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At least 55 records · Page 3

Type IV-like pili facilitate transformation in naturally competent archaea

Naturally competent organisms are capable of DNA uptake directly from the environment through the process of transformation. Despite the importance of transformation to microbial evolution, DNA uptake remains poorly characterized outside the bacterial domain. Here, we identify the pilus as a necessary component of the transformation machinery in archaea. We describe two naturally competent organisms: Methanococcus maripaludis and Methanoculleus thermophilus. In M. maripaludis, replicative vectors were transferred with an average efficiency of 2.4 x 10 3 transformants μg -1 DNA. In M. thermophilus, integrative vectors were transferred with an average efficiency of 2.7 x 10 3 transformants μg -1 DNA. Additionally, natural transformation of M. thermophilus could be used to introduce chromosomal mutations. To our knowledge, this is the first demonstration of a method to introduce targeted mutations in a member of the order Methanomicrobiales. For both organisms, mutants lacking structural components of the type IV-like pilus filament were defective for DNA uptake, demonstrating the importance of pili for natural transformation. Interestingly, competence could be induced in a non-competent strain of M. maripaludis by expressing pilin genes from a replicative vector. Furthermore, these results expand the known natural competence pili to include examples from the archaeal domain and highlight the importance of pili for DNA uptake in diverse microbial organisms.

59 BASIC BIOLOGICAL SCIENCES↗

Physiological potential and evolutionary trajectories of syntrophic sulfate-reducing bacterial partners of 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 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.

59 BASIC BIOLOGICAL SCIENCES↗

A fast comparative genome browser for diverse bacteria and archaea

Genome sequencing has revealed an incredible diversity of bacteria and archaea, but there are no fast and convenient tools for browsing across these genomes. It is cumbersome to view the prevalence of homologs for a protein of interest, or the gene neighborhoods of those homologs, across the diversity of the prokaryotes. We developed a web-based tool, fast . genomics , that uses two strategies to support fast browsing across the diversity of prokaryotes. First, the database of genomes is split up. The main database contains one representative from each of the 6,377 genera that have a high-quality genome, and additional databases for each taxonomic order contain up to 10 representatives of each species. Second, homologs of proteins of interest are identified quickly by using accelerated searches, usually in a few seconds. Once homologs are identified, fast . genomics can quickly show their prevalence across taxa, view their neighboring genes, or compare the prevalence of two different proteins. Fast . genomics is available at https://fast.genomics.lbl.gov .

59 BASIC BIOLOGICAL SCIENCES↗

Genome editing in Archaea

Methods of RNA-guided DNA endonuclease-mediated genome editing in Bacteria and Archaea are provided.

Metcalf, William W.↗

Activity-based cell sorting reveals responses of uncultured archaea and bacteria to substrate amendment

Metagenomic 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.

59 BASIC BIOLOGICAL SCIENCES↗

Biochemical and genetic studies define the functions of methylthiotransferases in methanogenic and methanotrophic archaea

Methylthiotransferases (MTTases) are radical S-adenosylmethionine (SAM) enzymes that catalyze the addition of a methylthio (-SCH 3 ) group to an unreactive carbon center. These enzymes are responsible for the production of 2-methylthioadenosine (ms 2 A) derivatives found at position A37 of select tRNAs in all domains of life. Additionally, some bacteria contain the RimO MTTase that catalyzes the methylthiolation of the S12 ribosomal protein. Although the functions of MTTases in bacteria and eukaryotes have been established via detailed genetic and biochemical studies, MTTases from the archaeal domain of life are understudied and the substrate specificity determinants of MTTases remain unclear. Here, we report the in vitro enzymatic activities of an MTTase (C4B56_06395) from a thermophilic Ca. Methanophagales anaerobic methanotroph (ANME) as well as the MTTase from a hyperthermophilic methanogen – MJ0867 from Methanocaldococcus jannaschii. Both enzymes catalyze the methylthiolation of N 6 -threonylcarbamoyladenosine (t 6 A) and N 6 -hydroxynorvalylcarbamoyladenosine (hn 6 A) residues to produce 2-methylthio-N 6 -threonylcarbamoyladenosine (ms 2 t 6 A) and 2-methylthio-N 6 -hydroxynorvalylcarbamoyladenosine (ms 2 hn 6 A), respectively. To further assess the function of archaeal MTTases, we analyzed select tRNA modifications in a model methanogen – Methanosarcina acetivorans – and generated a deletion of the MTTase-encoding gene (MA1153). We found that M. acetivorans produces ms 2 hn 6 A in exponential phase of growth, but does not produce ms 2 t 6 A in detectable amounts. Upon deletion of MA1153, the ms 2 A modification was absent, thus confirming the function of MtaB-family MTTases in generating ms 2 hn 6 A modified nucleosides in select tRNAs.

59 BASIC BIOLOGICAL SCIENCES↗

Reductive dissolution of pyrite by methanogenic archaea

Abstract The formation and fate of pyrite (FeS2) modulates global iron, sulfur, carbon, and oxygen biogeochemical cycles and has done so since early in Earth’s geological history. A longstanding paradigm is that FeS2 is stable at low temperature and is unavailable to microorganisms in the absence of oxygen and oxidative weathering. Here, we show that methanogens can catalyze the reductive dissolution of FeS2 at low temperature (≤38 °C) and utilize dissolution products to meet cellular iron and sulfur demands associated with the biosynthesis of simple and complex co-factors. Direct access to FeS2 is required to catalyze its reduction and/or to assimilate iron monosulfide that likely forms through coupled reductive dissolution and precipitation, consistent with close associations observed between cells and FeS2. These findings demonstrate that FeS2 is bioavailable to anaerobic methanogens and can be mobilized in low temperature anoxic environments. Given that methanogens evolved at least 3.46 Gya, these data indicate that the microbial contribution to the iron and sulfur cycles in ancient and contemporary anoxic environments may be more complex and robust than previously recognized, with impacts on the sources and sinks of iron and sulfur and other bio-essential and thiophilic elements such as nickel and cobalt.

59 BASIC BIOLOGICAL SCIENCES↗

An essential role for tungsten in the ecology and evolution of a previously uncultivated lineage of anaerobic, thermophilic Archaea

Trace metals have been an important ingredient for life throughout Earth’s history. Here, we describe the genome-guided cultivation of a member of the elusive archaeal lineage Caldarchaeales (syn. Aigarchaeota), Wolframiiraptor gerlachensis, and its growth dependence on tungsten. A metagenome-assembled genome (MAG) of W. gerlachensis encodes putative tungsten membrane transport systems, as well as pathways for anaerobic oxidation of sugars probably mediated by tungsten-dependent ferredoxin oxidoreductases that are expressed during growth. Catalyzed reporter deposition-fluorescence in-situ hybridization (CARD-FISH) and nanoscale secondary ion mass spectrometry (nanoSIMS) show that W. gerlachensis preferentially assimilates xylose. Phylogenetic analyses of 78 high-quality Wolframiiraptoraceae MAGs from terrestrial and marine hydrothermal systems suggest that tungsten-associated enzymes were present in the last common ancestor of extant Wolframiiraptoraceae. Our observations imply a crucial role for tungsten-dependent metabolism in the origin and evolution of this lineage, and hint at a relic metabolic dependence on this trace metal in early anaerobic thermophiles.

59 BASIC BIOLOGICAL SCIENCES↗

Diversity and evolution of nitric oxide reduction in bacteria and archaea

Nitrous oxide is a potent greenhouse gas whose production is catalyzed by nitric oxide reductase (NOR) members of the heme-copper oxidoreductase (HCO) enzyme superfamily. We identified several previously uncharacterized HCO families, four of which (eNOR, sNOR, gNOR, and nNOR) appear to perform NO reduction. These families have novel active-site structures and several have conserved proton channels, suggesting that they might be able to couple NO reduction to energy conservation. We isolated and biochemically characterized a member of the eNOR family from the bacterium Rhodothermus marinus and found that it performs NO reduction. These recently identified NORs exhibited broad phylogenetic and environmental distributions, greatly expanding the diversity of microbes in nature capable of NO reduction. Phylogenetic analyses further demonstrated that NORs evolved multiple times independently from oxygen reductases, supporting the view that complete denitrification evolved after aerobic respiration.

Science & Technology - Other Topics↗

CABO-16S—a Combined Archaea, Bacteria, Organelle 16S rRNA database framework for amplicon analysis of prokaryotes and eukaryotes in environmental samples

Abstract Identification of both prokaryotic and eukaryotic microorganisms in environmental samples is currently challenged by the need for additional sequencing to obtain separate 16S and 18S ribosomal RNA (rRNA) amplicons or the constraints imposed by “universal” primers. Organellar 16S rRNA sequences are amplified and sequenced along with prokaryote 16S rRNA and provide an alternative method to identify eukaryotic microorganisms. CABO-16S combines bacterial and archaeal sequences from the SILVA database with 16S rRNA sequences of plastids and other organelles from the PR2 database to enable identification of all 16S rRNA sequences. Comparison of CABO-16S with SILVA 138.2 results in equivalent taxonomic classification of mock communities and increased classification of diverse environmental samples. In particular, identification of phototrophic eukaryotes in shallow seagrass environments, marine waters, and lake waters was increased. The CABO-16S framework allows users to add custom sequences for further classification of underrepresented clades and can be easily updated with future releases of reference databases. Addition of sequences obtained from Sanger sequencing of methane seep sediments and curated sequences of the polyphyletic SEEP-SRB1 clade resulted in differentiation of syntrophic and non-syntrophic SEEP-SRB1 in hydrothermal vent sediments. CABO-16S highlights the benefit of combining and amending existing training sets when studying microorganisms in diverse environments.

Eitel, Eryn M. (ORCID:0009000723919297)↗

Crystal Ball: A Naturalist Perspective of Microbiology: Examples from Methanogenic Archaea

Storytelling has been the primary means of knowledge transfer over human history. The effectiveness and reach of stories are improved when the message is appropriate for the target audience. Oftentimes, the stories that are most well received and recounted are those that have a clear purpose and that are told from a variety of perspectives that touch on the varied interests of the target audience. Whether scientists realize or not, they are accustomed to telling stories of their own scientific discoveries through the preparation of manuscripts, presentations, and lectures. Perhaps less frequently, scientists prepare review articles or book chapters that summarize a body of knowledge on a given subject matter, meant to be more holistic recounts of a body of literature. Yet, by necessity, such summaries are often still narrow in their scope and are told from the perspective of a particular discipline. In other words, interdisciplinary reviews or book chapters tend to be the rarity rather than the norm. Naturalists are scientists that tell stories of organisms and their natural environment within a historical context and from an interdisciplinary perspective. Such stories interweave observations of biological subjects from disparate disciplines into a cohesive story. To this end, the success of naturalists in recounting compelling natural history stories is enhanced when they allow themselves to become vulnerable by knowingly entering a literature base full of unfamiliar ways of observing and describing such subjects. When successful, this approach can broaden the scope of the story, making it of interest to a larger audience and attracting further recounting of that story (e.g., citations). To this end, when we look into our crystal ball, we see a need to tell more holistic stories of microorganisms and their functions from an interdisciplinary, naturalist perspective that combines what is known of their contemporary ecology, physiology, and biochemistry with their evolutionary history thereby placing them within a historical Earth context. With this conviction in mind, we take the opportunity to look back on the natural history of methanogens to identify some of their most profound biochemical innovations and their impact on the trajectory of life and the planet. Our approach was to integrate knowledge from the fields of biochemistry, physiology, ecology, evolution, geochemistry, and atmospheric sciences. We start with the origin of the first autotrophic cells and how this set Earth on a path towards biomass expansion and functional diversification. Next, we discuss the importance of iron and sulfur in methanogen metabolism, and how their innovative ways of accessing these elements allowed for expanded use of iron-sulfur clusters ([Fe-S]) as nanoscale catalysts that power(ed) Earth’s biochemical cycles. We next discuss the origin and evolution of [NiFe]-hydrogenase and how this versatile enzyme chassis was co-opted by a variety of anaerobes and aerobes as the foundation for respiratory complexes capable of sustaining higher forms of life, including humans. Lastly, we discuss the evolution of the enzyme molybdenum nitrogenase, and how this allowed the biosphere to overcome fixed nitrogen limitation by tapping the seemingly endless reserve of atmospheric dinitrogen (N 2 ). In telling these stories, we aimed to generate template(s) to motivate future interdisciplinary natural history studies focused on other organisms and processes. Further, we hope to stimulate scientific discourse and provide impetus to become vulnerable and peruse literature of relevance to understanding the causes and consequences of the evolution of your favorite microorganism or biochemical process.

59 BASIC BIOLOGICAL SCIENCES↗

Precipitation of low-temperature disordered dolomite induced by extracellular polymeric substances of methanogenic Archaea Methanosarcina barkeri : Implications for sedimentary dolomite formation

Abstract A correlation between methanogenesis and dolomite formation has been reported; however, the mechanism underlying this association is not fully understood. In this study, we conducted forced carbonate precipitation experiments at room temperature in calcite-seeded Ca/Mg carbonate solutions containing either purified non-living biomass or bound extracellular polymeric substances (EPS) of the methanogen Methanosarcina barkeri. Purified non-living biomass and bound EPS was used so as to avoid the possible influence of the complex components of the growing microbial culture on carbonate crystallization. Our results demonstrated that non-living biomass of M. Barkeri can enhance the Mg incorporation into calcitic structure and induce the crystallization of disordered dolomite. In the presence of ~113 mg L–1 of non-living biomass, disordered dolomite with ~41 and 45 mol% of MgCO3 was precipitated in solutions with initial Mg:Ca ratios of 5:1 and 8:1, respectively. A systematic increase in the MgCO3 contents of the precipitated Ca-Mg carbonates was also observed with the increased non-living biomass concentration. Bound EPS was shown to be the component of non-living biomass that catalyzed the precipitation of disordered dolomite. At only ~25 mg L–1 of bound EPS, disordered dolomite with ~47 and 48 mol% of MgCO3 was precipitated in solutions with initial Mg:Ca ratios of 5:1 and 8:1, respectively. We propose that adsorption of bound EPS to growing carbonate surfaces through hydrogen bonding is the key to catalyzing disordered dolomite crystallization, and that this mechanism is also applicable to natural EPS-induced dolomite formation. This study provides significant insight into the formation mechanism of microbial-induced dolomite with high δ13C values.

Geochemistry & Geophysics↗

Inference and reconstruction of the heimdallarchaeial ancestry of eukaryotes

Abstract In the ongoing debates about eukaryogenesis—the series of evolutionary events leading to the emergence of the eukaryotic cell from prokaryotic ancestors—members of the Asgard archaea play a key part as the closest archaeal relatives of eukaryotes 1 . However, the nature and phylogenetic identity of the last common ancestor of Asgard archaea and eukaryotes remain unresolved 2–4 . Here we analyse distinct phylogenetic marker datasets of an expanded genomic sampling of Asgard archaea and evaluate competing evolutionary scenarios using state-of-the-art phylogenomic approaches. We find that eukaryotes are placed, with high confidence, as a well-nested clade within Asgard archaea and as a sister lineage to Hodarchaeales, a newly proposed order within Heimdallarchaeia. Using sophisticated gene tree and species tree reconciliation approaches, we show that analogous to the evolution of eukaryotic genomes, genome evolution in Asgard archaea involved significantly more gene duplication and fewer gene loss events compared with other archaea. Finally, we infer that the last common ancestor of Asgard archaea was probably a thermophilic chemolithotroph and that the lineage from which eukaryotes evolved adapted to mesophilic conditions and acquired the genetic potential to support a heterotrophic lifestyle. Our work provides key insights into the prokaryote-to-eukaryote transition and a platform for better understanding the emergence of cellular complexity in eukaryotic cells.

Science & Technology - Other Topics↗

Global Archaeal Diversity Revealed Through Massive Data Integration: Uncovering Just Tip of Iceberg

The domain of Archaea has gathered significant interest for its ecological and biotechnological potential and its role in helping us to understand the evolutionary history of Eukaryotes. In comparison to the bacterial domain, the number of adequately described members in Archaea is relatively low, with less than 1000 species described. It is not clear whether this is solely due to the cultivation difficulty of its members or, indeed, the domain is characterized by evolutionary constraints that keep the number of species relatively low. Based on molecular evidence that bypasses the difficulties of formal cultivation and characterization, several novel clades have been proposed, enabling insights into their metabolism and physiology. Given the extent of global sampling and sequencing efforts, it is now possible and meaningful to question the magnitude of global archaeal diversity based on molecular evidence. To do so, we extracted all sequences classified as Archaea from 500 thousand amplicon samples available in public repositories. After processing through our highly conservative pipeline, we named this comprehensive resource the ‘Global Archaea Diversity’ (GAD), which encompassed nearly 3 million molecular species clusters at 97% similarity, and organized it into over 500 thousand genera and nearly 100 thousand families. Saline environments have contributed the most to the novel taxa of this previously unseen diversity. The majority of those 16S rRNA gene sequence fragments were verified by matches in metagenomic datasets from IMG/M. These findings reveal a vast and previously overlooked diversity within the Archaea, offering insights into their ecological roles and evolutionary importance while establishing a foundation for the future study and characterization of this intriguing domain of life.

59 BASIC BIOLOGICAL SCIENCES↗