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At least 289 records · Page 16
All sourcesNASA NTRSDOE OSTI
DOE OSTI2023

The minimal SUF system is not required for Fe–S cluster biogenesis in the methanogenic archaeon Methanosarcina acetivorans

Abstract Iron–sulfur (Fe–S) proteins are essential for the ability of methanogens to carry out methanogenesis and biological nitrogen fixation (diazotrophy). Nonetheless, the factors involved in Fe–S cluster biogenesis in methanogens remain largely unknown. The minimal SUF Fe–S cluster biogenesis system (i.e., SufBC) is postulated to serve as the primary system in methanogens. Here, the role of SufBC in Methanosarcina acetivorans , which contains two sufCB gene clusters, was investigated. The CRISPRi-dCas9 and CRISPR-Cas9 systems were utilized to repress or delete sufC1B1 and sufC2B2 , respectively. Neither the dual repression of sufC1B1 and sufC2B2 nor the deletion of both sufC1B1 and sufC2B2 affected the growth of M. acetivorans under any conditions tested, including diazotrophy. Interestingly, deletion of only sufC1B1 led to a delayed-growth phenotype under all growth conditions, suggesting that the deletion of sufC2B2 acts as a suppressor mutation in the absence of sufC1B1 . In addition, the deletion of sufC1B1 and/or sufC2B2 did not affect the total Fe–S cluster content in M. acetivorans cells. Overall, these results reveal that the minimal SUF system is not required for Fe–S cluster biogenesis in M. acetivorans and challenge the universal role of SufBC in Fe–S cluster biogenesis in methanogens.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2024

Crystal structure of the 4-hydroxybutyryl-CoA synthetase (ADP-forming) from nitrosopumilus maritimus

The 3-hydroxypropionate/4-hydroxybutyrate (3HP/4HB) cycle from ammonia-oxidizing Thaumarchaeota is currently considered the most energy-efficient aerobic carbon fixation pathway. The Nitrosopumilus maritimus 4-hydroxybutyryl-CoA synthetase (ADP-forming; Nmar_0206) represents one of several enzymes from this cycle that exhibit increased efficiency over crenarchaeal counterparts. This enzyme reduces energy requirements on the cell, reflecting thaumarchaeal success in adapting to low-nutrient environments. Here we show the structure of Nmar_0206 from Nitrosopumilus maritimus SCM1, which reveals a highly conserved interdomain linker loop between the CoA-binding and ATP-grasp domains. Phylogenetic analysis suggests the widespread prevalence of this loop and highlights both its underrepresentation within the PDB and structural importance within the (ATP-forming) acyl-CoA synthetase (ACD) superfamily. This linker is shown to have a possible influence on conserved interface interactions between domains, thereby influencing homodimer stability. These results provide a structural basis for the energy efficiency of this key enzyme in the modified 3HP/4HB cycle of Thaumarchaeota.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Complex subsurface hydrothermal fluid mixing at a submarine arc volcano supports distinct and highly diverse microbial communities

Hydrothermally active submarine volcanoes are mineral-rich biological oases contributing significantly to chemical fluxes in the deep sea, yet little is known about the microbial communities inhabiting these systems. Here we investigate the diversity of microbial life in hydrothermal deposits and their metagenomics-inferred physiology in light of the geological history and resulting hydrothermal fluid paths in the subsurface of Brothers submarine volcano north of New Zealand on the southern Kermadec arc. From metagenome-assembled genomes we identified over 90 putative bacterial and archaeal genomic families and nearly 300 previously unknown genera, many potentially endemic to this submarine volcanic environment. While magmatically influenced hydrothermal systems on the volcanic resurgent cones of Brothers volcano harbor communities of thermoacidophiles and diverse members of the superphylum “DPANN,” two distinct communities are associated with the caldera wall, likely shaped by two different types of hydrothermal circulation. The communities whose phylogenetic diversity primarily aligns with that of the cone sites and magmatically influenced hydrothermal systems elsewhere are characterized predominately by anaerobic metabolisms. These populations are probably maintained by fluids with greater magmatic inputs that have interacted with different (deeper) previously altered mineral assemblages. However, proximal (a few meters distant) communities with gene-inferred aerobic, microaerophilic, and anaerobic metabolisms are likely supported by shallower seawater-dominated circulation. Furthermore, mixing of fluids from these two distinct hydrothermal circulation systems may have an underlying imprint on the high microbial phylogenomic diversity. Collectively our results highlight the importance of considering geologic evolution and history of subsurface processes in studying microbial colonization and community dynamics in volcanic environments.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Soil microbial community response to corrinoids is shaped by a natural reservoir of vitamin B12

Abstract Soil microbial communities perform critical ecosystem services through the collective metabolic activities of numerous individual organisms. Most microbes use corrinoids, a structurally diverse family of cofactors related to vitamin B12. Corrinoid structure influences the growth of individual microbes, yet how these growth responses scale to the community level remains unknown. Analysis of metagenome-assembled genomes suggests that corrinoids are supplied to the community by members of the archaeal and bacterial phyla Thermoproteota, Actinobacteria, and Proteobacteria. Corrinoids were found largely adhered to the soil matrix in a grassland soil, at levels exceeding those required by cultured bacteria. Enrichment cultures and soil microcosms seeded with different corrinoids showed distinct shifts in bacterial community composition, supporting the hypothesis that corrinoid structure can shape communities. Environmental context influenced both community- and taxon-specific responses to specific corrinoids. These results implicate corrinoids as key determinants of soil microbiome structure and suggest that environmental micronutrient reservoirs promote community stability.

58 GEOSCIENCES↗
DOE OSTI2025

Differential Expression of Core Metabolic Functions in Candidatus Altiarchaeum Inhabiting Distinct Subsurface Ecosystems

Candidatus Altiarchaea are widespread across aquatic subsurface ecosystems and possess a highly conserved core genome, yet adaptations of this core genome to different biotic and abiotic factors based on gene expression remain unknown. Here, we investigated the metatranscriptome of two Ca. Altiarchaeum populations that thrive in two substantially different subsurface ecosystems. In Crystal Geyser, a high-CO2 groundwater system in the USA, Ca. Altiarchaeum crystalense co-occurs with the symbiont Ca. Huberiarchaeum crystalense, while in the Muehlbacher sulfidic spring in Germany, an artesian spring high in sulfide concentration, Ca. A. hamiconexum is heavily infected with viruses. We here mapped metatranscriptome reads against their genomes to analyse the in situ expression profile of their core genomes. Out of 537 shared gene clusters, 331 were functionally annotated and 130 differed significantly in expression between the two sites. Main differences were related to genes involved in cell defence like CRISPR-Cas, virus defence, replication, transcription and energy and carbon metabolism. Our results demonstrate that altiarchaeal populations in the subsurface are likely adapted to their environment while influenced by other biological entities that tamper with their core metabolism. We consequently posit that viruses and symbiotic interactions can be major energy sinks for organisms in the deep biosphere.

archaea↗
DOE OSTI2024

Phenotypic and genomic characterization of Methanothermobacter wolfeii strain BSEL, a CO 2 -capturing archaeon with minimal nutrient requirements

A new variant of Methanothermobacter wolfeii was isolated from an anaerobic digester using enrichment cultivation in anaerobic conditions. Here, the new isolate was taxonomically identified via 16S rRNA gene sequencing and tagged as M. wolfeii BSEL. The whole genome of the new variant was sequenced and de novo assembled. Genomic variations between the BSEL strain and the type strain were discovered, suggesting evolutionary adaptations of the BSEL strain that conferred advantages while growing under a low concentration of nutrients. M. wolfeii BSEL displayed the highest specific growth rate ever reported for the wolfeii species (0.27 ± 0.03 h –1 ) using carbon dioxide (CO 2 ) as unique carbon source and hydrogen (H 2 ) as electron donor. M. wolfeii BSEL grew at this rate in an environment with ammonium (NH 4 + ) as sole nitrogen source. The minerals content required to cultivate the BSEL strain was relatively low and resembled the ionic background of tap water without mineral supplements. Optimum growth rate for the new isolate was observed at 64°C and pH 8.3. In this work, it was shown that wastewater from a wastewater treatment facility can be used as a low-cost alternative medium to cultivate M. wolfeii BSEL. Continuous gas fermentation fed with a synthetic biogas mimic along with H 2 in a bubble column bioreactor using M. wolfeii BSEL as biocatalyst resulted in a CO 2 conversion efficiency of 97% and a final methane (CH 4 ) titer of 98.5%v, demonstrating the ability of the new strain for upgrading biogas to renewable natural gas.

09 BIOMASS FUELS↗
DOE OSTI2024

Impact of mineral and non-mineral sources of iron and sulfur on the metalloproteome of Methanosarcina barkeri

Methanogens often inhabit sulfidic environments that favor the precipitation of transition metals such as iron (Fe) as metal sulfides, including mackinawite (FeS) and pyrite (FeS 2 ). These metal sulfides have historically been considered biologically unavailable. Nonetheless, methanogens are commonly cultivated with sulfide (HS - ) as a sulfur source, a condition that would be expected to favor metal precipitation and thus limit metal availability. Recent studies have shown that methanogens can access Fe and sulfur (S) from FeS and FeS 2 to sustain growth. As such, medium supplied with FeS 2 should lead to higher availability of transition metals when compared to medium supplied with HS - . Here, we examined how transition metal availability under sulfidic (i.e., cells provided with HS - as sole S source) versus non-sulfidic (cells provided with FeS 2 as sole S source) conditions impact the metalloproteome of Methanosarcina barkeri Fusaro. To achieve this, we employed size exclusion chromatography coupled with inductively coupled plasma mass spectrometry and shotgun proteomics. Significant changes were observed in the composition and abundance of iron, cobalt, nickel, zinc, and molybdenum proteins. Among the differences were alterations in the stoichiometry and abundance of multisubunit protein complexes involved in methanogenesis and electron transport chains. Furthermore, our data suggest that M. barkeri utilizes the minimal iron-sulfur cluster complex and canonical cysteine biosynthesis proteins when grown on FeS 2 but uses the canonical Suf pathway in conjunction with the tRNA-Sep cysteine pathway for iron-sulfur cluster and cysteine biosynthesis under sulfidic growth conditions.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Ecological Dichotomies Arise in Microbial Communities Due to Mixing of Deep Hydrothermal Waters and Atmospheric Gas in a Circumneutral Hot Spring

Understanding the source and availability of energy capable of supporting life in hydrothermal environments is central to predicting the ecology of microbial life on early Earth when volcanic activity was more widespread. Little is known of the substrates supporting microbial life in circumneutral to alkaline springs, despite their relevance to early Earth habitats.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Pathways of Iron and Sulfur Acquisition, Cofactor Assembly, Destination, and Storage in Diverse Archaeal Methanogens and Alkanotrophs

Archaeal methanogens, methanotrophs, and alkanotrophs, argued to be among the most ancient forms of life, have a high demand for iron (Fe) and sulfur (S) for cofactor biosynthesis, among other uses. Here, using comparative bioinformatic approaches applied to 326 genomes, we show that major differences in Fe/S acquisition, trafficking, deployment, and storage exist in this group.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

RecJ3/4-aRNase J form a Ubl-associated nuclease complex functioning in survival against DNA damage in Haloferax volcanii

Nucleases are strictly regulated and often localized in the cell to avoid the uncontrolled degradation of DNA and RNA. Here, a new type of nuclease complex, composed of RecJ3, RecJ4, and aRNase J, was identified through its ATP-dependent association with the ubiquitin-like SAMP1 and AAA-ATPase Cdc48a. The complex was discovered in Haloferax volcanii , an archaeon lacking an RNA exosome. Genetic analysis revealed aRNase J to be essential and RecJ3, RecJ4, and Cdc48a to function in the recovery from DNA damage including genotoxic agents that generate double-strand breaks. The RecJ3:RecJ4:aRNase J complex (isolated in 2:2:1 stoichiometry) functioned primarily as a 3′−5′ exonuclease in hydrolyzing RNA and ssDNA, with the mechanism non-processive for ssDNA. aRNase J could also be purified as a homodimer that catalyzed endoribonuclease activity and, thus, was not restricted to the 5′−3′ exonuclease activity typical of aRNase J homologs. Moreover, RecJ3 and RecJ4 could be purified as a 560-kDa subcomplex in equimolar subunit ratio with nuclease activities mirroring the full RecJ3/4-aRNase J complex. These findings prompted reconstitution assays that suggested RecJ3/4 could suppress, alter, and/or outcompete the nuclease activities of aRNase J. Based on the phenotypic results, this control mechanism of aRNase J by RecJ3/4 is not necessary for cell growth but instead appears important for DNA repair. IMPORTANCE Nucleases are critical for various cellular processes including DNA replication and repair. Here, a dynamic type of nuclease complex is newly identifiedidentifiedidentifiedin the archaeon Haloferax volcanii , which is missing the canonical RNA exosome. The complex, composed of RecJ3, RecJ4, and aRNase J, functions primarily as a 3′−5′ exonuclease and was discovered through its ATP-dependent association with the ubiquitin-like SAMP1 and Cdc48a. aRNase J alone forms a homodimer that has endonuclease function and, thus, is not restricted to 5′−3′ exonuclease activity typical of other aRNase J enzymes. RecJ3/4 appears to suppress, alter, and/or outcompete the nuclease activities of aRNase J. While aRNase J is essential for growth, RecJ3/4, Cdc48a, and SAMPs are important for recovery against DNA damage. These biological distinctions may correlate with the regulated nuclease activity of aRNase J in the RecJ3/4-aRNaseJ complex.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Methyl-Based Methanogenesis: an Ecological and Genomic Review

Methyl-based methanogenesis is one of three broad categories of archaeal anaerobic methanogenesis, including both the methyl dismutation (methylotrophic) pathway and the methyl-reducing (also known as hydrogen-dependent methylotrophic) pathway. Methyl-based methanogenesis is increasingly recognized as an important source of methane in a variety of environments. Here, we provide an overview of methyl-based methanogenesis research, including the conditions under which methyl-based methanogenesis can be a dominant source of methane emissions, experimental methods for distinguishing different pathways of methane production, molecular details of the biochemical pathways involved, and the genes and organisms involved in these processes. In this study, we also identify the current gaps in knowledge and present a genomic and metagenomic survey of methyl-based methanogenesis genes, highlighting the diversity of methyl-based methanogens at multiple taxonomic levels and the widespread distribution of known methyl-based methanogenesis genes and families across different environments.

methanogens↗
DOE OSTI2022

HT-SIP: a semi-automated stable isotope probing pipeline identifies cross-kingdom interactions in the hyphosphere of arbuscular mycorrhizal fungi

Abstract Background Linking the identity of wild microbes with their ecophysiological traits and environmental functions is a key ambition for microbial ecologists. Of many techniques that strive for this goal, Stable-isotope probing—SIP—remains among the most comprehensive for studying whole microbial communities in situ. In DNA-SIP, actively growing microorganisms that take up an isotopically heavy substrate build heavier DNA, which can be partitioned by density into multiple fractions and sequenced. However, SIP is relatively low throughput and requires significant hands-on labor. We designed and tested a semi-automated, high-throughput SIP (HT-SIP) pipeline to support well-replicated, temporally resolved amplicon and metagenomics experiments. We applied this pipeline to a soil microhabitat with significant ecological importance—the hyphosphere zone surrounding arbuscular mycorrhizal fungal (AMF) hyphae. AMF form symbiotic relationships with most plant species and play key roles in terrestrial nutrient and carbon cycling. Results Our HT-SIP pipeline for fractionation, cleanup, and nucleic acid quantification of density gradients requires one-sixth of the hands-on labor compared to manual SIP and allows 16 samples to be processed simultaneously. Automated density fractionation increased the reproducibility of SIP gradients compared to manual fractionation, and we show adding a non-ionic detergent to the gradient buffer improved SIP DNA recovery. We applied HT-SIP to 13 C-AMF hyphosphere DNA from a 13 CO 2 plant labeling study and created metagenome-assembled genomes (MAGs) using high-resolution SIP metagenomics (14 metagenomes per gradient). SIP confirmed the AMF Rhizophagus intraradices and associated MAGs were highly enriched (10–33 atom% 13 C), even though the soils’ overall enrichment was low (1.8 atom% 13 C). We assembled 212 13 C-hyphosphere MAGs; the hyphosphere taxa that assimilated the most AMF-derived 13 C were from the phyla Myxococcota, Fibrobacterota, Verrucomicrobiota, and the ammonia-oxidizing archaeon genus Nitrososphaera . Conclusions Our semi-automated HT-SIP approach decreases operator time and improves reproducibility by targeting the most labor-intensive steps of SIP—fraction collection and cleanup. We illustrate this approach in a unique and understudied soil microhabitat—generating MAGs of actively growing microbes living in the AMF hyphosphere (without plant roots). The MAGs’ phylogenetic composition and gene content suggest predation, decomposition, and ammonia oxidation may be key processes in hyphosphere nutrient cycling.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Global patterns of diversity and metabolism of microbial communities in deep-sea hydrothermal vent deposits

When deep-sea hydrothermal fluids mix with cold oxygenated fluids, minerals precipitate out of solution and form hydrothermal deposits. These actively venting deep-sea hydrothermal deposits support a rich diversity of thermophilic microorganisms which are involved in a range of carbon, sulfur, nitrogen, and hydrogen metabolisms. Global patterns of thermophilic microbial diversity in deep-sea hydrothermal ecosystems have illustrated the strong connectivity between geological processes and microbial colonization, but little is known about the genomic diversity and physiological potential of these novel taxa. Here we explore this genomic diversity in 42 metagenomes from four deep-sea hydrothermal vent fields and a deep-sea volcano collected from 2004 to 2018 and document their potential implications in biogeochemical cycles. Our dataset represents 3635 metagenome-assembled genomes encompassing 511 novel and recently identified genera from deep-sea hydrothermal settings. Some of the novel bacterial (107) and archaeal genera (30) that were recently reported from the deep-sea Brothers volcano were also detected at the deep-sea hydrothermal vent fields, while 99 bacterial and 54 archaeal genera were endemic to the deep-sea Brothers volcano deposits. We report some of the first examples of medium- (≥ 50% complete, ≤ 10% contaminated) to high-quality (> 90% complete, < 5% contaminated) MAGs from phyla and families never previously identified, or poorly sampled, from deep-sea hydrothermal environments. We greatly expand the novel diversity of Thermoproteia, Patescibacteria (Candidate Phyla Radiation, CPR), and Chloroflexota found at deep-sea hydrothermal vents and identify a small sampling of two potentially novel phyla, designated JALSQH01 and JALWCF01. Metabolic pathway analysis of metagenomes provides insights into the prevalent carbon, nitrogen, sulfur, and hydrogen metabolic processes across all sites and illustrates sulfur and nitrogen metabolic “handoffs” in community interactions. We confirm that Campylobacteria and Gammaproteobacteria occupy similar ecological guilds but their prevalence in a particular site is driven by shifts in the geochemical environment. Our study of globally distributed hydrothermal vent deposits provides a significant expansion of microbial genomic diversity associated with hydrothermal vent deposits and highlights the metabolic adaptation of taxonomic guilds. Collectively, our results illustrate the importance of comparative biodiversity studies in establishing patterns of shared phylogenetic diversity and physiological ecology, while providing many targets for enrichment and cultivation of novel and endemic taxa.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

SPRUCE Quantitative PCR (qPCR) of Microbial Gene Copy Numbers, 2021-2022

This dataset provides the results for quantitative polymerase chain reaction (qPCR) of peat samples collected from ambient and experimental plots in the Spruce and Peatland Responses Under Climatic and Environmental Change (SPRUCE) experiment site in June and August of 2021, and June of 2022. SPRUCE is located within the Marcell Experimental Forest in northern Minnesota, USA. The dataset includes bacterial, archaeal, fungal gene copy numbers, along with corresponding logarithmic values, at 11 depth increments of two-meter deep peat cores taken from 12 sampling sites locations inside SPRUCE plots (10 chambered and 2 ambient plots). The sampling, sample prep and analysis followed standard methods outlined in prior publications (Wilson et al. 2016; Kluber et al. 2020) except that a higher yielding Omega Bio-Tek Mag-Bind Environmental DNA 96 Kit was used for extractions and DNA was quantified using Qubit dsDNA High Sensitivity Assay Kit. qPCR subsamples of peat cores from the SPRUCE plots characterize changes in the abundance and composition of microbial communities of peat seasonally showing how composition varies under multiple levels of experimental peat warming and atmospheric CO2 concentrations. This dataset contains one data file in comma-separated values (.csv) format. Additional metadata are provided: one data dictionary and a file-level metadata file in comma-separated values (.csv) format and a user guide in PDF (*.pdf) format. On 2026-07-07 this dataset was updated to add three columns to the data file: ‘Fungal_copy_dry’, ‘Log_fungal_copy_dry’, ‘Fungal_copy_wet’. No previously released data values were altered. Additionally, the abstract, data dictionary, and user guide were updated, and a file-level metadata file was added.

Archaea↗
DOE OSTI2021

The Hyperthermophilic Restriction-Modification Systems of Thermococcus kodakarensis Protect Genome Integrity

Thermococcus kodakarensis (T. kodakarensis), a hyperthermophilic, genetically accessible model archaeon, encodes two putative restriction modification (R-M) defense systems, TkoI and TkoII. TkoI is encoded by TK1460 while TkoII is encoded by TK1158. Bioinformative analysis suggests both R-M enzymes are large, fused methyltransferase (MTase)-endonuclease polypeptides that contain both restriction endonuclease (REase) activity to degrade foreign invading DNA and MTase activity to methylate host genomic DNA at specific recognition sites. In this work, we demonsrate T. kodakarensis strains deleted for either or both R-M enzymes grow more slowly but display significantly increased competency compared to strains with intact R-M systems, suggesting that both TkoI and TkoII assist in maintenance of genomic integrity in vivo and likely protect against viral- or plasmid-based DNA transfers. Pacific Biosciences single molecule real-time (SMRT) sequencing of T. kodakarensis strains containing both, one or neither R-M systems permitted assignment of the recognition sites for TkoI and TkoII and demonstrated that both R-M enzymes are TypeIIL; TkoI and TkoII methylate the N 6 position of adenine on one strand of the recognition sequences GTGAAG and TTCAAG, respectively. Further in vitro biochemical characterization of the REase activities reveal TkoI and TkoII cleave the DNA backbone GTGAAG(N) 20 /(N) 18 and TTCAAG(N) 10 /(N) 8 , respectively, away from the recognition sequences, while in vitro characterization of the MTase activities reveal transfer of tritiated S-adenosyl methionine by TkoI and TkoII to their respective recognition sites. Together these results demonstrate TkoI and TkoII restriction systems are important for protecting T. kodakarensis genome integrity from invading foreign DNA.

59 BASIC BIOLOGICAL SCIENCES↗