Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 1.091
Filtrar
1.
Nat Commun ; 12(1): 2009, 2021 03 31.
Artículo en Inglés | MEDLINE | ID: mdl-33790294

RESUMEN

Microorganisms play crucial roles in water recycling, pollution removal and resource recovery in the wastewater industry. The structure of these microbial communities is increasingly understood based on 16S rRNA amplicon sequencing data. However, such data cannot be linked to functional potential in the absence of high-quality metagenome-assembled genomes (MAGs) for nearly all species. Here, we use long-read and short-read sequencing to recover 1083 high-quality MAGs, including 57 closed circular genomes, from 23 Danish full-scale wastewater treatment plants. The MAGs account for ~30% of the community based on relative abundance, and meet the stringent MIMAG high-quality draft requirements including full-length rRNA genes. We use the information provided by these MAGs in combination with >13 years of 16S rRNA amplicon sequencing data, as well as Raman microspectroscopy and fluorescence in situ hybridisation, to uncover abundant undescribed lineages belonging to important functional groups.


Asunto(s)
Genoma Bacteriano/genética , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Metagenoma/genética , Metagenómica/métodos , ARN Ribosómico 16S/genética , Aguas del Alcantarillado/microbiología , Bacterias/clasificación , Bacterias/genética , Reactores Biológicos/microbiología , Dinamarca , Microbiota/genética , Filogenia , ARN Ribosómico 23S/genética , ARN Ribosómico 5S/genética , Aguas Residuales/microbiología , Purificación del Agua/métodos
2.
Nat Commun ; 12(1): 2404, 2021 04 23.
Artículo en Inglés | MEDLINE | ID: mdl-33893309

RESUMEN

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.


Asunto(s)
Archaea/genética , Ciclo del Carbono/genética , Genoma Arqueal/genética , Metagenoma/genética , Filogenia , Archaea/clasificación , Archaea/metabolismo , Carbono/metabolismo , China , Geografía , Sedimentos Geológicos/microbiología , Manantiales de Aguas Termales/microbiología , Respiraderos Hidrotermales/microbiología , Metano/metabolismo , ARN Ribosómico 16S/genética , Especificidad de la Especie
3.
Microbiome ; 9(1): 58, 2021 03 03.
Artículo en Inglés | MEDLINE | ID: mdl-33658077

RESUMEN

BACKGROUND: Microbial eukaryotes are found alongside bacteria and archaea in natural microbial systems, including host-associated microbiomes. While microbial eukaryotes are critical to these communities, they are challenging to study with shotgun sequencing techniques and are therefore often excluded. RESULTS: Here, we present EukDetect, a bioinformatics method to identify eukaryotes in shotgun metagenomic sequencing data. Our approach uses a database of 521,824 universal marker genes from 241 conserved gene families, which we curated from 3713 fungal, protist, non-vertebrate metazoan, and non-streptophyte archaeplastida genomes and transcriptomes. EukDetect has a broad taxonomic coverage of microbial eukaryotes, performs well on low-abundance and closely related species, and is resilient against bacterial contamination in eukaryotic genomes. Using EukDetect, we describe the spatial distribution of eukaryotes along the human gastrointestinal tract, showing that fungi and protists are present in the lumen and mucosa throughout the large intestine. We discover that there is a succession of eukaryotes that colonize the human gut during the first years of life, mirroring patterns of developmental succession observed in gut bacteria. By comparing DNA and RNA sequencing of paired samples from human stool, we find that many eukaryotes continue active transcription after passage through the gut, though some do not, suggesting they are dormant or nonviable. We analyze metagenomic data from the Baltic Sea and find that eukaryotes differ across locations and salinity gradients. Finally, we observe eukaryotes in Arabidopsis leaf samples, many of which are not identifiable from public protein databases. CONCLUSIONS: EukDetect provides an automated and reliable way to characterize eukaryotes in shotgun sequencing datasets from diverse microbiomes. We demonstrate that it enables discoveries that would be missed or clouded by false positives with standard shotgun sequence analysis. EukDetect will greatly advance our understanding of how microbial eukaryotes contribute to microbiomes. Video abstract.


Asunto(s)
Eucariontes/genética , Eucariontes/aislamiento & purificación , Metagenoma/genética , Metagenómica/métodos , Metagenómica/normas , Animales , Eucariontes/clasificación , Humanos , Análisis de Secuencia de ADN
4.
Microbiome ; 9(1): 72, 2021 03 25.
Artículo en Inglés | MEDLINE | ID: mdl-33766108

RESUMEN

BACKGROUND: In octocorals (Cnidaria Octocorallia), the functional relationship between host health and its symbiotic consortium has yet to be determined. Here, we employed comparative metagenomics to uncover the distinct functional and phylogenetic features of the microbiomes of healthy Eunicella gazella, Eunicella verrucosa, and Leptogorgia sarmentosa tissues, in contrast with the microbiomes found in seawater and sediments. We further explored how the octocoral microbiome shifts to a pathobiome state in E. gazella. RESULTS: Multivariate analyses based on 16S rRNA genes, Clusters of Orthologous Groups of proteins (COGs), Protein families (Pfams), and secondary metabolite-biosynthetic gene clusters annotated from 20 Illumina-sequenced metagenomes each revealed separate clustering of the prokaryotic communities of healthy tissue samples of the three octocoral species from those of necrotic E. gazella tissue and surrounding environments. While the healthy octocoral microbiome was distinguished by so-far uncultivated Endozoicomonadaceae, Oceanospirillales, and Alteromonadales phylotypes in all host species, a pronounced increase of Flavobacteriaceae and Alphaproteobacteria, originating from seawater, was observed in necrotic E. gazella tissue. Increased abundances of eukaryotic-like proteins, exonucleases, restriction endonucleases, CRISPR/Cas proteins, and genes encoding for heat-shock proteins, inorganic ion transport, and iron storage distinguished the prokaryotic communities of healthy octocoral tissue regardless of the host species. An increase of arginase and nitric oxide reductase genes, observed in necrotic E. gazella tissues, suggests the existence of a mechanism for suppression of nitrite oxide production by which octocoral pathogens may overcome the host's immune system. CONCLUSIONS: This is the first study to employ primer-less, shotgun metagenome sequencing to unveil the taxonomic, functional, and secondary metabolism features of prokaryotic communities in octocorals. Our analyses reveal that the octocoral microbiome is distinct from those of the environmental surroundings, is host genus (but not species) specific, and undergoes large, complex structural changes in the transition to the dysbiotic state. Host-symbiont recognition, abiotic-stress response, micronutrient acquisition, and an antiviral defense arsenal comprising multiple restriction endonucleases, CRISPR/Cas systems, and phage lysogenization regulators are signatures of prokaryotic communities in octocorals. We argue that these features collectively contribute to the stabilization of symbiosis in the octocoral holobiont and constitute beneficial traits that can guide future studies on coral reef conservation and microbiome therapy. Video Abstract.


Asunto(s)
Antozoos/microbiología , Bacterias/clasificación , Bacterias/genética , Interacciones Huésped-Patógeno , Metagenoma/genética , Metagenómica , Filogenia , Animales , Disbiosis , ARN Ribosómico 16S/genética
6.
Nat Commun ; 12(1): 1106, 2021 02 17.
Artículo en Inglés | MEDLINE | ID: mdl-33597514

RESUMEN

Gut microbiota plays an important role in pig health and production. Still, availability of sequenced genomes and functional information for most pig gut microbes remains limited. Here we perform a landscape survey of the swine gut microbiome, spanning extensive sample sources by deep metagenomic sequencing resulting in an expanded gene catalog named pig integrated gene catalog (PIGC), containing 17,237,052 complete genes clustered at 90% protein identity from 787 gut metagenomes, of which 28% are unknown proteins. Using binning analysis, 6339 metagenome-assembled genomes (MAGs) were obtained, which were clustered to 2673 species-level genome bins (SGBs), among which 86% (2309) SGBs are unknown based on current databases. Using the present gene catalog and MAGs, we identified several strain-level differences between the gut microbiome of wild boars and commercial Duroc pigs. PIGC and MAGs provide expanded resources for swine gut microbiome-related research.


Asunto(s)
Microbioma Gastrointestinal/genética , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Metagenoma/genética , Metagenómica/métodos , Animales , Bacterias/clasificación , Bacterias/genética , Femenino , Genes Microbianos/genética , Filogenia , Especificidad de la Especie , Porcinos
7.
Curr Opin Biotechnol ; 67: 184-191, 2021 02.
Artículo en Inglés | MEDLINE | ID: mdl-33592536

RESUMEN

Ability to directly sequence DNA from the environment permanently changed microbial ecology. Here, we review the new insights to microbial life gleaned from the applications of metagenomics, as well as the extensive set of analytical tools that facilitate exploration of diversity and function of complex microbial communities. While metagenomics is shaping our understanding of microbial functions in ecosystems via gene-centric and genome-centric methods, annotating functions, metagenome assembly and binning in heterogeneous samples remains challenging. Development of new analysis and sequencing platforms generating high-throughput long-read sequences and functional screening opportunities will aid in harnessing metagenomes to increase our understanding of microbial taxonomy, function, ecology, and evolution in the environment.


Asunto(s)
Metagenómica , Microbiota , Ecología , Secuenciación de Nucleótidos de Alto Rendimiento , Metagenoma/genética , Microbiota/genética , Análisis de Secuencia de ADN
8.
Microbiome ; 9(1): 32, 2021 01 31.
Artículo en Inglés | MEDLINE | ID: mdl-33517907

RESUMEN

BACKGROUND: While indoor microbiomes impact our health and well-being, much remains unknown about taxonomic and functional transitions that occur in human-derived microbial communities once they are transferred away from human hosts. Toothbrushes are a model to investigate the potential response of oral-derived microbiota to conditions of the built environment. Here, we characterize metagenomes of toothbrushes from 34 subjects to define the toothbrush microbiome and resistome and possible influential factors. RESULTS: Toothbrush microbiomes often comprised a dominant subset of human oral taxa and less abundant or site-specific environmental strains. Although toothbrushes contained lower taxonomic diversity than oral-associated counterparts (determined by comparison with the Human Microbiome Project), they had relatively broader antimicrobial resistance gene (ARG) profiles. Toothbrush resistomes were enriched with a variety of ARGs, notably those conferring multidrug efflux and putative resistance to triclosan, which were primarily attributable to versatile environmental taxa. Toothbrush microbial communities and resistomes correlated with a variety of factors linked to personal health, dental hygiene, and bathroom features. CONCLUSIONS: Selective pressures in the built environment may shape the dynamic mixture of human (primarily oral-associated) and environmental microbiota that encounter each other on toothbrushes. Harboring a microbial diversity and resistome distinct from human-associated counterparts suggests toothbrushes could potentially serve as a reservoir that may enable the transfer of ARGs. Video abstract.


Asunto(s)
Entorno Construido , Microbiota , Boca/microbiología , Cepillado Dental , Adolescente , Adulto , Anciano , Farmacorresistencia Microbiana/efectos de los fármacos , Farmacorresistencia Microbiana/genética , Humanos , Metagenoma/efectos de los fármacos , Metagenoma/genética , Microbiota/efectos de los fármacos , Microbiota/genética , Persona de Mediana Edad , Boca/efectos de los fármacos , Triclosán/farmacología , Adulto Joven
9.
Microbiome ; 9(1): 55, 2021 02 23.
Artículo en Inglés | MEDLINE | ID: mdl-33622394

RESUMEN

BACKGROUND: The intestinal microbiota plays a crucial role in protecting the host from pathogenic microbes, modulating immunity and regulating metabolic processes. We studied the simplified human intestinal microbiota (SIHUMIx) consisting of eight bacterial species with a particular focus on the discovery of novel small proteins with less than 100 amino acids (= sProteins), some of which may contribute to shape the simplified human intestinal microbiota. Although sProteins carry out a wide range of important functions, they are still often missed in genome annotations, and little is known about their structure and function in individual microbes and especially in microbial communities. RESULTS: We created a multi-species integrated proteogenomics search database (iPtgxDB) to enable a comprehensive identification of novel sProteins. Six of the eight SIHUMIx species, for which no complete genomes were available, were sequenced and de novo assembled. Several proteomics approaches including two earlier optimized sProtein enrichment strategies were applied to specifically increase the chances for novel sProtein discovery. The search of tandem mass spectrometry (MS/MS) data against the multi-species iPtgxDB enabled the identification of 31 novel sProteins, of which the expression of 30 was supported by metatranscriptomics data. Using synthetic peptides, we were able to validate the expression of 25 novel sProteins. The comparison of sProtein expression in each single strain versus a multi-species community cultivation showed that six of these sProteins were only identified in the SIHUMIx community indicating a potentially important role of sProteins in the organization of microbial communities. Two of these novel sProteins have a potential antimicrobial function. Metabolic modelling revealed that a third sProtein is located in a genomic region encoding several enzymes relevant for the community metabolism within SIHUMIx. CONCLUSIONS: We outline an integrated experimental and bioinformatics workflow for the discovery of novel sProteins in a simplified intestinal model system that can be generically applied to other microbial communities. The further analysis of novel sProteins uniquely expressed in the SIHUMIx multi-species community is expected to enable new insights into the role of sProteins on the functionality of bacterial communities such as those of the human intestinal tract. Video abstract.


Asunto(s)
Proteínas Bacterianas/análisis , Proteínas Bacterianas/química , Comunicación Celular , Microbioma Gastrointestinal , Bacterias/clasificación , Bacterias/genética , Bacterias/aislamiento & purificación , Bacterias/metabolismo , Proteínas Bacterianas/genética , Microbioma Gastrointestinal/genética , Humanos , Intestinos/química , Intestinos/microbiología , Metagenoma/genética , Espectrometría de Masas en Tándem
10.
Methods Mol Biol ; 2243: 123-141, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33606256

RESUMEN

In this chapter, we will present an outline of a typical experimental and bioinformatic workflow for identification of bacterial amplicon sequence variants (ASVs) present in a set of samples. This chapter is written from a bioinformatic point of view; therefore, the specific experimental protocols are not detailed, but rather the impact of various experimental decisions on the downstream analysis is described. Emphasis is made on the transition from reads to ASVs, describing the Deblur algorithm.


Asunto(s)
Variación Genética/genética , Microbiota/genética , ARN Ribosómico 16S/genética , Algoritmos , Animales , Bacterias/genética , Biología Computacional/métodos , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Humanos , Metagenoma/genética , Flujo de Trabajo
11.
Methods Mol Biol ; 2243: 107-122, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33606255

RESUMEN

Microbial communities are found across diverse environments, including within and across the human body. As many microbes are unculturable in the lab, much of what is known about a microbiome-a collection of bacteria, fungi, archaea, and viruses inhabiting an environment--is from the sequencing of DNA from within the constituent community. Here, we provide an introduction to whole-metagenome shotgun sequencing studies, a ubiquitous approach for characterizing microbial communities, by reviewing three major research areas in metagenomics: assembly, community profiling, and functional profiling. Though not exhaustive, these areas encompass a large component of the metagenomics literature. We discuss each area in depth, the challenges posed by whole-metagenome shotgun sequencing, and approaches fundamental to the solutions of each. We conclude by discussing promising areas for future research. Though our emphasis is on the human microbiome, the methods discussed are broadly applicable across study systems.


Asunto(s)
Metagenoma/genética , Microbiota/genética , Archaea/genética , Bacterias/genética , Humanos , Metagenómica/métodos , Análisis de Secuencia de ADN/métodos , Virus/genética
12.
Elife ; 102021 01 19.
Artículo en Inglés | MEDLINE | ID: mdl-33461660

RESUMEN

While traditional microbiological freshwater tests focus on the detection of specific bacterial indicator species, including pathogens, direct tracing of all aquatic DNA through metagenomics poses a profound alternative. Yet, in situ metagenomic water surveys face substantial challenges in cost and logistics. Here, we present a simple, fast, cost-effective and remotely accessible freshwater diagnostics workflow centred around the portable nanopore sequencing technology. Using defined compositions and spatiotemporal microbiota from surface water of an example river in Cambridge (UK), we provide optimised experimental and bioinformatics guidelines, including a benchmark with twelve taxonomic classification tools for nanopore sequences. We find that nanopore metagenomics can depict the hydrological core microbiome and fine temporal gradients in line with complementary physicochemical measurements. In a public health context, these data feature relevant sewage signals and pathogen maps at species level resolution. We anticipate that this framework will gather momentum for new environmental monitoring initiatives using portable devices.


Asunto(s)
Agua Dulce/microbiología , Metagenoma/genética , Metagenómica/métodos , Microbiota/genética , Secuenciación de Nanoporos/métodos , Microbiología del Agua , Bacterias/clasificación , Bacterias/genética , Secuencia de Bases , Análisis por Conglomerados , Biología Computacional/métodos , Monitoreo del Ambiente/métodos , Geografía , ARN Ribosómico 16S/genética , Ríos/microbiología , Homología de Secuencia de Ácido Nucleico , Especificidad de la Especie , Reino Unido
13.
Microbiome ; 9(1): 4, 2021 01 09.
Artículo en Inglés | MEDLINE | ID: mdl-33422152

RESUMEN

BACKGROUND: Widespread bioinformatic resource development generates a constantly evolving and abundant landscape of workflows and software. For analysis of the microbiome, workflows typically begin with taxonomic classification of the microorganisms that are present in a given environment. Additional investigation is then required to uncover the functionality of the microbial community, in order to characterize its currently or potentially active biological processes. Such functional analysis of metagenomic data can be computationally demanding for high-throughput sequencing experiments. Instead, we can directly compare sequencing reads to a functionally annotated database. However, since reads frequently match multiple sequences equally well, analyses benefit from a hierarchical annotation tree, e.g. for taxonomic classification where reads are assigned to the lowest taxonomic unit. RESULTS: To facilitate functional microbiome analysis, we re-purpose well-known taxonomic classification tools to allow us to perform direct functional sequencing read classification with the added benefit of a functional hierarchy. To enable this, we develop and present a tree-shaped functional hierarchy representing the molecular function subset of the Gene Ontology annotation structure. We use this functional hierarchy to replace the standard phylogenetic taxonomy used by the classification tools and assign query sequences accurately to the lowest possible molecular function in the tree. We demonstrate this with simulated and experimental datasets, where we reveal new biological insights. CONCLUSIONS: We demonstrate that improved functional classification of metagenomic sequencing reads is possible by re-purposing a range of taxonomic classification tools that are already well-established, in conjunction with either protein or nucleotide reference databases. We leverage the advances in speed, accuracy and efficiency that have been made for taxonomic classification and translate these benefits for the rapid functional classification of microbiomes. While we focus on a specific set of commonly used methods, the functional annotation approach has broad applicability across other sequence classification tools. We hope that re-purposing becomes a routine consideration during bioinformatic resource development. Video abstract.


Asunto(s)
Clasificación/métodos , Biología Computacional/métodos , Secuenciación de Nucleótidos de Alto Rendimiento , Metagenoma/genética , Metagenómica/métodos , Microbiota/genética , Programas Informáticos , Filogenia
14.
PLoS One ; 16(1): e0244876, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33411719

RESUMEN

Characterizing the gut microbiota in terms of their capacity to interfere with drug metabolism is necessary to achieve drug efficacy and safety. Although examples of drug-microbiome interactions are well-documented, little has been reported about a computational pipeline for systematically identifying and characterizing bacterial enzymes that process particular classes of drugs. The goal of our study is to develop a computational approach that compiles drugs whose metabolism may be influenced by a particular class of microbial enzymes and that quantifies the variability in the collective level of those enzymes among individuals. The present paper describes this approach, with microbial ß-glucuronidases as an example, which break down drug-glucuronide conjugates and reactivate the drugs or their metabolites. We identified 100 medications that may be metabolized by ß-glucuronidases from the gut microbiome. These medications included morphine, estrogen, ibuprofen, midazolam, and their structural analogues. The analysis of metagenomic data available through the Sequence Read Archive (SRA) showed that the level of ß-glucuronidase in the gut metagenomes was higher in males than in females, which provides a potential explanation for the sex-based differences in efficacy and toxicity for several drugs, reported in previous studies. Our analysis also showed that infant gut metagenomes at birth and 12 months of age have higher levels of ß-glucuronidase than the metagenomes of their mothers and the implication of this observed variability was discussed in the context of breastfeeding as well as infant hyperbilirubinemia. Overall, despite important limitations discussed in this paper, our analysis provided useful insights on the role of the human gut metagenome in the variability in drug response among individuals. Importantly, this approach exploits drug and metagenome data available in public databases as well as open-source cheminformatics and bioinformatics tools to predict drug-metagenome interactions.


Asunto(s)
Predicción/métodos , Microbioma Gastrointestinal/efectos de los fármacos , Metagenómica/métodos , Adulto , Bacterias/genética , Biología Computacional/métodos , Manejo de Datos , Femenino , Microbioma Gastrointestinal/genética , Glucuronidasa/genética , Glucuronidasa/metabolismo , Humanos , Recién Nacido , Masculino , Metagenoma/efectos de los fármacos , Metagenoma/genética , Microbiota/efectos de los fármacos , Microbiota/genética , Madres
15.
Nat Med ; 27(2): 321-332, 2021 02.
Artículo en Inglés | MEDLINE | ID: mdl-33432175

RESUMEN

The gut microbiome is shaped by diet and influences host metabolism; however, these links are complex and can be unique to each individual. We performed deep metagenomic sequencing of 1,203 gut microbiomes from 1,098 individuals enrolled in the Personalised Responses to Dietary Composition Trial (PREDICT 1) study, whose detailed long-term diet information, as well as hundreds of fasting and same-meal postprandial cardiometabolic blood marker measurements were available. We found many significant associations between microbes and specific nutrients, foods, food groups and general dietary indices, which were driven especially by the presence and diversity of healthy and plant-based foods. Microbial biomarkers of obesity were reproducible across external publicly available cohorts and in agreement with circulating blood metabolites that are indicators of cardiovascular disease risk. While some microbes, such as Prevotella copri and Blastocystis spp., were indicators of favorable postprandial glucose metabolism, overall microbiome composition was predictive for a large panel of cardiometabolic blood markers including fasting and postprandial glycemic, lipemic and inflammatory indices. The panel of intestinal species associated with healthy dietary habits overlapped with those associated with favorable cardiometabolic and postprandial markers, indicating that our large-scale resource can potentially stratify the gut microbiome into generalizable health levels in individuals without clinically manifest disease.


Asunto(s)
Microbioma Gastrointestinal/genética , Metagenoma/genética , Microbiota/genética , Obesidad/microbiología , Adulto , Biomarcadores/metabolismo , Blastocystis/genética , Glucemia/metabolismo , Niño , Dieta/efectos adversos , Ayuno/metabolismo , Conducta Alimentaria , Femenino , Microbiología de Alimentos , Glucosa/genética , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Masculino , Persona de Mediana Edad , Obesidad/genética , Obesidad/metabolismo , Periodo Posprandial/genética , Prevotella/genética , Prevotella/aislamiento & purificación
16.
Methods Mol Biol ; 2232: 1-21, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33161534

RESUMEN

Recent studies indicate that seed microbiomes affect germination and plant performance. However, the interplay between seed microbiota and plant health is still poorly understood. To get a complete picture of the system, a comprehensive analysis is required, comprising culture-dependent and culture-independent techniques. In this chapter, we provide a combination of methods that are established and optimized for the analysis of the seed microbiome. These include methods to: (1) activate and cultivate dormant seed microbiota, (2) analyze microbiota in germinated seeds (with and without substrate), (3) quantify microbial DNA via real-time PCR, (4) deplete host DNA for amplicon and metagenome analysis, and (5) visualize seed endophytes in microtomed sections using fluorescent in situ hybridization (FISH) and confocal laser scanning microscopy (CLSM). A deep understanding of the seed microbiome and its functions can help in developing new seed treatments and breeding strategies for sustainable agriculture.


Asunto(s)
Hibridación Fluorescente in Situ/métodos , Microbiota/genética , Plantas/genética , Semillas/genética , Endófitos/genética , Endófitos/crecimiento & desarrollo , Germinación/genética , Metagenoma/genética , Plantas/microbiología , ARN Ribosómico 16S/genética , Semillas/microbiología
17.
Methods Mol Biol ; 2232: 147-153, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33161546

RESUMEN

DNA sequencing has become a common tool in environmental microbial ecology, facilitating characterization of microbial populations as well as complex microbial communities by circumventing culture bottlenecks. However, certain samples especially from host-associated environments (rhizosphere, human tissue) or complex communities (soils) can contain a high degree of DNA sequences derived from hosts (plants, human) or other organisms of non-interest (arthropods, unicellular eukaryotes). This chapter presents a simple in silico method to remove contaminating sequences in metagenomes based on aligning sequences to reference genomes of the target organism.


Asunto(s)
Secuencia de Bases/genética , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Metagenoma/genética , Metagenómica/métodos , Animales , Artrópodos/microbiología , Eucariontes/genética , Humanos , Microbiota , Plantas/microbiología , Análisis de Secuencia de ADN , Microbiología del Suelo
18.
Int J Biol Macromol ; 170: 239-250, 2021 Feb 15.
Artículo en Inglés | MEDLINE | ID: mdl-33316338

RESUMEN

Lignocellulosic biomass (LCB) is a prominent option for second-generation biofuels production. Cellulase hydrolyses cellulose, a component of LCB by attacking the ß-1,4-glycosidic bonds, thus liberating mono, di, and oligosaccharides, which subsequently, can be converted to biofuel. In this study, a novel cellulase (Cel-3.1) of 1593 bp which encodes a 530 amino acid protein was identified from buffalo rumen metagenomic fosmid library, and functional expression was achieved through transformation into Escherichia coli. The molecular weight was estimated as 58 kDa on SDS-PAGE. Cel-3.1 belongs to glycosyl hydrolase family-5 (GH-5) and is predicted to have 14 α-helices and 15 ß-strands. The optimal temperature and pH for Cel-3.1 were experimentally determined as 5.0 and 50 °C respectively. The synergistic effect of Ca2+ with K+ ions improved Cel-3.1 activity significantly (25%) and 1% Polyethylene Glycol (PEG-400), 1% ß-mercaptoethanol enhanced the relative activity Cel-3.1 by 31.68%, 12.03% respectively. Further, the enzymatic (Cel-3.1) hydrolysis of pretreated rice straw and corncob released 13.41 ± 0.26 mg/mL and 15.04 ± 0.08 mg/mL reducing sugars respectively. High Performance Liquid Chromatography (HPLC), Scanning Electron Microscope (SEM), and Fourier Transformation Infrared spectroscopy (FTIR) analysis revealed the capability of Cel-3.1 for the breakdown and hydrolysis of both rice straw and corncob to generate various fermentable sugars.


Asunto(s)
Celulasa/genética , Celulasa/aislamiento & purificación , Rumen/metabolismo , Animales , Biocombustibles , Biomasa , Búfalos/metabolismo , Celulasa/metabolismo , Celulosa/metabolismo , Clonación Molecular/métodos , Fermentación , Concentración de Iones de Hidrógeno , Hidrólisis , Lignina/química , Metagenoma/genética , Metagenómica , Especificidad por Sustrato
19.
Sci Rep ; 10(1): 22092, 2020 12 16.
Artículo en Inglés | MEDLINE | ID: mdl-33328537

RESUMEN

Human milk is the optimal nutrition source for infants, and oligosaccharides represent the third most abundant component in milk after lactose and fat. Human milk oligosaccharides (HMO) are favorable macromolecules which are, interestingly, indigestible by the infant but serve as substrates for bacteria. Hypothesizing that the maternal diet itself might influence HMO composition, we sought to directly determine the effect maternal diet on HMO and the milk bacteria. Employing a human cross-over study design, we demonstrate that distinct maternal dietary carbohydrate and energy sources preferentially alter milk concentrations of HMO, including fucosylated species. We find significant associations between the concentration of HMO-bound fucose and the abundance of fucosidase (a bacterial gene that digests fucose moieties) harbored by milk bacteria. These studies reveal a successive mechanism by which the maternal diet during lactation alters milk HMO composition, which in turn shapes the functional milk microbiome prior to infant ingestion.


Asunto(s)
Lactancia Materna , Metagenoma/genética , Leche Humana/química , Oligosacáridos/química , Animales , Estudios Cruzados , Dieta , Femenino , Humanos , Lactante , Lactancia/genética , Lactosa/genética , Lactosa/metabolismo , Microbiota/genética , Leche Humana/metabolismo , Estado Nutricional , Oligosacáridos/genética , Oligosacáridos/aislamiento & purificación
20.
Sci Rep ; 10(1): 22284, 2020 12 17.
Artículo en Inglés | MEDLINE | ID: mdl-33335272

RESUMEN

Birds, notably wild ducks, are reservoirs of pathogenic and zoonotic viruses such as influenza viruses and coronaviruses. In the current study, we used metagenomics to detect and characterise avian DNA and RNA viruses from wild Pacific black ducks, Chestnut teals and Grey teals collected at different time points from a single location. We characterised a likely new species of duck aviadenovirus and a novel duck gyrovirus. We also report what, to the best of our knowledge, is the first finding of an avian orthoreovirus from Pacific black ducks and a rotavirus F from Chestnut teals. Other viruses characterised from the samples from these wild ducks belong to the virus families Astroviridae, Caliciviridae and Coronaviridae. Some of the viruses may have potential cross-species transmissibility, while others indicated a wide genetic diversity of duck viruses within a genus. The study also showed evidence of potential transmission of viruses along the East Asian-Australasian Flyway; potentially facilitated by migrating shorebirds. The detection and characterisation of several avian viruses not previously described, and causing asymptomatic but potentially also symptomatic infections suggest the need for more virus surveillance studies for pathogenic and potential zoonotic viruses in wildlife reservoirs.


Asunto(s)
Patos/virología , Gyrovirus/genética , Virus de la Influenza A/genética , Gripe Aviar/genética , Animales , Animales Salvajes/virología , Aves/virología , Patos/genética , Gyrovirus/aislamiento & purificación , Virus de la Influenza A/aislamiento & purificación , Gripe Aviar/virología , Metagenoma/genética , Metagenómica , Filogenia
SELECCIÓN DE REFERENCIAS
DETALLE DE LA BÚSQUEDA
...