RESUMO
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 eukaryotes1. However, the nature and phylogenetic identity of the last common ancestor of Asgard archaea and eukaryotes remain unresolved2-4. 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.
Assuntos
Archaea , Eucariotos , Filogenia , Archaea/classificação , Archaea/citologia , Archaea/genética , Eucariotos/classificação , Eucariotos/citologia , Eucariotos/genética , Células Eucarióticas/classificação , Células Eucarióticas/citologia , Células Procarióticas/classificação , Células Procarióticas/citologia , Conjuntos de Dados como Assunto , Duplicação Gênica , Evolução MolecularRESUMO
Automated genome annotation is essential for extracting biological information from sequence data. The identification and annotation of tRNA genes is frequently performed by the software package tRNAscan-SE, the output of which is listed for selected genomes in the Genomic tRNA database (GtRNAdb). Here, we highlight a pervasive error in prokaryotic tRNA gene sets on GtRNAdb: the mis-categorization of partial, non-canonical tRNA genes as standard, canonical tRNA genes. Firstly, we demonstrate the issue using the tRNA gene sets of 20 organisms from the archaeal taxon Thermococcaceae. According to GtRNAdb, these organisms collectively deviate from the expected set of tRNA genes in 15 instances, including the listing of eleven putative canonical tRNA genes. However, after detailed manual annotation, only one of these eleven remains; the others are either partial, non-canonical tRNA genes resulting from the integration of genetic elements or CRISPR-Cas activity (seven instances), or attributable to ambiguities in input sequences (three instances). Secondly, we show that similar examples of the mis-categorization of predicted tRNA sequences occur throughout the prokaryotic sections of GtRNAdb. While both canonical and non-canonical prokaryotic tRNA gene sequences identified by tRNAscan-SE are biologically interesting, the challenge of reliably distinguishing between them remains. We recommend employing a combination of (i) screening input sequences for the genetic elements typically associated with non-canonical tRNA genes, and ambiguities, (ii) activating the tRNAscan-SE automated pseudogene detection function, and (iii) scrutinizing predicted tRNA genes with low isotype scores. These measures greatly reduce manual annotation efforts, and lead to improved prokaryotic tRNA gene set predictions.
Assuntos
Genoma , RNA de Transferência , RNA de Transferência/genéticaRESUMO
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.
Assuntos
Fontes Hidrotermais/microbiologia , Consórcios Microbianos/fisiologia , Água do Mar/microbiologia , Erupções Vulcânicas , Archaea/genética , Bactérias/genética , Biodiversidade , Concentração de Íons de Hidrogênio , Metagenoma , Nova Zelândia , Oxirredução , Oceano Pacífico , Filogenia , Sulfetos/químicaRESUMO
Organic matter degradation in marine environments is essential for the recycling of nutrients, especially under conditions of anoxia where organic matter tends to accumulate. However, little is known about the diversity of the microbial communities responsible for the mineralization of organic matter in the absence of oxygen, as well as the factors controlling their activities. Here, we determined the active heterotrophic prokaryotic community in the sulphidic water column of the Black Sea, an ideal model system, where a tight coupling between carbon, nitrogen and sulphur cycles is expected. Active microorganisms degrading both dissolved organic matter (DOM) and protein extracts were determined using quantitative DNA stable isotope probing incubation experiments. These results were compared with the metabolic potential of metagenome-assembled genomes obtained from the water column. Organic matter incubations showed that groups like Cloacimonetes and Marinimicrobia are generalists degrading DOM. Based on metagenomic profiles the degradation proceeds in a potential interaction with members of the Deltaproteobacteria and Chloroflexi Dehalococcoidia. On the other hand, microbes with small genomes like the bacterial phyla Parcubacteria, Omnitrophica and of the archaeal phylum Woesearchaeota, were the most active, especially in protein-amended incubations, revealing the potential advantage of streamlined microorganisms in highly reduced conditions.
Assuntos
Microbiota , Archaea/genética , Bactérias/genética , Mar Negro , MetagenomaRESUMO
Roots and leaves of healthy plants host taxonomically structured bacterial assemblies, and members of these communities contribute to plant growth and health. We established Arabidopsis leaf- and root-derived microbiota culture collections representing the majority of bacterial species that are reproducibly detectable by culture-independent community sequencing. We found an extensive taxonomic overlap between the leaf and root microbiota. Genome drafts of 400 isolates revealed a large overlap of genome-encoded functional capabilities between leaf- and root-derived bacteria with few significant differences at the level of individual functional categories. Using defined bacterial communities and a gnotobiotic Arabidopsis plant system we show that the isolates form assemblies resembling natural microbiota on their cognate host organs, but are also capable of ectopic leaf or root colonization. While this raises the possibility of reciprocal relocation between root and leaf microbiota members, genome information and recolonization experiments also provide evidence for microbiota specialization to their respective niche.
Assuntos
Arabidopsis/microbiologia , Microbiota/fisiologia , Folhas de Planta/microbiologia , Raízes de Plantas/microbiologia , Bactérias/classificação , Bactérias/genética , Bactérias/isolamento & purificação , Genoma Bacteriano/genética , Vida Livre de Germes , Microbiota/genética , Análise de Sequência de DNA , Microbiologia do SoloRESUMO
Microbial communities inhabit algae cell surfaces and produce a variety of compounds that can impact the fitness of the host. These interactions have been studied via culturing, single-gene diversity and metagenomic read survey methods that are limited by culturing biases and fragmented genetic characterizations. Higher-resolution frameworks are needed to resolve the physiological interactions within these algal-bacterial communities. Here, we infer the encoded metabolic capabilities of four uncultured bacterial genomes (reconstructed using metagenomic assembly and binning) associated with the marine dinoflagellates Gambierdiscus carolinianus and G. caribaeus. Phylogenetic analyses revealed that two of the genomes belong to the commonly algae-associated families Rhodobacteraceae and Flavobacteriaceae. The other two genomes belong to the Phycisphaeraceae and include the first algae-associated representative within the uncultured SM1A02 group. Analyses of all four genomes suggest these bacteria are facultative aerobes, with some capable of metabolizing phytoplanktonic organosulfur compounds including dimethylsulfoniopropionate and sulfated polysaccharides. These communities may biosynthesize compounds beneficial to both the algal host and other bacteria, including iron chelators, B vitamins, methionine, lycopene, squalene and polyketides. These findings have implications for marine carbon and nutrient cycling and provide a greater depth of understanding regarding the genetic potential for complex physiological interactions between microalgae and their associated bacteria.
Assuntos
Dinoflagellida/microbiologia , Flavobacteriaceae/genética , Genoma Bacteriano/genética , Microalgas/microbiologia , Rhodobacteraceae/genética , Fenômenos Bioquímicos , Metagenoma , Metagenômica , Microbiota/genética , Filogenia , Fitoplâncton/microbiologiaRESUMO
Anoxic subsurface sediments contain communities of heterotrophic microorganisms that metabolize organic carbon at extraordinarily low rates. In order to assess the mechanisms by which subsurface microorganisms access detrital sedimentary organic matter, we measured kinetics of a range of extracellular peptidases in anoxic sediments of the White Oak River Estuary, NC. Nine distinct peptidase substrates were enzymatically hydrolyzed at all depths. Potential peptidase activities (Vmax) decreased with increasing sediment depth, although Vmax expressed on a per-cell basis was approximately the same at all depths. Half-saturation constants (Km ) decreased with depth, indicating peptidases that functioned more efficiently at low substrate concentrations. Potential activities of extracellular peptidases acting on molecules that are enriched in degraded organic matter (d-phenylalanine and l-ornithine) increased relative to enzymes that act on l-phenylalanine, further suggesting microbial community adaptation to access degraded organic matter. Nineteen classes of predicted, exported peptidases were identified in genomic data from the same site, of which genes for class C25 (gingipain-like) peptidases represented more than 40% at each depth. Methionine aminopeptidases, zinc carboxypeptidases, and class S24-like peptidases, which are involved in single-stranded-DNA repair, were also abundant. These results suggest a subsurface heterotrophic microbial community that primarily accesses low-quality detrital organic matter via a diverse suite of well-adapted extracellular enzymes.IMPORTANCE Burial of organic carbon in marine and estuarine sediments represents a long-term sink for atmospheric carbon dioxide. Globally, â¼40% of organic carbon burial occurs in anoxic estuaries and deltaic systems. However, the ultimate controls on the amount of organic matter that is buried in sediments, versus oxidized into CO2, are poorly constrained. In this study, we used a combination of enzyme assays and metagenomic analysis to identify how subsurface microbial communities catalyze the first step of proteinaceous organic carbon degradation. Our results show that microbial communities in deeper sediments are adapted to access molecules characteristic of degraded organic matter, suggesting that those heterotrophs are adapted to life in the subsurface.
Assuntos
Estuários , Sedimentos Geológicos/química , Microbiota , Peptídeo Hidrolases/isolamento & purificação , Peptídeo Hidrolases/metabolismo , Carbono/química , Processos Heterotróficos , Cinética , Metagenoma , North Carolina , Compostos Orgânicos/químicaRESUMO
Plants host at the contact zone with soil a distinctive root-associated bacterial microbiota believed to function in plant nutrition and health. We investigated the diversity of the root microbiota within a phylogenetic framework of hosts: three Arabidopsis thaliana ecotypes along with its sister species Arabidopsis halleri and Arabidopsis lyrata, as well as Cardamine hirsuta, which diverged from the former â¼ 35 Mya. We surveyed their microbiota under controlled environmental conditions and of A. thaliana and C. hirsuta in two natural habitats. Deep 16S rRNA gene profiling of root and corresponding soil samples identified a total of 237 quantifiable bacterial ribotypes, of which an average of 73 community members were enriched in roots. The composition of this root microbiota depends more on interactions with the environment than with host species. Interhost species microbiota diversity is largely quantitative and is greater between the three Arabidopsis species than the three A. thaliana ecotypes. Host species-specific microbiota were identified at the levels of individual community members, taxonomic groups, and whole root communities. Most of these signatures were observed in the phylogenetically distant C. hirsuta. However, the branching order of host phylogeny is incongruent with interspecies root microbiota diversity, indicating that host phylogenetic distance alone cannot explain root microbiota diversification. Our work reveals within 35 My of host divergence a largely conserved and taxonomically narrow root microbiota, which comprises stable community members belonging to the Actinomycetales, Burkholderiales, and Flavobacteriales.
Assuntos
Brassicaceae/microbiologia , Microbiota/genética , Raízes de Plantas/microbiologia , Actinomycetales/genética , Bacteroidetes/genética , Sequência de Bases , Betaproteobacteria/genética , Primers do DNA/genética , Alemanha , Interações Hospedeiro-Patógeno , Dados de Sequência Molecular , Filogenia , RNA Ribossômico 16S/genética , Análise de Sequência de DNA , Especificidade da EspécieRESUMO
In Arabidopsis roots, the transcription factor MYB72 plays a dual role in the onset of rhizobacteria-induced systemic resistance (ISR) and plant survival under conditions of limited iron availability. Previously, it was shown that MYB72 coordinates the expression of a gene module that promotes synthesis and excretion of iron-mobilizing phenolic compounds in the rhizosphere, a process that is involved in both iron acquisition and ISR signaling. Here, we show that volatile organic compounds (VOCs) from ISR-inducing Pseudomonas bacteria are important elicitors of MYB72. In response to VOC treatment, MYB72 is co-expressed with the iron uptake-related genes FERRIC REDUCTION OXIDASE 2 (FRO2) and IRON-REGULATED TRANSPORTER 1 (IRT1) in a manner that is dependent on FER-LIKE IRON DEFICIENCY TRANSCRIPTION FACTOR (FIT), indicating that MYB72 is an intrinsic part of the plant's iron-acquisition response that is typically activated upon iron starvation. However, VOC-induced MYB72 expression is activated independently of iron availability in the root vicinity. Moreover, rhizobacterial VOC-mediated induction of MYB72 requires photosynthesis-related signals, while iron deficiency in the rhizosphere activates MYB72 in the absence of shoot-derived signals. Together, these results show that the ISR- and iron acquisition-related transcription factor MYB72 in Arabidopsis roots is activated by rhizobacterial volatiles and photosynthesis-related signals, and enhances the iron-acquisition capacity of roots independently of the iron availability in the rhizosphere. This work highlights the role of MYB72 in plant processes by which root microbiota simultaneously stimulate systemic immunity and activate the iron-uptake machinery in their host plants.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Deficiências de Ferro , Raízes de Plantas/metabolismo , Rhizobium/química , Compostos Orgânicos Voláteis/farmacologia , Arabidopsis/efeitos dos fármacos , Proteínas de Arabidopsis/genética , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Regulação da Expressão Gênica de Plantas/genética , Fotossíntese/efeitos dos fármacos , Raízes de Plantas/efeitos dos fármacosRESUMO
Here, we present the draft genome sequence of Alteromonas gracilis strain J4, isolated from the green macroalga Caulerpa prolifera. The draft genome is 4,492,914 bp in size and contains 4,719 coding DNA sequences, 67 tRNAs, and 16 rRNA-coding genes. Strain J4 may exhibit host growth-promoting properties.
RESUMO
DPANN archaea are a diverse group of microorganisms characterised by small cells and reduced genomes. To date, all cultivated DPANN archaea are ectosymbionts that require direct cell contact with an archaeal host species for growth and survival. However, these interactions and their impact on the host species are poorly understood. Here, we show that a DPANN archaeon (Candidatus Nanohaloarchaeum antarcticus) engages in parasitic interactions with its host (Halorubrum lacusprofundi) that result in host cell lysis. During these interactions, the nanohaloarchaeon appears to enter, or be engulfed by, the host cell. Our results provide experimental evidence for a predatory-like lifestyle of an archaeon, suggesting that at least some DPANN archaea may have roles in controlling host populations and their ecology.
Assuntos
Halorubrum , Simbiose , Halorubrum/genética , Halorubrum/fisiologia , Archaea/genética , Archaea/fisiologia , Nanoarchaeota/genética , Nanoarchaeota/fisiologia , Genoma Arqueal , FilogeniaRESUMO
The nature of the last universal common ancestor (LUCA), its age and its impact on the Earth system have been the subject of vigorous debate across diverse disciplines, often based on disparate data and methods. Age estimates for LUCA are usually based on the fossil record, varying with every reinterpretation. The nature of LUCA's metabolism has proven equally contentious, with some attributing all core metabolisms to LUCA, whereas others reconstruct a simpler life form dependent on geochemistry. Here we infer that LUCA lived ~4.2 Ga (4.09-4.33 Ga) through divergence time analysis of pre-LUCA gene duplicates, calibrated using microbial fossils and isotope records under a new cross-bracing implementation. Phylogenetic reconciliation suggests that LUCA had a genome of at least 2.5 Mb (2.49-2.99 Mb), encoding around 2,600 proteins, comparable to modern prokaryotes. Our results suggest LUCA was a prokaryote-grade anaerobic acetogen that possessed an early immune system. Although LUCA is sometimes perceived as living in isolation, we infer LUCA to have been part of an established ecological system. The metabolism of LUCA would have provided a niche for other microbial community members and hydrogen recycling by atmospheric photochemistry could have supported a modestly productive early ecosystem.
RESUMO
Ocean plastic pollution is a severe environmental problem but most of the plastic that has been released to the ocean since the 1950s is unaccounted for. Although fungal degradation of marine plastics has been suggested as a potential sink mechanism, unambiguous proof of plastic degradation by marine fungi, or other microbes, is scarce. Here we applied stable isotope tracing assays with 13C-labeled polyethylene to measure biodegradation rates and to trace the incorporation of plastic-derived carbon into individual cells of the yeast Rhodotorula mucilaginosa, which we isolated from the marine environment. 13C accumulation in the CO2 pool during 5-day incubation experiments with R. mucilaginosa and UV-irradiated 13C-labeled polyethylene as a sole energy and carbon source translated to degradation rates of 3.8% yr-1 of the initially added substrate. Furthermore, nanoSIMS measurements revealed substantial incorporation of polyethylene-derived carbon into fungal biomass. Our results demonstrate the potential of R. mucilaginosa to mineralize and assimilate carbon from plastics and suggest that fungal plastic degradation may be an important sink for polyethylene litter in the marine environment.
RESUMO
The timing of early cellular evolution, from the divergence of Archaea and Bacteria to the origin of eukaryotes, is poorly constrained. The ATP synthase complex is thought to have originated prior to the Last Universal Common Ancestor (LUCA) and analyses of ATP synthase genes, together with ribosomes, have played a key role in inferring and rooting the tree of life. We reconstruct the evolutionary history of ATP synthases using an expanded taxon sampling set and develop a phylogenetic cross-bracing approach, constraining equivalent speciation nodes to be contemporaneous, based on the phylogenetic imprint of endosymbioses and ancient gene duplications. This approach results in a highly resolved, dated species tree and establishes an absolute timeline for ATP synthase evolution. Our analyses show that the divergence of ATP synthase into F- and A/V-type lineages was a very early event in cellular evolution dating back to more than 4 Ga, potentially predating the diversification of Archaea and Bacteria. Our cross-braced, dated tree of life also provides insight into more recent evolutionary transitions including eukaryogenesis, showing that the eukaryotic nuclear and mitochondrial lineages diverged from their closest archaeal (2.67-2.19 Ga) and bacterial (2.58-2.12 Ga) relatives at approximately the same time, with a slightly longer nuclear stem-lineage.
Assuntos
Archaea , Bactérias , Filogenia , Bactérias/genética , Archaea/genética , Mitocôndrias/genética , Trifosfato de Adenosina , Evolução Molecular , Eucariotos/genética , Evolução BiológicaRESUMO
Deltaproteobacteria, now proposed to be the phyla Desulfobacterota, Myxococcota, and SAR324, are ubiquitous in marine environments and play essential roles in global carbon, sulfur, and nutrient cycling. Despite their importance, our understanding of these bacteria is biased towards cultured organisms. Here we address this gap by compiling a genomic catalog of 1 792 genomes, including 402 newly reconstructed and characterized metagenome-assembled genomes (MAGs) from coastal and deep-sea sediments. Phylogenomic analyses reveal that many of these novel MAGs are uncultured representatives of Myxococcota and Desulfobacterota that are understudied. To better characterize Deltaproteobacteria diversity, metabolism, and ecology, we clustered ~1 500 genomes based on the presence/absence patterns of their protein families. Protein content analysis coupled with large-scale metabolic reconstructions separates eight genomic clusters of Deltaproteobacteria with unique metabolic profiles. While these eight clusters largely correspond to phylogeny, there are exceptions where more distantly related organisms appear to have similar ecological roles and closely related organisms have distinct protein content. Our analyses have identified previously unrecognized roles in the cycling of methylamines and denitrification among uncultured Deltaproteobacteria. This new view of Deltaproteobacteria diversity expands our understanding of these dominant bacteria and highlights metabolic abilities across diverse taxa.
Assuntos
Deltaproteobacteria , Metagenoma , Bactérias/genética , Deltaproteobacteria/genética , Genômica , Humanos , FilogeniaRESUMO
Core gene phylogenies provide a window into early evolution, but different gene sets and analytical methods have yielded substantially different views of the tree of life. Trees inferred from a small set of universal core genes have typically supported a long branch separating the archaeal and bacterial domains. By contrast, recent analyses of a broader set of non-ribosomal genes have suggested that Archaea may be less divergent from Bacteria, and that estimates of inter-domain distance are inflated due to accelerated evolution of ribosomal proteins along the inter-domain branch. Resolving this debate is key to determining the diversity of the archaeal and bacterial domains, the shape of the tree of life, and our understanding of the early course of cellular evolution. Here, we investigate the evolutionary history of the marker genes key to the debate. We show that estimates of a reduced Archaea-Bacteria (AB) branch length result from inter-domain gene transfers and hidden paralogy in the expanded marker gene set. By contrast, analysis of a broad range of manually curated marker gene datasets from an evenly sampled set of 700 Archaea and Bacteria reveals that current methods likely underestimate the AB branch length due to substitutional saturation and poor model fit; that the best-performing phylogenetic markers tend to support longer inter-domain branch lengths; and that the AB branch lengths of ribosomal and non-ribosomal marker genes are statistically indistinguishable. Furthermore, our phylogeny inferred from the 27 highest-ranked marker genes recovers a clade of DPANN at the base of the Archaea and places the bacterial Candidate Phyla Radiation (CPR) within Bacteria as the sister group to the Chloroflexota.
Assuntos
Archaea , Bactérias , Archaea/genética , Bactérias/metabolismo , Evolução Molecular , Filogenia , Proteínas Ribossômicas/genética , Proteínas Ribossômicas/metabolismoRESUMO
Micrarchaeota is a distinctive lineage assigned to the DPANN archaea, which includes poorly characterised microorganisms with reduced genomes that likely depend on interactions with hosts for growth and survival. Here, we report the enrichment of a stable co-culture of a member of the Micrarchaeota (Ca. Micrarchaeum harzensis) together with its Thermoplasmatales host (Ca. Scheffleriplasma hospitalis), as well as the isolation of the latter. We show that symbiont-host interactions depend on biofilm formation as evidenced by growth experiments, comparative transcriptomic analyses and electron microscopy. In addition, genomic, metabolomic, extracellular polymeric substances and lipid content analyses indicate that the Micrarchaeon symbiont relies on the acquisition of metabolites from its host. Our study of the cell biology and physiology of a Micrarchaeon and its host adds to our limited knowledge of archaeal symbioses.
Assuntos
Thermoplasmales , Archaea/genética , Biofilmes , Genoma Arqueal , Filogenia , Thermoplasmales/genética , Thermoplasmales/metabolismoRESUMO
Profiling diverse microbiomes is revolutionizing our understanding of biological mechanisms and ecologically relevant problems, including metaorganism (host + microbiome) assembly, functions and adaptation. Amplicon sequencing of multiple conserved, phylogenetically informative loci has therefore become an instrumental tool for many researchers. Investigations in many systems are hindered, however, since essential sequencing depth can be lost by amplification of nontarget DNA from hosts or overabundant microorganisms. Here, we introduce "blocking oligos", a low-cost and flexible method using standard oligonucleotides to block amplification of diverse nontargets and software to aid their design. We apply them primarily in leaves, where exceptional challenges with host amplification prevail. A. thaliana-specific blocking oligos applied in eight different target loci reduce undesirable host amplification by up to 90%. To expand applicability, we designed universal 16S and 18S rRNA gene plant blocking oligos for targets that are conserved in diverse plant species and demonstrate that they efficiently block five plant species from five orders spanning monocots and dicots (Bromus erectus, Plantago lanceolata, Lotus corniculatus, Amaranth sp., Arabidopsis thaliana). These can increase alpha diversity discovery without biasing beta diversity patterns and do not compromise microbial load information inherent to plant-derived 16S rRNA gene amplicon sequencing data. Finally, we designed and tested blocking oligos to avoid amplification of 18S rRNA genes of a sporulating oomycete pathogen, demonstrating their effectiveness in applications well beyond plants. Using these tools, we generated a survey of the A. thaliana leaf microbiome based on eight loci targeting bacterial, fungal, oomycete and other eukaryotic microorganisms and discuss complementarity of commonly used amplicon sequencing regions for describing leaf microbiota. This approach has potential to make questions in a variety of study systems more tractable by making amplicon sequencing more targeted, leading to deeper, systems-based insights into microbial discovery. For fast and easy design for blocking oligos for any nontarget DNA in other study systems, we developed a publicly available R package.
Assuntos
Microbiota , Plantas/microbiologia , Bactérias/classificação , Fungos/classificação , Sequenciamento de Nucleotídeos em Larga Escala , Oomicetos/classificação , RNA Ribossômico 16S/genética , Análise de Sequência de DNARESUMO
Geothermal environments, such as hot springs and hydrothermal vents, are hotspots for carbon cycling and contain many poorly described microbial taxa. Here, we reconstructed 15 archaeal metagenome-assembled genomes (MAGs) from terrestrial hot spring sediments in China and deep-sea hydrothermal vent sediments in Guaymas Basin, Gulf of California. Phylogenetic analyses of these MAGs indicate that they form a distinct group within the TACK superphylum, and thus we propose their classification as a new phylum, 'Brockarchaeota', named after Thomas Brock for his seminal research in hot springs. Based on the MAG sequence information, we infer that some Brockarchaeota are uniquely capable of mediating non-methanogenic anaerobic methylotrophy, via the tetrahydrofolate methyl branch of the Wood-Ljungdahl pathway and reductive glycine pathway. The hydrothermal vent genotypes appear to be obligate fermenters of plant-derived polysaccharides that rely mostly on substrate-level phosphorylation, as they seem to lack most respiratory complexes. In contrast, hot spring lineages have alternate pathways to increase their ATP yield, including anaerobic methylotrophy of methanol and trimethylamine, and potentially use geothermally derived mercury, arsenic, or hydrogen. Their broad distribution and their apparent anaerobic metabolic versatility indicate that Brockarchaeota may occupy previously overlooked roles in anaerobic carbon cycling.