Scholarly Work - Chemistry & Biochemistry
Permanent URI for this collectionhttps://scholarworks.montana.edu/handle/1/8714
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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 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 Diversity and function of methyl-coenzyme M reductase-encoding archaea in Yellowstone hot springs revealed by metagenomics and mesocosm experiments(Springer Science and Business Media LLC, 2023-03) Lynes, Mackenzie M.; Krukenberg, Viola; Jay, Zackary J.; Kohtz, Anthony J.; Gobrogge, Christine A.; Lange Spietz, Rachel K.; Hatzenpichler, RolandMetagenomic studies on geothermal environments have been central in recent discoveries on the diversity of archaeal methane and alkane metabolism. Here, we investigated methanogenic populations inhabiting terrestrial geothermal features in Yellowstone National Park (YNP) by combining amplicon sequencing with metagenomics and mesocosm experiments. Detection of methyl-coenzyme M reductase subunit A (mcrA) gene amplicons demonstrated a wide diversity of Mcr-encoding archaea inhabit geothermal features with differing physicochemical regimes across YNP. From three selected hot springs we recovered twelve Mcr-encoding metagenome assembled genomes (MAGs) affiliated with lineages of cultured methanogens as well as Candidatus (Ca.) Methanomethylicia, Ca. Hadesarchaeia, and Archaeoglobi. These MAGs encoded the potential for hydrogenotrophic, aceticlastic, hydrogen-dependent methylotrophic methanogenesis, or anaerobic short-chain alkane oxidation. While Mcr-encoding archaea represent minor fractions of the microbial community of hot springs, mesocosm experiments with methanogenic precursors resulted in the stimulation of methanogenic activity and the enrichment of lineages affiliated with Methanosaeta and Methanothermobacter as well as with uncultured Mcr-encoding archaea including Ca. Korarchaeia, Ca. Nezhaarchaeia, and Archaeoglobi. We revealed that diverse Mcr-encoding archaea with the metabolic potential to produce methane from different precursors persist in the geothermal environments of YNP and can be enriched under methanogenic conditions. This study highlights the importance of combining environmental metagenomics with laboratory-based experiments to expand our understanding of uncultured Mcr-encoding archaea and their potential impact on microbial carbon transformations in geothermal environments and beyond.Item Activity-based cell sorting reveals responses of uncultured archaea and bacteria to substrate amendment(2020) Reichart, Nicholas J.; Jay, Zackary J.; Krukenberg, Viola; Parker, Albert E.; Lange Spietz, Rachel K.; Hatzenpichler, RolandMetagenomic studies have revolutionized our understanding of the metabolic potential of uncultured microorganisms in various ecosystems. However, many of these genomic predictions have yet to be experimentally tested, and the functional expression of genomic potential often remains unaddressed. In order to obtain a more thorough understanding of cell physiology, novel techniques capable of testing microbial metabolism under close to in situ conditions must be developed. Here, we provide a benchmark study to demonstrate that bioorthogonal non-canonical amino acid tagging (BONCAT) in combination with fluorescence-activated cell sorting (FACS) and 16S rRNA gene sequencing can be used to identify anabolically active members of a microbial community incubated in the presence of various growth substrates or under changing physicochemical conditions. We applied this approach to a hot spring sediment microbiome from Yellowstone National Park (Wyoming, USA) and identified several microbes that changed their activity levels in response to substrate addition, including uncultured members of the phyla Thaumarchaeota, Acidobacteria, and Fervidibacteria. Because shifts in activity in response to substrate amendment or headspace changes are indicative of microbial preferences for particular growth conditions, results from this and future BONCAT-FACS studies could inform the development of cultivation media to specifically enrich uncultured microbes. Most importantly, BONCAT-FACS is capable of providing information on the physiology of uncultured organisms at as close to in situ conditions as experimentally possible.