Biology subjects

Hatzenpichler, R.

Publications and source records attributed to Hatzenpichler, R..

15 recordsLinked to original sources

bioRxiv2026

Proteomic stress response by a novel methanogen enriched from the Great Salt Lake

Methanogenic archaea affect the climate through their production of the greenhouse gas, methane. However, it is unclear how a changing climate and other anthropogenic influences impact methanogen physiology and consequent methane flux. The Great Salt Lake (GSL) is an environment that has been heavily impacted by human activity; more than doubling its salt concentration since the last methanogen was cultured from it in 1985. In this study, we enriched a novel methanogen, for which we propose the name Candidatus Methanohalophilus hillemani, from the GSL at a time when its salinity reached a historical high. Interestingly, Ca. M. hillemani does not increase expression of energy-conservation or osmo-tolerance proteins when challenged with salinity or oxygen. In contrast, Ca. M. hillemani prioritizes trace metal uptake and immune functions in response to the presence of the sulfate-reducing bacterium Desulfovermiculus. 16S rRNA gene amplicon data from GSL shore soils with extremely high and variable methane flux indicated the presence of Ca. M. hillemani. Our results show that Ca. M. hillemani is active when challenged with environmental stressors and contributes to the methane flux emanating from the GSL. ImportanceMethanogens are microbes that affect the climate through their production of the greenhouse gas, methane. Changes in climate and land-use patterns are drying up saline lakes, damaging their unique economic and ecological value. As lake levels across the globe fall, it is unclear how methanogens and the amount of methane they produce will concurrently shift. In this study, we measured high methane output from the Great Salt Lake (GSL) across seasons and identified a novel methanogen as part of a larger methanogenic community that is responsible for these methane emissions. We cultured this novel methanogen from GSL sediments and determined that its methane production was largely unaffected by stress conditions. Our findings indicate that methanogens in saline environments, including a novel cultivated species, may be important sources of methane and will continue to produce methane as salinity increases.

microbiology↗

bioRxiv2025

Tracking active heterotrophic microbial communities in the Guaymas Basin deep biosphere with BONCAT-FACS

The marine deep biosphere harbors microbial communities that drive organic matter transformations and biogeochemical cycles. Previous work on these communities has focused either on genomic characterization or metabolic activity measurements. However, to understand microbial ecophysiology in the deep biosphere taxonomic identity and metabolic function must be connected on both single-cell and ecosystem scales. In this work, we optimized a bioorthogonal non-canonical amino acid tagging fluorescence-activated cell sorting (BONCAT-FACS) workflow for low-biomass deep-biosphere sediments obtained during International Ocean Discovery Program Expedition 385 (IODP 385). BONCAT-FACS with 16S rRNA gene amplicon sequencing as well as metagenomics of sediment communities was applied to characterize translationally active communities in hydrothermally altered subsurface sediments of the Guaymas Basin. Our results revealed a heterotrophic microbial population throughout all sediments examined, with taxa translationally active down to our deepest sampling point, 154 meters below the seafloor. Based on 16S rRNA gene identities, the translationally active microbial community was dominated by heterotrophic members of the Gammaproteobacteria, Bacilli, Deinococci, and Alphaproteobacteria. These taxa are likely key contributors to cycling the large quantities of hydrothermally altered organic matter in Guaymas Basin sediments. To further elucidate the metabolic capacity of active taxa, we mapped 16S rRNA gene amplicons to metagenome assembled genomes (MAGs) previously obtained from IODP 385. These MAGs contained genes associated with C1 metabolism, carbohydrate degradation, and fermentation, indicating that active taxa leverage these metabolisms for energy conservation. Our results demonstrate that BONCAT-FACS provides high-throughput and single-cell insights into the metabolic activity of microbes in the low-biomass marine subsurface.

microbiology↗

bioRxiv2025

Ecology of methyl-coenzyme M reductase encoding Thermoproteota

The recent demonstration that members of at least three classes of archaea affiliated with the Thermoproteota superphylum are involved in the production of the climate-active gas methane has sparked discussions into how well we understand the diversity of methanogens. Here we show that members of all three of these lineages, as well as several other, yet uncultured and physiologically uncharacterized groups within the Thermoproteota that encode the key enzyme of anaerobic methane cycling, methyl-coenzyme M reductase (MCR), are widely distributed in anoxic ecosystems. We postulate that the taxonomic, metabolic, and ecological diversity of MCR-encoding Thermoproteota is poorly understood, and that the contribution of methylotrophic and thermoproteotal methanogenesis to methane production is largely unknown. We hypothesize that thermoproteotal methanogens contribute, potentially substantially, to methane emissions in many anoxic environments that harbor methylated precursors, including wetlands, sediments, peat, rice paddies, wastewater sludge, and geothermal systems. We highlight the necessity to experimentally test the (eco)physiology of these widely distributed archaea using both culture-dependent (in vitro) and culture-independent (in situ) approaches to assess their potential contribution to methane emissions. Last, we stress the importance of remaining agnostic about the physiology of MCR-encoding Thermoproteota in the absence of experimental data because most of these archaea also carry the genetic potential to grow non-methanogenically.

microbiology↗

bioRxiv2025

Cultivation of Methanonezhaarchaeia, the third class of methanogens within the phylum Thermoproteota

Methane is a potent greenhouse gas that is largely produced through the activity of methanogenic archaea, which contribute to Earths dynamic climate and biogeochemical cycles. In the past decade, metagenomics revealed that lineages outside of the traditional Euryarchaeota superphylum encode genes for methanogenesis. This was recently confirmed through the cultivation of two classes of methanogenic Thermoproteota. Thus far, all methanogens within the Thermoproteota are predicted or were shown to be methylotrophic. The only exception to this is the Nezhaarchaea, which, based on metagenomics, are predicted to be CO2-reducing methanogens. Here, we demonstrate methanogenic activity in a third class of Thermoproteota, the Methanonezhaarchaeia. We expand the metabolic diversity of this class by cultivating a methylotrophic species, Candidatus Methanonezhaarchaeum fastidiosum YNP3N. We describe novel genes involved in methanogenesis that are not found in other methanogens. We investigate the metabolic diversity of Methanonezhaarchaeia, including metabolic modifications accompanying frequent loss of methanogenesis in this class. This highlights gaps in our understanding of the biochemistry, diversity, and evolution of non-traditional methanogens and their contributions to carbon cycling. TeaserThe cultivation of a new group of methanogens illuminates their metabolic diversity and evolution of archaea.

microbiology↗

bioRxiv2024

Comparing Raman and NanoSIMS for heavy water labeling of single cells

Stable isotope probing (SIP) experiments in conjunction with Raman microspectroscopy (Raman) or nano-scale secondary ion mass spectrometry (NanoSIMS) are frequently used to explore single cell metabolic activity in pure cultures as well as complex microbiomes. Despite the increasing popularity of these techniques, the comparability of isotope incorporation measurements using both Raman and NanoSIMS directly on the same cell remains largely unexplored. This knowledge gap creates uncertainty about the consistency of single-cell SIP data obtained independently from each method. Here, we conducted a comparative analysis of 543 Escherichia coli cells grown in M9 minimal medium in the absence or presence of heavy water (2H2O) using correlative Raman and NanoSIMS measurements to quantify the results between the two approaches. We demonstrate that Raman and NanoSIMS yield highly comparable measurements of 2H incorporation, with varying degrees of similarity based on the mass ratios analyzed using NanoSIMS. The 12C2H/12C1H and 12C22H/12C21H mass ratios provide targeted measurements of C-H bonds but may suffer from biases and background interference, while the 2H/1H ratio captures all hydrogen with lower detection limits, making it suitable for applications requiring comprehensive 2H quantification. Importantly, despite its higher mass resolution requirements, the use of C22H/C21H may be a viable alternative to using C2H/C1H due to lower background and higher overall count rates. Furthermore, using an empirical approach to determining Raman wavenumber ranges via the 2nd derivative improved the data equivalency of 2H quantification between Raman and NanoSIMS, highlighting its potential for enhancing cross-technique comparability. These findings provide a robust framework for leveraging both techniques, enabling informed experimental design and data interpretation. By enhancing cross-technique comparability, this work advances SIP methodologies for investigating microbial metabolism and interactions in diverse systems. ImportanceAccurate and reliable measurements of cellular properties are fundamental to understanding the function and activity of microbes. This study addresses to what extent Raman microspectroscopy and nano-scale secondary ion mass spectrometry (NanoSIMS) measurements of single cell anabolic activity can be compared. Here, we study the relationship of the incorporation of a stable isotope (2H through incorporation of 2H2O) as determined by the two techniques and calculate a correlation coefficient to support the use of either technique when analyzing cells incubated with 2H2O. The ability to discern between the comparative strengths and limitations of these techniques is invaluable in refining experimental protocols, enhancing data comparability between studies, data interpretation, and ultimately advancing the quality and reliability of outcomes in microbiome research.

microbiology↗

bioRxiv2024

A roadmap for fair reuse of public microbiome data

Science benefits from rapid, open data sharing but samples for sequencing data are expensive for data creators to acquire and process. Current guidelines for data reuse were established two decades ago, when databases were several million times smaller, necessitating an update. This article presents a roadmap to establish best practices for sequence data reuse, developed in consultation with a data consortium of 167 microbiome scientists. It introduces a Data Reuse Information tag (DRI) for public sequencing data, which will be associated with at least one Open Researcher and Contributor ID (ORCID) account. The machine-readable DRI tag indicates that the data creators prefer to be contacted prior to data reuse, and simultaneously provides data consumers with a mechanism to get in touch with the data creators. Ideally, the DRI will facilitate and foster collaborations, and serve as a guideline that can be expanded to other data types.

microbiology↗

bioRxiv2023

Multicellular magnetotactic bacterial consortia are metabolically differentiated and not clonal

Consortia of multicellular magnetotactic bacteria (MMB) are currently the only known example of bacteria without a unicellular stage in their life cycle. Because of their recalcitrance to cultivation, most previous studies of MMB have been limited to microscopic observations. To study the biology of these unique organisms in more detail, we use multiple culture-independent approaches to analyze the genomics and physiology of MMB consortia at single cell resolution. We separately sequenced the metagenomes of 22 individual MMB consortia, representing eight new species, and quantified the genetic diversity within each MMB consortium. This revealed that, counter to conventional views, cells within MMB consortia are not clonal. Single consortia metagenomes were then used to reconstruct the species-specific metabolic potential and infer the physiological capabilities of MMB. To validate genomic predictions, we performed stable isotope probing (SIP) experiments and interrogated MMB consortia using fluorescence in situ hybridization (FISH) combined with nano-scale secondary ion mass spectrometry (NanoSIMS). By coupling FISH with bioorthogonal non-canonical amino acid tagging (BONCAT) we explored their in situ activity as well as variation of protein synthesis within cells. We demonstrate that MMB consortia are mixotrophic sulfate reducers and that they exhibit metabolic differentiation between individual cells, suggesting that MMB consortia are more complex than previously thought. These findings expand our understanding of MMB diversity, ecology, genomics, and physiology, as well as offer insights into the mechanisms underpinning the multicellular nature of their unique lifestyle. Significance statementThe emergence of multicellular lifeforms represents a pivotal milestone in Earths history, ushering in a new era of biological complexity. Because of the relative scarcity of multicellularity in the domains Bacteria and Archaea, research on the evolution of multicellularity has predominantly focused on eukaryotic model organisms. In this study, we explored the complexity of the only known bacteria without a unicellular stage in their life cycle, consortia of multicellular magnetotactic bacteria (MMB). Genomic and physiological analyses revealed that cells within individual MMB consortia are not clonal and exhibit metabolic differentiation. This implies a higher level of complexity than previously assumed for MMB consortia, prompting a reevaluation of the evolutionary factors that have led to the emergence of multicellularity. Because of their unique biology MMB consortia are ideally suited to become a model system to explore the underpinnings of bacterial multicellularity.

microbiology↗

bioRxiv2023

Methylotrophic methanogenesis in the Archaeoglobi: Cultivation of Ca. Methanoglobus hypatiae from a Yellowstone hot spring

Over the past decade, environmental metagenomics and PCR-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 euryarchaeotes 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 {degrees}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.

microbiology↗

bioRxiv2022

Physiological adaptation of sulfate reducing bacteria in syntrophic partnership with anaerobic methanotrophic archaea

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 explore the metabolic adaptation of four syntrophic SRB clades (HotSeep-1, Seep-SRB2, Seep-SRB1a and Seep-SRB1g) from a phylogenomics perspective, tracing the evolution of conserved proteins in the syntrophic SRB clades, and comparing the genomes of syntrophic SRB to their nearest evolutionary neighbors in the phylum Desulfobacterota. We note several examples of gain, loss or biochemical adaptation of proteins within pathways involved in extracellular electron transfer, electron transport chain, nutrient sharing, biofilm formation and cell adhesion. We demonstrate that the metabolic adaptations in each of these syntrophic clades are unique, suggesting that they have independently evolved, converging to a syntrophic partnership with ANME. Within the clades we also investigated the specialization of different syntrophic SRB species to partnerships with different ANME clades, using metagenomic sequences obtained from ANME and SRB partners in individual consortia after fluorescent-sorting of cell aggregates from anaerobic sediments. In one instance of metabolic adaptation to different partnerships, we show that Seep-SRB1a partners of ANME-2c appear to lack nutritional auxotrophies, while the related Seep-SRB1a partners of a different methanotrophic archaeal lineage, ANME-2a, are missing the cobalamin synthesis pathway, suggesting that the Seep-SRB1a partners of ANME-2a may have a nutritional dependence on its partner. Together, our paired genomic analysis of AOM consortia highlights the specific adaptation and diversification of syntrophic SRB clades linked to their associated ANME lineages.

microbiology↗

bioRxiv2022

Diversity and function of methyl-coenzyme M reductase-encoding archaea in Yellowstone hot springs revealed by metagenomics and mesocosm experiments

Metagenomic studies on geothermal environments have been central in recent discoveries on the diversity of archaeal methane and alkane metabolism. Here, we investigated the methanogenic populations inhabiting terrestrial geothermal features in Yellowstone National Park (YNP) by combining amplicon sequencing with metagenomics and mesocosm experiments. Detection of gene amplicons of methyl-coenzyme M reductase subunit A (mcrA) indicated a wide diversity of Mcr-encoding archaea across geothermal features with differing physicochemical regimes. 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, or hydrogen-dependent methylotrophic methanogenesis, or anaerobic short-chain alkane oxidation. While Mcr-encoding archaea represented a minor fraction of the microbial community of hot springs, mesocosm experiments with methanogenic precursors resulted in 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. Altogether, we revealed that diverse Mcr-encoding populations with the metabolic potential to produce methane from different precursors persist in the geothermal environments of YNP. 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.

microbiology↗

bioRxiv2022

Culexarchaeia, a novel archaeal class of anaerobic generalists inhabiting geothermal environments

Geothermal 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.

microbiology↗

bioRxiv2022

Correlative SIP-FISH-SEM-Raman-NanoSIMS links identity, morphology, biochemistry, and physiology of environmental microbes

Microscopic and spectroscopic techniques are commonly applied to study microbial cells but are typically used on separate samples, resulting in population-level datasets that are integrated across different cells with little spatial resolution. To address this shortcoming, we developed a workflow that correlates several microscopic and spectroscopic techniques to generate an in-depth analysis of individual cells. By combining stable isotope probing (SIP), fluorescence in situ hybridization (FISH), scanning electron microscopy (SEM), confocal Raman microspectroscopy (Raman), and nano-scale secondary ion mass spectrometry (NanoSIMS), we illustrate how individual cells can be thoroughly interrogated to obtain information about their taxonomic identity, structure, physiology, and metabolic activity. Analysis of an artificial microbial community demonstrated that our correlative approach was able to resolve the activity of single cells using heavy water SIP in conjunction with Raman and/or NanoSIMS and establish their taxonomy and morphology using FISH and SEM. This workflow was then applied to a sample of yet uncultured multicellular magnetotactic bacteria (MMB). In addition to establishing their identity and activity, backscatter electron microscopy (BSE), NanoSIMS, and energy-dispersive X-ray spectroscopy (EDS) were employed to characterize the magnetosomes within the cells. By integrating these techniques, we demonstrate a cohesive approach to thoroughly study environmental microbes on a single cell level.

microbiology↗

bioRxiv2021

Diversity and evolution of nitric oxide reduction

Nitrogen is an essential element for life, with the availability of fixed nitrogen limiting productivity in many ecosystems. The return of oxidized nitrogen species to the atmospheric N2 pool is predominately catalyzed by microbial denitrification (NO3- [->] NO2- [->] NO [->] N2O [->] N2)1. Incomplete denitrification can produce N2O as a terminal product, leading to an increase in atmospheric N2O, a potent greenhouse and ozone-depleting gas2. The production of N2O is catalyzed by nitric oxide reductase (NOR) members of the heme-copper oxidoreductase (HCO) superfamily3. Here we use phylogenomics to identify a number of previously uncharacterized HCO families and propose that many of them (eNOR, sNOR, gNOR, and nNOR) perform nitric oxide reduction. These families have novel active-site structures and several have conserved proton channels, suggesting that they might be able to couple nitric oxide reduction to energy conservation. We isolated and biochemically characterized a member of the eNOR family from Rhodothermus marinus, verifying that it performs nitric oxide reduction both in vitro and in vivo. These newly identified NORs exhibit broad phylogenetic and environmental distributions, expanding the diversity of microbes that can perform denitrification. Phylogenetic analyses of the HCO superfamily demonstrate that nitric oxide reductases evolved multiple times independently from oxygen reductases, suggesting that complete denitrification evolved after aerobic respiration.

biochemistry↗

bioRxiv2021

Comparative genomics reveals electron transfer and syntrophic mechanisms differentiating methanotrophic and methanogenic archaea

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 which 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.

microbiology↗

bioRxiv2020

Spatially-resolved correlative microscopy and microbial identification reveals dynamic depth- and mineral-dependent anabolic activity in salt marsh sediment

Coastal salt marshes are key sites of biogeochemical cycling and ideal systems in which to investigate the community structure of complex microbial communities. Here, we clarify structural-functional relationships among microorganisms and their mineralogical environment, revealing previously undescribed metabolic activity patterns and precise spatial arrangements within salt marsh sediment. Following 3.7-day in situ incubations with a non-canonical amino acid that was incorporated into new biomass, samples were embedded and analyzed by correlative fluorescence and electron microscopy to map the microscale arrangements of anabolically active and inactive organisms alongside mineral grains. Parallel sediment samples were examined by fluorescence-activated cell sorting and 16S rRNA gene sequencing to link anabolic activity to taxonomic identity. Both approaches demonstrated a rapid decline in the proportion of anabolically active cells with depth into salt marsh sediment, from [~]60% in the top cm to 10-25% between 2-7 cm. From the top to the bottom, the most prominent active community members shifted from sulfur cycling phototrophic consortia, to sulfate-reducing bacteria likely oxidizing organic compounds, to fermentative lineages. Correlative microscopy revealed more abundant (and more anabolically active) organisms around non-quartz minerals including rutile, orthoclase, and plagioclase. Microbe-mineral relationships appear to be dynamic and context-dependent arbiters of biogeochemical cycling. Statement of SignificanceMicroscale spatial relationships dictate critical aspects of a microbiomes inner workings and emergent properties, such as evolutionary pathways, niche development, and community structure and function. However, many commonly used methods in microbial ecology neglect this parameter - obscuring important microbe-microbe and microbe-mineral interactions - and instead employ bulk-scale methodologies that are incapable of resolving these intricate relationships. This benchmark study presents a compelling new approach for exploring the anabolic activity of a complex microbial community by mapping the precise spatial configuration of anabolically active organisms within mineralogically heterogeneous sediment through in situ incubation, resin embedding, and correlative fluorescence and electron microscopy. In parallel, active organisms were identified through fluorescence-activated cell sorting and 16S rRNA gene sequencing, enabling a powerful interpretive framework connecting location, identity, activity, and putative biogeochemical roles of microbial community members. We deploy this novel approach in salt marsh sediment, revealing quantitative insights into the fundamental principles that govern the structure and function of sediment-hosted microbial communities. In particular, at different sediment horizons, we observed striking changes in the proportion of anabolically active cells, the identities of the most prominent active community members, and the nature of microbe-mineral affiliations. Improved approaches for understanding microscale ecosystems in a new light, such as those presented here, reveal environmental parameters that promote or constrain metabolic activity and clarify the impact that microbial communities have on our world.

microbiology↗