Novel endolithic bacteria of phylum Chloroflexota reveal a myriad of potential survival strategies in the Antarctic desert.

The ice-free McMurdo Dry Valleys of Antarctica are dominated by nutrient-poor mineral soil and rocky outcrops. The principal habitat for microorganisms is within rocks (endolithic). In this environment, microorganisms are provided with protection against sub-zero temperatures, rapid thermal fluctuations, extreme dryness, and ultraviolet and solar radiation. Endolithic communities include lichen, algae, fungi, and a diverse array of bacteria. Chloroflexota is among the most abundant bacterial phyla present in these communities. Among the Chloroflexota are four novel classes of bacteria, here named Candidatus Spiritibacteria class. nov. (=UBA5177), Candidatus Martimicrobia class. nov. (=UBA4733), Candidatus Tarhunnaeia class. nov. (=UBA6077), and Candidatus Uliximicrobia class. nov. (=UBA2235). We retrieved 17 high-quality metagenome-assembled genomes (MAGs) that represent these four classes. Based on genome predictions, all these bacteria are inferred to be aerobic heterotrophs that encode enzymes for the catabolism of diverse sugars. These and other organic substrates are likely derived from lichen, algae, and fungi, as metabolites (including photosynthate), cell wall components, and extracellular matrix components. The majority of MAGs encode the capacity for trace gas oxidation using high-affinity uptake hydrogenases, which could provide energy and metabolic water required for survival and persistence. Furthermore, some MAGs encode the capacity to couple the energy generated from H2 and CO oxidation to support carbon fixation (atmospheric chemosynthesis). All encode mechanisms for the detoxification and efflux of heavy metals. Certain MAGs encode features that indicate possible interactions with other organisms, such as Tc-type toxin complexes, hemolysins, and macroglobulins.IMPORTANCEThe ice-free McMurdo Dry Valleys of Antarctica are the coldest and most hyperarid desert on Earth. It is, therefore, the closest analog to the surface of the planet Mars. Bacteria and other microorganisms survive by inhabiting airspaces within rocks (endolithic). We identify four novel classes of phylum Chloroflexota, and, based on interrogation of 17 metagenome-assembled genomes, we predict specific metabolic and physiological adaptations that facilitate the survival of these bacteria in this harsh environment-including oxidation of trace gases and the utilization of nutrients (including sugars) derived from lichen, algae, and fungi. We propose that such adaptations allow these endolithic bacteria to eke out an existence in this cold and extremely dry habitat.

Distinct and Temporally Stable Assembly Mechanisms Shape Bacterial and Fungal Communities in Vineyard Soils.

Microbial communities in agricultural soils are fundamental for plant growth and in vineyard ecosystems contribute to defining regional wine quality. Managing soil microbes towards beneficial outcomes requires knowledge of how community assembly processes vary across taxonomic groups, spatial scales, and through time. However, our understanding of microbial assembly remains limited. To quantify the contributions of stochastic and deterministic processes to bacterial and fungal assembly across spatial scales and through time, we used 16 s rRNA gene and ITS sequencing in the soil of an emblematic wine-growing region of Italy.Combining null- and neutral-modelling, we found that assembly processes were consistent through time, but bacteria and fungi were governed by different processes. At the within-vineyard scale, deterministic selection and homogenising dispersal dominated bacterial assembly, while neither selection nor dispersal had clear influence over fungal assembly. At the among-vineyard scale, the influence of dispersal limitation increased for both taxonomic groups, but its contribution was much larger for fungal communities. These null-model-based inferences were supported by neutral modelling, which estimated a dispersal rate almost two orders-of-magnitude lower for fungi than bacteria.This indicates that while stochastic processes are important for fungal assembly, bacteria were more influenced by deterministic selection imposed by the biotic and/or abiotic environment. Managing microbes in vineyard soils could thus benefit from strategies that account for dispersal limitation of fungi and the importance of environmental conditions for bacteria. Our results are consistent with theoretical expectations whereby larger individual size and smaller populations can lead to higher levels of stochasticity.

Tissue age, orchard location and disease management influence the composition of fungal and bacterial communities present on the bark of apple trees.

Plants host microbial communities that can be affected by environmental conditions and agronomic practices. Despite the role of bark as a reservoir of plant pathogens and beneficial microorganisms, no information is available on the effects of disease management on the taxonomic composition of the bark-associated communities of apple trees. We assessed the impact of disease management strategies on fungal and bacterial communities on the bark of a scab-resistant apple cultivar in two orchard locations and for two consecutive seasons. The amplicon sequencing revealed that bark age and orchard location strongly affected fungal and bacterial diversity. Microbiota dissimilarity between orchards evolved during the growing season and showed specific temporal series for fungal and bacterial populations in old and young bark. Disease management did not induce global changes in the microbial populations across locations and seasons, but specifically affected the abundance of some taxa according to bark age, orchard location and sampling time. Therefore, the disease management applied to scab-resistant cultivars, which is based on a limited use of fungicides, partially changed the taxonomic composition of bark-associated fungal and bacterial communities, suggesting the need for a more accurate risk assessment regarding possible pathogen outbreaks.

A promoter-level mammalian expression atlas.

Regulated transcription controls the diversity, developmental pathways and spatial organization of the hundreds of cell types that make up a mammal. Using single-molecule cDNA sequencing, we mapped transcription start sites (TSSs) and their usage in human and mouse primary cells, cell lines and tissues to produce a comprehensive overview of mammalian gene expression across the human body. We find that few genes are truly 'housekeeping', whereas many mammalian promoters are composite entities composed of several closely separated TSSs, with independent cell-type-specific expression profiles. TSSs specific to different cell types evolve at different rates, whereas promoters of broadly expressed genes are the most conserved. Promoter-based expression analysis reveals key transcription factors defining cell states and links them to binding-site motifs. The functions of identified novel transcripts can be predicted by coexpression and sample ontology enrichment analyses. The functional annotation of the mammalian genome 5 (FANTOM5) project provides comprehensive expression profiles and functional annotation of mammalian cell-type-specific transcriptomes with wide applications in biomedical research.

Diversity and Cyclical Seasonal Transitions in the Bacterial Community in a Large and Deep Perialpine Lake.

High-throughput sequencing (HTS) was used to analyze the seasonal variations in the bacterioplankton community composition (BCC) in the euphotic layer of a large and deep lake south of the Alps (Lake Garda). The BCC was analyzed throughout two annual cycles by monthly samplings using the amplification and sequencing of the V3-V4 hypervariable region of the 16S rRNA gene by the MiSeq Illumina platform. The dominant and most diverse bacterioplankton phyla were among the more frequently reported in freshwater ecosystems, including the Proteobacteria, Cyanobacteria, Bacteroidetes, Verrucomicrobia, Actinobacteria, and Planctomycetes. As a distinctive feature, the development of the BCC showed a cyclical temporal pattern in the two analyzed years and throughout the euphotic layer. The recurring temporal development was controlled by the strong seasonality in water temperature and thermal stratification, and by cyclical temporal changes in nutrients and, possibly, by the remarkable annual cyclical development of cyanobacteria and eukaryotic phytoplankton hosting bacterioplankton that characterizes Lake Garda. Further downstream analyses of operational taxonomic units associated to cyanobacteria allowed confirming the presence of the most abundant taxa previously identified by microscopy and/or phylogenetic analyses, as well as the presence of other small Synechococcales/Chroococcales and rare Nostocales never identified so far in the deep lakes south of the Alps. The implications of the high diversity and strong seasonality are relevant, opening perspectives for the definition of common and discriminating patterns characterizing the temporal and spatial distribution in the BCC, and for the application of the new sequencing technologies in the monitoring of water quality in large and deep lakes.

Influence of essential oils in diet and life-stage on gut microbiota and fillet quality of rainbow trout (Oncorhynchus mykiss).

Developing fish farming to meet the demands of food security and sustainability in the 21st century will require new farming systems and improved feeds. Diet and microbe interactions in the gut is an important variable with the potential to make a significant impact on future fish farming diets and production systems. It was monitored the gut microbiota of farmed rainbow trout using 16S rRNA profiling over 51 weeks during standard rearing conditions and feeding diet with supplementation of an essential oils (MixOil) mixture from plants (at a concentration in diet of 200 mg/kg). Gut microbiota 16S rRNA profiling indicated that the fish gut was dominated by Actinobacteria, Proteobacteria, Bacteroidetes and Firmicutes. Although the dietary supplementation with MixOil had no impact on either the composition or architecture of gut microbiota, significant changes in alpha and beta diversity and relative abundance of groups of gut bacteria were evident during growth stages on test feeds, especially upon prolonged growth on finishing feed. Fish fillet quality to guarantee palatability and safety for human consumption was also evaluated. Significant differences within the gut microbiota of juvenile and adult trout under the same rearing conditions were observed, The addition of essential oil blend affected some physicochemical characteristics of trout fillets, including their resistance to oxidative damage and their weight loss (as liquid loss and water holding capacity) during the first period of storage, that are two important parameters related to product shelf life and susceptibility to spoilage. The results highlighted the need for further studies concern dietary microbiome modulation at different life stages and its influence on animal health, growth performance and final product quality.

Temperature drives the assembly of endophytic communities' seasonal succession.

Endophytic microorganisms asymptomatically colonise plant tissues. Exploring the assembly dynamics of bacterial endophytic communities is essential to understand the functioning of the plant holobiont and to optimise their possible use as biopesticides or plant biostimulants. The variation in endophytic communities in above and below-ground organs in Vitis vinifera in the field were studied. To understand the specific effect of temperature on endophytic communities, a separate experiment was set up where grapevine cuttings were grown under controlled conditions at three different temperatures. The findings revealed the succession of endophytic communities over the year. Endophytic communities of roots and stems differ in terms of composition and dynamic response to temperature. Noticeably, compositional differences during the seasons affected bacterial taxa more in stems than in roots, suggesting that roots offer a more stable and less easily perturbed environment. Correlation abundance networks showed that the presence of several taxa (including Bradyrhizobium, Burkholderia, Dyella, Mesorhizobium, Propionibacterium and Ralstonia) is linked in both the field and the greenhouse.

Explaining diversity in metagenomic datasets by phylogenetic-based feature weighting.

Metagenomics is revolutionizing our understanding of microbial communities, showing that their structure and composition have profound effects on the ecosystem and in a variety of health and disease conditions. Despite the flourishing of new analysis methods, current approaches based on statistical comparisons between high-level taxonomic classes often fail to identify the microbial taxa that are differentially distributed between sets of samples, since in many cases the taxonomic schema do not allow an adequate description of the structure of the microbiota. This constitutes a severe limitation to the use of metagenomic data in therapeutic and diagnostic applications. To provide a more robust statistical framework, we introduce a class of feature-weighting algorithms that discriminate the taxa responsible for the classification of metagenomic samples. The method unambiguously groups the relevant taxa into clades without relying on pre-defined taxonomic categories, thus including in the analysis also those sequences for which a taxonomic classification is difficult. The phylogenetic clades are weighted and ranked according to their abundance measuring their contribution to the differentiation of the classes of samples, and a criterion is provided to define a reduced set of most relevant clades. Applying the method to public datasets, we show that the data-driven definition of relevant phylogenetic clades accomplished by our ranking strategy identifies features in the samples that are lost if phylogenetic relationships are not considered, improving our ability to mine metagenomic datasets. Comparison with supervised classification methods currently used in metagenomic data analysis highlights the advantages of using phylogenetic information.

Evaluation of the effects of intrapartum antibiotic prophylaxis on newborn intestinal microbiota using a sequencing approach targeted to multi hypervariable 16S rDNA regions.

Different factors are known to influence the early gut colonization in newborns, among them the perinatal use of antibiotics. On the other hand, the effect on the baby of the administration of antibiotics to the mother during labor, referred to as intrapartum antibiotic prophylaxis (IAP), has received less attention, although routinely used in group B Streptococcus positive women to prevent the infection in newborns. In this work, the fecal microbiota of neonates born to mothers receiving IAP and of control subjects were compared taking advantage for the first time of high-throughput DNA sequencing technology. Seven different 16S rDNA hypervariable regions (V2, V3, V4, V6 + V7, V8, and V9) were amplified and sequenced using the Ion Torrent Personal Genome Machine. The results obtained showed significant differences in the microbial composition of newborns born to mothers who had received IAP, with a lower abundance of Actinobacteria and Bacteroidetes as well as an overrepresentation of Proteobacteria. Considering that the seven hypervariable regions showed different discriminant ability in the taxonomic identification, further analyses were performed on the V4 region evidencing in IAP infants a reduced microbial richness and biodiversity, as well as a lower number of bacterial families with a predominance of Enterobacteriaceae members. In addition, this analysis pointed out a significant reduction in Bifidobacterium spp. strains. The reduced abundance of these beneficial microorganisms, together with the increased amount of potentially pathogenic bacteria, may suggest that IAP infants are more exposed to gastrointestinal or generally health disorders later in age.

Minerva and minepy: a C engine for the MINE suite and its R, Python and MATLAB wrappers.

UNLABELLED: We introduce a novel implementation in ANSI C of the MINE family of algorithms for computing maximal information-based measures of dependence between two variables in large datasets, with the aim of a low memory footprint and ease of integration within bioinformatics pipelines. We provide the libraries minerva (with the R interface) and minepy for Python, MATLAB, Octave and C++. The C solution reduces the large memory requirement of the original Java implementation, has good upscaling properties and offers a native parallelization for the R interface. Low memory requirements are demonstrated on the MINE benchmarks as well as on large ( = 1340) microarray and Illumina GAII RNA-seq transcriptomics datasets. AVAILABILITY AND IMPLEMENTATION: Source code and binaries are freely available for download under GPL3 licence at http://minepy.sourceforge.net for minepy and through the CRAN repository http://cran.r-project.org for the R package minerva. All software is multiplatform (MS Windows, Linux and OSX).

Ecological "Windows of opportunity" influence biofilm prokaryotic diversity differently in glacial and non-glacial Alpine streams.

In glacier-fed streams, the Windows of Opportunity (WOs) are periods of mild environmental conditions supporting the seasonal development of benthic microorganisms. WOs have been defined based on changes in biofilm biomass, but the responses of microbial diversity to WOs in Alpine streams have been overlooked. A two year (2017-2018) metabarcoding of epilithic and epipsammic biofilm prokaryotes was conducted in Alpine streams fed by glaciers (kryal), rock glaciers (rock glacial), or groundwater/precipitation (krenal) in two catchments of the Central-Eastern European Alps (Italy), aiming at testing the hypothesis that: 1) environmental WOs enhance not only the biomass but also the α-diversity of the prokaryotic biofilm in all stream types, 2) diversity and phenology of prokaryotic biofilm are mainly influenced by the physical habitat in glacial streams, and by water chemistry in the other two stream types. The study confirmed kryal and krenal streams as endmembers of epilithic and sediment prokaryotic α- and ß-diversity, with rock glacial streams sharing a large proportion of taxa with the two other stream types. Alpha-diversity appeared to respond to ecological WOs, but, contrary to expectations, seasonality was less pronounced in the turbid kryal than in the clear streams. This was attributed to the small size of the glaciers feeding the studied kryal streams, whose discharge dynamics were those typical of the late phase of deglaciation. Prokaryotic α-diversity of non-glacial streams tended to be higher in early summer than in early autumn. Our findings, while confirming that high altitude streams are heavily threatened by climate change, underscore the still neglected role of rock glacier runoffs as climate refugia for the most stenothermic benthic aquatic microorganism. This advocates the need to define and test strategies for protecting these ecosystems for preserving, restoring, and connecting cold Alpine aquatic biodiversity in the context of the progressing global warming.

Integration and holistic analysis of multiple multidimensional soil data sets.

Complex matrices such as soil have a range of measurable characteristics, and thus data to describe them can be considered multidimensional. These characteristics can be strongly influenced by factors that introduce confounding effects that hinder analyses. Traditional statistical approaches lack the flexibility and granularity required to adequately evaluate such matrices, particularly those with large dataset of varying data types (i.e. quantitative non-compositional, quantitative compositional). We present a statistical workflow designed to effectively analyse complex, multidimensional systems, even in the presence of confounding variables. The developed methodology involves exploratory analysis to identify the presence of confounding variables, followed by data decomposition (including strategies for both compositional and non-compositional quantitative data) to minimise the influence of these confounding factors such as sampling site/location. These data processing methods then allow for common patterns to be highlighted in the data, including the identification of biomarkers and determination of non-trivial associations between variables. We demonstrate the utility of this statistical workflow by jointly analysing the chemical composition and fungal biodiversity of New Zealand vineyard soils that have been managed with either organic low-input or conventional input approaches. By applying this pipeline, we were able to identify biomarkers that distinguish viticultural soil from both approaches and also unearth links and associations between the chemical and metagenomic profiles. While soil is an example of a system that can require this type of statistical methodology, there are a range of biological and ecological systems that are challenging to analyse due to the complex interplay of global and local effects. Utilising our developed pipeline will greatly enhance the way that these systems can be studied and the quality and impact of insight gained from their analysis.

Metagenomics untangles potential adaptations of Antarctic endolithic bacteria at the fringe of habitability.

Survival and growth strategies of Antarctic endolithic microbes residing in Earth's driest and coldest desert remain virtually unknown. From 109 endolithic microbiomes, 4539 metagenome-assembled genomes were generated, 49.3 % of which were novel candidate bacterial species. We present evidence that trace gas oxidation and atmospheric chemosynthesis may be the prevalent strategies supporting metabolic activity and persistence of these ecosystems at the fringe of life and the limits of habitability.

Highly diverse and unknown viruses may enhance Antarctic endoliths' adaptability.

BACKGROUND: Rock-dwelling microorganisms are key players in ecosystem functioning of Antarctic ice free-areas. Yet, little is known about their diversity and ecology, and further still, viruses in these communities have been largely unexplored despite important roles related to host metabolism and nutrient cycling. To begin to address this, we present a large-scale viral catalog from Antarctic rock microbial communities. RESULTS: We performed metagenomic analyses on rocks from across Antarctica representing a broad range of environmental and spatial conditions, and which resulted in a predicted viral catalog comprising > 75,000 viral operational taxonomic units (vOTUS). We found largely undescribed, highly diverse and spatially structured virus communities which had predicted auxiliary metabolic genes (AMGs) with functions indicating that they may be potentially influencing bacterial adaptation and biogeochemistry. CONCLUSION: This catalog lays the foundation for expanding knowledge of virosphere diversity, function, spatial ecology, and dynamics in extreme environments. This work serves as a step towards exploring adaptability of microbial communities in the face of a changing climate. Video Abstract.

A pile of pipelines: An overview of the bioinformatics software for metabarcoding data analyses.

Environmental DNA (eDNA) metabarcoding has gained growing attention as a strategy for monitoring biodiversity in ecology. However, taxa identifications produced through metabarcoding require sophisticated processing of high-throughput sequencing data from taxonomically informative DNA barcodes. Various sets of universal and taxon-specific primers have been developed, extending the usability of metabarcoding across archaea, bacteria and eukaryotes. Accordingly, a multitude of metabarcoding data analysis tools and pipelines have also been developed. Often, several developed workflows are designed to process the same amplicon sequencing data, making it somewhat puzzling to choose one among the plethora of existing pipelines. However, each pipeline has its own specific philosophy, strengths and limitations, which should be considered depending on the aims of any specific study, as well as the bioinformatics expertise of the user. In this review, we outline the input data requirements, supported operating systems and particular attributes of thirty-two amplicon processing pipelines with the goal of helping users to select a pipeline for their metabarcoding projects.

Genome sequencing provides new insights on the distribution of Erwinia amylovora lineages in northern Italy.

Erwinia amylovora is a Gram-negative bacterium that colonizes a wide variety of plant species causing recurrent local outbreaks of fire blight in crops of the Rosaceae family. Recent genomic surveys have documented the limited genomic diversity of this species, possibly related to a recent evolutionary bottleneck and a strong correlation between geography and phylogenetic structure of the species. Despite its economic importance, little is known about the genetic variability of co-circulating strains during local outbreaks. Here, we report the genome sequences of 82 isolates of E. amylovora, collected from different host plants in a period of 16 years in Trentino, a small region in the Northeastern Italian Alps that has been characterized by recurrent outbreaks of fire blight in apple orchards. While the genome isolated before 2018 are closely related to other strains already present in Europe, we found a novel subclade composed only by isolates that were sampled starting from 2018 and demonstrate that the endemic population of this pathogen can be composed by mixture of strains.

Rocks support a distinctive and consistent mycobiome across contrasting dry regions of Earth.

Rock-dwelling fungi play critical ecological roles in drylands, including soil formation and nutrient cycling; however, we know very little about the identity, function and environmental preferences of these important organisms, and the mere existence of a consistent rock mycobiome across diverse arid regions of the planet remains undetermined. To address this knowledge gap, we conducted a meta-analysis of rock fungi and spatially associated soil communities, surveyed across 28 unique sites spanning four major biogeographic regions (North America, Arctic, Maritime and Continental Antarctica) including contrasting climates, from cold and hot deserts to semiarid drylands. We show that rocks support a consistent and unique mycobiome that was different from that found in surrounding soils. Lichenized fungi from class Lecanoromycetes were consistently indicative of rocks across contrasting regions, together with ascomycetous representatives of black fungi in Arthoniomycetes, Dothideomycetes and Eurotiomycetes. In addition, compared with soil, rocks had a lower proportion of saprobes and plant symbiotic fungi. The main drivers structuring rock fungi distribution were spatial distance and, to a larger extent, climatic factors regulating moisture and temperature (i.e. mean annual temperature and mean annual precipitation), suggesting that these paramount and unique communities might be particularly sensitive to increases in temperature and desertification.

Large-scale quality assessment of prokaryotic genomes with metashot/prok-quality.

Metagenomic sequencing allows large-scale identification and genomic characterization. Binning is the process of recovering genomes from complex mixtures of sequence fragments (metagenome contigs) of unknown bacteria and archaeal species. Assessing the quality of genomes recovered from metagenomes requires the use of complex pipelines involving many independent steps, often difficult to reproduce and maintain. A comprehensive, automated and easy-to-use computational workflow for the quality assessment of draft prokaryotic genomes, based on container technology, would greatly improve reproducibility and reusability of published results. We present metashot/prok-quality, a container-enabled Nextflow pipeline for quality assessment and genome dereplication. The metashot/prok-quality tool produces genome quality reports that are compliant with the Minimum Information about a Metagenome-Assembled Genome (MIMAG) standard, and can run out-of-the-box on any platform that supports Nextflow, Docker or Singularity, including computing clusters or batch infrastructures in the cloud. metashot/prok-quality is part of the metashot collection of analysis pipelines. Workflow and documentation are available under GPL3 licence on GitHub.

Genome Recovery, Functional Profiling, and Taxonomic Classification from Metagenomes.

Recovering and annotating bacterial genomes from metagenomes involves a series of complex computational tools that are often difficult to use for researches without a specialistic bioinformatic background. In this chapter we review all the steps that lead from raw reads to a collection of quality-controlled, functionally annotated bacterial genomes and propose a working protocol using state-of-the-art, open source software tools.

Power Play of Commensal Bacteria in the Buccal Cavity of Female Nile Tilapia.

Fish are widely exposed to higher microbial loads compared to land and air animals. It is known that the microbiome plays an essential role in the health and development of the host. The oral microbiome is vital in females of different organisms, including the maternal mouthbrooding species such as Nile tilapia (Oreochromis niloticus). The present study reports for the first time the microbial composition in the buccal cavity of female and male Nile tilapia reared in a recirculating aquaculture system. Mucus samples were collected from the buccal cavity of 58 adult fish (∼1 kg), and 16S rRNA gene amplicon sequencing was used to profile the microbial communities in females and males. The analysis revealed that opportunistic pathogens such as Streptococcus sp. were less abundant in the female buccal cavity. The power play of certain bacteria such as Acinetobacter, Acidobacteria (GP4 and GP6), and Saccharibacteria that have known metabolic advantages was evident in females compared to males. Association networks inferred from relative abundances showed few microbe-microbe interactions of opportunistic pathogens in female fish. The findings of opportunistic bacteria and their interactions with other microbes will be valuable for improving Nile tilapia rearing practices. The presence of bacteria with specific functions in the buccal cavity of female fish points to their ability to create a protective microbial ecosystem for the offspring.