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Item Methyl-reducing methanogenesis by a thermophilic culture of Korarchaeia(Springer Science and Business Media LLC, 2024-07) Krukenberg, Viola; Kohtz, Anthony J.; Jay, Zackary J.; Hatzenpichler, RolandMethanogenesis mediated by archaea is the main source of methane, a strong greenhouse gas, and thus is critical for understanding Earth’s climate dynamics. Recently, genes encoding diverse methanogenesis pathways have been discovered in metagenome-assembled genomes affiliated with several archaeal phyla1,2,3,4,5,6,7. However, all experimental studies on methanogens are at present restricted to cultured representatives of the Euryarchaeota. Here we show methanogenic growth by a member of the lineage Korarchaeia within the phylum Thermoproteota (TACK superphylum)5,6,7. Following enrichment cultivation of ‘Candidatus Methanodesulfokora washburnenis’ strain LCB3, we used measurements of metabolic activity and isotope tracer conversion to demonstrate methanol reduction to methane using hydrogen as an electron donor. Analysis of the archaeon’s circular genome and transcriptome revealed unique modifications in the energy conservation pathways linked to methanogenesis, including enzyme complexes involved in hydrogen and sulfur metabolism. The cultivation and characterization of this new group of archaea is critical for a deeper evaluation of the diversity, physiology and biochemistry of methanogens.Item Cultivation and visualization of a methanogen of the phylum Thermoproteota(Springer Science and Business Media LLC, 2024-07) Kohtz, Anthony J.; Petrosian, Nikolai; Krukenberg, Viola; Jay, Zackary J.; Pihofer, Martin; Hatzenpichler, RolandMethane is the second most abundant climate-active gas, and understanding its sources and sinks is an important endeavour in microbiology, biogeochemistry, and climate sciences1,2. For decades, it was thought that methanogenesis, the ability to conserve energy coupled to methane production, was taxonomically restricted to a metabolically specialized group of archaea, the Euryarchaeota1. The discovery of marker genes for anaerobic alkane cycling in metagenome-assembled genomes obtained from diverse habitats has led to the hypothesis that archaeal lineages outside the Euryarchaeota are also involved in methanogenesis3,4,5,6. Here we cultured Candidatus Methanosuratincola verstraetei strain LCB70, a member of the archaeal class Methanomethylicia (formerly Verstraetearchaeota) within the phylum Thermoproteota, from a terrestrial hot spring. Growth experiments combined with activity assays, stable isotope tracing, and genomic and transcriptomic analyses demonstrated that this thermophilic archaeon grows by means of methyl-reducing hydrogenotrophic methanogenesis. Cryo-electron tomography revealed that Ca. M. verstraetei are coccoid cells with archaella and chemoreceptor arrays, and that they can form intercellular bridges connecting two to three cells with continuous cytoplasm and S-layer. The wide environmental distribution of Ca. M. verstraetei suggests that they might play important and hitherto overlooked roles in carbon cycling within diverse anoxic habitats.Item MicrobioRaman: an open-access web repository for microbiological Raman spectroscopy data(Springer Science and Business Media LLC, 2024-05) Lee, Kang Soo et al.; Hatzenpichler, RolandHere we present the establishment of an open-access web-based repository for microbiological Raman spectroscopy data. The data collection, called ‘MicrobioRaman’ (https://www.ebi.ac.uk/biostudies/MicrobioRaman/studies), was inspired by the great success and usefulness of research databases such as GenBank and UniProt. This centralized repository, residing within the BioStudies database1 — which is maintained by a public institution, the European Bioinformatics Institute — minimizes the risk of data loss or eventual abandonment, offering a long-term common reference for analysis with advantages in accessibility and transparency over commercial data analysis tools. We feel that MicrobioRaman will provide a foundation for this growing field by serving as an open-access repository for sharing microbiological Raman data and through the codification of a set of reporting standards.Item Diversity and evolution of nitric oxide reduction in bacteria and archaea(Proceedings of the National Academy of Sciences, 2024-06) Murali, Ranjani; Pace, Laura A.; Sanford, Robert A.; Ward, L. M.; Lynes, Mackenzie M.; Hatzenpichler, Roland; Lingappa, Usha F.; Fischer, Woodward W.; Gennis, Robert B.; Hemp, JamesWith the advent of culture-independent techniques for studying environmental microbes, our knowledge of their diversity has exploded, uncovering unique organisms, pathways, and proteins carrying out important processes in the biosphere. Novel biochemical reactions are often proposed based on sequence data, but experimental validation is difficult and rare. In this work, we used environmental sequence data to find enzymes that produce the greenhouse gas N2O from NO and validated our hypothesis with experiments. These new enzymes likely contribute to global N2O fluxes and expand the breadth of nitrogen cycling. We also demonstrated that these enzymes evolved multiple times from oxygen reductases, indicating that the evolutionary histories of aerobic respiration and denitrification—and more broadly the oxygen and nitrogen cycles—are tightly connected.Item Methylotrophic methanogenesis in the Archaeoglobi revealed by cultivation of Ca. Methanoglobus hypatiae from a Yellowstone hot spring(Oxford University Press, 2024-03) Lynes, Mackenzie M.; Jay, Zackary J.; Kohtz, Anthony J.; Hatzenpichler, RolandOver the past decade, environmental metagenomics and polymerase chain reaction-based marker gene surveys have revealed that several lineages beyond just a few well-established groups within the Euryarchaeota superphylum harbor the genetic potential for methanogenesis. One of these groups are the Archaeoglobi, a class of thermophilic Euryarchaeota that have long been considered to live non-methanogenic lifestyles. Here, we enriched Candidatus Methanoglobus hypatiae, a methanogen affiliated with the family Archaeoglobaceae, from a hot spring in Yellowstone National Park. The enrichment is sediment-free, grows at 64–70°C and a pH of 7.8, and produces methane from mono-, di-, and tri-methylamine. Ca. M. hypatiae is represented by a 1.62 Mb metagenome-assembled genome with an estimated completeness of 100% and accounts for up to 67% of cells in the culture according to fluorescence in situ hybridization. Via genome-resolved metatranscriptomics and stable isotope tracing, we demonstrate that Ca. M. hypatiae expresses methylotrophic methanogenesis and energy-conserving pathways for reducing monomethylamine to methane. The detection of Archaeoglobi populations related to Ca. M. hypatiae in 36 geochemically diverse geothermal sites within Yellowstone National Park, as revealed through the examination of previously published gene amplicon datasets, implies a previously underestimated contribution to anaerobic carbon cycling in extreme ecosystems.Item Physiological potential and evolutionary trajectories of syntrophic sulfate-reducing bacterial partners of anaerobic methanotrophic archaea(Public Library of Science, 2023-09) Murali, Ranjani; Yu, Hang; Speth, Daan R.; Wu, Fabai; Metcalfe, Kyle S.; Crémière, Antoine; Laso-Pèrez, Rafael; Malmstrom, Rex R.; Goudeau, Danielle; Woyke, Tanja; Hatzenpichler, Roland; Chadwick, Grayson L.; Connon, Stephanie A.; Orphan, Victoria J.Sulfate-coupled anaerobic oxidation of methane (AOM) is performed by multicellular consortia of anaerobic methanotrophic archaea (ANME) in obligate syntrophic partnership with sulfate-reducing bacteria (SRB). Diverse ANME and SRB clades co-associate but the physiological basis for their adaptation and diversification is not well understood. In this work, we used comparative metagenomics and phylogenetics to investigate the metabolic adaptation among the 4 main syntrophic SRB clades (HotSeep-1, Seep-SRB2, Seep-SRB1a, and Seep-SRB1g) and identified features associated with their syntrophic lifestyle that distinguish them from their non-syntrophic evolutionary neighbors in the phylum Desulfobacterota. We show that the protein complexes involved in direct interspecies electron transfer (DIET) from ANME to the SRB outer membrane are conserved between the syntrophic lineages. In contrast, the proteins involved in electron transfer within the SRB inner membrane differ between clades, indicative of convergent evolution in the adaptation to a syntrophic lifestyle. Our analysis suggests that in most cases, this adaptation likely occurred after the acquisition of the DIET complexes in an ancestral clade and involve horizontal gene transfers within pathways for electron transfer (CbcBA) and biofilm formation (Pel). We also provide evidence for unique adaptations within syntrophic SRB clades, which vary depending on the archaeal partner. Among the most widespread syntrophic SRB, Seep-SRB1a, subclades that specifically partner ANME-2a are missing the cobalamin synthesis pathway, suggestive of nutritional dependency on its partner, while closely related Seep-SRB1a partners of ANME-2c lack nutritional auxotrophies. Our work provides insight into the features associated with DIET-based syntrophy and the adaptation of SRB towards it.Item Natural and anthropogenic carbon input affect microbial activity in salt marsh sediment(Frontiers Media SA, 2023-09) Frates, Erin S.; Lange Spietz, Rachel K.; Silverstein, Michael R.; Girguis, Peter; Hatzenpichler, Roland; Marlow, Jeffrey J.Salt marshes are dynamic, highly productive ecosystems positioned at the interface between terrestrial and marine systems. They are exposed to large quantities of both natural and anthropogenic carbon input, and their diverse sediment-hosted microbial communities play key roles in carbon cycling and remineralization. To better understand the effects of natural and anthropogenic carbon on sediment microbial ecology, several sediment cores were collected from Little Sippewissett Salt Marsh (LSSM) on Cape Cod, MA, USA and incubated with either Spartina alterniflora cordgrass or diesel fuel. Resulting shifts in microbial diversity and activity were assessed via bioorthogonal non-canonical amino acid tagging (BONCAT) combined with fluorescence-activated cell sorting (FACS) and 16S rRNA gene amplicon sequencing. Both Spartina and diesel amendments resulted in initial decreases of microbial diversity as well as clear, community-wide shifts in metabolic activity. Multi-stage degradative frameworks shaped by fermentation were inferred based on anabolically active lineages. In particular, the metabolically versatile Marinifilaceae were prominent under both treatments, as were the sulfate-reducing Desulfovibrionaceae, which may be attributable to their ability to utilize diverse forms of carbon under nutrient limited conditions. By identifying lineages most directly involved in the early stages of carbon processing, we offer potential targets for indicator species to assess ecosystem health and highlight key players for selective promotion of bioremediation or carbon sequestration pathways.Item Functional and Phylogenetic Diversity of Cas10 Proteins(Mary Ann Liebert Inc, 2023-04) Wiegand, Tanner; Wilkinson, Royce; Santiago-Frangos, Andrew; Lynes, Mackenzie; Hatzenpichler, Roland; Wiedenheft, BlakeCas10 proteins are large subunits of type III CRISPR RNA (crRNA)-guided surveillance complexes, many of which have nuclease and cyclase activities. Here, we use computational and phylogenetic methods to identify and analyze 2014 Cas10 sequences from genomic and metagenomic databases. Cas10 proteins cluster into five distinct clades that mirror previously established CRISPR-Cas subtypes. Most Cas10 proteins (85.0%) have conserved polymerase active-site motifs, while HD-nuclease domains are less well conserved (36.0%). We identify Cas10 variants that are split over multiple genes or genetically fused to nucleases activated by cyclic nucleotides (i.e., NucC) or components of toxin–antitoxin systems (i.e., AbiEii). To clarify the functional diversification of Cas10 proteins, we cloned, expressed, and purified five representatives from three phylogenetically distinct clades. None of the Cas10s are functional cyclases in isolation, and activity assays performed with polymerase domain active site mutants indicate that previously reported Cas10 DNA-polymerase activity may be a result of contamination. Collectively, this work helps clarify the phylogenetic and functional diversity of Cas10 proteins in type III CRISPR systems.Item Culexarchaeia, a novel archaeal class of anaerobic generalists inhabiting geothermal environments(Springer Science and Business Media LLC, 2022-09) Kohtz, Anthony J.; Jay, Zackary J.; Lynes, Mackenzie M.; Krukenberg, Viola; Hatzenpichler, RolandGeothermal environments, including terrestrial hot springs and deep-sea hydrothermal sediments, often contain many poorly understood lineages of archaea. Here, we recovered ten metagenome-assembled genomes (MAGs) from geothermal sediments and propose that they constitute a new archaeal class within the TACK superphylum, “Candidatus Culexarchaeia”, named after the Culex Basin in Yellowstone National Park. Culexarchaeia harbor distinct sets of proteins involved in key cellular processes that are either phylogenetically divergent or are absent from other closely related TACK lineages, with a particular divergence in cell division and cytoskeletal proteins. Metabolic reconstruction revealed that Culexarchaeia have the capacity to metabolize a wide variety of organic and inorganic substrates. Notably, Culexarchaeia encode a unique modular, membrane associated, and energy conserving [NiFe]-hydrogenase complex that potentially interacts with heterodisulfide reductase (Hdr) subunits. Comparison of this [NiFe]-hydrogenase complex with similar complexes from other archaea suggests that interactions between membrane associated [NiFe]-hydrogenases and Hdr may be more widespread than previously appreciated in both methanogenic and non-methanogenic lifestyles. The analysis of Culexarchaeia further expands our understanding of the phylogenetic and functional diversity of lineages within the TACK superphylum and the ecology, physiology, and evolution of these organisms in extreme environments.Item Comparative genomics reveals electron transfer and syntrophic mechanisms differentiating methanotrophic and methanogenic archaea(Public Library of Science, 2022-01) Chadwick, Grayson L.; Skennerton, Connor T.; Laso-Pérez, Rafael; Leu, Andy O.; Speth, Daan R.; Yu, Hang; Morgan-Lang, Connor; Hatzenpichler, Roland; Goudeau, Danielle; Malmstrom, Rex; Brazelton, William J.; Woyke, Tanja; Hallam, Steven J.; Tyson, Gene W.; Wegener, Gunter; Boetius, Antje; Orphan, Victoria J.The anaerobic oxidation of methane coupled to sulfate reduction is a microbially mediated process requiring a syntrophic partnership between anaerobic methanotrophic (ANME) archaea and sulfate-reducing bacteria (SRB). Based on genome taxonomy, ANME lineages are polyphyletic within the phylum Halobacterota, none of which have been isolated in pure culture. Here, we reconstruct 28 ANME genomes from environmental metagenomes and flow sorted syntrophic consortia. Together with a reanalysis of previously published datasets, these genomes enable a comparative analysis of all marine ANME clades. We review the genomic features that separate ANME from their methanogenic relatives and identify what differentiates ANME clades. Large multiheme cytochromes and bioenergetic complexes predicted to be involved in novel electron bifurcation reactions are well distributed and conserved in the ANME archaea, while significant variations in the anabolic C1 pathways exists between clades. Our analysis raises the possibility that methylotrophic methanogenesis may have evolved from a methanotrophic ancestor.
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