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.

Gut microbiota variations in wild yellow baboons (Papio cynocephalus) are associated with sex and habitat disturbance.

Although male and female mammals differ in biological traits and functional needs, the contribution of this sexual dimorphism to variations in gut bacteria and fungi (gut microbiota) in relation to habitat type has not been fully examined. To understand whether the combination of sex and habitat affects gut microbiota variation, we analyzed 40 fecal samples of wild yellow baboons (Papio cynocephalus) living in contrasting habitat types (intact, well-protected vs. fragmented, less protected forests) in the Udzungwa Mountains of Tanzania. Sex determination was performed using the marker genes SRY (Sex-determining Region Y) and DDX3X-DDX3Y (DEAD-Box Helicase 3). Samples were attributed to 34 individuals (19 females and 15 males) belonging to five social groups. Combining the results of sex determination with two amplicon sequencing datasets on bacterial (V1-V3 region of the 16S rRNA gene) and fungal (ITS2) gut communities, we found that overall, baboon females had a significantly higher gut bacterial richness compared to males. Beta diversity estimates indicated that bacterial composition was significantly different between males and females, and this was true for individuals from both well- and less protected forests. Our results highlight the combined role of sex and habitat type in shaping variation in gut microbial communities in wild non-human primates.

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.

Draft genome sequence of the anatoxin-a producing cyanobacterium Tychonema bourrellyi B0820 isolated from the epilimnion of the deep Alpine Lake Garda.

We report the draft genome sequence of strain B0820 of the cyanobacterium Tychonema bourrellyi isolated from the epilimnion of Lake Garda and assembled from a metagenome of a non-axenic culture. The strain analyzed was shown to produce anatoxin-a, a potent neurotoxin that can cause fatal intoxication in exposed organisms.

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.

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.

Apyrase-mediated amplification of secretory IgA promotes intestinal homeostasis.

Secretory immunoglobulin A (SIgA) interaction with commensal bacteria conditions microbiota composition and function. However, mechanisms regulating reciprocal control of microbiota and SIgA are not defined. Bacteria-derived adenosine triphosphate (ATP) limits T follicular helper (Tfh) cells in the Peyer's patches (PPs) via P2X7 receptor (P2X7R) and thereby SIgA generation. Here we show that hydrolysis of extracellular ATP (eATP) by apyrase results in amplification of the SIgA repertoire. The enhanced breadth of SIgA in mice colonized with apyrase-releasing Escherichia coli influences topographical distribution of bacteria and expression of genes involved in metabolic versus immune functions in the intestinal epithelium. SIgA-mediated conditioning of bacteria and enterocyte function is reflected by differences in nutrient absorption in mice colonized with apyrase-expressing bacteria. Apyrase-induced SIgA improves intestinal homeostasis and attenuates barrier impairment and susceptibility to infection by enteric pathogens in antibiotic-induced dysbiosis. Therefore, amplification of SIgA by apyrase can be leveraged to restore intestinal fitness in dysbiotic conditions.

Intergenerational Transfer of Persistent Bacterial Communities in Female Nile Tilapia.

Resident microbial communities that can support various host functions play a key role in their development and health. In fishes, microbial symbionts are vertically transferred from the parents to their progeny. Such transfer of microbes in mouthbrooder fish species has not been reported yet. Here, we employed Nile tilapia (Oreochromis niloticus) to investigate the vertical transmission of microbes across generations using a 16S rRNA amplicon sequencing approach, based on the presence of bacteria in different generations. Our analysis revealed that the core microbiome in the buccal cavity and posterior intestine of parents shapes the gut microbiome of the progeny across generations. We speculate that the route of this transmission is via the buccal cavity. The identified core microbiome bacteria, namely Nocardioides, Propionibacterium, and Sphingomonas have been reported to play an essential role in the health and development of offspring. These core microbiome members could have specific functions in fish, similar to mammals.

DNA sequence and taxonomic gap analyses to quantify the coverage of aquatic cyanobacteria and eukaryotic microalgae in reference databases: Results of a survey in the Alpine region.

The taxonomic identification of organisms based on the amplification of specific genetic markers (metabarcoding) implicitly requires adequate discriminatory information and taxonomic coverage of environmental DNA sequences in taxonomic databases. These requirements were quantitatively examined by comparing the determination of cyanobacteria and microalgae obtained by metabarcoding and light microscopy. We used planktic and biofilm samples collected in 37 lakes and 22 rivers across the Alpine region. We focused on two of the most used and best represented genetic markers in the reference databases, namely the 16S rRNA and 18S rRNA genes. A sequence gap analysis using blastn showed that, in the identity range of 99-100%, approximately 30% (plankton) and 60% (biofilm) of the sequences did not find any close counterpart in the reference databases (NCBI GenBank). Similarly, a taxonomic gap analysis showed that approximately 50% of the cyanobacterial and eukaryotic microalgal species identified by light microscopy were not represented in the reference databases. In both cases, the magnitude of the gaps differed between the major taxonomic groups. Even considering the species determined under the microscope and represented in the reference databases, 22% and 26% were still not included in the results obtained by the blastn at percentage levels of identity ≥95% and ≥97%, respectively. The main causes were the absence of matching sequences due to amplification and/or sequencing failure and potential misidentification in the microscopy step. Our results quantitatively demonstrated that in metabarcoding the main obstacles in the classification of 16S rRNA and 18S rRNA sequences and interpretation of high-throughput sequencing biomonitoring data were due to the existence of important gaps in the taxonomic completeness of the reference databases and the short length of reads. The study focused on the Alpine region, but the extent of the gaps could be much greater in other less investigated geographic areas.

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.

On the Origin and Propagation of the COVID-19 Outbreak in the Italian Province of Trento, a Tourist Region of Northern Italy.

BACKGROUND: Trentino is an Italian province with a tourism-based economy, bordering the regions of Lombardy and Veneto, where the two earliest and largest outbreaks of COVID-19 occurred in Italy. The earliest cases in Trentino were reported in the first week of March 2020, with most of the cases occurring in the winter sport areas in the Dolomites mountain range. The number of reported cases decreased over the summer months and was followed by a second wave in the autumn and winter of 2020. METHODS: we performed high-coverage Oxford Nanopore sequencing of 253 positive SARS-CoV-2 swabs collected in Trentino between March and December 2020. RESULTS: in this work, we analyzed genome sequences to trace the routes through which the virus entered the area, and assessed whether the autumnal resurgence could be attributed to lineages persisting undetected during summer, or as a consequence of new introductions. CONCLUSIONS: Comparing the draft genomes analyzed with a large selection of European sequences retrieved from GISAID we found that multiple introductions of the virus occurred at the early stage of the epidemics; the two epidemic waves were unrelated; the second wave was due to reintroductions of the virus in summer when traveling restrictions were uplifted.

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.

Interactions between parasitic helminths and gut microbiota in wild tropical primates from intact and fragmented habitats.

The mammalian gastrointestinal tract harbours a highly complex ecosystem composed of a variety of micro- (bacteria, fungi, viruses, protozoans) and macro-organisms (helminths). Although most microbiota research focuses on the variation of single gut components, the crosstalk between components is still poorly characterized, especially in hosts living under natural conditions. We investigated the gut micro-biodiversity (bacteria, fungi and helminths) of 158 individuals of two wild non-human primates, the Udzungwa red colobus (Procolobus gordonorum) and the yellow baboon (Papio cynocephalus). These species have contrasting diets and lifestyles, but live sympatrically in both human-impacted and pristine forests in the Udzungwa Mountains of Tanzania. Using non-invasive faecal pellets, helminths were identified using standard microscopy while bacteria and fungi were characterized by sequencing the V1-V3 variable region of the 16S rRNA gene for bacteria and the ITS1-ITS2 fragment for fungi. Our results show that both diversity and composition of bacteria and fungi are associated with variation in helminth presence. Although interactions differed by habitat type, in both primates we found that Strongyloides was negatively associated and Trichuris was positively associated with bacterial and fungal richness. To our knowledge, this is one of the few studies demonstrating an interaction between helminth and gut microbiota communities in wild non-human primates.

Breeding Strategy Shapes the Composition of Bacterial Communities in Female Nile Tilapia Reared in a Recirculating Aquaculture System.

In industrial animal production, breeding strategies are essential to produce offspring of better quality and vitality. It is also known that host microbiome has a bearing on its health. Here, we report for the first time the influence of crossbreeding strategy, inbreeding or outbreeding, on the buccal and intestinal bacterial communities in female Nile tilapia (Oreochromis niloticus). Crossbreeding was performed within a family and between different fish families to obtain the inbred and outbred study groups, respectively. The genetic relationship and structure analysis revealed significant genetic differentiation between the inbred and outbred groups. We also employed a 16S rRNA gene sequencing technique to understand the significant differences between the diversities of the bacterial communities of the inbred and outbred groups. The core microbiota composition in the mouth and the intestine was not affected by the crossbreeding strategy but their abundance varied between the two groups. Furthermore, opportunistic bacteria were abundant in the buccal cavity and intestine of the outbred group, whereas beneficial bacteria were abundant in the intestine of the inbred group. The present study indicates that crossbreeding can influence the abundance of beneficial bacteria, core microbiome and the inter-individual variation in the microbiome.

Responses of cucumber (Cucumis sativus L.) rhizosphere microbial community to some agronomic management practices.

The microbial communities associated to the rhizosphere (the rhizomicrobiome) have a substantial impact on plant growth and yield. Understanding the effects of agricultural management on the rhizomicrobiome is very important for selecting efficient practices. By sequencing the V4 region of 16S rRNA for bacteria and the ITS1 regions and fungi, we investigated the influences of agronomic practices, including cucumber grafting on cucurbit hybrid (Cucurbita moschata × C. maxima), cucumber-garlic intercropping, and treatment with fungicide iprodione-carbendazim on cucumber rhizosphere microbial communities during plant growth. Soil dehydrogenase activity (DHA) and plant vegetative parameters were assessed as an indicator of overall soil microbial activity. We found that both treatments and growth stage induced significant shifts in microbial community structure. Grafting had the highest number of differentially abundant OTUs compared to control samples, followed by intercropping and fungicide treatment, while plant development stage affected both alpha and beta diversities indices and composition of the rhizomicrobiome. DHA was more dependent on plant growth stages than on treatments. Among the assessed factors, grafting and plant developmental stage resulted in the greatest changes in the microbial community composition. Grafting also increased the plant growth parameters, suggesting that this method should be further investigated in vegetable production systems.

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.

Pre-Cambrian roots of novel Antarctic cryptoendolithic bacterial lineages.

BACKGROUND: Cryptoendolithic communities are microbial ecosystems dwelling inside porous rocks that are able to persist at the edge of the biological potential for life in the ice-free areas of the Antarctic desert. These regions include the McMurdo Dry Valleys, often accounted as the closest terrestrial counterpart of the Martian environment and thought to be devoid of life until the discovery of these cryptic life-forms. Despite their interest as a model for the early colonization by living organisms of terrestrial ecosystems and for adaptation to extreme conditions of stress, little is known about the evolution, diversity, and genetic makeup of bacterial species that reside in these environments. Using the Illumina Novaseq platform, we generated the first metagenomes from rocks collected in Continental Antarctica over a distance of about 350 km along an altitudinal transect from 834 up to 3100 m above sea level (a.s.l.). RESULTS: A total of 497 draft bacterial genome sequences were assembled and clustered into 269 candidate species that lack a representative genome in public databases. Actinobacteria represent the most abundant phylum, followed by Chloroflexi and Proteobacteria. The "Candidatus Jiangella antarctica" has been recorded across all samples, suggesting a high adaptation and specialization of this species to the harshest Antarctic desert environment. The majority of these new species belong to monophyletic bacterial clades that diverged from related taxa in a range from 1.2 billion to 410 Ma and are functionally distinct from known related taxa. CONCLUSIONS: Our findings significantly increase the repertoire of genomic data for several taxa and, to date, represent the first example of bacterial genomes recovered from endolithic communities. Their ancient origin seems to not be related to the geological history of the continent, rather they may represent evolutionary remnants of pristine clades that evolved across the Tonian glaciation. These unique genomic resources will underpin future studies on the structure, evolution, and function of these ecosystems at the edge of life. Video abstract.

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.

Shed Light in the DaRk LineagES of the Fungal Tree of Life-STRES.

The polyphyletic group of black fungi within the Ascomycota (Arthoniomycetes, Dothideomycetes, and Eurotiomycetes) is ubiquitous in natural and anthropogenic habitats. Partly because of their dark, melanin-based pigmentation, black fungi are resistant to stresses including UV- and ionizing-radiation, heat and desiccation, toxic metals, and organic pollutants. Consequently, they are amongst the most stunning extremophiles and poly-extreme-tolerant organisms on Earth. Even though ca. 60 black fungal genomes have been sequenced to date, [mostly in the family Herpotrichiellaceae (Eurotiomycetes)], the class Dothideomycetes that hosts the largest majority of extremophiles has only been sparsely sampled. By sequencing up to 92 species that will become reference genomes, the "Shed light in The daRk lineagES of the fungal tree of life" (STRES) project will cover a broad collection of black fungal diversity spread throughout the Fungal Tree of Life. Interestingly, the STRES project will focus on mostly unsampled genera that display different ecologies and life-styles (e.g., ant- and lichen-associated fungi, rock-inhabiting fungi, etc.). With a resequencing strategy of 10- to 15-fold depth coverage of up to ~550 strains, numerous new reference genomes will be established. To identify metabolites and functional processes, these new genomic resources will be enriched with metabolomics analyses coupled with transcriptomics experiments on selected species under various stress conditions (salinity, dryness, UV radiation, oligotrophy). The data acquired will serve as a reference and foundation for establishing an encyclopedic database for fungal metagenomics as well as the biology, evolution, and ecology of the fungi in extreme environments.

Large scale genome reconstructions illuminate Wolbachia evolution.

Wolbachia is an iconic example of a successful intracellular bacterium. Despite its importance as a manipulator of invertebrate biology, its evolutionary dynamics have been poorly studied from a genomic viewpoint. To expand the number of Wolbachia genomes, we screen over 30,000 publicly available shotgun DNA sequencing samples from 500 hosts. By assembling over 1000 Wolbachia genomes, we provide a substantial increase in host representation. Our phylogenies based on both core-genome and gene content provide a robust reference for future studies, support new strains in model organisms, and reveal recent horizontal transfers amongst distantly related hosts. We find various instances of gene function gains and losses in different super-groups and in cytoplasmic incompatibility inducing strains. Our Wolbachia-host co-phylogenies indicate that horizontal transmission is widespread at the host intraspecific level and that there is no support for a general Wolbachia-mitochondrial synchronous divergence.