Metagenomics of two aquifers with thermal anomalies in Mallorca Island, and proposal of new uncultivated taxa named following the rules of SeqCode.

Groundwater offers an intriguing blend of distinctive physical and chemical conditions, constituting a challenge for microbial life. In Mallorca, the largest island of Balearic archipelago, harbours a variety of thermal anomalies (i.e., geothermal manifestation where surface aquifers exhibiting temperatures exceeding the regional average). The metagenomes of two aquifers in the centre and southern of the island showed Pseudomonadota to be the most represented phylum when using extracted 16S rRNA gene sequences. However, the microbial structures within and between aquifers were remarkably diverse but similar in their metabolic profiles as revealed by the metagenome-assembled genomes (MAGs) pointing to a prevalence of aerobic chemolithoautotrophic and heterotrophic metabolisms, especially in the Llucmajor aquifer. Also, some evidences of anaerobic lifestyles were detected, which would indicate that these environments either could suffer episodes of oxygen depletion or the anaerobes had been transported from deeper waters. We believe that the local environmental factors (temperature, external inputs or chemistry) seem to be more relevant than the connection and, eventually, transport of microbial cells within the aquifer in determining the highly divergent structures. Notably, most of the reconstructed genomes belonged to undescribed bacterial lineages and from them two high-quality MAGs could be classified as novel taxa named following the rules of the Code for Nomenclature of Prokaryotes Described from Sequence Data (SeqCode). Accordingly, we propose the new species and genus Costitxia debesea gen. nov., sp. nov., affiliated with the novel family Costitxiaceae fam. nov., order Costitxiales ord. nov. and class Costitxiia class. nov.; and the new new species and genus Lloretia debesea gen. nov. sp. nov. affiliated with the novel family Lloretiaceae fam. nov.

Towards estimating the number of strains that make up a natural bacterial population.

What a strain is and how many strains make up a natural bacterial population remain elusive concepts despite their apparent importance for assessing the role of intra-population diversity in disease emergence or response to environmental perturbations. To advance these concepts, we sequenced 138 randomly selected Salinibacter ruber isolates from two solar salterns and assessed these genomes against companion short-read metagenomes from the same samples. The distribution of genome-aggregate average nucleotide identity (ANI) values among these isolates revealed a bimodal distribution, with four-fold lower occurrence of values between 99.2% and 99.8% relative to ANI >99.8% or <99.2%, revealing a natural "gap" in the sequence space within species. Accordingly, we used this ANI gap to define genomovars and a higher ANI value of >99.99% and shared gene-content >99.0% to define strains. Using these thresholds and extrapolating from how many metagenomic reads each genomovar uniquely recruited, we estimated that -although our 138 isolates represented about 80% of the Sal. ruber population- the total population in one saltern pond is composed of 5,500 to 11,000 genomovars, the great majority of which appear to be rare in-situ. These data also revealed that the most frequently recovered isolate in lab media was often not the most abundant genomovar in-situ, suggesting that cultivation biases are significant, even in cases that cultivation procedures are thought to be robust. The methodology and ANI thresholds outlined here should represent a useful guide for future microdiversity surveys of additional microbial species.

An ANI gap within bacterial species that advances the definitions of intra-species units.

IMPORTANCE: Bacterial strains and clonal complexes are two cornerstone concepts for microbiology that remain loosely defined, which confuses communication and research. Here we identify a natural gap in genome sequence comparisons among isolate genomes of all well-sequenced species that has gone unnoticed so far and could be used to more accurately and precisely define these and related concepts compared to current methods. These findings advance the molecular toolbox for accurately delineating and following the important units of diversity within prokaryotic species and thus should greatly facilitate future epidemiological and micro-diversity studies across clinical and environmental settings.

Impact of dilution on stochastically driven methanogenic microbial communities of hypersaline anoxic sediments.

Sediments underlying the solar salterns of S'Avall are anoxic hypersaline ecosystems dominated by anaerobic prokaryotes, and with the especial relevance of putative methanogenic archaea. Slurries from salt-saturated sediments, diluted in a gradient of salinity and incubated for > 4 years revealed that salt concentration was the major selection force that deterministically structured microbial communities. The dominant archaea in the original communities showed a decrease in alpha diversity with dilution accompanied by the increase of bacterial alpha diversity, being highest at 5% salts. Correspondingly, methanogens decreased and in turn sulfate reducers increased with decreasing salt concentrations. Methanogens especially dominated at 25%. Different concentrations of litter of Posidonia oceanica seagrass added as a carbon substrate, did not promote any clear relevant effect. However, the addition of ampicillin as selection pressure exerted important effects on the assemblage probably due to the removal of competitors or enhancers. The amended antibiotic enhanced methanogenesis in the concentrations ≤ 15% of salts, whereas it was depleted at salinities ≥ 20% revealing key roles of ampicillin-sensitive bacteria.

Mycoplasma bradburyae sp. nov. isolated from the trachea of sea birds.

In the search for mollicutes in wild birds, six Mycoplasma strains were isolated from tracheal swabs taken from four different species of seabirds. Four strains originated from three Yellow-legged gulls (Larus michahellis) and a Cory's shearwater (Calonectris borealis) from Spain, one from a South African Kelp gull (Larus dominicanus), and one from an Italian Black-headed gull (Chroicocephalus ridibundus). These Mycoplasma strains presented 99 % 16S rRNA gene sequence similarity values with Mycoplasma (M.) gallisepticum. Phylogenetic analyses of marker genes (16S rRNA gene and rpoB) confirmed the close relationship of the strains to M. gallisepticum and M. tullyi. The seabirds' strains grew well in modified Hayflick medium, and colonies showed typical fried egg morphology. They produced acid from glucose and mannose but did not hydrolyze arginine or urea. Transmission electron microscopy revealed a cell morphology characteristic of mycoplasmas, presenting spherical to flask-shaped cells with an attachment organelle. Gliding motility was also observed. Furthermore, serological tests, MALDI-ToF mass spectrometry and genomic studies demonstrated that the strains were different to any known Mycoplasma species, for which the name Mycoplasma bradburyae sp. nov. is proposed; the type strain is T158T (DSM 110708 = NCTC 14398).

Disparate behavioral types in wild and reared juveniles of gilthead seabream.

Fish differ consistently in behavior within the same species and population, reflecting distinct behavioral types (BTs). Comparing the behavior of wild and reared individuals provides an excellent opportunity to delve into the ecological and evolutionary consequences of BTs. In this work, we evaluated the behavioral variation of wild and reared juvenile gilthead seabreams, Sparus aurata, a highly relevant species for aquaculture and fisheries. We quantified behavioral variation along the five major axes of fish behavioral traits (exploration-avoidance, aggressiveness, sociability, shyness-boldness, and activity) using standardized behavioral tests and a deep learning tracking algorithm for behavioral annotation. Results revealed significant repeatability in all five behavior traits, suggesting high consistency of individual behavioral variation across the different axes in this species. We found reared fish to be more aggressive, social and active compared to their wild conspecifics. Reared individuals also presented less variance in their aggressiveness, lacking very aggressive and very tame individuals. Phenotypic correlation decomposition between behavioral types revealed two different behavioral syndromes: exploration-sociability and exploration-activity. Our work establishes the first baseline of repeatability scores in wild and reared gilthead seabreams, providing novel insight into the behavior of this important commercial species with implications for fisheries and aquaculture.

Thermocaproicibacter melissae gen. nov., sp. nov., a thermophilic chain-elongating bacterium, producing n-caproate from polymeric carbohydrates.

Strain MDTJ8T is a chain-elongating thermophilic bacterium isolated from a thermophilic acidogenic anaerobic digestor treating human waste while producing the high commodity chemical n-caproate. The strain grows and produces formate, acetate, n-butyrate, n-caproate and lactate from mono-, di- and polymeric saccharides at 37-60 °C (optimum, 50-55 °C) and at pH 5.0-7.0 (optimum, pH 6.5). The organism is an obligate anaerobe, is motile and its cells form rods (0.3-0.5×1.0-3.0 µm) that stain Gram-positive and occur primarily as chains. Phylogenetic analysis of both the 16S rRNA gene and full genome sequence shows that strain MDTJ8T belongs to a group that consists of mesophylic chain-elongating bacteria within the family Oscillospiraceae, being nearest to Caproicibacter fermentans EA1T (94.8 %) and Caproiciproducens galactitolivorans BS-1T (93.7 %). Its genome (1.96 Mbp) with a G+C content of 49.6 mol% is remarkably smaller than those of other chain-elongating bacteria of the family Oscillospiraceae. Pairwise average nucleotide identity and DNA-DNA hybridization values between strain MDJT8T and its mesophilic family members are less than 70 and 35 %, respectively, while pairwise average amino acid identity values are less than 68 %. In addition, strain MDJT8T uses far less carbohydrate and non-carbohydrate substrates compared to its nearest family members. The predominant cellular fatty acids of strain MDTJ8T are C14 : 0, C14 : 0 DMA (dimethyl acetal) and C16 : 0, while its polar lipid profile shows three unidentified glycophospholipids, 11 glycolipids, 13 phospholipids and six unidentified lipids. No respiratory quinones and polyamines are detected. Based on its phylogenetic, genotypic, morphological, physiological, biochemical and chemotaxonomic characteristics, strain MDTJ8T represents a novel species and novel genus of the family Oscillospiraceae and Thermocaproicibacter melissae gen. nov., sp. nov. is proposed as its name. The type strain is MDTJ8T (=DSM 114174T=LMG 32615T=NCCB 100883T).

Food determines ephemerous and non-stable gut microbiome communities in juvenile wild and farmed Mediterranean fish.

Novel insights were provided by contrasting the composition of wild and farmed fish gut microbiomes because the latter had essentially different environmental conditions from those in the wild. This was reflected in the gut microbiome of the wild Sparus aurata and Xyrichtys novacula studied here, which showed highly diverse microbial community structures, dominated by Proteobacteria, mostly related to an aerobic or microaerophilic metabolism, but with some common shared major species, such as Ralstonia sp. On the other hand, farmed non-fasted S. aurata individuals had a microbial structure that mirrored the microbial composition of their food source, which was most likely anaerobic, since several members of the genus Lactobacillus, probably revived from the feed and enriched in the gut, dominated the communities. The most striking observation was that after a short fasting period (86 h), farmed gilthead seabream almost lost their whole gut microbiome, and the resident community associated with the mucosa had a very much reduced diversity that was highly dominated by a single potentially aerobic species Micrococcus sp., closely related to M. flavus. The results pointed to the fact that, at least for the juvenile S. aurata studied, most of the microbes in the gut were transient and highly dependent on the feed source, and that only after fasting for at least 2 days could the resident microbiome in the intestinal mucosa be determined. Since an important role of this transient microbiome in relation to fish metabolism could not be discarded, the methodological approach needs to be well designed in order not to bias the results. The results have important implications for fish gut studies that could explain the diversity and occasional contradictory results published in relation to the stability of marine fish gut microbiomes, and might provide important information for feed formulation in the aquaculture industry.

Microbiota Phenotype Promotes Anastomotic Leakage in a Model of Rats with Ischemic Colon Resection.

Anastomotic leakage (AL) is a major cause of morbidity and mortality after colorectal surgery, but the mechanism behind this complication is still not fully understood. Despite the advances in surgical techniques and perioperative care, the complication rates have remained steady. Recently, it has been suggested that colon microbiota may be involved in the development of complications after colorectal surgery. The aim of this study was to evaluate the association of gut microbiota in the development of colorectal AL and their possible virulence strategies to better understand the phenomenon. Using 16S rRNA sequencing of samples collected on the day of surgery and the sixth day following surgery, we analyzed the changes in tissue-associated microbiota at anastomotic sites created in a model of rats with ischemic colon resection. We discovered a trend for lower microbial diversity in the AL group compared to non-leak anastomosis (NLA). There were no differences in relative abundance in the different types of microbial respiration between these groups and the high abundance of the facultative anaerobic Gemella palaticanis is a marker species that stands out as a distinctive feature.

Description of two cultivated and two uncultivated new Salinibacter species, one named following the rules of the bacteriological code: Salinibacter grassmerensis sp. nov.; and three named following the rules of the SeqCode: Salinibacter pepae sp. nov., Salinibacter abyssi sp. nov., and Salinibacter pampae sp. nov.

Current -omics methods allow the collection of a large amount of information that helps in describing the microbial diversity in nature. Here, and as a result of a culturomic approach that rendered the collection of thousands of isolates from 5 different hypersaline sites (in Spain, USA and New Zealand), we obtained 21 strains that represent two new Salinibacter species. For these species we propose the names Salinibacter pepae sp. nov. and Salinibacter grassmerensis sp. nov. (showing average nucleotide identity (ANI) values < 95.09% and 87.08% with Sal. ruber M31T, respectively). Metabolomics revealed species-specific discriminative profiles. Sal. ruber strains were distinguished by a higher percentage of polyunsaturated fatty acids and specific N-functionalized fatty acids; and Sal. altiplanensis was distinguished by an increased number of glycosylated molecules. Based on sequence characteristics and inferred phenotype of metagenome-assembled genomes (MAGs), we describe two new members of the genus Salinibacter. These species dominated in different sites and always coexisted with Sal. ruber and Sal. pepae. Based on the MAGs from three Argentinian lakes in the Pampa region of Argentina and the MAG of the Romanian lake Fara Fund, we describe the species Salinibacter pampae sp. nov. and Salinibacter abyssi sp. nov. respectively (showing ANI values 90.94% and 91.48% with Sal. ruber M31T, respectively). Sal. grassmerensis sp. nov. name was formed according to the rules of the International Code for Nomenclature of Prokaryotes (ICNP), and Sal. pepae, Sal. pampae sp. nov. and Sal. abyssi sp. nov. are proposed following the rules of the newly published Code of Nomenclature of Prokaryotes Described from Sequence Data (SeqCode). This work constitutes an example on how classification under ICNP and SeqCode can coexist, and how the official naming a cultivated organism for which the deposit in public repositories is difficult finds an intermediate solution.

Discovery of the Streamlined Haloarchaeon Halorutilus salinus, Comprising a New Order Widespread in Hypersaline Environments across the World.

The class Halobacteria is one of the most diverse groups within the Euryarchaeota phylum, whose members are ubiquitously distributed in hypersaline environments, where they often constitute the major population. Here, we report the discovery and isolation of a new halophilic archaeon, strain F3-133T exhibiting ≤86.3% 16S rRNA gene identity to any previously cultivated archaeon, and, thus, representing a new order. Analysis of available 16S rRNA gene amplicon and metagenomic data sets showed that the new isolate represents an abundant group in intermediate-to-high salinity ecosystems and is widely distributed across the world. The isolate presents a streamlined genome, which probably accounts for its ecological success in nature and its fastidious growth in culture. The predominant osmoprotection mechanism appears to be the typical salt-in strategy used by other haloarchaea. Furthermore, the genome contains the complete gene set for nucleotide monophosphate degradation pathway through archaeal RuBisCO, being within the first halophilic archaea representatives reported to code this enzyme. Genomic comparisons with previously described representatives of the phylum Euryarchaeota were consistent with the 16S rRNA gene data in supporting that our isolate represents a novel order within the class Halobacteria for which we propose the names Halorutilales ord. nov., Halorutilaceae fam. nov., Halorutilus gen. nov. and Halorutilus salinus sp. nov. IMPORTANCE The discovery of the new halophilic archaeon, Halorutilus salinus, representing a novel order, family, genus, and species within the class Halobacteria and phylum Euryarchaeota clearly enables insights into the microbial dark matter, expanding the current taxonomical knowledge of this group of archaea. The in-depth comparative genomic analysis performed on this new taxon revealed one of the first known examples of an Halobacteria representative coding the archaeal RuBisCO gene and with a streamlined genome, being ecologically successful in nature and explaining its previous non-isolation. Altogether, this research brings light into the understanding of the physiology of the Halobacteria class members, their ecological distribution, and capacity to thrive in hypersaline environments.

Microbial Motility at the Bottom of North America: Digital Holographic Microscopy and Genomic Motility Signatures in Badwater Spring, Death Valley National Park.

Motility is widely distributed across the tree of life and can be recognized by microscopy regardless of phylogenetic affiliation, biochemical composition, or mechanism. Microscopy has thus been proposed as a potential tool for detection of biosignatures for extraterrestrial life; however, traditional light microscopy is poorly suited for this purpose, as it requires sample preparation, involves fragile moving parts, and has a limited volume of view. In this study, we deployed a field-portable digital holographic microscope (DHM) to explore microbial motility in Badwater Spring, a saline spring in Death Valley National Park, and complemented DHM imaging with 16S rRNA gene amplicon sequencing and shotgun metagenomics. The DHM identified diverse morphologies and distinguished run-reverse-flick and run-reverse types of flagellar motility. PICRUSt2- and literature-based predictions based on 16S rRNA gene amplicons were used to predict motility genotypes/phenotypes for 36.0-60.1% of identified taxa, with the predicted motile taxa being dominated by members of Burkholderiaceae and Spirochaetota. A shotgun metagenome confirmed the abundance of genes encoding flagellar motility, and a Ralstonia metagenome-assembled genome encoded a full flagellar gene cluster. This study demonstrates the potential of DHM for planetary life detection, presents the first microbial census of Badwater Spring and brine pool, and confirms the abundance of mobile microbial taxa in an extreme environment.

Isolation and characterization of a thermophilic chain elongating bacterium that produces the high commodity chemical n-caproate from polymeric carbohydrates.

A thermophilic chain elongating bacterium, strain MDTJ8, was isolated from a thermophilic acidogenic anaerobic digestor producing n-caproate from human waste, growing optimally at 50-55 °C and pH 6.5. 16S rRNA gene analysis suggests that MDTJ8 represents a new species/genus within a group currently composed of mesophilic chain elongators of the Oscillospiraceae family. Genome analysis showed that strain MDTJ8 contains homologues of genes encoding for chain elongation and energy conservation but also indicated n-caproate production from carbohydrates including polymeric substances. This was confirmed by culturing experiments in which MDTJ8 converted, at pH 6.5 and 55 °C, mono-, di- and polymeric carbohydrates (starch and hemicellulose) to n-caproate reaching concentrations up to 283 mg/L and accounting for up to 10 % of the measured fermentation products. MDTJ8 is the first axenic organism that thermophilically performs chain elongation, opening doors to understand and intensify thermophilic bioprocesses targeting anaerobic digestion towards the production of the value-added chemical n-caproate.

Litter Management Strategies and Their Impact on the Environmental and Respiratory Microbiome Might Influence Health in Poultry.

Aerial and respiratory tract-associated bacterial diversity has been scarcely studied in broiler production systems. This study examined the relationship between the environmental air and birds' respiratory microbiome, considering a longitudinal sampling. Total viable bacteria and coliforms in the air were quantified, and the 16S rRNA gene was sequenced from tracheal and air samples obtained through a novelty protocol. Air results showed a decrease in coliforms over time. However, at week 3, we reported an increase in coliforms (from 143 to 474 CFUc/m3) associated with litter management. Additionally, 16S rRNA gene results indicated a distinctive air microbial community, associated primarily with Bacillota phylum particularly of the Bacilli class (>58%), under all conditions. Tracheal results indicated a predominance of Escherichia coli/Shigella at the beginning of the productive cycle, shifting toward the middle and end of the cycle to Gallibacterium. However, at week 3, the dominance of Escherichia coli/Shigella (>99.5%) associated with litter aeration by tumbling stood out. Tracheal and air samples displayed a statistically different community structure, but shared differentially abundant features through time: Enterococcus, Gallibacterium, and Romboutsia ilealis. These results indicate the impact of production management protocols on the birds' respiratory system that should be considered a breakpoint in poultry farm health.

Toward quantifying the adaptive role of bacterial pangenomes during environmental perturbations.

Metagenomic surveys have revealed that natural microbial communities are predominantly composed of sequence-discrete, species-like populations but the genetic and/or ecological processes that maintain such populations remain speculative, limiting our understanding of population speciation and adaptation to perturbations. To address this knowledge gap, we sequenced 112 Salinibacter ruber isolates and 12 companion metagenomes from four adjacent saltern ponds in Mallorca, Spain that were experimentally manipulated to dramatically alter salinity and light intensity, the two major drivers of this ecosystem. Our analyses showed that the pangenome of the local Sal. ruber population is open and similar in size (~15,000 genes) to that of randomly sampled Escherichia coli genomes. While most of the accessory (noncore) genes were isolate-specific and showed low in situ abundances based on the metagenomes compared to the core genes, indicating that they were functionally unimportant and/or transient, 3.5% of them became abundant when salinity (but not light) conditions changed and encoded for functions related to osmoregulation. Nonetheless, the ecological advantage of these genes, while significant, was apparently not strong enough to purge diversity within the population. Collectively, our results provide an explanation for how this immense intrapopulation gene diversity is maintained, which has implications for the prokaryotic species concept.

Solar salterns as model systems to study the units of bacterial diversity that matter for ecosystem functioning.

Microbial communities often harbor overwhelming species and gene diversity, making it challenging to determine the important units to study this diversity. We argue that the reduced, and thus tractable, microbial diversity of manmade salterns provides an ideal system to advance this cornerstone issue. We review recent time-series genomic and metagenomic studies of the saltern-dominating bacterial and archaeal taxa to show that these taxa form persistent, sequence-discrete, species-like populations. While these populations harbor extensive intra-population gene diversity, even within a single saltern site, only a small minority of these genes appear to be functionally important during environmental perturbations. We outline an approach to detect and track such populations and their ecologically important genes that should be broadly applicable.

Comparative genome analysis of the genus Hydrotalea and proposal of the novel species Hydrotalea lipotrueae sp. nov., isolated from a groundwater aquifer in the south of Mallorca Island, Spain.

From a collection of > 140 strains isolated from groundwater with thermal anomalies for the purpose of obtaining good candidates with applications in the cosmetic industry, two strains were selected because of their taxonomic novelty. Among the isolates, strains TMF_100T and TFM_099 stood out for their potential biotechnological relevance, and a comparative analysis of 16S rRNA gene sequences indicated that these strains represented a new species of the genus Hydrotalea. In addition, from the public genomic databases, metagenome-assembled genomes (MAGs) and single-cell amplified genomes (SAGs) could be retrieved that affiliated with this genus. These MAGs and SAGs had been obtained from different environmental samples, such as acid mine drainage or marine sediments. In addition to the description of the new species, the ecological relevance of the members of this genus was demonstrated by means of denitrification, CRISPR-Cas system diversity and heavy metal resistance, as well as their wide geographical distribution and environmental versatility. Supported by the taxonomic study, together with physiological and morphological differences and ecological features, we concluded that strain TMF_100T represented a novel species within the genus Hydrotalea, for which we propose the name Hydrotalea lipotrueae sp. nov.

Inverted microbial community stratification and spatial-temporal stability in hypersaline anaerobic sediments from the S'Avall solar salterns.

The anaerobic hypersaline sediments of an ephemeral pond from the S'Avall solar salterns constituted an excellent study system because of their easy accessibility, as well as the analogy of their microbial assemblages with some known deep-sea hypersaline anaerobic brines. By means of shotgun metagenomics and 16S rRNA gene amplicon sequencing, the microbial composition of the sediment was shown to be stable in time and space. The communities were formed by prokaryote representatives with a clear inferred anaerobic metabolism, mainly related to the methane, sulfur and nitrate cycles. The most conspicuous finding was the inverted nature of the vertical stratification. Contrarily to what could be expected, a methanogenic archaeal metabolism was found to dominate in the upper layers, whereas Bacteria with fermentative and anaerobic respiration metabolisms increased with depth. We could demonstrate the methanogenic nature of the members of candidate lineages DHVE2 and MSBL1, which were present in high abundance in this system, and described, for the first time, viruses infecting these lineages. Members of the putatively active aerobic genera Salinibacter and Halorubrum were detected especially in the deepest layers for which we hypothesize that either oxygen could be sporadically available, or they could perform anaerobic metabolisms. We also report a novel repertoire of virus species thriving in these sediments, which had special relevance because of their lysogenic lifestyles.

Release LTP_12_2020, featuring a new ARB alignment and improved 16S rRNA tree for prokaryotic type strains.

The new release of the All-Species Living Tree Project (LTP) represents an important step forward in the reconstruction of 16S rRNA gene phylogenies, since we not only provide an updated set of type strain sequences until December 2020, but also a series of improvements that increase the quality of the database. An improved universal alignment has been introduced that is implemented in the ARB format. In addition, all low-quality sequences present in the previous releases have been substituted by new entries with higher quality, many of them as a result of whole genome sequencing. Altogether, the improvements in the dataset and 16S rRNA sequence alignment allowed us to reconstruct robust phylogenies. The trees made available through this current LTP release feature the best topologies currently achievable. The given nomenclature and taxonomic hierarchy reflect all the changes available up to December 2020. The aim is to regularly update the validly published nomenclatural classification changes and new taxa proposals. The new release can be found at the following URL: https://imedea.uib-csic.es/mmg/ltp/.

Ancient saltern metagenomics: tracking changes in microbes and their viruses from the underground to the surface.

Microbial communities in hypersaline underground waters derive from ancient organisms trapped within the evaporitic salt crystals and are part of the poorly known subterranean biosphere. Here, we characterized the viral and prokaryotic assemblages present in the hypersaline springs that dissolve Triassic-Keuper evaporite rocks and feed the Añana Salt Valley (Araba/Alava, Basque Country, Spain). Four underground water samples (around 23% total salinity) with different levels of exposure to the open air were analysed by means of microscopy and metagenomics. Cells and viruses in the spring water had lower concentrations than what are normally found in hypersaline environments and seemed to be mostly inactive. Upon exposure to the open air, there was an increase in activity of both cells and viruses as well as a selection of phylotypes. The underground water was inhabited by a rich community harbouring a diverse set of genes coding for retinal binding proteins. A total of 35 viral contigs from 15 to 104 kb, representing partial or total viral genomes, were assembled and their evolutionary changes through the spring system were followed by SNP analysis and metagenomic island tracking. Overall, both the viral and the prokaryotic assemblages changed quickly upon exposure to the open air conditions.