archaeal SdhC, we proposed that the slr0201 encodes a SDH function-relevant protein and is most likely the SdhC, a membrane anchoring subunit, which, while being genetically distinct from those in traditional bacterial SDH, belongs to the C subunit of novel archaeal SDH.

The Synechocystis sp. PCC 6803 open reading frame slr0201 that is homologous to sdhC from Archaea codes for a [2Fe-2S] protein

A hypothetical protein encoded by Synechocystis sp. PCC 6803 open reading frame slr0201 shows high sequence similarity to the C subunit of a group of unusual succinate dehydrogenases found in some archaeal species. Slr0201 was originally annotated as HdrB, the B subunit of heterodisulfide reductase, but appears to be SdhC instead. This protein was overexpressed in E. coli by cloning the PCR-derived slr0201 open reading frame into a pET16b-based expression vector. The overproduced Slr0201 accumulated predominantly in inclusion bodies with an apparent molecular mass of 33 kDa. The protein contained at least one [2Fe-2S] cluster based on UV-visible absorbance and CD spectra and EPR spectroscopy, in conjunction with stoichiometric analysis of protein-bound iron and sulfur content. Redox titration showed a midpoint potential (Em) of + 17 mV at pH 7.0, which is consistent with Slr0201 serving a role in transferring electrons between succinate and plastoquinone. Slr0201 was also overproduced in Synechocystis sp. PCC 6803 by introducing an additional, His-tagged slr0201 into the Synechocystis genome replacing psbA3, creating the slr0201+-His overexpression strain. Immunoblot analysis shows that Slr0201 is membrane-associated in the wild type. However, in the Slr0201+-His strain, immunoreaction occurred in both the membrane and soluble fractions, possibly as a consequence of processing near the N-terminus. The results obtained with Slr0201 are discussed in the light of one of the cyanobacterial SdhB subunits, which shares redox commonalities with archaeal SdhB.

Metagenomic Insights Into Ecological and Phylogenetic Significances of Candidatus Natranaeroarchaeales, a Novel Abundant Archaeal Order in Soda Lake Sediment

BackgroundArchaea were originally discovered in extreme environments, and thrive in many extreme habitats including soda lakes with high pH and salinity. Characteristic and diverse archaeal community played a significant role in biogeochemical cycles; however, the archaeal community and their functions are still less-studied in the intricate sediment of soda lakes. ResultsIn this article, the archaeal community of the deep sediment (40-50 cm depth) of five artificially-separated ponds with a salinity range from 7.0% to 33.0% in a soda saline lake was systematically surveyed using culture-independent metagenomics combined with the next-generation sequencing of the archaeal 16S rRNA amplicons. Nine archaeal phyla were detected, which accounted for 2.2% to 35.73% of microbial community in the five deep sediments. Besides the well-known class Halobacteria, one novel archaeal order (Candidatus Natranaeroarchaeales) of the class Thermoplasmata was even more abundant than Halobacteria in some deep sediment samples. Of 69 dereplicated archaeal metagenome-assembled genomes (MAGs), 30 MAGs belonged to Ca. Natranaeroarchaeales. Different genera of the Ca. Natranaeroarchaeales preferred to inhabit in the different salinities, and the divergent halophilic adaptation strategies (salt-out or salt-in) suggested the fast evolution adaptation within this lineage. Most high-quality MAGs had the genes of Wood-Ljungdahl pathway, organic acid fermentation and sulfur respiration, suggesting the putative functions in carbon fixation and sulfur reduction. Interestingly, heterodisulfide reductase and F420-non-reducing hydrogenase complex HdrABC-MvhADG were widely distributed in Ca. Natranaeroarchaeales and may play the core roles in energy metabolism from hydrogen. The regeneration of CoM-S-S-CoB was coupled to succinate or 2-oxoglutarate production in Ca. Natranaeroarchaeales instead of methanogenesis in the close related Methanomassiliicoccales. It suggested that methyl-coenzyme M reductase in Methanomassiliicoccales may be laterally transferred from other methanogens. ConclusionA novel archaeal order Ca. Natranaeroarchaeales of Thermoplasmata was found by culture-independent approaches. This order was the most abundant archaeal lineage in the deep sediment of soda lakes, with the characteristic environmental adaptation and biogeochemical potentials in carbon fixation and sulfur reduction. The difference in fermentation products coupled to energy metabolism between methanogens and Ca. Natranaeroarchaeales provided additional insights into the origination of methanogenesis in Thermoplasmata from the energy metabolism perspective.

Formate dependent heterodisulfide reduction in a Methanomicrobiales archaeon

Hydrogenotrophic methanogens produce CH4 using H2 as an electron donor to reduce CO2. In the absence of H2, many are able to use formate or alcohols as alternate electron donors. Methanogens from the order Methanomicrobiales are capable of growth with H2, but many lack genes encoding hydrogenases that are typically found in other hydrogenotrophic methanogens. In an effort to better understand electron flow in methanogens from the Methanomicrobiales, we undertook a genetic and biochemical study of heterodisulfide reductase (Hdr) in Methanoculleus thermophilus. Hdr catalyzes an essential reaction by coupling the first and last steps of methanogenesis through flavin-based electron bifurcation. Hdr from M. thermophilus co-purified with formate dehydrogenase (Fdh) and only displayed activity when formate was supplied as an electron donor. We found no evidence of an Hdr associated hydrogenase, and H2 could not function as an electron donor, even with Hdr purified from cells grown on H2. We found that cells catalyze a formate hydrogenlyase activity that is likely essential for generating the formate needed for the Hdr reaction. Together, these results highlight the importance of formate as an electron donor for methanogenesis and suggest the ability to use formate is closely integrated into the methanogenic pathway in organisms from the order Methanomicrobiales. Importance Methanogens from the order Methanomicrobiales are thought to prefer H2 as an electron donor for growth. They are ubiquitous in anaerobic environments such as in wastewater treatment facilities, anaerobic digesters, and the rumen where they catalyze the terminal steps in the breakdown of organic matter. However, despite their importance, the metabolism of these organisms remains understudied. Using a genetic and biochemical approach, we show that formate metabolism is closely integrated into methanogenesis in Methanoculleus thermophilus. This is due to a requirement for formate as the electron donor to heterodisulfide reductase (Hdr), an enzyme responsible for catalyzing essential reactions in methanogenesis by linking the initial CO2 fixing step to the exergonic, terminal reaction of the pathway. These results suggest that hydrogen is not necessarily the preferred electron donor for all hydrogenotrophic methanogens and provide insight into the metabolism of methanogens from the order Methanomicrobiales.

Responses of Methanosarcina barkeri to acetate stress

Background Anaerobic digestion of easily degradable biowaste can lead to the accumulation of volatile fatty acids, which will cause environmental stress to the sensitive methanogens consequently. The metabolic characteristics of methanogens under acetate stress can affect the overall performance of mixed consortia. Nevertheless, there exist huge gaps in understanding the responses of the dominant methanogens to the stress, e.g., Methanosarcinaceae. Such methanogens are resistant to environmental deterioration and able to utilize multiple carbon sources. In this study, transcriptomic and proteomic analyses were conducted to explore the responses of Methanosarcina barkeri strain MS at different acetate concentrations of 10, 25, and 50 mM. Results The trend of OD600 and the regulation of the specific genes in 50 mM acetate, indicated that high concentration of acetate promoted the acclimation of M. barkeri to acetate stress. Acetate stress hindered the regulation of quorum sensing and thereby eliminated the advantages of cell aggregation, which was beneficial to resist stress. Under acetate stress, M. barkeri allocated more resources to enhance the uptake of iron to maintain the integrities of electron-transport chains and other essential biological processes. Comparing with the initial stages of different acetate concentrations, most of the genes participating in acetoclastic methanogenesis did not show significantly different expressions except hdrB1C1, an electron-bifurcating heterodisulfide reductase participating in energy conversion and improving thermodynamic efficiency. Meanwhile, vnfDGHK and nifDHK participating in nitrogen fixation pathway were upregulated. Conclusion In this work, transcriptomic and proteomic analyses are combined to reveal the responses of M. barkeri to acetate stress in terms of central metabolic pathways, which provides basic clues for exploring the responses of other specific methanogens under high organics load. Moreover, the results can also be used to gain insights into the complex interactions and geochemical cycles among natural or engineered populations. Furthermore, these findings also provide the potential for designing effective and robust anaerobic digesters with high organic loads.

Genetic Evidence for Two Carbon Fixation Pathways (the Calvin-Benson-Bassham Cycle and the Reverse Tricarboxylic Acid Cycle) in Symbiotic and Free-Living Bacteria

ABSTRACT Very few bacteria are able to fix carbon via both the reverse tricarboxylic acid (rTCA) and the Calvin-Benson-Bassham (CBB) cycles, such as symbiotic, sulfur-oxidizing bacteria that are the sole carbon source for the marine tubeworm Riftia pachyptila, the fastest-growing invertebrate. To date, the coexistence of these two carbon fixation pathways had not been found in a cultured bacterium and could thus not be studied in detail. Moreover, it was not clear if these two pathways were encoded in the same symbiont individual, or if two symbiont populations, each with one of the pathways, coexisted within tubeworms. With comparative genomics, we show that Thioflavicoccus mobilis, a cultured, free-living gammaproteobacterial sulfur oxidizer, possesses the genes for both carbon fixation pathways. Here, we also show that both the CBB and rTCA pathways are likely encoded in the genome of the sulfur-oxidizing symbiont of the tubeworm Escarpia laminata from deep-sea asphalt volcanoes in the Gulf of Mexico. Finally, we provide genomic and transcriptomic data suggesting a potential electron flow toward the rTCA cycle carboxylase 2-oxoglutarate:ferredoxin oxidoreductase, via a rare variant of NADH dehydrogenase/heterodisulfide reductase in the E. laminata symbiont. This electron-bifurcating complex, together with NAD(P)+ transhydrogenase and Na+ translocating Rnf membrane complexes, may improve the efficiency of the rTCA cycle in both the symbiotic and the free-living sulfur oxidizer. IMPORTANCE Primary production on Earth is dependent on autotrophic carbon fixation, which leads to the incorporation of carbon dioxide into biomass. Multiple metabolic pathways have been described for autotrophic carbon fixation, but most autotrophic organisms were assumed to have the genes for only one of these pathways. Our finding of a cultivable bacterium with two carbon fixation pathways in its genome, the rTCA and the CBB cycle, opens the possibility to study the potential benefits of having these two pathways and the interplay between them. Additionally, this will allow the investigation of the unusual and potentially very efficient mechanism of electron flow that could drive the rTCA cycle in these autotrophs. Such studies will deepen our understanding of carbon fixation pathways and could provide new avenues for optimizing carbon fixation in biotechnological applications.

Lipoate-binding proteins and specific lipoate-protein ligases in microbial sulfur oxidation reveal an atpyical role for an old cofactor

Many Bacteria and Archaea employ the heterodisulfide reductase (Hdr)-like sulfur oxidation pathway. The relevant genes are inevitably associated with genes encoding lipoate-binding proteins (LbpA). Here, deletion of the gene identified LbpA as an essential component of the Hdr-like sulfur-oxidizing system in the Alphaproteobacterium Hyphomicrobium denitrificans. Thus, a biological function was established for the universally conserved cofactor lipoate that is markedly different from its canonical roles in central metabolism. LbpAs likely function as sulfur-binding entities presenting substrate to different catalytic sites of the Hdr-like complex, similar to the substrate-channeling function of lipoate in carbon-metabolizing multienzyme complexes, for example pyruvate dehydrogenase. LbpAs serve a specific function in sulfur oxidation, cannot functionally replace the related GcvH protein in Bacillus subtilis and are not modified by the canonical E. coli and B. subtilis lipoyl attachment machineries. Instead, LplA-like lipoate-protein ligases encoded in or in immediate vicinity of hdr-lpbA gene clusters act specifically on these proteins.