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Microbial activity is a major contributor to the biogeochemical cycles that make up the life support system of planet Earth. A 613 m deep geomicrobiological perforation and a systematic multi-analytical characterization revealed an unexpected diversity associated with the rock matrix microbiome that operates in the subsurface of the Iberian Pyrite Belt (IPB). Members of 1 class and 16 genera were deemed the most representative microorganisms of the IPB deep subsurface and selected for a deeper analysis. The use of fluorescence in situ hybridization allowed not only the identification of microorganisms but also the detection of novel activities in the subsurface such as anaerobic ammonium oxidation (ANAMMOX) and anaerobic methane oxidation, the co-occurrence of microorganisms able to maintain complementary metabolic activities and the existence of biofilms. The use of enrichment cultures sensed the presence of five different complementary metabolic activities along the length of the borehole and isolated 29 bacterial species. Genomic analysis of nine isolates identified the genes involved in the complete operation of the light-independent coupled C, H, N, S and Fe biogeochemical cycles. This study revealed the importance of nitrate reduction microorganisms in the oxidation of iron in the anoxic conditions existing in the subsurface of the IPB, FP7 Ideas: European Research Council, Grant/Award Number: ERC Advanced Grant #250-350

Microbial communities are complex and dynamic entities, and their structure arises from the interplay of a multitude of factors, including the interactions of microorganisms with each other and with the environment. Since each extant community has a unique eco-evolutionary history, it might appear that contingency rather than general rules govern their assembly. In spite of this, there is evidence that some general assembly principles exist, at least to a certain extent. In this work, we sought to identify those principles by performing a cross-study, cross-biome meta-analysis of microbial occurrence data in more than 5,000 samples from ten different environmental groups. We adopted a novel algorithm that allows the same taxa to aggregate with different partners in different habitats, capturing the complexity of interactions inherent to natural microbial communities. We tried to decouple function from phylogeny, the environment, and genome size, in order to provide an unbiased characterization of phylogenetic and functional redundancy in environmental microbial assemblages. We then examined the phylogenetic and functional composition of the resulting inferred communities, and searched for global patterns of assembly both at the community level and in individual metabolic pathways.Our analysis of the resulting microbial assemblages highlighted that environmental communities are more functionally redundant than expected by chance. This effect is greater for communities appearing in more than one environment, suggesting a link between functional redundancy and environmental adaptation. In spite of this, certain pathways are observed in fewer taxa than expected by chance, suggesting the presence of auxotrophy, and presumably cooperation among community members, which is supported by our analysis of amino acid biosynthesis pathways. Furthermore, this hypothetical cooperation may play a role in genome reduction, since we observed a negative relationship between the size of bacterial genomes and the number of taxa of the community they belong to.Overall, our results provide a global characterization of environmental microbial communities, and offer design principles for engineering robust bacterial communities.

Recent years have witnessed the detection of an increasing number of complex organic molecules in interstellar space, some of them being of prebiotic interest. Disentangling the origin of interstellar prebiotic chemistry and its connection to biochemistry and ultimately to biology is an enormously challenging scientific goal where the application of complexity theory and network science has not been fully exploited. Encouraged by this idea, we present a theoretical and computational framework to model the evolution of simple networked structures toward complexity. In our environment, complex networks represent simplified chemical compounds, and interact optimizing the dynamical importance of their nodes. We describe the emergence of a transition from simple networks toward complexity when the parameter representing the environment reaches a critical value. Notably, although our system does not attempt to model the rules of real chemistry, nor is dependent on external input data, the results describe the emergence of complexity in the evolution of chemical diversity in the interstellar medium. Furthermore, they reveal an as yet unknown relationship between the abundances of molecules in dark clouds and the potential number of chemical reactions that yield them as products, supporting the ability of the conceptual framework presented here to shed light on real scenarios. Our work reinforces the notion that some of the properties that condition the extremely complex journey from the chemistry in space to prebiotic chemistry and finally to life could show relatively simple and universal patterns., Comment: Published in PNAS. The Supplementary Information can be found at https://github.com/MiguelGarciaSanchez/NetWorld/blob/main/garcia_sanchez_et_al_Supp_Info_2022.pdf

15 pages, 4 figures, 3 tables, supporting Information https://doi.org/10.1111/1755-0998.13687.-- Data availability statement: All metagenomes analysed in this study are in the public domain as described in Table 2, Mercury (Hg) methylation genes (hgcAB) mediate the formation of the toxic methylmercury and have been identified from diverse environments, including freshwater and marine ecosystems, Arctic permafrost, forest and paddy soils, coal-ash amended sediments, chlor-alkali plants discharges and geothermal springs. Here we present the first attempt at a standardized protocol for the detection, identification and quantification of hgc genes from metagenomes. Our Hg-cycling microorganisms in aquatic and terrestrial ecosystems (Hg-MATE) database, a catalogue of hgc genes, provides the most accurate information to date on the taxonomic identity and functional/metabolic attributes of microorganisms responsible for Hg methylation in the environment. Furthermore, we introduce “marky-coco”, a ready-to-use bioinformatic pipeline based on de novo single-metagenome assembly, for easy and accurate characterization of hgc genes from environmental samples. We compared the recovery of hgc genes from environmental metagenomes using the marky-coco pipeline with an approach based on coassembly of multiple metagenomes. Our data show similar efficiency in both approaches for most environments except those with high diversity (i.e., paddy soils) for which a coassembly approach was preferred. Finally, we discuss the definition of true hgc genes and methods to normalize hgc gene counts from metagenomes, This work was funded by the Severo Ochoa Excellence Programme postdoctoral fellowship awarded in 2021 to Eric Capo (CEX2019-000928-S), the Swedish Research Council Formas (grant 2018-01031), the EMFF-Blue Economy project MER-CLUB (grant 863584). [...]. Benjamin Peterson was funded as a postdoctoral research associate by the National Science Foundation (award 1935173) during the work in this manuscript.[...]. Oak Ridge National Laboratory is managed by UT-Battelle, LLC under contract no. DE-AC05-00OR22725 with the U.S. Department of Energy (DOE)

Phytochelatins (PCs) are cysteine-rich small peptides, enzymatically synthesized from reduced glutathione (GSH) by cytosolic enzyme phytochelatin synthase (PCS). The open reading frame (ORF) of the

At the genome level, microorganisms are highly adaptable both in terms of allele and gene composition. Such heritable traits emerge in response to different environmental niches and can have a profound influence on microbial community dynamics. As a consequence of this, any individual genome or clonal population will contain merely a fraction of the total genetic diversity of any operationally defined “species”, with the collective from that group presenting the broader genomic diversity known as the pangenome. Pangenomes are valuable concepts for studying evolution and adaptation in microorganisms, as they partition genomes into core regions (present in all the genomes, and responsible for housekeeping and species-level niche adaptation) and accessory regions (present only in some genomes, and responsible for ecotype divergence).Here we present SuperPang, an algorithm capable of producing pangenome assemblies from a set of input genomes of varying quality, including metagenome-assembled genomes or MAGs. SuperPang runs in linear time and its results are complete, non-redundant, preserve gene ordering and contain both coding and non-coding regions. Our approach provides a modular view of the pangenome, identifying operons and genomic islands, and allowing to track their prevalence in different populations. We illustrate our approach by analyzing the intra-species diversity of Polynucleobacter, a clade of ubiquitous freshwater microorganisms characterized by their streamlined genomes and their ecological versatility. We show how SuperPang facilitates the simultaneous analysis of allelic and gene content variation under different environmental pressures, allowing us to study the drivers of microbial diversification at unprecedented resolution.

Microbial assemblages under sea ice of Dease Strait, Canadian Arctic, were sequenced for metagenomes and metatranscriptomes of the small size fraction (0.2 to 3 µm). The community from early March was typical for this season, with Alpha- and Gammaproteobacteria as the dominant taxa, followed by Thaumarchaeota and Bacteroidetes. Towards summer, Bacteroidetes and particularly the genus Polaribacter, became increasingly dominant, followed by the Gammaproteobacteria. Analysis of genes responsible for microbial acquisition of iron showed an abundance of ABC transporters for divalent cations and ferrous iron. The most abundant transporters, however, were the outer membrane TonB dependent transporters of iron-siderophore complexes. The abundance of iron acquisition genes suggested this element was essential for the microbial assemblage. Interestingly, Gammaproteobacteria were responsible for most of the siderophore synthesis genes. On the contrary, Bacteroidetes did not synthesize siderophores but accounted for most of the transporters, suggesting a role as cheaters in the competition for siderophores as public goods. Likely, this cheating ability of the Bacteroidetes contributed to their dominance in summer.

14 Pág. Departamento de Mejora Genetica Animal, Mitigating the effects of global warming has become the main challenge for humanity in recent decades. Livestock farming contributes to greenhouse gas emissions, with an important output of methane from enteric fermentation processes, mostly in ruminants. Because ruminal microbiota is directly involved in digestive fermentation processes and methane biosynthesis, understanding the ecological relationships between rumen microorganisms and their active metabolic pathways is essential for reducing emissions. This study analysed whole rumen metagenome using long reads and considering its compositional nature in order to disentangle the role of rumen microbes in methane emissions., This research was financed by RTA2015-00022-C03-02 (METALGEN) project from the National Plan of Research, Development and Innovation 2013–2020 and the Department of Economic Development and Competitiveness (Madrid, Spain). A.L.G. was funded by FPI-INIA grant with reference FPI-SGIT2016-06.

17 Pág. Departamento de ​Mejora Genética Animal (INIA), The rumen is a complex microbial system of substantial importance in terms of greenhouse gas emissions and feed efficiency. This study proposes combining metagenomic and host genomic data for selective breeding of the cow hologenome toward reduced methane emissions. We analyzed nanopore long reads from the rumen metagenome of 437 Holstein cows from 14 commercial herds in 4 northern regions in Spain. After filtering, data were treated as compositional. The large complexity of the rumen microbiota was aggregated, through principal component analysis (PCA), into few principal components (PC) that were used as proxies of the core metagenome. The PCA allowed us to condense the huge and fuzzy taxonomical and functional information from the metagenome into a few PC. Bivariate animal models were applied using these PC and methane production as phenotypes. The variability condensed in these PC is controlled by the cow genome, with heritability estimates for the first PC of ~0.30 at all taxonomic levels, with a large probability (>83%) of the posterior distribution being >0.20 and with the 95% highest posterior density interval (95%HPD) not containing zero. Most genetic correlation estimates between PC1 and methane were large (≥0.70), with most of the posterior distribution (>82%) being >0.50 and with its 95%HPD not containing zero. Enteric methane production was positively associated with relative abundance of eukaryotes (protozoa and fungi) through the first component of the PCA at phylum, class, order, family, and genus. Nanopore long reads allowed the characterization of the core rumen metagenome using whole-metagenome sequencing, and the purposed aggregated variables could be used in animal breeding programs to reduce methane emissions in future generations., This research was financed by the METALGEN project (RTA2015-00022-C03) from the national plan for research, development, and innovation 2013–2020 and the Department of Economic Development and Competitiveness (Madrid, Spain).

Summary Advances in sequencing technologies have triggered the development of many bioinformatic tools aimed to analyze 16S rDNA sequencing data. As these tools need to be tested, it is important to simulate datasets that resemble samples from different environments. Here, we introduce M&Ms, a user-friendly open-source bioinformatic tool to produce different 16S rDNA datasets from reference sequences, based on pragmatic ecological parameters. It creates sequence libraries for ‘in silico’ microbial communities with user-controlled richness, evenness, microdiversity and source environment. M&Ms allows the user to generate simple to complex read datasets based on real parameters that can be used in developing bioinformatic software or in benchmarking current tools. Availability and implementation The source code of M&Ms is freely available at https://github.com/ggnatalia/MMs (GPL-3.0 License). Supplementary information Supplementary data are available at Bioinformatics online.

MotivationAdvances in sequencing technologies have triggered the development of many bioinformatic tools aimed to analyze these data. As these tools need to be tested, it is important to simulate datasets that resemble realistic conditions. Although there is a large amount of software dedicated to produce reads from ‘in silico’ microbial communities, often the simulated data diverge widely from real situations.ResultsHere, we introduce M&Ms, a user-friendly open-source bioinformatic tool to produce realistic amplicon datasets from reference sequences, based on pragmatic ecological parameters. This tool creates sequence libraries for ‘in silico’ microbial communities with user-controlled richness, evenness, microdiversity, and source environment. M&Ms allows the user to generate simple to complex read datasets based on real parameters that can be used in developing bioinformatic software or in benchmarking current tools. M&Ms also provides additional figures and files with extensive details on how each synthetic community is composed, so that users can make informed choices when designing their benchmarking pipelines.AvailabilityThe source code of M&Ms is freely available from https://github.com/ggnatalia/MMsContactngarcia@cnb.csic.es

In this work, we characterize the geochemistry and microbial community of Bajo las Peñas, a neutral (pH 6.5-7.4), hot spring (T = 62.0-68.0°C) located near Turrialba Volcano, Costa Rica. The microbiota at its two sources belongs mainly to the family Aquificae, comprising OTUs closely related to the genera Sulfurihydrogenibium, Thermosulfidibacter, Thermodesulfovibrio and Thermocrinis which is consistent with the presence of moderate levels of sulfate (243-284 mg/L) along the stream. We determined a dramatic shift in the microbial community just a few meters downstream of the sources of the hot spring (15-20 meters), with a change from sulfur related chemoautotrophic (e.g. Sulfurihydrogenibium and an OTU closely related to Thermodesulfovibrio) to chemoheterotrophic prokaryotes (e.g. Meiothermus, Nitrososphaera, Thermoflexus, Thermus). Thus, in this neutral hot spring, the first level of the trophic chain is associated with photosynthesis as well other anaerobic CO2 fixing bacteria. Then, thermotolerant chemoheterotrophic bacteria colonize the environment to degrade organic matter and use fermentative products from the first level of the trophic chain. Our data demonstrate how quickly the microbial community of an ecosystem can change in response to environmental variables and sheds light on the microbial ecology of less common circumneutral pH hot springs. Universidad de Costa Rica/[809-B6-524]/UCR/Costa Rica Ministerio de Ciencia, Tecnología y Telecomunicaciones/[FI-255B-17]/MICITT/Costa Rica Ministerio Federal de Educación e Investigación/[FI-255B-17]/BMBF/Alemania European Research Council/[ERC250350-IPBSL]/ERC/Unión Europea Ministerio de Economía y Competitividad/[CTM2016-80095-C2-1-R]/MINECO/España 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 Química UCR::Vicerrectoría de Docencia::Ciencias Básicas::Facultad de Ciencias::Escuela Centroamericana de Geología UCR::Vicerrectoría de Docencia::Ciencias Básicas::Facultad de Ciencias::Escuela de Biología

Aquatic environments of volcanic origin provide an exceptional opportunity to study the adaptations of microorganisms to early planet life conditions. Here, we characterized the prokaryotic communities and physicochemical properties of seepage sites at the bottom of the Poas Volcano crater and the Agrio River, two geologically related extremely acidic environments located in Costa Rica. Both locations hold a low pH (1.79-2.20) and have high sulfate and iron concentrations (Fe = 47-206 mg/L, SO

Aquatic environments of volcanic origin provide an exceptional opportunity to study the adaptations of microbial communities to early planet life conditions such as high temperatures, high metal concentrations, and low pH. Here, we characterized the prokaryotic communities and physicochemical properties of seepage sites at the bottom of the Poas Volcano crater and the Agrio River, two geologically related extremely acidic environments located in the Central Volcanic mountain range of Costa Rica. Both locations hold a very low pH (pH 1.79-2.20) and have high sulfate and iron concentrations (Fe = 47-206 mg/L, SO42- = 1170-2460 mg/L measured as S), but significant differences in their temperature (90.0–95.0°C in the seepages at Poas Volcano versus 19.1–26.6 °C in Agrio River) and in the abundance of elemental sulfur. Based on the analysis of 16S rRNA gene sequences, we determined that Sulfobacillus spp., sulfur-oxidizing bacteria, represented more than half (58.4–78.4%) of the sequences in Poas Volcano seepage sites, while Agrio River was dominated by the iron- and sulfur-oxidizing Leptospirillum (7.4–55.5%) and members of the archeal order Thermoplasmatales (16.0-58.2%). Both environments share some chemical characteristics and part of their microbiota, however the temperature and the presence of reduced sulfur are likely the main distinguishing feature ultimately shaping their microbial communities. Our data suggest that in the Poas Volcano-Agrio River system there is a common metabolism but with specialization of species that adapt to the physicochemical conditions of each environment. Universidad de Costa Rica/[809-B6-524]/UCR/Costa Rica Ministerio Federal de Educación e Investigación/[FI-255B-17]/BMBF/Alemania Ministerio de Ciencia, Tecnología y Telecomunicaciones/[FI-255B-17]/MICITT/Costa Rica European Research Council/[ERC250350-IPBSL]/ERC/Unión Europea 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 Química UCR::Vicerrectoría de Docencia::Ciencias Básicas::Facultad de Ciencias::Escuela Centroamericana de Geología UCR::Vicerrectoría de Docencia::Ciencias Básicas::Facultad de Ciencias::Escuela de Biología UCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias Básicas::Centro de Investigación en Electroquímica y Energía Química (CELEQ)

We describe the geochemistry and microbial diversity of a pristine environment that resembles an acid rock drainage (ARD) but it is actually the result of hydrothermal and volcanic influences. We designate this environment, and other comparable sites, as volcanic influenced acid rock drainage (VARD) systems. The metal content and sulfuric acid in this ecosystem stem from the volcanic milieu and not from the product of pyrite oxidation. Based on the analysis of 16S rRNA gene amplicons, we report the microbial community structure in the pristine San Cayetano Costa Rican VARD environment (pH = 2.94–3.06, sulfate ~ 0.87–1.19 g L−1, iron ~ 35–61 mg L−1 (waters), and ~ 8–293 g kg−1 (sediments)). San Cayetano was found to be dominated by microorganisms involved in the geochemical cycling of iron, sulfur, and nitrogen; however, the identity and abundance of the species changed with the oxygen content (0.40–6.06 mg L−1) along the river course. The hypoxic source of San Cayetano is dominated by a putative anaerobic sulfate-reducing Deltaproteobacterium. Sulfur-oxidizing bacteria such as Acidithiobacillus or Sulfobacillus are found in smaller proportions with respect to typical ARD. In the oxic downstream, we identified aerobic iron-oxidizers (Leptospirillum, Acidithrix, Ferrovum) and heterotrophic bacteria (Burkholderiaceae bacterium, Trichococcus, Acidocella). Thermoplasmatales archaea closely related to environmental phylotypes found in other ARD niches were also observed throughout the entire ecosystem. Overall, our study shows the differences and similarities in the diversity and distribution of the microbial communities between an ARD and a VARD system at the source and along the oxygen gradient that establishes on the course of the river., This work was supported by The Vice-rectory of Research of Universidad de Costa Rica (project number VI 809-B6-524), the Costa Rican Ministry of Science, Technology and Telecommunication (MICITT) and Federal Ministry of Education and Research (BMBF) (project VolcanZyme contract No FI-255B-17), and the ERC grant IPBSL (ERC250350-IPBSL). MM acknowledges government funding from the Transitorio I of the National Law 8488 for Emergencies and Risk Prevention in Costa Rica. F.P-S. is supported by grant IJC2018-035180-I from the Spanish Ministry of Science and Innovation.

© The Author(s) 2020., We have analyzed the bacterial community of a large Saharan dust event in the Iberian Peninsula and, for the first time, we offer new insights regarding the bacterial distribution at different altitudes of the lower troposphere and the replacement of the microbial airborne structure as the dust event receeds. Samples from different open-air altitudes (surface, 100 m and 3 km), were obtained onboard the National Institute for Aerospace Technology (INTA) C-212 aircrafts. Samples were collected during dust and dust-free air masses as well two weeks after the dust event. Samples related in height or time scale seems to show more similar community composition patterns compared with unrelated samples. The most abundant bacterial species during the dust event, grouped in three different phyla: (a) Proteobacteria: Rhizobiales, Sphingomonadales, Rhodobacterales, (b) Actinobacteria: Geodermatophilaceae; (c) Firmicutes: Bacillaceae. Most of these taxa are well known for being extremely stress-resistant. After the dust intrusion, Rhizobium was the most abundant genus, (40–90% total sequences). Samples taken during the flights carried out 15 days after the dust event were much more similar to the dust event samples compared with the remaining samples. In this case, Brevundimonas, and Methylobacterium as well as Cupriavidus and Mesorizobium were the most abundant genera., This work was funded by the Spanish Economy and Competitiveness Ministry (MINECO) grants CGL2015-69758-P, CGL2017-92086-EXP, ESP2016-79612-C3-1-R, RTI2018-094867-BI00 and i-LINK 1151 grant founded by the Council for Scientific Research (CSIC). FPS was supported by the MINECO grant CTM2016-80095-C2-1-R.

Cyanobacteria are ecologically versatile microorganisms inhabiting most environments, ranging from marine systems to arid deserts. Although they possess several pathways for light-independent energy generation, until now their ecological range appeared to be restricted to environments with at least occasional exposure to sunlight. Here we present molecular, microscopic, and metagenomic evidence that cyanobacteria predominate in deep subsurface rock samples from the Iberian Pyrite Belt Mars analog (southwestern Spain). Metagenomics showed the potential for a hydrogen-based lithoautotrophic cyanobacterial metabolism. Collectively, our results suggest that they may play an important role as primary producers within the deep-Earth biosphere. Our description of this previously unknown ecological niche for cyanobacteria paves the way for models on their origin and evolution, as well as on their potential presence in current or primitive biospheres in other planetary bodies, and on the extant, primitive, and putative extraterrestrial biospheres., This work was supported by the European Research Council Advanced Grant ERC250350-IPBSL, and the Spanish Ministerio de Economía y Competitividad/Fondo Europeo de Desarrollo Regional Grants AYA2011-24803 and ESP2015-69540-R. F.P.-S. is currently funded by the Spanish Ministerio de Economía y Competitividad/Fondo Europeo de Desarrollo Regional Grant CTM2016-80095-C2-1-R.

Oligotrophic glacial lakes in the Andes Mountains serve as models to study the effects of climate change on natural biological systems. The persistent high UV regime and evolution of the lake biota due to deglaciation make Andean lake ecosystems potential analogues in the search for life on other planetary bodies. Our objective was to identify microbial biomarkers and metabolic patterns that represent time points in the evolutionary history of Andean glacial lakes, as these may be used in long-term studies as microscale indicators of climate change processes. We investigated a variety of microbial markers in shallow sediments from Laguna Negra and Lo Encañado lakes (Región Metropolitana, Chile). An on-site immunoassay-based Life Detector Chip (LDChip) revealed the presence of sulfate-reducing bacteria, methanogenic archaea, and exopolymeric substances from Gammaproteobacteria. Bacterial and archaeal 16S rRNA gene sequences obtained from field samples confirmed the results from the immunoassays and also revealed the presence of Alpha-, Beta-, Gamma-, and Deltaproteobacteria, as well as cyanobacteria and methanogenic archaea. The complementary immunoassay and phylogenetic results indicate a rich microbial diversity with active sulfate reduction and methanogenic activities along the shoreline and in shallow sediments. Sulfate inputs from the surrounding volcanic terrains during deglaciation may explain the observed microbial biomarker and metabolic patterns, which differ with depth and between the two lakes. A switch from aerobic and heterotrophic metabolisms to anaerobic ones such as sulfate reduction and methanogenesis in the shallow shores likely reflects the natural evolution of the lake sediments due to deglaciation. Hydrodynamic deposition of sediments creates compartmentalization (e.g., sediments with different structure and composition surrounded by oligotrophic water) that favors metabolic transitions. Similar phenomena would be expected to occur on other planetary lakes, such as those of Titan, where watery niches fed by depositional events would be surrounded by a "sea" of hydrocarbons. Key Words: Glacier lakes-Sedimentation-Prokaryotic metabolisms and biomarkers-Deglaciation-Life detection-Planetary exploration. Astrobiology 18, 586-606.

© María Ángeles Lezcano et al. 2019., Substrate–atmosphere interfaces in Antarctic geothermal environments are hot–cold regions that constitute thin habitable niches for microorganisms with possible counterparts in ancient Mars. Cerro Caliente hill in Deception Island (active volcano in the South Shetland Islands) is affected by ascending hydrothermal fluids that form a band of warm substrates buffered by low air temperatures. We investigated the influence of temperature on the community structure and metabolism of three microbial mats collected along the geothermal band of Cerro Caliente registering 88°C, 8°C, and 2°C at the time of collection. High-throughput sequencing of small subunit ribosomal ribonucleic acid (SSU rRNA) genes and Life Detector Chip (LDChip) microarray immunoassays revealed different bacterial, archaeal, and eukaryotic composition in the three mats. The mat at 88°C showed the less diverse microbial community and a higher proportion of thermophiles (e.g., Thermales). In contrast, microbial communities in the mats at 2°C and 8°C showed relatively higher diversity and higher proportion of psychrophiles (e.g., Flavobacteriales). Despite this overall association, similar microbial structures at the phylum level (particularly the presence of Cyanobacteria) and certain hot- and cold-tolerant microorganisms were identified in the three mats. Daily thermal oscillations recorded in the substrate over the year (4.5–76°C) may explain the coexistence of microbial fingerprints with different thermal tolerances. Stable isotope composition also revealed metabolic differences among the microbial mats. Carbon isotopic ratios suggested the Calvin–Benson–Bassham cycle as the major pathway for carbon dioxide fixation in the mats at 2°C and 8°C, and the reductive tricarboxylic acid cycle and/or the 3-hydroxypropionate bicycle for the mat at 88°C, indicating different metabolisms as a function of the prevailing temperature of each mat. The comprehensive biomarker profile on the three microbial mats from Cerro Caliente contributes to unravel the diversity, composition, and metabolism in geothermal polar sites and highlights the relevance of geothermal-cold environments to create habitable niches with interest in other planetary environments., This study has been funded by the Spanish Research Agency (AEI) from the Ministry of Science Innovation and Universities and the European FEDER Grants Nos. ESP2015-69540-R, RYC2014-19446, and CGL2015-74254-JIN; the AEI Project No. MDM-2017-0737 Unidad de Excelencia ‘‘Marı´a de Maeztu,’’ and the European Research Council Starting Grant (ERC StG) No. 307496. M.A´ . Lezcano and M.A´ . Fernández Martínez were supported by a postdoctoral fellowship by the Youth Employment Initiative from the European Union and implanted in Comunidad de Madrid.

This work aimed to use 16S ribosomal RNA sequencing with the Illumina MiSeq platform to describe the milk microbiota from 50 healthy Assaf ewes. The global observed microbial community for clinically healthy milk samples analysed was complex and showed a vast diversity. The core microbiota of the sheep milk includes five genera: Staphylococcus, Lactobacillus, Corynebacterium, Streptococcus and Escherichia/Shigella. Although there are some differences, some of these genera are common with the microbiota core pattern of milk from other species, especially with dairy cows. The microbial composition of the studied samples, based on the definition of amplicon sequence variants, was analysed through a correlation network. A preliminary analysis by grouping the milk samples based on their somatic cell count (SCC), which is considered an indicator of subclinical mastitis (SM), showed certain differences for the core of the samples identified as SM. The differences in the microbiota diversity pattern among samples might also suggest that subclinical mastitis would be associated with the significant increase in some genera that are inhabitants of the mammary gland and a remarkable concomitant reduction in the microbial diversity. Additionally, we have also presented here a preliminary analysis to assess the impact of the sheep milk microbiome on SCC, as an indicator of subclinical mastitis. The results here reported provide a first characterization of the sheep milk microbiota and settle the basis for future studies in this field., Ministerio de Economía y Competitividad, Grant/Award Number: AGL2015‐66035‐R

Recent studies report the presence of fungal species in breast milk of healthy mothers, suggesting a potential role in infant mycobiome development. In the present work, we aimed to determine whether the healthy human breast milk mycobiota is influenced by geographical location and mode of delivery, as well as to investigate its interaction with bacterial profiles in the same samples. A total of 80 mature breast milk samples from 4 different countries were analyzed by Illumina sequencing of the internal transcribed spacer 1 (ITS1) region, joining the 18S and 5.8S regions of the fungal rRNA region. Basidiomycota and Ascomycota were found to be the dominant phyla, with Malassezia and Davidiella being the most prevalent genera across countries. A core formed by Malassezia, Davidiella, Sistotrema, and Penicillium was shared in the milk samples from the different origins, although specific shifts in mycobiome composition were associated with geographic location and delivery mode. The presence of fungi in the breast milk samples was further confirmed by culture and isolate characterization, and fungal loads were estimated by quantitative PCR (qPCR) targeting the fungal ITS1 region. Cooccurrence network analysis of bacteria and fungi showed complex interactions that were influenced by geographical location, mode of delivery, maternal age, and pregestational body mass index. The presence of a breast milk mycobiome was confirmed in all samples analyzed, regardless of the geographic origin. IMPORTANCE During recent years, human breast milk has been documented as a potential source of bacteria for the newborn. Recently, we have reported the presence of fungi in breast milk from healthy mothers. It is well known that environmental and perinatal factors can affect milk bacteria; however, the impact on milk fungi is still unknown. The current report describes fungal communities (mycobiota) in breast milk samples across different geographic locations and the influence of the mode of delivery. We also provide novel insights on bacterium-fungus interactions, taking into account environmental and perinatal factors. We identified a core of four genera shared across locations, consisting of Malassezia, Davidiella, Sistotrema, and Penicillium, which have been reported to be present in the infant gut. Our data confirm the presence of fungi in breast milk across continents and support the potential role of breast milk in the initial seeding of fungal species in the infant gut., A.B.-A. and M.C.C. acknowledge the European Research Council (ERC) under the European Union's Horizon 2020 research and innovation program (ERC Starting Grant project no. 639226). F.P.-S. was supported by Ministerio de Economía y Competitividad (MINECO) grant no. CTM2016-80095-C2-1-R (NOVAMAR). The Chinese group acknowledges support from Key Projects of Beijing Science and Technology (D141100004814002) and the Natural Scientific Foundation of Beijing (Z140001).

Microdiversity can lead to different ecotypes within the same species. These are assumed to provide stability in time and space to those species. However, the role of microdiversity in the stability of whole microbial communities remains underexplored. Understanding the drivers of microbial community stability is necessary to predict community response to future disturbances. Here, we analyzed 16S rRNA gene amplicons from eight different temperate bog lakes at the 97% OTU and amplicon sequence variant (ASV) levels and found ecotypes within the same OTU with different distribution patterns in space and time. We observed that these ecotypes are adapted to different values of environmental factors such as water temperature and oxygen concentration. Our results showed that the existence of several ASVs within a OTU favored its persistence across changing environmental conditions. We propose that microdiversity aids the stability of microbial communities in the face of fluctuations in environmental factors., This work was funded by grant CTM2016-80095-C2-1-R from the Spanish Ministerio de Economía, Industria y Competitividad. NG-G was funded by a student scholarship from the Severo Ochoa Program at CNB (SEV-2013-0347-17-2).

Intra-species diversity comprises different ecotypes within the same species. These are assumed to provide stability in time and space to those species. However, the role that microdiversity plays in the stability of whole microbial communities remains underexplored. Understanding the drivers of microbial community stability is necessary to predict community response to future disturbances. Here, we analyzed 16S rRNA gene amplicons from eight different temperate bog lakes at OTU-97% and amplicon sequence variant (ASV) levels, and we found ecotypes within the same species with different distribution patterns in space and time. We observed that these ecotypes are adapted to different values of environmental factors such as water temperature and oxygen concentration. Our results showed that the existence of several ASVs within a species favored its persistence across changing environmental conditions. We propose that microdiversity aids the stability of microbial communities in the face of fluctuations in environmental factors.

Here we report the chemical and microbial characterization of the surface water of a CO2-rich hydrothermal vent known in Costa Rica as Borbollones, located at Tenorio Volcano National Park. The Borbollones showed a temperature surrounding 60 °C, a pH of 2.4 and the gas released has a composition of ~ 97% CO2, ~ 0.07% H2S, ~ 2.3% N2 and ~ 0.12% CH4. Other chemical species such as sulfate and iron were found at high levels with respect to typical fresh water bodies. Analysis by 16S rRNA gene metabarcoding revealed that in Borbollones predominates an archaeon from the order Thermoplasmatales and one bacterium from the genus Sulfurimonas. Other sulfur- (genera Thiomonas, Acidithiobacillus, Sulfuriferula, and Sulfuricurvum) and iron-oxidizing bacteria (genera Sideroxydans, Gallionella, and Ferrovum) were identified. Our results show that CO2-influenced surface water of Borbollones contains microorganisms that are usually found in acid rock drainage environments or sulfur-rich hydrothermal vents. To our knowledge, this is the first microbiological characterization of a CO2-dominated hydrothermal spring from Central America and expands our understanding of those extreme ecosystems., The Vice-rectory of Research of Universidad de Costa Rica (809-B6-524), CENIBiot and by the ERC grant IPBSL (ERC250350-IPBSL) supported this research. Data Intensive Academic Grid, which is supported by the USA National Science Foundation (0959894) provided computational resources. F.P-S. is supported by the Spanish Economy and Competitiveness Ministry (MINECO) grant CTM2016-80095-C2-1-R. MMC acknowledges government funding from the Transitorio I of the National Law 8488 for Emergencies and Risk Prevention in Costa Rica. JMdM gratefully acknowledges support from the Deep Carbon Observatory Biology Meets Subduction project.

Here we report the chemical and microbial characterization of the surface water of a CO

The ability to establish biofilms is a key trait for microorganisms growing in extreme environments. The extracellular polymeric substances (EPS) present in biofilms provide not only surface attachment, but also protection against all kinds of environmental stressors, including desiccation, salinity, temperature or heavy metal pollution. The acquisition of suitable biofilm characteristics might thus be an important process mediating the adaptation of microorganisms to novel environmental conditions. In this work we have characterized the EPS of 20 phylogenetically diverse biofilms collected in situ from five contrasting extreme environments, including two geothermal areas (Copahue, Argentina; Seltun, Iceland), two cold areas (Pastoruri glacier, Peru; Byers Peninsula, Antarctica) and one extremely acidic river (Río Tinto, Spain). Biofilms were subjected to biochemical characterization, glycan profiling and immunoprofiling with an antibody microarray. Our results showed that environmental conditions strongly influence biofilm characteristics, with microorganisms from the same environment achieving similar EPS compositions regardless of the phylogeny of their main species. The concentration of some monosaccharides in the EPS could be related to environmental conditions such as temperature or heavy metal toxicity, suggesting that in some cases stress resistance can be mediated by specific sugars. Overall, our results highlight the existence of conserved EPS compositional patterns for each extreme environment, which could in turn be exploited to engineer ecological adaptations in genetically modified microorganisms., This work was funded by the Spanish “Secretaría de Estado de Investigación, Desarrollo e Innovación” from the Economy and Competitiveness Ministry (MINECO) grants N° AYA2011-24803, EPS2014-58494-R, ESP2015-69540-R, ESP2014-55811-C2-2, and ESP2014-51989-P. FPS was supported by the MINECO grant CTM2016-80095-C2-1-R.

The improvement of sequencing technologies has facilitated generalization of metagenomic sequencing, which has become a standard procedure for analyzing the structure and functionality of microbiomes. Bioinformatic analysis of sequencing results poses a challenge because it involves many different complex steps. SqueezeMeta is a fully automatic pipeline for metagenomics/metatranscriptomics, covering all steps of the analysis. SqueezeMeta includes multi-metagenome support that enables co-assembly of related metagenomes and retrieval of individual genomes via binning procedures. SqueezeMeta features several unique characteristics: co-assembly procedure or co-assembly of unlimited number of metagenomes via merging of individual assembled metagenomes, both with read mapping for estimation of the abundances of genes in each metagenome. It also includes binning and bin checking for retrieving individual genomes. Internal checks for the assembly and binning steps provide information about the consistency of contigs and bins. Moreover, results are stored in a MySQL database, where they can be easily exported and shared, and can be inspected anywhere using a flexible web interface that allows simple creation of complex queries. We illustrate the potential of SqueezeMeta by analyzing 32 gut metagenomes in a fully automatic way, enabling retrieval of several million genes and several hundreds of genomic bins. One of the motivations in the development of SqueezeMeta was producing a software capable of running in small desktop computers and thus amenable to all users and settings. We were also able to co-assemble two of these metagenomes and complete the full analysis in less than one day using a simple laptop computer. This reveals the capacity of SqueezeMeta to run without high-performance computing infrastructure and in absence of any network connectivity. It is therefore adequate for in situ, real time analysis of metagenomes produced by nanopore sequencing. SqueezeMeta can be downloaded from https://github.com/jtamames/SqueezeMeta., Ministerio de Economía y Competitividad

Gloeocapsopsis sp. strain AAB1 is an extremely desiccation-tolerant cyanobacterium isolated from translucent quartz stones from the Atacama Desert (Chile). Here, we report its draft genome sequence, which consists of 137 contigs with an ∼5.4-Mb genome size. The annotation revealed 5,641 coding DNA sequences, 38 tRNA genes, and 5 rRNA genes., F.P.-S. was supported by the Spanish Ministerio de Economía y Competitividad grant CTM2016-80095-C2-1-R. A.A.-B. thanks the European Research Council for starting grant 307496. This work was partially supported by the Spanish Ministerio de Economía y Competitividad grant ESP2015-69540-R.

To better understand heavy metal tolerance in Chlamydomonas acidophila, an extremophilic green alga, we assembled its transcriptome and measured transcriptomic expression before and after Cd exposure in this and the neutrophilic model microalga Chlamydomonas reinhardtii. Genes possibly related to heavy metal tolerance and detoxification were identified and analyzed as potential key innovations that enable this species to live in an extremely acid habitat with high levels of heavy metals. In addition we provide a data set of single orthologous genes from eight green algal species as a valuable resource for comparative studies including eukaryotic extremophiles. Our results based on differential gene expression, detection of unique genes and analyses of codon usage all indicate that there are important genetic differences in C. acidophila compared to C. reinhardtii. Several efflux family proteins were identified as candidate key genes for adaptation to acid environments. This study suggests for the first time that exposure to cadmium strongly increases transposon expression in green algae, and that oil biosynthesis genes are induced in Chlamydomonas under heavy metal stress. Finally, the comparison of the transcriptomes of several acidophilic and non-acidophilic algae showed that the Chlamydomonas genus is polyphyletic and that acidophilic algae have distinctive aminoacid usage patterns.

High concentrations of heavy metals are typical of acidic environments. Therefore, studies on acidophilic organisms in their natural environments improve our understanding on the evolution of heavy metal tolerance and detoxification in plants. Here we sequenced the transcriptome of the extremophilic microalga Chlamydomonas acidophila cultivated in control conditions and with 500 μM of copper for 24 h. High-throughput 454 sequencing was followed by de novo transcriptome assembly. The reference transcriptome was annotated and genes related to heavy metal tolerance and abiotic stress were identified. Analyses of differentially expressed transcripts were used to detect genes involved in metabolic pathways related to abiotic stress tolerance, focusing on effects caused by increased levels of copper. Both transcriptomic data and observations from PAM fluorometry analysis suggested that the photosynthetic activity of C. acidophila is not adversely affected by addition of high amounts of copper. Up-regulated transcripts include several transcripts related to photosynthesis and carbohydrate metabolism, transcripts coding for general stress response, and a transcript annotated as homologous to the oil-body-associated protein HOGP coding gene. The first de novo assembly of C. acidophila significantly increases transcriptomic data available on extremophiles and green algae and thus provides an important reference for further molecular genetic studies. The differences between differentially expressed transcripts detected in our study suggest that the response to heavy metal exposure in C. acidophila is different from other studied green algae. © 2015, Springer Japan.

The search for biomarkers of present or past life is one of the major challenges for in situ planetary exploration. Multiple constraints limit the performance and sensitivity of remote in situ instrumentation. In addition, the structure, chemical, and mineralogical composition of the sample may complicate the analysis and interpretation of the results. The aim of this work is to highlight the main constraints, performance, and complementarity of several techniques that have already been implemented or are planned to be implemented on Mars for detection of organic and molecular biomarkers on a best-case sample scenario. We analyzed a 1000-year-old desiccated and mummified microbial mat from Antarctica by Raman and IR (infrared) spectroscopies (near- and mid-IR), thermogravimetry (TG), differential thermal analysis, mass spectrometry (MS), and immunological detection with a life detector chip. In spite of the high organic content (ca. 20% wt/wt) of the sample, the Raman spectra only showed the characteristic spectral peaks of the remaining beta-carotene biomarker and faint peaks of phyllosilicates over a strong fluorescence background. IR spectra complemented the mineralogical information from Raman spectra and showed the main molecular vibrations of the humic acid functional groups. The TG-MS system showed the release of several volatile compounds attributed to biopolymers. An antibody microarray for detecting cyanobacteria (CYANOCHIP) detected biomarkers from Chroococcales, Nostocales, and Oscillatoriales orders. The results highlight limitations of each technique and suggest the necessity of complementary approaches in the search for biomarkers because some analytical techniques might be impaired by sample composition, presentation, or processing. Key Words: Planetary exploration—Life detection—Microbial mat—Life detector chip—Thermogravimetry—Raman spectroscopy—NIR—DRIFTS. Astrobiology 17, 984–996.

Whether the extreme conditions of acidity and heavy metal pollution of streams and rivers originating in pyritic formations are caused primarily by mining activities or by natural activities of metal-oxidizing microbes living within the geological formations is a subject of considerable controversy. Most microbiological studies of such waters have so far focused on acid mine drainage sites, which are heavily human-impacted environments, so it has been problematic to eliminate the human factor in the question of the origin of the key metal compounds. We have studied the physico-chemistry and microbiology of the Río Sucio in the Braulio Carrillo National Park of Costa Rica, 22 km from its volcanic rock origin. Neither the remote origin, nor the length of the river to the sampling site, have experienced human activity and are thus pristine. The river water had a characteristic brownish-yellow color due to high iron-dominated minerals, was slightly acidic, and rich in chemolithoautotrophic iron- and sulfur-oxidizing bacteria, dominated by Gallionella spp. Río Sucio is thus a natural acid-rock drainage system whose metal-containing components are derived primarily from microbial activities. Universidad de Costa Rica/[809-B4-282]/UCR/Costa Rica European Commission-Science and Technology Development for in situ detection and characterization of subsurface life on the Iberian Pyritic Belt/[ERC250350-IPBSL]/ERC IPBSL/Unión Europea USA National Science Foundation/[0959894]//Estados Unidos Consejo Superior de Investigaciones Científicas//CSIC/España European Union FP7 programme /[607346]/EU/ 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 Química UCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias Básicas::Centro de Investigación en Biología Celular y Molecular (CIBCM)

Transcriptomic sequencing together with bioinformatic analyses and an automated annotation process led us to identify novel phytochelatin synthase (PCS) genes from two extremophilic green algae (Chlamydomonas acidophila and Dunaliella acidophila). These genes are of intermediate length compared to known PCS genes from eukaryotes and PCS-like genes from prokaryotes. A detailed phylogenetic analysis gives new insight into the complicated evolutionary history of PCS genes and provides evidence for multiple horizontal gene transfer events from bacteria to eukaryotes within the gene family. A separate subgroup containing PCS-like genes within the PCS gene family is not supported since the PCS genes are monophyletic only when the PCS-like genes are included. The presence and functionality of the novel genes in the organisms were verified by genomic sequencing and qRT-PCR. Furthermore, the novel PCS gene in Chlamydomonas acidophila showed very strong induction by cadmium. Cloning and expression of the gene in Escherichia coli clearly improves its cadmium resistance. The gene in Dunaliella was not induced, most likely due to gene duplication.

The type strain of Tessaracoccus lapidicaptus was isolated from the deep subsurface of the Iberian Pyrite Belt (southwest Spain). Here, we report its draft genome, consisting of 27 contigs with a ~3.1-Mb genome size. The annotation revealed 2,905 coding DNA sequences, 45 tRNA genes, and three rRNA genes.

Heavy metals are toxic compounds known to cause multiple and severe cellular damage. However, acidophilic extremophiles are able to cope with very high concentrations of heavy metals. This study investigated the stress response under natural environmental heavy metal concentrations in an acidophilic Dunaliella acidophila. We employed Illumina sequencing for a de novo transcriptome assembly and to identify changes in response to high cadmium concentrations and natural metal-rich water. The photosynthetic performance was also estimated by pulse amplitude-modulated (PAM) fluorescence. Transcriptomic analysis highlights a number of processes mainly related to a high constitutive expression of genes involved in oxidative stress and response to reactive oxygen species (ROS), even in the absence of heavy metals. Photosynthetic activity seems to be unaltered under short-term exposition to Cd and chronic exposure to natural metal-rich water, probably due to an increase in the synthesis of structural photosynthetic components preserving their functional integrity. An overrepresentation of Gene Ontology (GO) terms related to metabolic activities, transcription, and proteosomal catabolic process was observed when D. acidophila grew under chronic exposure to natural metal-rich water. GO terms involved in carbohydrate metabolic process, reticulum endoplasmic and Golgi bodies, were also specifically overrepresented in natural metal-rich water library suggesting an endoplasmic reticulum stress response. © 2016, Springer Science+Business Media New York.

The daily photosynthetic performance of a natural biofilm of the extreme acidophilic Euglena mutabilis from Rio Tinto (SW, Spain) under full solar radiation was analyzed by means of pulse amplitude-modulated (PAM) fluorescence measurements and metatrascriptomic analysis. Natural E. mutabilis biofilms undergo large-scale transcriptomic reprogramming during midday due to a dynamic photoinhibition and solar radiation stress. Photoinhibition is due to UV radiation and not to light intensity, as revealed by PAM fluorometry analysis. In order to minimize the negative effects of solar radiation, our data supports the presence of a circadian rhythm in this euglenophyte that increases their opportunity to survive. Differential gene expression throughout the day (at 12:00, 20:00 and night) was monitored by massive Illumina parallel sequencing of metatranscriptomic libraries. The transcription pattern was altered in genes involved in Photosystem II stability and repair, UV damaged DNA repair, non-photochemical quenching and oxidative stress, supporting the photoinhibition detected by PAM fluorometry at midday.

A novel actinobacterium, designated IPBSL-7T, was isolated from a drilling core 297 m deep obtained from the Iberian Pyrite Belt. The strain was isolated anaerobically using nitrate as the electron acceptor. 16S rRNA gene sequence analysis revealed that it was related to Tessaracoccus flavescens SST-39T (95.7 % similarity), Tessaracoccus bendigoensis Ben 106T (95.7 %), Tessaracoccus lubricantis KSS-17SeT (95.6 %) and Tessaracoccus oleiagri SL014B-20A1T (95.0 %), while its similarity to any other member of the family Propionibacteriaceae was less than 94 %. Cells were non-motile, non-spore-forming, Gram-positive, oval to rod-shaped, and often appeared in pairs or small groups. The strain was facultatively anaerobic, oxidase-negative, catalase-positive and capable of reducing nitrate. Colonies were circular, convex, smooth and colourless. The organism could grow at between 15 and 40 °C, with an optimal growth at 37 °C. The pH range for growth was from pH 6 to 9, with pH 8 being the optimal value. Strain IPBSL-7T had peptidoglycan type A3-γ′, with ll-diaminopimelic acid as the diagnostic diamino-acid and glycine at position 1 of the peptide subunit. The dominant menaquinone was MK-9(H4) (93.8 %). The major cellular fatty acid was anteiso-C15 : 0 (55.0 %). The DNA G+C content was 70.3 mol%. On the basis of phenotypic and phylogenetic results, strain IPBSL-7T can be differentiated from previously described species of the genus Tessaracoccus and, therefore, represents a novel species, for which the name Tessaracoccus lapidicaptus sp. nov. is proposed. The type strain is IPBSL-7T ( = CECT 8385T = DSM 27266T).