Draft Genome Sequences of "Candidatus Synechococcus spongiarum," Cyanobacterial Symbionts of the Mediterranean Sponge Aplysina aerophoba

We report here four draft genome sequences belonging to clade F of the cyanobacterium “Candidatus Synechococcus spongiarum” of the marine sponge Aplysina aerophoba, which were collected from two nearby locations in the northern Adriatic Sea. The sequences provide the basis for within-clade comparisons between members of this widespread group of cyanobacterial sponge symbionts

Exploring the microbiome of the Mediterranean sponge Aplysina aerophoba by single-cell and metagenomics

Sponges (phylum Porifera) are evolutionary ancient, sessile filter-feeders that harbor a largely diverse microbial community within their internal mesohyl matrix. Throughout this thesis project, I aimed at exploring the adaptations of these symbionts to life within their sponge host by sequencing and analyzing the genomes of a variety of bacteria from the microbiome of the Mediterranean sponge Aplysina aerophoba. Employed methods were fluorescence-activated cell sorting with subsequent multiple displacement amplification and single-cell / ‘mini-metagenome’ sequencing, and metagenomic sequencing followed by differential coverage binning. These two main approaches both aimed at obtaining genome sequences of bacterial symbionts of A. aerophoba, that were then compared to each other and to references from other environments, to gain information on adaptations to the host sponge environment and on possible interactions with the host and within the microbial community. Cyanobacteria are frequent members of the sponge microbial community. My ‘mini-metagenome’ sequencing project delivered three draft genomes of “Candidatus Synechococcus spongiarum,” the cyanobacterial symbiont of A. aerophoba and many more sponges inhabiting the photic zone. The most complete of these genomes was compared to other clades of this symbiont and to closely related free-living cyanobacterial references in a collaborative project published in Burgsdorf I*, Slaby BM* et al. (2015; *shared first authorship). Although the four clades of “Ca. Synechococcus spongiarum” from the four sponge species A. aerophoba, Ircinia variabilis, Theonella swinhoei, and Carteriospongia foliascens were approximately 99% identical on the level of 16S rRNA gene sequences, they greatly differed on the genomic level. Not only the genome sizes were different from clade to clade, but also the gene content and a number of features including proteins containing the eukaryotic-type domains leucine-rich repeats or tetratricopeptide repeats. On the other hand, the four clades shared a number of features such as ankyrin repeat domain-containing proteins that seemed to be conserved also among other microbial phyla in different sponge hosts and from different geographic locations. A possible novel mechanism for host phagocytosis evasion and phage resistance by means of an altered O antigen of the lipopolysaccharide was identified. To test previous hypotheses on adaptations of sponge-associated bacteria on a broader spectrum of the microbiome of A. aerophoba while also taking a step forward in methodology, I developed a bioinformatic pipeline to combine metagenomic Illumina short-read sequencing data with PacBio long-read data. At the beginning of this project, no pipelines to combine short-read and long-read data for metagenomics were published, and at time of writing, there are still no projects published with a comparable aim of un-targeted assembly, binning and analysis of a metagenome. I tried a variety of assembly programs and settings on a simulated test dataset reflecting the properties of the real metagenomic data. The developed assembly pipeline improved not only the overall assembly statistics, but also the quality of the binned genomes, which was evaluated by comparison to the originally published genome assemblies. The microbiome of A. aerophoba was studied from various angles in the recent years, but only genomes of the candidate phylum Poribacteria and the cyanobacterial sequences from my above-described project have been published to date. By applying my newly developed assembly pipeline to a metagenomic dataset of A. aerophoba consisting of a PacBio long-read dataset and six Illumina short-read datasets optimized for subsequent differential coverage binning, I aimed at sequencing a larger number and greater diversity of symbionts. The results of this project are currently in review by The ISME Journal. The complementation of Illumina short-read with PacBio long-read sequencing data for binning of this highly complex metagenome greatly improved the overall assembly statistics and improved the quality of the binned genomes. Thirty-seven genomes from 13 bacterial phyla and candidate phyla were binned representing the most prominent members of the microbiome of A. aerophoba. A statistical comparison revealed an enrichment of genes involved in restriction modification and toxin-antitoxin systems in most symbiont genomes over selected reference genomes. Both are defense features against incoming foreign DNA, which may be important for sponge symbionts due to the sponge’s filtration and phagocytosis activity that exposes the symbionts to high levels of free DNA. Also host colonization and matrix utilization features were significantly enriched. Due to the diversity of the binned symbiont genomes, a within-symbionts genome comparison was possible, that revealed three guilds of symbionts characterized by i) nutritional specialization on the metabolization of carnitine, ii) specialization on sulfated polysaccharides, and iii) apparent nutritional generalism. Both carnitine and sulfated polysaccharides are abundant in the sponge extracellular matrix and therefore available to the sponge symbionts as substrates. In summary, the genomes of the diverse community of symbionts in A. aerophoba were united in their defense features, but specialized regarding their nutritional preferences

Genomic blueprints of sponge-prokaryote symbiosis are shared by low abundant and cultivatable Alphaproteobacteria

Marine sponges are early-branching, filter-feeding metazoans that usually host complex microbiomes comprised of several, currently uncultivatable symbiotic lineages. Here, we use a low-carbon based strategy to cultivate low-abundance bacteria from Spongia officinalis. This approach favoured the growth of Alphaproteobacteria strains in the genera Anderseniella, Erythrobacter, Labrenzia, Loktanella, Ruegeria, Sphingorhabdus, Tateyamaria and Pseudovibrio, besides two likely new genera in the Rhodobacteraceae family. Mapping of complete genomes against the metagenomes of S. officinalis, seawater, and sediments confirmed the rare status of all the above-mentioned lineages in the marine realm. Remarkably, this community of low-abundance Alphaproteobacteria possesses several genomic attributes common to dominant, presently uncultivatable sponge symbionts, potentially contributing to host fitness through detoxification mechanisms (e.g. heavy metal and metabolic waste removal, degradation of aromatic compounds), provision of essential vitamins (e.g. B6 and B12 biosynthesis), nutritional exchange (especially regarding the processing of organic sulphur and nitrogen) and chemical defence (e.g. polyketide and terpenoid biosynthesis). None of the studied taxa displayed signs of genome reduction, indicative of obligate mutualism. Instead, versatile nutrient metabolisms along with motility, chemotaxis, and tight-adherence capacities - also known to confer environmental hardiness - were inferred, underlying dual host-associated and free-living life strategies adopted by these diverse sponge-associated Alphaproteobacteria.PTDC/MAR-BIO/1547/2014; full PhD scholarship from the Erasmus Mundus Programme/SALAM EMA2 lot7/SALA1206422info:eu-repo/semantics/publishedVersio

The sponge holobiont in a changing ocean: from microbes to ecosystems

The recognition that all macroorganisms live in symbiotic association with microbial communities has opened up a new field in biology. Animals, plants, and algae are now considered holobionts, complex ecosystems consisting of the host, the microbiota, and the interactions among them. Accordingly, ecological concepts can be applied to understand the host-derived and microbial processes that govern the dynamics of the interactive networks within the holobiont. In marine systems, holobionts are further integrated into larger and more complex communities and ecosystems, a concept referred to as “nested ecosystems.” In this review, we discuss the concept of holobionts as dynamic ecosystems that interact at multiple scales and respond to environmental change. We focus on the symbiosis of sponges with their microbial communities—a symbiosis that has resulted in one of the most diverse and complex holobionts in the marine environment. In recent years, the field of sponge microbiology has remarkably advanced in terms of curated databases, standardized protocols, and information on the functions of the microbiota. Like a Russian doll, these microbial processes are translated into sponge holobiont functions that impact the surrounding ecosystem. For example, the sponge-associated microbial metabolisms, fueled by the high filtering capacity of the sponge host, substantially affect the biogeochemical cycling of key nutrients like carbon, nitrogen, and phosphorous. Since sponge holobionts are increasingly threatened by anthropogenic stressors that jeopardize the stability of the holobiont ecosystem, we discuss the link between environmental perturbations, dysbiosis, and sponge diseases. Experimental studies suggest that the microbial community composition is tightly linked to holobiont health, but whether dysbiosis is a cause or a consequence of holobiont collapse remains unresolved. Moreover, the potential role of the microbiome in mediating the capacity for holobionts to acclimate and adapt to environmental change is unknown. Future studies should aim to identify the mechanisms underlying holobiont dynamics at multiple scales, from the microbiome to the ecosystem, and develop management strategies to preserve the key functions provided by the sponge holobiont in our present and future oceans

Degradation of Perfluorinated Ether Lubricants on Pure Aluminum Surfaces: Semiempirical Quantum Chemical Modeling

The AM1 semiempirical quantum chemical method was used to model the interaction of perfluoroethers with aluminum surfaces. Perfluorodimethoxymethane and perfluorodimethyl ether were studied interacting with aluminum surfaces, which were modeled by a five-atom cluster and a nine-atom cluster. Interactions were studied for edge (high index) sites and top (low index) sites of the clusters. Both dissociative binding and nondissociative binding were found, with dissociative binding being stronger. The two different ethers bound and dissociated on the clusters in different ways: perfluorodimethoxymethane through its oxygen atoms, but perfluorodimethyl ether through its fluorine atoms. The acetal linkage of perfluorodimeth-oxymethane was the key structural feature of this molecule in its binding and dissociation on the aluminum surface models. The high-index sites of the clusters caused the dissociation of both ethers. These results are consistent with the experimental observation that perfluorinated ethers decompose in contact with sputtered aluminum surfaces

Fan-Intake interaction under high incidence

In this paper, we present an extensive numerical study on the interaction between the downstream fan and the flow separating over an intake under high incidence. The objectives of this investigation are twofold: (a) to gain qualitative insight into the mechanism of fan–intake interaction and (b) to quantitatively examine the effect of the proximity of the fan on the inlet distortion. The fan proximity is altered using the key design parameter, L/D, where D is the diameter of the intake, and L is the distance of the fan from the intake lip. Both steady and unsteady Reynolds-averaged numerical simulations (RANS) were carried out. For the steady calculations, a low-order fan model has been used, while a full 3D geometry has been used for the unsteady RANS. The numerical methodology is also thoroughly validated against the measurements for the intake-only and fan-only configurations on a high bypass ratio turbofan intake and fan, respectively. To systematically study the effect of fan on the intake separation and explore the design criteria, a simplified intake–fan configuration has been considered. In this fan–intake model, the proximity of the fan to the intake separation (L/D) can be conveniently altered without affecting other parameters. The key results indicate that, depending on L/D, the fan has either suppressed the level of the postseparation distortion or increased the separation-free operating range. At the lowest L/D (∼0.17), around a 5 deg increase in the separation-free angle of incidence was achieved. This delay in the separation-free angle of incidence decreased with increasing L/D. At the largest L/D (∼0.44), the fan was effective in suppressing the postseparation distortion rather than entirely eliminating the separation. Isentropic Mach number distribution over the intake lip for different L/D's revealed that the fan accelerates the flow near the casing upstream of the fan face, thereby decreasing the distortion level in the immediate vicinity. However, this acceleration effect decayed rapidly with increasing upstream distance from the fan-face.</jats:p

Microbial diversity of the glass sponge Vazella pourtalesii in response to anthropogenic activities

Establishment of adequate conservation areas represents a challenging but crucial task in the conservation of genetic diversity and biological variability. Anthropogenic pressures on marine ecosystems and organisms are steadily increasing. Whether and to what extent these pressures influence marine genetic biodiversity is only starting to be revealed. Using 16S rRNA gene amplicon sequencing, we analysed the microbial community structure of 33 individuals of the habitat-forming glass sponge Vazella pourtalesii, as well as reference seawater, sediment, and biofilm samples. We assessed how two anthropogenic impacts, i.e. habitat destruction by trawling and artificial substrate provision (moorings made of composite plastic), correspond with in situ V. pourtalesii microbiome variability. In addition, we evaluated the role of two bottom fishery closures in preserving sponge-associated microbial diversity on the Scotian Shelf, Canada. Our results illustrate that V. pourtalesii sponges collected from pristine sites within fishery closures contained distinct and taxonomically largely novel microbial communities. At the trawled site we recorded significant quantitative differences in distinct microbial phyla, such as a reduction in Nitrospinae in sponges and environmental references. Individuals of V. pourtalesii growing on the mooring were significantly enriched in Bacteroidetes, Verrucomicrobia and Cyanobacteria in comparison to sponge individuals growing on the natural seabed. Due to a concomitant enrichment of these taxa in the mooring biofilm, we propose that biofilms on artificial substrates may ‘prime’ sponge-associated microbial communities when small sponges settle on such substrates. These observations likely have relevant management implications when considering the increase of artificial substrates in the marine environment, e.g., marine litter, off-shore wind parks, and petroleum platforms

Microbial strategies for survival in the glass sponge Vazella pourtalesii

Few studies have thus far explored the microbiomes of glass sponges (Hexactinellida). The present study seeks to elucidate the composition of the microbiota associated with the glass sponge Vazella pourtalesii and the functional strategies of the main symbionts. We combined microscopic approaches with metagenome-guided microbial genome reconstruction and amplicon community profiling towards this goal. Microscopic imaging revealed that the host and microbial cells appeared within dense biomass patches that are presumably syncytial tissue aggregates. Based on abundances in amplicon libraries and metagenomic data, SAR324 bacteria, Crenarchaeota, Patescibacteria and Nanoarchaeota were identified as abundant members of the V. pourtalesii microbiome and their genomic potentials were thus analyzed in detail. A general pattern emerged in that the V. pourtalesii symbionts had very small genome sizes in the range of 0.5-2.2 Mb and low GC contents, even below those of seawater relatives. Based on functional analyses of metagenome-assembled genomes (MAGs), we propose two major microbial strategies: the “givers”, namely Crenarchaeota and SAR324, heterotrophs and facultative anaerobes, produce and partly secrete all required amino acids and vitamins. The “takers”, Nanoarchaeota and Patescibacteria, are anaerobes with reduced genomes that tap into the microbial community for resources, e.g., lipids and DNA, likely using pili-like structures. We posit that the existence of microbial cells in sponge syncytia together with the low-oxygen conditions in the seawater environment are factors that shape the unique compositional and functional properties of the microbial community associated with V. pourtalesii . Importance: We investigated the microbial community of V. pourtalesii that forms globally unique, monospecific sponge grounds under low-oxygen conditions on the Scotian Shelf, where it plays a key role for its vulnerable ecosystem. The microbial community was found to be concentrated within biomass patches and is dominated by small cells (<1 μm). MAG analyses showed consistently small genome sizes and low GC contents, which is unusual in comparison to known sponge symbionts. These properties as well as the (facultatively) anaerobic metabolism and a high degree of interdependence between the dominant symbionts regarding amino acid and vitamin synthesis are likely adaptations to the unique conditions within the syncytial tissue of their hexactinellid host and the low-oxygen environment

A Microbial Nitrogen Engine Modulated by Bacteriosyncytia in Hexactinellid Sponges: Ecological Implications for Deep-Sea Communities

Hexactinellid sponges are common in the deep sea, but their functional integration into those ecosystems remains poorly understood. The phylogenetically related species Schaudinnia rosea and Vazella pourtalesii were herein incubated for nitrogen and phosphorous, returning markedly different nutrient fluxes. Transmission electron microscopy (TEM) revealed S. rosea to host a low abundance of extracellular microbes, while Vazella pourtalesii showed higher microbial abundance and hosted most microbes within bacteriosyncytia, a novel feature for Hexactinellida. Amplicon sequences of the microbiome corroborated large between-species differences, also between the sponges and the seawater of their habitats. Metagenome-assembled genome of the V. pourtalesii microbiota revealed genes coding for enzymes operating in nitrification, denitrification, dissimilatory nitrate reduction to ammonium, nitrogen fixation, and ammonia/ammonium assimilation. In the nitrification and denitrification pathways some enzymes were missing, but alternative bridging routes allow the microbiota to close a N cycle in the holobiont. Interconnections between aerobic and anaerobic pathways may facilitate the sponges to withstand the low-oxygen conditions of deep-sea habitats. Importantly, various N pathways coupled to generate ammonium, which, through assimilation, fosters the growth of the sponge microbiota. TEM showed that the farmed microbiota is digested by the sponge cells, becoming an internal food source. This microbial farming demands more ammonium that can be provided internally by the host sponges and some 2.6 million kg of ammonium from the seawater become annually consumed by the aggregations of V. pourtalesii. Such ammonium removal is likely impairing the development of the free-living bacterioplankton and the survival chances of other sponge species that feed on bacterioplankton. Such nutritional competitive exclusion would favor the monospecific character of the V. pourtalesii aggregations. These aggregations also affect the surrounding environment through an annual release of 27.3 million kg of nitrite and, in smaller quantities, of nitrate and phosphate. The complex metabolic integration among the microbiota and the sponge suggests that the holobiont depends critically on the correct functioning of its N-driven microbial engine. The metabolic intertwining is so delicate that it changed after moving the sponges out of their habitat for a few days, a serious warning on the conservation needs of these sponge aggregations

Expression of miRNA-106b in conventional renal cell carcinoma is a potential marker for prediction of early metastasis after nephrectomy

<p>Abstract</p> <p>Background</p> <p>MicroRNAs are endogenously expressed regulatory noncoding RNAs. Previous studies have shown altered expression levels of several microRNAs in renal cell carcinoma.</p> <p>Methods</p> <p>We examined the expression levels of selected microRNAs in 38 samples of conventional renal cell carcinoma (RCC) and 10 samples of non-tumoral renal parenchyma using TaqMan real-time PCR method.</p> <p>Results</p> <p>The expression levels of miRNA-155 (p < 0.0001), miRNA-210 (p < 0.0001), miRNA-106a (p < 0.0001) and miRNA-106b (p < 0.0001) were significantly over-expressed in tumor tissue, whereas the expression of miRNA-141 (p < 0.0001) and miRNA-200c (p < 0.0001) were significantly decreased in RCC samples. There were no significant differences between expression levels of miRNA-182 and miRNA-200b in tumor samples and renal parenchyma. Our data suggest that expression levels of miRNA-106b are significantly lower in tumors of patients who developed metastasis (p = 0.030) and miR-106b is a potential predictive marker of early metastasis after nephrectomy in RCC patients (long-rank p = 0.032).</p> <p>Conclusions</p> <p>We have confirmed previous observations obtained by miRNA microarray analysis using standardized real-time PCR method. For the first time, we have identified a prognostic significance of miRNA-106b, which, after validation on a larger group of patients, maybe useful as a promising biomarker in patients with RCC.</p