Benchmarking de métodos de binning a partir de un metagenoma conceptual

106 p.La metagenómica implica el estudio de comunidades microbianas enteras mediante el muestreo directo y la secuenciación de los genomas presentes en un entorno determinado. En los últimos años, la disponibilidad de secuencias metagenomicas ha crecido de manera significativa, debido principalmente al enorme impacto de las tecnologías de secuenciación de nueva generación (NGS). A diferencia del análisis clásico de un genoma, que implica la manipulación y ensamble de un número moderado de lecturas (20,000 – 30,000) largas (650-800 pb), la metagenómica tiene el reto de analizar millones de lecturas cortas (35-250 pb) de múltiples microorganismos, muchos de los cuales se desconocen. Uno de los problemas subyacentes del análisis metagenómico es la recuperación de los genomas individuales presentes en la muestra en estudio. La clasificación y el agrupamiento de lecturas o contigs de NGS en unidades taxonómicas operacionales putativos se conoce como binning. Varios métodos de bioinformática para binning se han descrito en los últimos años. Estos métodos se basan en principios diferentes (dependientes de la taxonomía e independientes de la taxonomía) y los softwares se ejecutan de forma local o a través de un servicio web. A pesar de la diversidad de métodos, no hay consenso sobre cuál es el mejor. Con esto en mente, 11 de los métodos de agrupación disponibles han sido evaluados utilizando un metagenoma conceptual construido con el software Grinder (*). Este metagenoma incluye 9 genomas procariontes completamente secuenciados y 28 genomas virales de diversa filiación taxonómica, consta de 2,5 millones de lecturas. El rendimiento de los softwares de binning se evaluó a través de cuatro criterios (el porcentaje de bases clasificadas, el porcentaje de bases correctamente clasificadas, el puntaje taxonómico y la distancia de la variación de la información). Los métodos de clasificación se evaluaron para todo el conjunto de datos, y las fracciones microbianas y virales. * //sourceforge.net/projects/biogrinder/ Se evaluaron cuarenta y siete programas de binning diferentes. De estos: 18 resultaron ser obsoletos, 16 fueron limitados en su aplicación y 13 estaban funcionales para realizar binning. De este último grupo, 2 no fueron evaluados por razones técnicas, dejando a 11 programas de binning que fueron evaluados en profundidad. Estos programas fueron clasificados de acuerdo a los criterios antes mencionados. Los tres programas de binning mejor evaluados fueron: DiScRIBinATE, MEGAN y Sort-ITEMS. El ranking completo se muestra en la Tabla 24./ABSTRACT: Metagenomics entails the study of entire microbial communities via direct sampling and sequencing of genomes present in a given environment. In recent years, the availability of metagenomic sequences has grown significantly, mainly due to the enormous impact of Next Generation Sequencing (NGS) technologies. Unlike classical genome analysis involving the handling and assembly of a moderate number (20,000 – 30,000) of long reads (650-800 bp), metagenomics is challenged to analyze millions of short reads (35-250 bp) of multiple microorganisms, many of which are unknown.One of the underlying problems of metagenomic analysis is the retrieval of the individual genomes present in the sample under study. Classification and grouping of NGS reads or contigs into putative operational taxonomic units is known as binning. Several bioinformatics methods for binning have been described in recent years. These methods are based on different principles (taxonomy dependent and taxonomy independent) and executable programs are run either locally or through a web service. Despite the diversity of methods there is no consensus on which is best.With this in mind, 11 of the available binning methods have been benchmarked using a conceptual metagenome built with Grinder (*). This metagenome includes 9 completely sequenced prokaryotic and 28 viral genomes of diverse taxonomic affiliation and consists of 2.5 million reads. The binning performance of each softwares was evaluated and ranked through four criteria (percent of classified bases, percent of correctly classified bases, taxonomic score and distance of variation of information). Best scoring methods for the whole data set, and the microbial and viral fractions are sourceforge.net/projects/biogrinder/ Fourty-seven different softwares programs for binning were evaluated. Of these: 18 were found to be obsolete, 16 were limited in their application and 13 were determined to be suitable for metagenomic binning. Of this latter group, 2 were not evaluated further for technical reasons, leaving 11 softwares programs that were evaluated in depth. These programs were ranked according to the aforementioned criteria. The top three programs according to correct binning were: DiScRIBinATE, MEGAN and SOrt-ITEMS. The complete ranking is shown in “Tabla 24”

Draft genome sequence of chloride-tolerant Leptospirillum ferriphilum Sp-Cl from industrial bioleaching operations in northern Chile

Indexación: Web of Science; PubMedLeptospirillum ferriphilum Sp-Cl is a Gram negative, thermotolerant, curved, rod- shaped bacterium, isolated from an industrial bioleaching operation in northern Chile, where chalcocite is the major copper mineral and copper hydroxychloride atacamite is present in variable proportions in the ore. This strain has unique features as compared to the other members of the species, namely resistance to elevated concentrations of chloride, sulfate and metals. Basic microbiological features and genomic properties of this biotechnologically relevant strain are described in this work. The 2,475,669 bp draft genome is arranged into 74 scaffolds of 74 contigs. A total of 48 RNA genes and 2,834 protein coding genes were predicted from its annotation; 55 % of these were assigned a putative function. Release of the genome sequence of this strain will provide further understanding of the mechanisms used by acidophilic bacteria to endure high osmotic stress and high chloride levels and of the role of chloride-tolerant iron-oxidizers in industrial bioleaching operations.https://standardsingenomics.biomedcentral.com/articles/10.1186/s40793-016-0142-

Nutrient structure dynamics and microbial communities at the water–sediment interface in an extremely acidic lake in northern Patagonia

Lake Caviahue (37° 50 ‘S and 71° 06’ W; Patagonia, Argentina) is an extreme case of a glacial, naturally acidic, aquatic environment (pH  ~  3). Knowledge of the bacterial communities in the water column of this lake, is incipient, with a basal quantification of the bacterioplankton abundance distribution in the North and South Basins of Lake Caviahue, and the described the presence of sulfur and iron oxidizing bacteria in the lake sediments. The role that bacterioplankton plays in nutrient utilization and recycling in this environment, especially in the phosphorus cycle, has not been studied. In this work, we explore this aspect in further depth by assessing the diversity of pelagic, littoral and sediment bacteria, using state of the art molecular methods and identifying the differences and commonalties in the composition of the cognate communities. Also, we investigate the interactions between the sediments of Lake Caviahue and the microbial communities present in both sediments, pore water and the water column, to comprehend the ecological relationships driving nutrient structure and fluxes, with a special focus on carbon, nitrogen, and phosphorus. Two major environmental patterns were observed: (a) one distinguishing the surface water samples due to temperature, Fe2+, and electrical conductivity, and (b) another distinguishing winter and summer samples due to the high pH and increasing concentrations of N-NH4 +, DOC and SO4 2−, from autumn and spring samples with high soluble reactive phosphorus (SRP) and iron concentrations. The largest bacterial abundance was found in autumn, alongside higher levels of dissolved phosphorus, iron forms, and increased conductivity. The highest values of bacterial biomass were found in the bottom strata of the lake, which is also where the greatest diversity in microbial communities was found. The experiments using continuous flow column microcosms showed that microbial growth over time, in both the test and control columns, was accompanied by a decrease in the concentration of dissolved nutrients (SRP and N-NH4 +), providing proof that sediment microorganisms are active and contribute significantly to nutrient utilization/mobilizationFil: Cuevas, Mayra. Universidad Nacional del Comahue. Centro Regional Universitario Bariloche; Argentina.Fil: Cuevas, Mayra. Consejo Nacional de Investigaciones Científicas y técnicas. Instituto de Investigaciones en Biodiversidad y Medioambiente; Argentina.Fil: Issotta, Francisco. Centro Científico y Tecnológico de Excelencia Ciencia & Vida. Fundación Ciencia & Vida; Chile.Fil: Issotta, Francisco. Universidad Católica de Chile. Department of Molecular Genetics and Microbiology. School of Biological Sciences; Chile.Fil: Díaz-González, Fernando. Centro Científico y Tecnológico de Excelencia Ciencia & Vida. Fundación Ciencia & Vida; Chile.Fil: Díaz-González, Fernando. Universidad San Sebastián. Facultad de Medicina y Ciencia; Chile.Fil: Diaz, Mónica. Universidad Nacional del Comahue. Centro Regional Universitario Bariloche; Argentina.Fil: Diaz, Mónica. Consejo Nacional de Investigaciones Científicas y técnicas. Instituto de Investigaciones en Biodiversidad y Medioambiente; Argentina.Fil: Quatrini, Raquel. Centro Científico y Tecnológico de Excelencia Ciencia & Vida. Fundación Ciencia & Vida; Chile.Fil: Quatrini, Raquel. Universidad San Sebastián. Facultad de Medicina y Ciencia; Chile.Fil: Beamud, Guadalupe. Universidad Nacional del Comahue. Centro Regional Universitario Bariloche; Argentina.Fil: Beamud, Guadalupe. Consejo Nacional de Investigaciones Científicas y técnicas. Instituto de Investigaciones en Biodiversidad y Medioambiente; Argentina.Fil: Fernando Pedrozo. Universidad Nacional del Comahue. Centro Regional Universitario Bariloche; Argentina.Fil: Fernando Pedrozo. Consejo Nacional de Investigaciones Científicas y técnicas. Instituto de Investigaciones en Biodiversidad y Medioambiente; Argentina.Fil: Temporett, Pedro. Universidad Nacional del Comahue. Centro Regional Universitario Bariloche; Argentina.Fil: Temporett, Pedro. Consejo Nacional de Investigaciones Científicas y técnicas. Instituto de Investigaciones en Biodiversidad y Medioambiente; Argentina

Multi-omics Reveals the Lifestyle of the Acidophilic, Mineral-Oxidizing Model Species Leptospirillum ferriphilumT.

Leptospirillum ferriphilum plays a major role in acidic, metal-rich environments, where it represents one of the most prevalent iron oxidizers. These milieus include acid rock and mine drainage as well as biomining operations. Despite its perceived importance, no complete genome sequence of the type strain of this model species is available, limiting the possibilities to investigate the strategies and adaptations that Leptospirillum ferriphilum DSM 14647T (here referred to as Leptospirillum ferriphilum T) applies to survive and compete in its niche. This study presents a complete, circular genome of Leptospirillum ferriphilum T obtained by PacBio single-molecule real-time (SMRT) long-read sequencing for use as a high-quality reference. Analysis of the functionally annotated genome, mRNA transcripts, and protein concentrations revealed a previously undiscovered nitrogenase cluster for atmospheric nitrogen fixation and elucidated metabolic systems taking part in energy conservation, carbon fixation, pH homeostasis, heavy metal tolerance, the oxidative stress response, chemotaxis and motility, quorum sensing, and biofilm formation. Additionally, mRNA transcript counts and protein concentrations were compared between cells grown in continuous culture using ferrous iron as the substrate and those grown in bioleaching cultures containing chalcopyrite (CuFeS2). Adaptations of Leptospirillum ferriphilum T to growth on chalcopyrite included the possibly enhanced production of reducing power, reduced carbon dioxide fixation, as well as elevated levels of RNA transcripts and proteins involved in heavy metal resistance, with special emphasis on copper efflux systems. Finally, the expression and translation of genes responsible for chemotaxis and motility were enhanced.IMPORTANCE Leptospirillum ferriphilum is one of the most important iron oxidizers in the context of acidic and metal-rich environments during moderately thermophilic biomining. A high-quality circular genome of Leptospirillum ferriphilum T coupled with functional omics data provides new insights into its metabolic properties, such as the novel identification of genes for atmospheric nitrogen fixation, and represents an essential step for further accurate proteomic and transcriptomic investigation of this acidophile model species in the future. Additionally, light is shed on adaptation strategies of Leptospirillum ferriphilum T for growth on the copper mineral chalcopyrite. These data can be applied to deepen our understanding and optimization of bioleaching and biooxidation, techniques that present sustainable and environmentally friendly alternatives to many traditional methods for metal extraction

Genomic evolution of the class Acidithiobacillia:deep-branching Proteobacteria living in extreme acidic conditions

Funding Information: Acknowledgements This work was supported the Agencia Nacional de Investigación y Desarrollo under Grants FONDECYT 1181251 (R.Q.), Programa de Apoyo a Centros con Financiamiento Basal AFB170004 (R.Q.), CONICYT-PFCHA/Doctorado Nacional/ 20171049 (A.M.B.), CONICYT-PFCHA/Doctorado Nacional/ 21160871 (F.I.), the ANID—Millennium Science Initiative Program— NCN17_093, granted by the Ministry of Economy, Development and Tourism from Chile (R.Q.), and Fundación Ciencia and Vida Hinge PostDoc Program (S.B.). Mónica Gonzales, Paulo Covarrubias and Hector Carrasco provided assistance with microbiological and molecular biology analyses at early stages of this study. Douglas Rawlings, Violaine Bonnefoy, Patricia Chiacharini, Jiri Kucera, Martin Mandl, Francisco Remonsellez, Pablo Ramirez Roca, and Mario Vera shared strains used in this study. We are grateful to Professor Aharon Oren (Hebrew University of Jerusalem Israel) for his help and advice on the projected names of novel bacterial genera and species. The authors thank the authorities of the Provincial Thermal Baths Agency (EPROTEN) and the Directorate of Protected Natural Areas (ANP) of the province of Neuquén Argentina for allowing access and sampling in the Copahue-Caviahue Provincial Park and Ing. Rubén Vargas (alias Caniche) for guidance during ascent to the Copahue volcano. Publisher Copyright: © 2021, The Author(s).Members of the genus Acidithiobacillus, now ranked within the class Acidithiobacillia, are model bacteria for the study of chemolithotrophic energy conversion under extreme conditions. Knowledge of the genomic and taxonomic diversity of Acidithiobacillia is still limited. Here, we present a systematic analysis of nearly 100 genomes from the class sampled from a wide range of habitats. Some of these genomes are new and others have been reclassified on the basis of advanced genomic analysis, thus defining 19 Acidithiobacillia lineages ranking at different taxonomic levels. This work provides the most comprehensive classification and pangenomic analysis of this deep-branching class of Proteobacteria to date. The phylogenomic framework obtained illuminates not only the evolutionary past of this lineage, but also the molecular evolution of relevant aerobic respiratory proteins, namely the cytochrome bo3 ubiquinol oxidases

Molecular systematics of the genus Acidithiobacillus:insights into the phylogenetic structure and diversification of the taxon

The acidithiobacilli are sulfur-oxidizing acidophilic bacteria that thrive in both natural and anthropogenic low pH environments. They contribute to processes that lead to the generation of acid rock drainage in several different geoclimatic contexts, and their properties have long been harnessed for the biotechnological processing of minerals. Presently, the genus is composed of seven validated species, described between 1922 and 2015: Acidithiobacillus thiooxidans, A. ferrooxidans, A. albertensis, A. caldus, A. ferrivorans, A. ferridurans, and A. ferriphilus. However, a large number of Acidithiobacillus strains and sequence clones have been obtained from a variety of ecological niches over the years, and many isolates are thought to vary in phenotypic properties and cognate genetic traits. Moreover, many isolates remain unclassified and several conflicting specific assignments muddle the picture from an evolutionary standpoint. Here we revise the phylogenetic relationships within this species complex and determine the phylogenetic species boundaries using three different typing approaches with varying degrees of resolution: 16S rRNA gene-based ribotyping, oligotyping, and multi-locus sequencing analysis (MLSA). To this end, the 580 16S rRNA gene sequences affiliated to the Acidithiobacillus spp. were collected from public and private databases and subjected to a comprehensive phylogenetic analysis. Oligotyping was used to profile high-entropy nucleotide positions and resolve meaningful differences between closely related strains at the 16S rRNA gene level. Due to its greater discriminatory power, MLSA was used as a proxy for genome-wide divergence in a smaller but representative set of strains. Results obtained indicate that there is still considerable unexplored diversity within this genus. At least six new lineages or phylotypes, supported by the different methods used herein, are evident within the Acidithiobacillus species complex. Although the diagnostic characteristics of these subgroups of strains are as yet unresolved, correlations to specific metadata hint to the mechanisms behind econiche-driven divergence of some of the species/phylotypes identified. The emerging phylogenetic structure for the genus outlined in this study can be used to guide isolate selection for future population genomics and evolutionary studies in this important acidophile model

Draft genome sequence of the type strain of the sulfur-oxidizing acidophile, Acidithiobacillus albertensis (DSM 14366)

Abstract Acidithiobacillus albertensis is an extremely acidophilic, mesophilic, obligatory autotrophic sulfur-oxidizer, with potential importance in the bioleaching of sulfidic metal ores, first described in the 1980s. Here we present the draft genome sequence of Acidithiobacillus albertensis DSM 14366T, thereby both filling a long-standing gap in the genomics of the acidithiobacilli, and providing further insight into the understanding of the biology of the non iron-oxidizing members of the Acidithiobacillus genus. The assembled genome is 3,1 Mb, and contains 47 tRNAs, tmRNA gene and 2 rRNA operons, along with 3149 protein-coding predicted genes. The Whole Genome Shotgun project was deposited in DDBJ/EMBL/GenBank under the accession MOAD00000000

Acidithiobacillia class members originating at sites within the Pacific Ring of Fire and other tectonically active locations and description of the novel genus ‘ Igneacidithiobacillus’

Recent studies have expanded the genomic contours of the Acidithiobacillia, highlighting important lacunae in our comprehension of the phylogenetic space occupied by certain lineages of the class. One such lineage is ‘Igneacidithiobacillus’, a novel genus-level taxon, represented by ‘Igneacidithiobacillus copahuensis’ VAN18-1T as its type species, along with two other uncultivated metagenome-assembled genomes (MAGs) originating from geothermally active sites across the Pacific Ring of Fire. In this study, we investigate the genetic and genomic diversity, and the distribution patterns of several uncharacterized Acidithiobacillia class strains and sequence clones, which are ascribed to the same 16S rRNA gene sequence clade. By digging deeper into this data and contributing to novel MAGs emerging from environmental studies in tectonically active locations, the description of this novel genus has been consolidated. Using state-of-the-art genomic taxonomy methods, we added to already recognized taxa, an additional four novel Candidate (Ca.) species, including ‘Ca. Igneacidithiobacillus chanchocoensis’ (mCHCt20-1TS), ‘Igneacidithiobacillus siniensis’ (S30A2T), ‘Ca. Igneacidithiobacillus taupoensis’ (TVZ-G3 TS), and ‘Ca. Igneacidithiobacillus waiarikiensis’ (TVZ-G4 TS). Analysis of published data on the isolation, enrichment, cultivation, and preliminary microbiological characterization of several of these unassigned or misassigned strains, along with the type species of the genus, plus the recoverable environmental data from metagenomic studies, allowed us to identify habitat preferences of these taxa. Commonalities and lineage-specific adaptations of the seven species of the genus were derived from pangenome analysis and comparative genomic metabolic reconstruction. The findings emerging from this study lay the groundwork for further research on the ecology, evolution, and biotechnological potential of the novel genus ‘Igneacidithiobacillus’

Multiple osmotic stress responses in acidihalobacter prosperus result in tolerance to chloride ions

Extremely acidophilic microorganisms (pH optima for growth of =3) are utilized for the extraction of metals from sulfide minerals in the industrial biotechnology of "biomining." A long term goal for biomining has been development of microbial consortia able to withstand increased chloride concentrations for use in regions where freshwater is scarce. However, when challenged by elevated salt, acidophiles experience both osmotic stress and an acidification of the cytoplasm due to a collapse of the inside positive membrane potential, leading to an influx of protons. In this study, we tested the ability of the halotolerant acidophile Acidihalobacter prosperus to grow and catalyze sulfide mineral dissolution in elevated concentrations of salt and identified chloride tolerance mechanisms in Ac. prosperus as well as the chloride susceptible species, Acidithiobacillus ferrooxidans. Ac. prosperus had optimum iron oxidation at 20 g L-1 NaCl while At. ferrooxidans iron oxidation was inhibited in the presence of 6 g L-1 NaCl. The tolerance to chloride in Ac. prosperus was consistent with electron microscopy, determination of cell viability, and bioleaching capability. The Ac. prosperus proteomic response to elevated chloride concentrations included the production of osmotic stress regulators that potentially induced production of the compatible solute, ectoine uptake protein, and increased iron oxidation resulting in heightened electron flow to drive proton export by the F0F1 ATPase. In contrast, At. ferrooxidans responded to low levels of Cl- with a generalized stress response, decreased iron oxidation, and an increase in central carbon metabolism. One potential adaptation to high chloride in the Ac. prosperus Rus protein involved in ferrous iron oxidation was an increase in the negativity of the surface potential of Rus Form I (and Form II) that could help explain how it can be active under elevated chloride concentrations. These data have been used to create a model of chloride tolerance in the salt tolerant and susceptible species Ac. prosperus and At. ferrooxidans, respectively. © 2017 The Authors