Environmental dissolved DNA harbours meaningful biological information on microbial community structure.

Extracellular DNA (eDNA) comprises all the DNA molecules outside cells. This component of microbial ecosystems may serve as a source of nutrients and genetic information. Hypersaline environments harbour one of the highest concentrations of eDNA reported for natural systems, which has been attributed to the physicochemical preservative effect of salts and to high viral abundance. Here, we compared centrifugation and filtration protocols for the extraction of dissolved DNA (dDNA, as opposed to eDNA that also includes DNA from free viral particles) from a solar saltern crystallizer pond (CR30) water sample. The crystallizer dDNA fraction has been characterized, for the first time, and compared with cellular and viral metagenomes from the same location. High-speed centrifugation affected CR30 dDNA concentration and composition due to cell lysis, highlighting that protocol optimization should be the first step in dDNA studies. Crystallizer dDNA, which accounted for lower concentrations than those previously reported for hypersaline anoxic sediments, had a mixed viral and cellular origin, was enriched in archaeal DNA and had a distinctive taxonomic composition compared to that from the cellular assemblage of the same sample. Bioinformatic analyses indicated that nanohaloarchaeal viruses could be a cause for these differences.

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.

Prokaryotic and viral community structure in the singular chaotropic salt lake Salar de Uyuni.

Salar de Uyuni (SdU) is the largest hypersaline salt flat and the highest lithium reservoir on Earth. In addition to extreme temperatures and high UV irradiance, SdU has high concentrations of chaotropic salts which can be important factors in controlling microbial diversity. Here, for the first time we characterize the viral diversity of this hypersaline environment during the two seasons, as well as the physicochemical characteristics and the prokaryotic communities of the analysed samples. Most of the selected samples showed a peculiar physicochemical composition and prokaryotic diversity, mostly different from each other even for samples from locations in close proximity or the same season. In contrast to most hypersaline systems Bacteria frequently outnumbered Archaea. Furthermore, an outstanding percentage of members of Salinibacter sp., likely a species different from the cosmopolitan Salinibacter ruber, was obtained in most of the samples. Viral communities displayed the morphologies normally found in hypersaline environments. Two seasonal samples were chosen for a detailed metagenomic analysis of the viral assemblage. Both viral communities shared common sequences but were dominated by sample-specific viruses, mirroring the differences also observed in physicochemical and prokaryotic community composition. These metaviromes were distinct from those detected in other hypersaline systems analysed to date.

Predominance of deterministic microbial community dynamics in salterns exposed to different light intensities.

While the dynamics of microbial community assembly driven by environmental perturbations have been extensively studied, our understanding is far from complete, particularly for light-induced perturbations. Extremely halophilic communities thriving in coastal solar salterns are mainly influenced by two environmental factors-salt concentrations and high sunlight irradiation. By experimentally manipulating light intensity through the application of shading, we showed that light acts as a deterministic factor that ultimately drives the establishment of recurrent microbial communities under near-saturation salt concentrations. In particular, the stable and highly change-resistant communities that established under high-light intensities were dominated (>90% of metagenomic reads) by Haloquadratum spp. and Salinibacter spp. On the other hand, under 37-fold lower light intensity, different, less stable and change-resistant communities were established, mainly dominated by yet unclassified haloarchaea and relatively diverse photosynthetic microorganisms. These communities harboured, in general, much lower carotenoid pigment content than their high-irradiation counterparts. Both assemblage types appeared to be highly resilient, re-establishing when favourable conditions returned after perturbation (i.e. high-irradiation for the former communities and low-irradiation for the latter ones). Overall, our results revealed that stochastic processes were of limited significance to explain these patterns.

Prokaryotic and viral community of the sulfate-rich crust from Peñahueca ephemeral lake, an astrobiology analogue.

Peñahueca is an athalassohaline hypersaline inland ephemeral lake originated under semiarid conditions in the central Iberian Peninsula (Spain). Its chemical composition makes it extreme for microbial life as well as a terrestrial analogue of other planetary environments. To investigate the persistence of microbial life associated with sulfate-rich crusts, we applied cultivation-independent methods (optical and electron microscopy, 16S rRNA gene profiling and metagenomics) to describe the prokaryotic community and its associated viruses. The diversity for Bacteria was very low and was vastly dominated by endospore formers related to Pontibacillus marinus of the Firmicutes phylum. The archaeal assemblage was more diverse and included taxa related to those normally found in hypersaline environments. Several 'metagenome assembled genomes' were recovered, corresponding to new species of Pontibacillus, several species from the Halobacteria and one new member of the Nanohaloarchaeota. The viral assemblage, although composed of the morphotypes typical of high salt systems, showed little similarity to previously isolated/reconstructed halophages. Several putative prophages of Pontibacillus and haloarchaeal hosts were identified. Remarkably, the Peñahueca sulfate-rich metagenome contained CRISPR-associated proteins and repetitions which were over 10-fold higher than in most hypersaline systems analysed so far.

The low diverse gastric microbiome of the jellyfish Cotylorhiza tuberculata is dominated by four novel taxa.

Cotylorhiza tuberculata is an important scyphozoan jellyfish producing population blooms in the Mediterranean probably due to pelagic ecosystem's decay. Its gastric cavity can serve as a simple model of microbial-animal digestive associations, yet poorly characterized. Using state-of-the-art metagenomic population binning and catalyzed reporter deposition fluorescence in situ hybridization (CARD-FISH), we show that only four novel clonal phylotypes were consistently associated with multiple jellyfish adults. Two affiliated close to Spiroplasma and Mycoplasma genera, one to chlamydial 'Candidatus Syngnamydia', and one to bacteroidetal Tenacibaculum, and were at least one order of magnitude more abundant than any other bacteria detected. Metabolic modelling predicted an aerobic heterotrophic lifestyle for the chlamydia, which were found intracellularly in Onychodromopsis-like ciliates. The Spiroplasma-like organism was predicted to be an anaerobic fermenter associated to some jellyfish cells, whereas the Tenacibaculum-like as free-living aerobic heterotroph, densely colonizing the mesogleal axis inside the gastric filaments. The association between the jellyfish and its reduced microbiome was close and temporally stable, and possibly related to food digestion and protection from pathogens. Based on the genomic and microscopic data, we propose three candidate taxa: 'Candidatus Syngnamydia medusae', 'Candidatus Medusoplasma mediterranei' and 'Candidatus Tenacibaculum medusae'.

Spiribacter aquaticus sp. nov., a novel member of the genus Spiribacter isolated from a saltern.

A moderately halophilic bacterium, designated strain SP30T, was isolated from a solar saltern located in Santa Pola, Alicante, on the East coast of Spain. It was a Gram-stain-negative, strictly aerobic bacterium, able to grow in 7.5-25 % (w/v) NaCl and optimally in 12.5 % (w/v) NaCl. Phylogenetic analyses, based on 16S rRNA gene sequences, showed that the novel isolate is a member of the genus Spiribacter, with the most closely related species being Spiribacter roseus SSL50T (99.9 % sequence similarity) and Spiribacter curvatus UAH-SP71T (99.4 % sequence similarity). The 16S rRNA gene sequence similarity with the type species Spiribacter salinus M19-40T was 96.6 %. The DNA-DNA relatedness value between strain SP30T and S. roseus SSL50T and S. curvatus UAH-SP71T was 40 and 55 %, respectively; these values are lower than the 70 % threshold accepted for species delineation. The major fatty acids were C16:0, C18 : 1ω7c, C19 : 0 cyclo ω8c and C12 : 0. Similarly to other species of the genus Spiribacter, strain SP30Twas observed as curved rods and spiral cells. Metabolic versatility was reduced to the utilization of a few organic compounds as the sole carbon and energy sources, as with other members of Spiribacter. However, it differed in terms of colony pigmentation (brownish-yellow instead of pink) and in having a higher growth rate. Based on these data and on the phenotypic, genotypic and chemotaxonomic characterization, we propose the classification of strain SP30T as a novel species within the genus Spiribacter, with the name Spiribacter aquaticus sp. nov. The type strain is SP30T (=CECT 9238T=LMG 30005T).

Structure and temporal dynamics of the bacterial communities associated to microhabitats of the coral Oculina patagonica.

Corals are known to contain a diverse microbiota that plays a paramount role in the physiology and health of holobiont. However, few studies have addressed the variability of bacterial communities within the coral host. In this study, bacterial community composition from the mucus, tissue and skeleton of the scleractinian coral Oculina patagonica were investigated seasonally at two locations in the Western Mediterranean Sea, to further understand how environmental conditions and the coral microbiome structure are related. We used denaturing gradient gel electrophoresis in combination with next-generation sequencing and electron microscopy to characterize the bacterial community. The bacterial communities were significantly different among coral compartments, and coral tissue displayed the greatest changes related to environmental conditions and coral health status. Species belonging to the Rhodobacteraceae and Vibrionaceae families form part of O. patagonica tissues core microbiome and may play significant roles in the nitrogen cycle. Furthermore, sequences related to the coral pathogens, Vibrio mediterranei and Vibrio coralliilyticus, were detected not only in bleached corals but also in healthy ones, even during cold months. This fact opens a new view onto unveiling the role of pathogens in the development of coral diseases in the future.

Retinal-binding proteins mirror prokaryotic dynamics in multipond solar salterns.

Microbial opsin (i.e. retinal-binding protein) dynamics has been studied along a salinity gradient in Santa Pola solar salterns (Alicante, Spain) by using culture-independent approaches and statistical analyses. Five ponds of salinities ranging from 18% to above 40% were sampled nine times along a year. Forty-three opsin-like sequences were retrieved by denaturing gradient gel electrophoresis and clustered into 18 different phylogroups, indicating that their diversity was higher than expected according to previous data. Moreover, the statistical correlation between environmental factors controlling microbial community structure and dynamics of environmental rhodopsin proteins indicated almost identical temporal fluctuations between the opsin-related sequences and their corresponding putative 'producers' in nature. Although most sequences were related to others previously detected in hypersaline environments, some pond-specific opsins putatively belonged to previously uncharacterized hosts. Furthermore, we propose that subtle changes in the bacteriorhodopsin 'retinal proton binding pocket', which is key in the photocycle function, could be the molecular basis behind a fine 'photocycle-tuning' mechanism to avoid inter/intraspecies light-competition in hypersaline environments.

Interactions between closely related bacterial strains are revealed by deep transcriptome sequencing.

Comparative genomics, metagenomics, and single-cell technologies have shown that populations of microbial species encompass assemblages of closely related strains. This raises the question of whether individual bacterial lineages respond to the presence of their close relatives by modifying their gene expression or, instead, whether assemblages simply act as the arithmetic addition of their individual components. Here, we took advantage of transcriptome sequencing to address this question. For this, we analyzed the transcriptomes of two closely related strains of the extremely halophilic bacterium Salinibacter ruber grown axenically and in coculture. These organisms dominate bacterial assemblages in hypersaline environments worldwide. The strains used here cooccurred in the natural environment and are 100% identical in their 16S rRNA genes, and each strain harbors an accessory genome representing 10% of its complete genome. Overall, transcriptomic patterns from pure cultures were very similar for both strains. Expression was detected along practically the whole genome albeit with some genes at low levels. A subset of genes was very highly expressed in both strains, including genes coding for the light-driven proton pump xanthorhodopsin, genes involved in the stress response, and genes coding for transcriptional regulators. Expression differences between pure cultures affected mainly genes involved in environmental sensing. When the strains were grown in coculture, there was a modest but significant change in their individual transcription patterns compared to those in pure culture. Each strain sensed the presence of the other and responded in a specific manner, which points to fine intraspecific transcriptomic modulation.

Coral mucus as a reservoir of bacteriophages targeting Vibrio pathogens.

The increasing trend in sea surface temperature promotes the spread of Vibrio species, which are known to cause diseases in a wide range of marine organisms. Among these pathogens, Vibrio mediterranei has emerged as a significant threat, leading to bleaching in the coral species Oculina patagonica. Bacteriophages, or phages, are viruses that infect bacteria, thereby regulating microbial communities and playing a crucial role in the coral's defense against pathogens. However, our understanding of phages that infect V. mediterranei is limited. In this study, we identified two phage species capable of infecting V. mediterranei by utilizing a combination of cultivation and metagenomic approaches. These phages are low-abundance specialists within the coral mucus layer that exhibit rapid proliferation in the presence of their hosts, suggesting a potential role in coral defense. Additionally, one of these phages possesses a conserved domain of a leucine-rich repeat protein, similar to those harbored in the coral genome, that plays a key role in pathogen recognition, hinting at potential coral-phage coevolution. Furthermore, our research suggests that lytic Vibrio infections could trigger prophage induction, which may disseminate genetic elements, including virulence factors, in the coral mucus layer. Overall, our findings underscore the importance of historical coral-phage interactions as a form of coral immunity against invasive Vibrio pathogens.

Extremely halophilic brine community manipulation shows higher robustness of microbiomes inhabiting human-driven solar saltern than naturally driven lake.

Hypersaline ecosystems display taxonomically similar assemblages with low diversities and highly dense accompanying viromes. The ecological implications of viral infection on natural microbial populations remain poorly understood, especially at finer scales of diversity. Here, we sought to investigate the influence of changes in environmental physicochemical conditions and viral predation pressure by autochthonous and allochthonous viruses on host dynamics. For this purpose, we transplanted two microbiomes coming from distant hypersaline systems (solar salterns of Es Trenc in Spain and the thalassohaline lake of Aran-Bidgol lake in Iran), by exchanging the cellular fractions with the sterile-filtered accompanying brines with and without the free extracellular virus fraction. The midterm exposure (1 month) of the microbiomes to the new conditions showed that at the supraspecific taxonomic range, the assemblies from the solar saltern brine more strongly resisted the environmental changes and viral predation than that of the lake. The metagenome-assembled genomes (MAGs) analysis revealed an intraspecific transition at the ecotype level, mainly driven by changes in viral predation pressure, by both autochthonous and allochthonous viruses. IMPORTANCE: Viruses greatly influence succession and diversification of their hosts, yet the effects of viral infection on the ecological dynamics of natural microbial populations remain poorly understood, especially at finer scales of diversity. By manipulating the viral predation pressure by autochthonous and allochthonous viruses, we uncovered potential phage-host interaction, and their important role in structuring the prokaryote community at an ecotype level.

Distribution, abundance, and ecogenomics of the Palauibacterales, a new cosmopolitan thiamine-producing order within the Gemmatimonadota phylum.

The phylum Gemmatimonadota comprises mainly uncultured microorganisms that inhabit different environments such as soils, freshwater lakes, marine sediments, sponges, or corals. Based on 16S rRNA gene studies, the group PAUC43f is one of the most frequently retrieved Gemmatimonadota in marine samples. However, its physiology and ecological roles are completely unknown since, to date, not a single PAUC43f isolate or metagenome-assembled genome (MAG) has been characterized. Here, we carried out a broad study of the distribution, abundance, ecotaxonomy, and metabolism of PAUC43f, for which we propose the name of Palauibacterales. This group was detected in 4,965 16S rRNA gene amplicon datasets, mainly from marine sediments, sponges, corals, soils, and lakes, reaching up to 34.3% relative abundance, which highlights its cosmopolitan character, mainly salt-related. The potential metabolic capabilities inferred from 52 Palauibacterales MAGs recovered from marine sediments, sponges, and saline soils suggested a facultative aerobic and chemoorganotrophic metabolism, although some members may also oxidize hydrogen. Some Palauibacterales species might also play an environmental role as N2O consumers as well as suppliers of serine and thiamine. When compared to the rest of the Gemmatimonadota phylum, the biosynthesis of thiamine was one of the key features of the Palauibacterales. Finally, we show that polysaccharide utilization loci (PUL) are widely distributed within the Gemmatimonadota so that they are not restricted to Bacteroidetes, as previously thought. Our results expand the knowledge about this cryptic phylum and provide new insights into the ecological roles of the Gemmatimonadota in the environment. IMPORTANCE Despite advances in molecular and sequencing techniques, there is still a plethora of unknown microorganisms with a relevant ecological role. In the last years, the mostly uncultured Gemmatimonadota phylum is attracting scientific interest because of its widespread distribution and abundance, but very little is known about its ecological role in the marine ecosystem. Here we analyze the global distribution and potential metabolism of the marine Gemmatimonadota group PAUC43f, for which we propose the name of Palauibacterales order. This group presents a saline-related character and a chemoorganoheterotrophic and facultatively aerobic metabolism, although some species might oxidize H2. Given that Palauibacterales is potentially able to synthesize thiamine, whose auxotrophy is the second most common in the marine environment, we propose Palauibacterales as a key thiamine supplier to the marine communities. This finding suggests that Gemmatimonadota could have a more relevant role in the marine environment than previously thought.

Culture-independent approaches for studying viruses from hypersaline environments.

Hypersaline close-to-saturation environments harbor an extremely high concentration of virus-like particles, but the number of haloviruses isolated so far is still very low. Haloviruses can be directly studied from natural samples by using different culture-independent techniques that include transmission electron microscopy, pulsed-field gel electrophoresis, and different metagenomic approaches. Here, we review the findings of these studies, with a main focus on the metagenomic approaches. The analysis of bulk viral nucleic acids directly retrieved from the environment allows estimations of viral diversity, activity, and dynamics and tentative host assignment. Results point to a diverse and active viral community in constant interplay with its hosts and to a "hypersalineness" quality common to viral assemblages present in hypersaline environments that are thousands of kilometers away from each other.

Virioplankton community structure in Tunisian solar salterns.

The microbial community inhabiting Sfax solar salterns on the east coast of Tunisia has been studied by means of different molecular and culture-dependent tools that have unveiled the presence of novel microbial groups as well as a community structure different from that of other coastal hypersaline environments. We have focused on the study of the viral assemblages of these salterns and their changes along the salinity gradient and over time. Viruses from three ponds (C4, M1, and TS) encompassing salinities from moderately hypersaline to saturated (around 14, 19, and 35%, respectively) were sampled in May and October 2009 and analyzed by transmission electron microscopy (TEM) and pulsed-field gel electrophoresis (PFGE). Additionally, for all three October samples and the May TS sample, viral metagenomic DNA was cloned in fosmids, end sequenced, and analyzed. Viral concentration, as well as virus-to-cell ratios, increased along the salinity gradient, with around 10(10) virus-like particles (VLPs)/ml in close-to-saturation ponds, which represents the highest viral concentration reported so far for aquatic systems. Four distinct morphologies could be observed with TEM (spherical, tailed, spindled, and filamentous) but with various proportions in the different samples. Metagenomic analyses indicated that every pond harbored a distinct viral assemblage whose G+C content could be roughly correlated with that of the active part of the microbial community that may have constituted the putative hosts. As previously reported for hypersaline metaviromes, most sequences did not have matches in the databases, although some were conserved among the Sfax metaviromes. BLASTx, BLASTp, and dinucleotide frequency analyses indicated that (i) factors additional to salinity could be structuring viral communities and (ii) every metavirome had unique gene contents and dinucleotide frequencies. Comparison with hypersaline metaviromes available in the databases indicated that the viral assemblages present in close-to-saturation environments located thousands of kilometers apart presented some common traits among them in spite of their differences regarding the putative hosts. A small core metavirome for close-to-saturation systems was found that contained 7 sequences of around 100 nucleotides (nt) whose function was not hinted at by in silico search results, although it most likely represents properties essential for hyperhalophilic viruses.

Spatial and seasonal prokaryotic community dynamics in ponds of increasing salinity of Sfax solar saltern in Tunisia.

The spatial and seasonal dynamics of the halophilic prokaryotic community was investigated in five ponds from Sfax solar saltern (Tunisia), covering a salinity gradient ranging from 20 to 36%. Fluorescence in situ hybridization indicated that, above 24% salinity, the prokaryotic community shifted from bacterial to archaeal dominance with a remarkable increase in the proportion of detected cells. Denaturing gradient gel electrophoresis (DGGE) profiles were rather similar in all the samples analyzed, except in the lowest salinity pond (around 20% salt) where several specific archaeal and bacterial phylotypes were detected. In spite of previous studies on these salterns, DGGE analysis unveiled the presence of microorganisms not previously described in these ponds, such as Archaea related to Natronomonas or bacteria related to Alkalimnicola, as well as many new sequences of Bacteroidetes. Some phylotypes, such as those related to Haloquadratum or to some Bacteroidetes, displayed a strong dependence of salinity and/or magnesium concentrations, which in the case of Haloquadratum could be related to the presence of ecotypes. Seasonal variability in the prokaryotic community composition was focused on two ponds with the lowest (20%) and the highest salinity (36%). In contrast to the crystallized pond, where comparable profiles between autumn 2007 and summer 2008 were obtained, the non-crystallized pond showed pronounced seasonal changes and a sharp succession of "species" during the year. Canonical correspondence analysis of biological and physicochemical parameters indicated that temperature was a strong factor structuring the prokaryotic community in the non-crystallizer pond, that had salinities ranging from 20 to 23.8% during the year.

Factors structuring microbial communities in highly impacted coastal marine sediments (Mar Menor lagoon, SE Spain).

Coastal marine lagoons are environments highly vulnerable to anthropogenic pressures such as agriculture nutrient loading or runoff from metalliferous mining. Sediment microorganisms, which are key components in the biogeochemical cycles, can help attenuate these impacts by accumulating nutrients and pollutants. The Mar Menor, located in the southeast of Spain, is an example of a coastal lagoon strongly altered by anthropic pressures, but the microbial community inhabiting its sediments remains unknown. Here, we describe the sediment prokaryotic communities along a wide range of environmental conditions in the lagoon, revealing that microbial communities were highly heterogeneous among stations, although a core microbiome was detected. The microbiota was dominated by Delta- and Gammaproteobacteria and members of the Bacteroidia class. Additionally, several uncultured groups such as Asgardarchaeota were detected in relatively high proportions. Sediment texture, the presence of Caulerpa or Cymodocea, depth, and geographic location were among the most important factors structuring microbial assemblages. Furthermore, microbial communities in the stations with the highest concentrations of potentially toxic elements (Fe, Pb, As, Zn, and Cd) were less stable than those in the non-contaminated stations. This finding suggests that bacteria colonizing heavily contaminated stations are specialists sensitive to change.

Response to adverse conditions in two strains of the extremely halophilic species Salinibacter ruber.

We have studied the response of the two closest relative strains M8 and M31 of Salinibacter ruber to environmental changes as the transition from exponential to stationary phase in a batch growth, and the submission to two different environmental stresses (dilution of the culture medium and temperature decrease). We monitored the changes in cultivability, ribosomal content by fluorescence in situ hybridization (FISH), and metabolic changes with high-field ion cyclotron Fourier transform mass spectrometry. In all cases, we could observe an important decrease in cultivability that was not accompanied by a decrease in FISH counts, pointing to a transition to viable but non-cultivable state rather than cell death. Furthermore, the metabolomic analyses indicated a common response of both strains to the different conditions assayed. Only a small portion of the detected masses could be annotated due to database constraints. Among them, the most remarkable changes could be attributed to modifications in the composition of the cell envelope, and especially in the cell membrane. We could track changes in the length or saturation of the fatty acids and in the composition of phospholipids involved in aminosugar, glycerolipid, and glycerophospholipid metabolic pathways.

Epibiosis of oxygenic phototrophs containing chlorophylls a, b, c, and d on the colonial ascidian Cystodytes dellechiajei.

The external surfaces of marine animals are colonized by a wide variety of epibionts. Here, we study the phototrophic epibiotic community attached to the colonial ascidian Cystodytes dellechiajei collected in the Mediterranean Sea. Epifluorescence microscopy analysis showed abundant filamentous cyanobacteria on the upper and basal parts of the ascidian that displayed autofluorescence, as well as some unicellular cyanobacteria, diatoms, and structures, which could belong to microscopic rhodophyte algae. In addition, high-performance liquid chromatography of the photosynthetic pigments confirmed that the phototrophic epibionts possess chlorophyll (Chl) d, as well as Chl a, b, and c, which enable them to use far-red light for photosynthesis in that peculiar microenvironment. Furthermore, laser scanning confocal microscopy showed the presence of a few small patches of cells on the basal part of the ascidian displaying fluorescence between 700 and 750 nm after excitement with a 635-nm red laser, typically within the range of Chl d. Denaturing gradient gel electrophoresis of the 16S rRNA gene polymerase chain reaction amplified using specific primers for Cyanobacteria detected sequences related with the genera Planktothricoides, Synechococcus, Phormidium, and Myxosarcina, as well as sequences of chloroplasts of diatoms and rhodophyte algae. Remarkably, only the sequences related to the filamentous cyanobacteria Planktothricoides spp. and some chloroplast sequences were found in almost all specimens collected under different macroecological conditions and geographical areas, suggesting thus certain specificity in the epibiotic association. On the other hand, Prochloron spp. and Acaryochloris marina, typically associated to tropical ascidians, were not detected by denaturing gradient gel electrophoresis. However, given the low abundance of cells displaying Chl d in C. dellechiajei and the fact that molecular fingerprinting techniques not always recover low abundance groups, the presence of these cyanobacteria cannot be ruled out. Nevertheless, our data indicate that tropical ascidians and C. dellechiajei differ in their phototrophic communities, although Chl d-containing cells are present in both microenvironments.

Metagenomics Unveils Posidonia oceanica "Banquettes" as a Potential Source of Novel Bioactive Compounds and Carbohydrate Active Enzymes (CAZymes).

Posidonia oceanica is a long-living and very slow-growing marine seagrass endemic to the Mediterranean Sea. It produces large amounts of leaf material and rhizomes, which can reach the shore and build important banks known as "banquettes." In recent years, interest in the potential uses of these P. oceanica banquettes has increased, and it was demonstrated that biomass extracts showed antioxidant, antifungal, and antiviral activities. The discovery of new compounds through the culture of microorganisms is limited, and to overcome this limitation, we performed a metagenomic study to investigate the microbial community associated with P. oceanica banquettes. Our results showed that the microbial community associated with P. oceanica banquettes was dominated by Alphaproteobacteria, Gammaproteobacteria, Bacteroidetes, and Cyanobacteria. Pseudoalteromonas was the dominant genus, followed by Alteromonas, Labrenzia, and Aquimarina. The metagenome reads were binned and assembled into 23 nearly complete metagenome-assembled genomes (MAGs), which belonged to new families of Cyanobacteria, Myxococcota, and Granulosicoccaceae and also to the novel genus recently described as Gammaproteobacteria family UBA10353. A comparative analysis with 60 published metagenomes from different environments, including seawater, marine biofilms, soils, corals, sponges, and hydrothermal vents, indicated that banquettes have numbers of natural products and carbohydrate active enzymes (CAZymes) similar to those found for soils and were only surpassed by marine biofilms. New proteins assigned to cellulosome modules and lignocellulose-degrading enzymes were also found. These results unveiled the diverse microbial composition of P. oceanica banquettes and determined that banquettes are a potential source of bioactive compounds and novel enzymes. IMPORTANCE Posidonia oceanica is a long-living and very slow-growing marine seagrass endemic to the Mediterranean Sea that forms large amounts of leaf material and rhizomes, which can reach the shore and build important banks known as "banquettes." These banquettes accumulate on the shore, where they can prevent erosion, although they also cause social concern due to their impact on beach use. Furthermore, Posidonia dry material has been considered a source of traditional remedies in several areas of the Mediterranean, and a few studies have been carried out to explore pharmacological activities of Posidonia extracts. The work presented here provides the first characterization of the microbiome associated with Posidonia banquettes. We carried out a metagenomic analysis together with an in-depth comparison of the banquette metagenome with 60 published metagenomes from different environments. This comparative analysis has unveiled the potential that Posidonia banquettes have for the synthesis of natural products, both in abundance (only surpassed by marine biofilms) and novelty. These products include mainly nonribosomal peptides and carbohydrate active enzymes. Thus, the interest of our work lies in the interest of Posidonia "waste" material as a source of new bioactive compounds and CAZymes.