Funcionalidad biológica de la resistencia al arsénico den la bacteria hipertolerante Pseudomonas putida KT2440

Tesis doctoral inédita. Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura: 30-10-200

Unraveling the functional dark matter through global metagenomics

30 pages, 4 figures, 1 table, supplementary information https://doi.org/10.1038/s41586-023-06583-7.-- Data availability: All of the analysed datasets along with their corresponding sequences are available from the IMG system (http://img.jgi.doe.gov/). A list of the datasets used in this study is provided in Supplementary Data 8. All data from the protein clusters, including sequences, multiple alignments, HMM profiles, 3D structure models, and taxonomic and ecosystem annotation, are available through NMPFamsDB, publicly accessible at www.nmpfamsdb.org. The 3D models are also available at ModelArchive under accession code ma-nmpfamsdb.-- Code availability: Sequence analysis was performed using Tantan (https://gitlab.com/mcfrith/tantan), BLAST (https://blast.ncbi.nlm.nih.gov/Blast.cgi), LAST (https://gitlab.com/mcfrith/last), HMMER (http://hmmer.org/) and HH-suite3 (https://github.com/soedinglab/hh-suite). Clustering was performed using HipMCL (https://bitbucket.org/azadcse/hipmcl/src/master/). Additional taxonomic annotation was performed using Whokaryote (https://github.com/LottePronk/whokaryote), EukRep (https://github.com/patrickwest/EukRep), DeepVirFinder (https://github.com/jessieren/DeepVirFinder) and MMseqs2 (https://github.com/soedinglab/MMseqs2). 3D modelling was performed using AlphaFold2 (https://github.com/deepmind/alphafold) and TrRosetta2 (https://github.com/RosettaCommons/trRosetta2). Structural alignments were performed using TMalign (https://zhanggroup.org/TM-align/) and MMalign (https://zhanggroup.org/MM-align/). All custom scripts used for the generation and analysis of the data are available at Zenodo (https://doi.org/10.5281/zenodo.8097349)Metagenomes encode an enormous diversity of proteins, reflecting a multiplicity of functions and activities1,2. Exploration of this vast sequence space has been limited to a comparative analysis against reference microbial genomes and protein families derived from those genomes. Here, to examine the scale of yet untapped functional diversity beyond what is currently possible through the lens of reference genomes, we develop a computational approach to generate reference-free protein families from the sequence space in metagenomes. We analyse 26,931 metagenomes and identify 1.17 billion protein sequences longer than 35 amino acids with no similarity to any sequences from 102,491 reference genomes or the Pfam database3. Using massively parallel graph-based clustering, we group these proteins into 106,198 novel sequence clusters with more than 100 members, doubling the number of protein families obtained from the reference genomes clustered using the same approach. We annotate these families on the basis of their taxonomic, habitat, geographical and gene neighbourhood distributions and, where sufficient sequence diversity is available, predict protein three-dimensional models, revealing novel structures. Overall, our results uncover an enormously diverse functional space, highlighting the importance of further exploring the microbial functional dark matterWith the institutional support of the ‘Severo Ochoa Centre of Excellence’ accreditation (CEX2019-000928-S)Peer reviewe

Cultivation and sequencing of rumen microbiome members from the Hungate1000 Collection

Productivity of ruminant livestock depends on the rumen microbiota, which ferment indigestible plant polysaccharides into nutrients used for growth. Understanding the functions carried out by the rumen microbiota is important for reducing greenhouse gas production by ruminants and for developing biofuels from lignocellulose. We present 410 cultured bacteria and archaea, together with their reference genomes, representing every cultivated rumen-associated archaeal and bacterial family. We evaluate polysaccharide degradation, short-chain fatty acid production and methanogenesis pathways, and assign specific taxa to functions. A total of 336 organisms were present in available rumen metagenomic data sets, and 134 were present in human gut microbiome data sets. Comparison with the human microbiome revealed rumen-specific enrichment for genes encoding de novo synthesis of vitamin B12, ongoing evolution by gene loss and potential vertical inheritance of the rumen microbiome based on underrepresentation of markers of environmental stress. We estimate that our Hungate genome resource represents ?75% of the genus-level bacterial and archaeal taxa present in the rumen.publishersversionPeer reviewe

The Standard European Vector Architecture (SEVA): a coherent platform for the analysis and deployment of complex prokaryotic phenotypes

The 'Standard European Vector Architecture' database (SEVA-DB, http://seva.cnb.csic.es) was conceived as a user-friendly, web-based resource and a material clone repository to assist in the choice of optimal plasmid vectors for de-constructing and re-constructing complex prokaryotic phenotypes. The SEVA-DB adopts simple design concepts that facilitate the swapping of functional modules and the extension of genome engineering options to microorganisms beyond typical laboratory strains. Under the SEVA standard, every DNA portion of the plasmid vectors is minimized, edited for flaws in their sequence and/or functionality, and endowed with physical connectivity through three inter-segment insulators that are flanked by fixed, rare restriction sites. Such a scaffold enables the exchangeability of multiple origins of replication and diverse antibiotic selection markers to shape a frame for their further combination with a large variety of cargo modules that can be used for varied end-applications. The core collection of constructs that are available at the SEVA-DB has been produced as a starting point for the further expansion of the formatted vector platform. We argue that adoption of the SEVA format can become a shortcut to fill the phenomenal gap between the existing power of DNA synthesis and the actual engineering of predictable and efficacious bacteria

ArsH protects Pseudomonas putida from oxidative damage caused by exposure to arsenic

The two As resistance arsRBC operons of Pseudomonas putida KT2440 are followed by a downstream gene called arsH that encodes an NADPH‐dependent flavin mononucleotide reductase. In this work, we show that the arsH1 and (to a lesser extent) arsH2 genes of P. putida KT2440 strengthened its tolerance to both inorganic As(V) and As(III) and relieved the oxidative stress undergone by cells exposed to either oxyanion. Furthermore, overexpression of arsH1 and arsH2 endowed P. putida with a high tolerance to the oxidative stress caused by diamide (a drainer of metabolic NADPH) in the absence of any arsenic. To examine whether the activity of ArsH was linked to a direct action on the arsenic compounds tested, arsH1 and arsH2 genes were expressed in Escherichia coli, which has an endogenous arsRBC operon but lacks an arsH ortholog. The resulting clones both deployed a lower production of reactive oxygen species (ROS) when exposed to As salts and had a superior endurance to physiological redox insults. These results suggest that besides the claimed direct action on organoarsenicals, ArsH contributes to relieve toxicity of As species by mediating reduction of ROS produced in vivo upon exposure to the oxyanion, e.g. by generating FMNH2 to fuel ROS‐quenching activities.UCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias Básicas::Centro de Investigaciones en Productos Naturales (CIPRONA)UCR::Vicerrectoría de Docencia::Ciencias Básicas::Facultad de Ciencias::Escuela de Químic

Metagenomic compendium of 189,680 DNA viruses from the human gut microbiome.

Bacteriophages have important roles in the ecology of the human gut microbiome but are under-represented in reference databases. To address this problem, we assembled the Metagenomic Gut Virus catalogue that comprises 189,680 viral genomes from 11,810 publicly available human stool metagenomes. Over 75% of genomes represent double-stranded DNA phages that infect members of the Bacteroidia and Clostridia classes. Based on sequence clustering we identified 54,118 candidate viral species, 92% of which were not found in existing databases. The Metagenomic Gut Virus catalogue improves detection of viruses in stool metagenomes and accounts for nearly 40% of CRISPR spacers found in human gut Bacteria and Archaea. We also produced a catalogue of 459,375 viral protein clusters to explore the functional potential of the gut virome. This revealed tens of thousands of diversity-generating retroelements, which use error-prone reverse transcription to mutate target genes and may be involved in the molecular arms race between phages and their bacterial hosts

The Standard European Vector Architecture (SEVA): a coherent platform for the analysis and deployment of complex prokaryotic phenotypes.

The 'Standard European Vector Architecture' database (SEVA-DB, http://seva.cnb.csic.es) was conceived as a user-friendly, web-based resource and a material clone repository to assist in the choice of optimal plasmid vectors for de-constructing and re-constructing complex prokaryotic phenotypes. The SEVA-DB adopts simple design concepts that facilitate the swapping of functional modules and the extension of genome engineering options to microorganisms beyond typical laboratory strains. Under the SEVA standard, every DNA portion of the plasmid vectors is minimized, edited for flaws in their sequence and/or functionality, and endowed with physical connectivity through three inter-segment insulators that are flanked by fixed, rare restriction sites. Such a scaffold enables the exchangeability of multiple origins of replication and diverse antibiotic selection markers to shape a frame for their further combination with a large variety of cargo modules that can be used for varied end-applications. The core collection of constructs that are available at the SEVA-DB has been produced as a starting point for the further expansion of the formatted vector platform. We argue that adoption of the SEVA format can become a shortcut to fill the phenomenal gap between the existing power of DNA synthesis and the actual engineering of predictable and efficacious bacteria

IMG/VR v3: an integrated ecological and evolutionary framework for interrogating genomes of uncultivated viruses.

Viruses are integral components of all ecosystems and microbiomes on Earth. Through pervasive infections of their cellular hosts, viruses can reshape microbial community structure and drive global nutrient cycling. Over the past decade, viral sequences identified from genomes and metagenomes have provided an unprecedented view of viral genome diversity in nature. Since 2016, the IMG/VR database has provided access to the largest collection of viral sequences obtained from (meta)genomes. Here, we present the third version of IMG/VR, composed of 18 373 cultivated and 2 314 329 uncultivated viral genomes (UViGs), nearly tripling the total number of sequences compared to the previous version. These clustered into 935 362 viral Operational Taxonomic Units (vOTUs), including 188 930 with two or more members. UViGs in IMG/VR are now reported as single viral contigs, integrated proviruses or genome bins, and are annotated with a new standardized pipeline including genome quality estimation using CheckV, taxonomic classification reflecting the latest ICTV update, and expanded host taxonomy prediction. The new IMG/VR interface enables users to efficiently browse, search, and select UViGs based on genome features and/or sequence similarity. IMG/VR v3 is available at https://img.jgi.doe.gov/vr, and the underlying data are available to download at https://genome.jgi.doe.gov/portal/IMG_VR

Influence of the polar light cycle on seasonal dynamics of an Antarctic lake microbial community.

BackgroundCold environments dominate the Earth's biosphere and microbial activity drives ecosystem processes thereby contributing greatly to global biogeochemical cycles. Polar environments differ to all other cold environments by experiencing 24-h sunlight in summer and no sunlight in winter. The Vestfold Hills in East Antarctica contains hundreds of lakes that have evolved from a marine origin only 3000-7000 years ago. Ace Lake is a meromictic (stratified) lake from this region that has been intensively studied since the 1970s. Here, a total of 120 metagenomes representing a seasonal cycle and four summers spanning a 10-year period were analyzed to determine the effects of the polar light cycle on microbial-driven nutrient cycles.ResultsThe lake system is characterized by complex sulfur and hydrogen cycling, especially in the anoxic layers, with multiple mechanisms for the breakdown of biopolymers present throughout the water column. The two most abundant taxa are phototrophs (green sulfur bacteria and cyanobacteria) that are highly influenced by the seasonal availability of sunlight. The extent of the Chlorobium biomass thriving at the interface in summer was captured in underwater video footage. The Chlorobium abundance dropped from up to 83% in summer to 6% in winter and 1% in spring, before rebounding to high levels. Predicted Chlorobium viruses and cyanophage were also abundant, but their levels did not negatively correlate with their hosts.ConclusionOver-wintering expeditions in Antarctica are logistically challenging, meaning insight into winter processes has been inferred from limited data. Here, we found that in contrast to chemolithoautotrophic carbon fixation potential of Southern Ocean Thaumarchaeota, this marine-derived lake evolved a reliance on photosynthesis. While viruses associated with phototrophs also have high seasonal abundance, the negative impact of viral infection on host growth appeared to be limited. The microbial community as a whole appears to have developed a capacity to generate biomass and remineralize nutrients, sufficient to sustain itself between two rounds of sunlight-driven summer-activity. In addition, this unique metagenome dataset provides considerable opportunity for future interrogation of eukaryotes and their viruses, abundant uncharacterized taxa (i.e. dark matter), and for testing hypotheses about endemic species in polar aquatic ecosystems. Video Abstract

On the Origin of Reverse Transcriptase-Using CRISPR-Cas Systems and Their Hyperdiverse, Enigmatic Spacer Repertoires.

Cas1 integrase is the key enzyme of the clustered regularly interspaced short palindromic repeat (CRISPR)-Cas adaptation module that mediates acquisition of spacers derived from foreign DNA by CRISPR arrays. In diverse bacteria, the cas1 gene is fused (or adjacent) to a gene encoding a reverse transcriptase (RT) related to group II intron RTs. An RT-Cas1 fusion protein has been recently shown to enable acquisition of CRISPR spacers from RNA. Phylogenetic analysis of the CRISPR-associated RTs demonstrates monophyly of the RT-Cas1 fusion, and coevolution of the RT and Cas1 domains. Nearly all such RTs are present within type III CRISPR-Cas loci, but their phylogeny does not parallel the CRISPR-Cas type classification, indicating that RT-Cas1 is an autonomous functional module that is disseminated by horizontal gene transfer and can function with diverse type III systems. To compare the sequence pools sampled by RT-Cas1-associated and RT-lacking CRISPR-Cas systems, we obtained samples of a commercially grown cyanobacterium-Arthrospira platensis Sequencing of the CRISPR arrays uncovered a highly diverse population of spacers. Spacer diversity was particularly striking for the RT-Cas1-containing type III-B system, where no saturation was evident even with millions of sequences analyzed. In contrast, analysis of the RT-lacking type III-D system yielded a highly diverse pool but reached a point where fewer novel spacers were recovered as sequencing depth was increased. Matches could be identified for a small fraction of the non-RT-Cas1-associated spacers, and for only a single RT-Cas1-associated spacer. Thus, the principal source(s) of the spacers, particularly the hypervariable spacer repertoire of the RT-associated arrays, remains unknown.IMPORTANCE While the majority of CRISPR-Cas immune systems adapt to foreign genetic elements by capturing segments of invasive DNA, some systems carry reverse transcriptases (RTs) that enable adaptation to RNA molecules. From analysis of available bacterial sequence data, we find evidence that RT-based RNA adaptation machinery has been able to join with CRISPR-Cas immune systems in many, diverse bacterial species. To investigate whether the abilities to adapt to DNA and RNA molecules are utilized for defense against distinct classes of invaders in nature, we sequenced CRISPR arrays from samples of commercial-scale open-air cultures of Arthrospira platensis, a cyanobacterium that contains both RT-lacking and RT-containing CRISPR-Cas systems. We uncovered a diverse pool of naturally occurring immune memories, with the RT-lacking locus acquiring a number of segments matching known viral or bacterial genes, while the RT-containing locus has acquired spacers from a distinct sequence pool for which the source remains enigmatic