Pyrosequencing Reveals Diverse and Distinct Sponge-Specific Microbial Communities in Sponges from a Single Geographical Location in Irish Waters

Marine sponges are host to numerically vast and phylogenetically diverse bacterial communities, with 26 major phyla to date having been found in close association with sponge species worldwide. Analyses of these microbial communities have revealed many sponge-specific novel genera and species. These endosymbiotic microbes are believed to play significant roles in sponge physiology including the production of an array of bioactive secondary metabolites. Here, we report on the use of culture-based and culture-independent (pyrosequencing) techniques to elucidate the bacterial community profiles associated with the marine sponges Raspailia ramosa and Stelligera stuposa sampled from a single geographical location in Irish waters and with ambient seawater. To date, little is known about the microbial ecology of sponges of these genera. Culture isolation grossly underestimated sponge-associated bacterial diversity. Four bacterial phyla (Actinobacteria, Bacteroidetes, Firmicutes, Proteobacteria) were represented amongst ~200 isolates, compared with ten phyla found using pyrosequencing. Long average read lengths of ~430 bp (V1-V3 region of 16S rRNA gene) allowed for robust resolution of sequences to genus level. Bacterial OTUs (2,109 total), at 95% sequence similarity, from ten bacterial phyla were recovered from R. ramosa, 349 OTUs were identified in S. stuposa representing eight phyla, while 533 OTUs from six phyla were found in surrounding seawater. Bacterial communities differed significantly between sponge species and the seawater. Analysis of the data for sponge-specific taxa revealed that 2.8% of classified reads from the sponge R. ramosa can be defined as sponge-specific, while 26% of S. stuposa sequences represent sponge-specific bacteria. Novel sponge-specific clusters were identified, whereas the majority of previously reported sponge-specific clusters (e.g. Poribacteria) were absent from these sponge species. This deep and robust analysis provides further evidence that the microbial communities associated with marine sponge species are highly diverse and divergent from one another and appear to be host-selected through as yet unknown processes.     

Physiology, phylogeny and in situ evidence for bacterial and archaeal nitrifiers in the marine sponge Aplysina aerophoba

The potential for nitrification in the Mediterranean sponge Aplysina aerophoba was assessed using a combined physiological and molecular approach. Nitrate excretion rates in whole sponges reached values of up to 344 nmol g(-1) dry weight (wt) h(-1) (unstimulated) and 1325 nmol g(-1) dry wt h(-1) (stimulated). Addition of nitrapyrin, a nitrification-specific inhibitor, effectively inhibited nitrate excretion. Ammonium was taken up by sponges in spring and excreted in fall, the sponges thus serving as either an ammonium sink or ammonium source. Nitrosospira cluster 1 and Crenarchaeota group I.1A 16S rRNA and amoA genes were recovered from A. aerophoba and other sponges from different world's oceans. The archaeal 16S rRNA genes formed a sponge-specific subcluster, indicating that their representatives are members of the stable microbial community of sponges. On the other hand, clustering was not evident for Nitrosospira rRNA genes which is consistent with their presence in sediment and seawater samples. The presence of both Nitrosospira cluster 1 and crenarchaeal group 1 phylotypes in sponge tissue was confirmed using fluorescently labelled 16S rRNA gene probes. This study contributes to an ongoing effort to link microbial diversity with metabolic functions in the phylogenetically diverse, elusive and so far uncultivated microbial communities of marine sponges.     

Molecular Microbial Diversity Survey of Sponge Reproductive Stages and Mechanistic Insights into Vertical Transmission of Microbial Symbionts

Many marine sponges, hereafter termed high-microbial-abundance (HMA) sponges, harbor large and complex microbial consortia, including bacteria and archaea, within their mesohyl matrices. To investigate vertical microbial transmission as a strategy to maintain these complex associations, an extensive phylogenetic analysis was carried out with the 16S rRNA gene sequences of reproductive (n = 136) and adult (n = 88) material from five different Caribbean species, as well as all published 16S rRNA gene sequences from sponge offspring (n = 116). The overall microbial diversity, including members of at least 13 bacterial phyla and one archaeal phylum, in sponge reproductive stages is high. In total, 28 vertical-transmission clusters, defined as clusters of phylotypes that are found both in adult sponges and their offspring, were identified. They are distributed among at least 10 bacterial phyla and one archaeal phylum, demonstrating that the complex adult microbial community is collectively transmitted through reproductive stages. Indications of host-species specificity and cospeciation were not observed. Mechanistic insights were provided using a combined electron microscopy and fluorescence in situ hybridization analysis, and an indirect mechanism of vertical transmission via nurse cells is proposed for the oviparous sponge Ectyoplasia ferox. Based on these phylogenetic and mechanistic results, we suggest the following symbiont transmission model: entire microbial consortia are vertically transmitted in sponges. While vertical transmission is clearly present, additional environmental transfer between adult individuals of the same and even different species might obscure possible signals of cospeciation. We propose that associations of HMA sponges with highly sponge-specific microbial communities are maintained by this combination of vertical and horizontal symbiont transmission.     

Biogeography and Host Fidelity of Bacterial Communities in Ircinia spp. from the Bahamas

Research on sponge microbial assemblages has revealed different trends in the geographic variability and specificity of bacterial symbionts. Here, we combined replicated terminal-restriction fragment length polymorphism (T-RFLP) and clone library analyses of 16S rRNA gene sequences to investigate the biogeographic and host-specific structure of bacterial communities in two congeneric and sympatric sponges: Ircinia strobilina, two color morphs of Ircinia felix and ambient seawater. Samples were collected from five islands of the Bahamas separated by 80 to 400 km. T-RFLP profiles revealed significant differences in bacterial community structure among sponge hosts and ambient bacterioplankton. Pairwise statistical comparisons of clone libraries confirmed the specificity of the bacterial assemblages to each host species and differentiated symbiont communities between color morphs of I. felix. Overall, differences in bacterial communities within each host species and morph were unrelated to location. Our results show a high degree of symbiont fidelity to host sponge across a spatial scale of up to 400 km, suggesting that host-specific rather than biogeographic factors play a primary role in structuring and maintaining sponge–bacteria relationships in Ircinia species from the Bahamas.     

Host-specificity among abundant and rare taxa in the sponge microbiome

Microbial communities have a key role in the physiology of the sponge host, and it is therefore essential to understand the stability and specificity of sponge–symbiont associations. Host-specific bacterial associations spanning large geographic distance are widely acknowledged in sponges. However, the full spectrum of specificity remains unclear. In particular, it is not known whether closely related sponges host similar or very different microbiota over wide bathymetric and geographic gradients, and whether specific associations extend to the rare members of the sponge microbiome. Using the ultra-deep Illumina sequencing technology, we conducted a comparison of sponge bacterial communities in seven closely related Hexadella species with a well-resolved host phylogeny, as well as of a distantly related sponge Mycale. These samples spanned unprecedentedly large bathymetric (15–960 m) gradients and varying European locations. In addition, this study included a bacterial community analysis of the local background seawater for both Mycale and the widespread deep-sea taxa Hexadella cf. dedritifera. We observed a striking diversity of microbes associated with the sponges, spanning 47 bacterial phyla. The data did not reveal any Hexadella microbiota co-speciation pattern, but confirmed sponge-specific and species-specific host–bacteria associations, even within extremely low abundant taxa. Oligotyping analysis also revealed differential enrichment preferences of closely related Nitrospira members in closely related sponges species. Overall, these results demonstrate highly diverse, remarkably specific and stable sponge–bacteria associations that extend to members of the rare biosphere at a very fine phylogenetic scale, over significant geographic and bathymetric gradients.     

Pyrosequencing reveals highly diverse and species-specific microbial communities in sponges from the Red Sea

Marine sponges are associated with a remarkable array of microorganisms. Using a tag pyrosequencing technology, this study was the first to investigate in depth the microbial communities associated with three Red Sea sponges, Hyrtios erectus, Stylissa carteri and Xestospongia testudinaria. We revealed highly diverse sponge-associated bacterial communities with up to 1000 microbial operational taxonomic units (OTUs) and richness estimates of up to 2000 species. Altogether, 26 bacterial phyla were detected from the Red Sea sponges, 11 of which were absent from the surrounding sea water and 4 were recorded in sponges for the first time. Up to 100 OTUs with richness estimates of up to 300 archaeal species were revealed from a single sponge species. This is by far the highest archaeal diversity ever recorded for sponges. A non-negligible proportion of unclassified reads was observed in sponges. Our results demonstrated that the sponge-associated microbial communities remained highly consistent in the same sponge species from different locations, although they varied at different degrees among different sponge species. A significant proportion of the tag sequences from the sponges could be assigned to one of the sponge-specific clusters previously defined. In addition, the sponge-associated microbial communities were consistently divergent from those present in the surrounding sea water. Our results suggest that the Red Sea sponges possess highly sponge-specific or even sponge-species-specific microbial communities that are resistant to environmental disturbance, and much of their microbial diversity remains to be explored.     

Sponge-specific clusters revisited: a comprehensive phylogeny of sponge-associated microorganisms

Marine sponges often contain diverse and abundant communities of microorganisms including bacteria, archaea and eukaryotic microbes. Numerous 16S rRNA-based studies have identified putative 'sponge-specific' microbes that are apparently absent from seawater and other (non-sponge) marine habitats. With more than 7500 sponge-derived rRNA sequences (from clone, isolate and denaturing gradient gel electrophoresis data) now publicly available, we sought to determine whether the current notion of sponge-specific sequence clusters remains valid. Comprehensive phylogenetic analyses were performed on the 7546 sponge-derived 16S and 18S rRNA sequences that were publicly available in early 2010. Overall, 27% of all sequences fell into monophyletic, sponge-specific sequence clusters. Such clusters were particularly well represented among the Chloroflexi, Cyanobacteria, 'Poribacteria', Betaproteobacteria and Acidobacteria, and in total were identified in at least 14 bacterial phyla, as well as the Archaea and fungi. The largest sponge-specific cluster, representing the cyanobacterium 'Synechococcus spongiarum', contained 245 sequences from 40 sponge species. These results strongly support the existence of sponge-specific microbes and provide a suitable framework for future studies of rare and abundant sponge symbionts, both of which can now be studied using next-generation sequencing technologies.     

Use of Real-Time qPCR to Quantify Members of the Unculturable Heterotrophic Bacterial Community in a Deep Sea Marine Sponge, <Emphasis Type="Italic">Vetulina</Emphasis> sp

In this report, real-time quantitative PCR (TaqMan qPCR) of the small subunit (SSU) 16S-like rRNA molecule, a universal phylogenetic marker, was used to quantify the relative abundance of individual bacterial members of a diverse, yet mostly unculturable, microbial community from a marine sponge. Molecular phylogenetic analyses of bacterial communities derived from Caribbean Lithistid sponges have shown a wide diversity of microbes that included at least six major subdivisions; however, very little overlap was observed between the culturable and unculturable microbial communities. Based on sequence data of three culture-independent Lithistid-derived representative bacteria, we designed probe/primer sets for TaqMan qPCR to quantitatively characterize selected microbial residents in a Lithistid sponge, Vetulina, metagenome. TaqMan assays included specificity testing, DNA limit of detection analysis, and quantification of specific microbial rRNA sequences such as Nitrospira-like microbes and Actinobacteria up to 172 million copies per microgram per Lithistid sponge metagenome. By contrast, qPCR amplification with probes designed for common previously cultured sponge-associated bacteria in the genera Rheinheimera and Marinomonas and a representative of the CFB group resulted in only minimal detection of the Rheiheimera in total DNA extracted from the sponge. These data verify that a large portion of the microbial community within Lithistid sponges may consist of currently unculturable microorganisms.     

Sponge-Associated Microorganisms: Evolution, Ecology, and Biotechnological Potential

Summary: Marine sponges often contain diverse and abundant microbial communities, including bacteria, archaea, microalgae, and fungi. In some cases, these microbial associates comprise as much as 40% of the sponge volume and can contribute significantly to host metabolism (e.g., via photosynthesis or nitrogen fixation). We review in detail the diversity of microbes associated with sponges, including extensive 16S rRNA-based phylogenetic analyses which support the previously suggested existence of a sponge-specific microbiota. These analyses provide a suitable vantage point from which to consider the potential evolutionary and ecological ramifications of these widespread, sponge-specific microorganisms. Subsequently, we examine the ecology of sponge-microbe associations, including the establishment and maintenance of these sometimes intimate partnerships, the varied nature of the interactions (ranging from mutualism to host-pathogen relationships), and the broad-scale patterns of symbiont distribution. The ecological and evolutionary importance of sponge-microbe associations is mirrored by their enormous biotechnological potential: marine sponges are among the animal kingdom's most prolific producers of bioactive metabolites, and in at least some cases, the compounds are of microbial rather than sponge origin. We review the status of this important field, outlining the various approaches (e.g., cultivation, cell separation, and metagenomics) which have been employed to access the chemical wealth of sponge-microbe associations.     

Sponge-associated microorganisms: evolution, ecology, and biotechnological potential.

Marine sponges often contain diverse and abundant microbial communities, including bacteria, archaea, microalgae, and fungi. In some cases, these microbial associates comprise as much as 40% of the sponge volume and can contribute significantly to host metabolism (e.g., via photosynthesis or nitrogen fixation). We review in detail the diversity of microbes associated with sponges, including extensive 16S rRNA-based phylogenetic analyses which support the previously suggested existence of a sponge-specific microbiota. These analyses provide a suitable vantage point from which to consider the potential evolutionary and ecological ramifications of these widespread, sponge-specific microorganisms. Subsequently, we examine the ecology of sponge-microbe associations, including the establishment and maintenance of these sometimes intimate partnerships, the varied nature of the interactions (ranging from mutualism to host-pathogen relationships), and the broad-scale patterns of symbiont distribution. The ecological and evolutionary importance of sponge-microbe associations is mirrored by their enormous biotechnological potential: marine sponges are among the animal kingdom's most prolific producers of bioactive metabolites, and in at least some cases, the compounds are of microbial rather than sponge origin. We review the status of this important field, outlining the various approaches (e.g., cultivation, cell separation, and metagenomics) which have been employed to access the chemical wealth of sponge-microbe associations.     

A Cross-Taxon Analysis of Insect-Associated Bacterial Diversity

Although it is well known that plants and animals harbor microbial symbionts that can influence host traits, the factors regulating the structure of these microbial communities often remain largely undetermined. This is particularly true for insect-associated microbial communities, as few cross-taxon comparisons have been conducted to date. To address this knowledge gap and determine how host phylogeny and ecology affect insect-associated microbial communities, we collected 137 insect specimens representing 39 species, 28 families, and 8 orders, and characterized the bacterial communities associated with each specimen via 16S rRNA gene sequencing. Bacterial taxa within the phylum Proteobacteria were dominant in nearly all insects sampled. On average, the insect-associated bacterial communities were not very diverse, with individuals typically harboring fewer than 8 bacterial phylotypes. Bacterial communities also tended to be dominated by a single phylotype; on average, the most abundant phylotype represented 54.7% of community membership. Bacterial communities were significantly more similar among closely related insects than among less-related insects, a pattern driven by within-species community similarity but detected at every level of insect taxonomy tested. Diet was a poor predictor of bacterial community composition. Individual insect species harbored remarkably unique communities: the distribution of 69.0% of bacterial phylotypes was limited to unique insect species, whereas only 5.7% of phylotypes were detected in more than five insect species. Together these results suggest that host characteristics strongly regulate the colonization and assembly of bacterial communities across insect lineages, patterns that are driven either by co-evolution between insects and their symbionts or by closely related insects sharing conserved traits that directly select for similar bacterial communities.     

Thermal stress responses in the bacterial biosphere of the Great Barrier Reef sponge,Rhopaloeides odorabile

Marine sponges are diverse, abundant and provide a crucial coupling point between benthic and pelagic habitats due to their high filtration rates. They also harbour extensive microbial communities, with many microbial phylotypes found exclusively in sponge hosts and not in the seawater or surrounding environment, i.e. so‐called sponge‐specific clusters (SCs) or sponge‐ and coral‐specific clusters (SCCs). We employed DNA (16S rRNA gene) and RNA (16S rRNA)‐based amplicon pyrosequencing to investigate the effects of sublethal thermal stress on the bacterial biosphere of the Great Barrier Reef sponge Rhopaloeides odorabile. A total of 8381 operational taxonomic units (OTUs) (97% sequence similarity) were identified, affiliated with 32 bacterial phyla from seawater samples, 23 bacterial phyla from sponge DNA extracts and 18 bacterial phyla from sponge RNA extracts. Sublethal thermal stress (31°C) had no effect on the present and/or active portions of the R. odorabile bacterial community but a shift in the bacterial assemblage was observed in necrotic sponges. Over two‐thirds of DNA and RNA sequences could be assigned to previously defined SCs/SCCs in healthy sponges whereas only 12% of reads from necrotic sponges could be assigned to SCs/SCCs. A rapid decline in host health over a 1°C temperature increment suggests that sponges such as R. odorabile may be highly vulnerable to the effects of global climate change.     

Spatial distribution of sponge-associated bacteria in the Mediterranean sponge Tethya aurantium

The local distribution of the bacterial community associated with the marine sponge Tethya aurantium Pallas 1766 was studied. Distinct bacterial communities were found to inhabit the endosome and cortex. Clear differences in the associated bacterial populations were demonstrated by denaturing gradient gel electrophoresis (DGGE) and analysis of 16S rRNA gene clone libraries. Specifically associated phylotypes were identified for both regions: a new phylotype of Flexibacteria was recovered only from the sponge cortex, while Synechococcus species were present mainly in the sponge endosome. Light conduction via radiate spicule bundles conceivably facilitates the unusual association of Cyanobacteria with the sponge endosome. Furthermore, a new monophyletic cluster of sponge-derived 16S rRNA gene sequences related to the Betaproteobacteria was identified using analysis of 16S rRNA gene clone libraries. Members of this cluster were specifically associated with both cortex and endosome of T. aurantium.     

Phylogenetic diversity of bacteria associated with the endemic freshwater sponge Lubomirskia baicalensis

In this study, for the first time the diversity of bacteria associated with the endemic freshwater sponge Lubomirskia baicalensis collected from the Sousern Basin of Lake Baikal was investigated employing cultivation-independent approaches. In total, 102 bacterial 16S rRNA clones were screened using restriction fragment length polymorphism (RFLP) and 30 were selected for sequencing. BLASTN and phylogenetic analysis based on near full length 16S rDNA sequences showed that 22 operational taxonomic units (OTUs) were clustered in six known phyla: Actinobacteria (8 OTUs), alpha-Proteobacteria (4 OTUs), beta-Proteobacteria (4 OTUs), Verrucomicrobia (4 OTUs), Nitrospiracea (1 OTU) and Bacteroidetes (1 OTU). Remarkably all phylotypes were affiliated to uncultured microorganisms, however, all alpha-Proteobacteria sequences were closely related to bacteria derived from the freshwater sponge Spongilla lacustris. Our results reveal a high diversity in the L. baicalensis bacterial community and provide an insight into microbial ecology and diversity within freshwater sponges inhabiting the ancient Lake Baikal ecosystem.     

A molecular systematic survey of cultured microbial associates of deep-water marine invertebrates

A taxonomic survey was conducted to determine the microbial diversity held within the Harbor Branch Oceanographic Marine Microbial Culture Collection (HBMMCC). The collection consists of approximately 17,000 microbial isolates, with 11,000 from a depth of greater than 150 ft seawater. A total of 2273 heterotrophic bacterial isolates were inventoried using the DNA fingerprinting technique amplified rDNA restriction analysis on approximately 750-800 base pairs (bp) encompassing hypervariable regions in the 5' portion of the small subunit (SSU) 16S rRNA gene. Restriction fragment length polymorphism patterns obtained from restriction digests with RsaI, HaeIII, and HhaI were used to infer taxonomic similarity. SSU 16S rDNA fragments were sequenced from a total of 356 isolates for more definitive taxonomic analysis. Sequence results show that this subset of the HBMMCC contains 224 different phylotypes from six major bacterial clades (Proteobacteria (Alpha, Beta, Gamma), Cytophaga, Flavobacteria, and Bacteroides (CFB), Gram + high GC content, Gram + low GC content). The 2273 microorganisms surveyed encompass 834 alpha-Proteobacteria (representing 60 different phylotypes), 25 beta-Proteobacteria (3 phylotypes), 767 gamma-Proteobacteria (77 phylotypes), 122 CFB (17 phylotypes), 327 Gram + high GC content (43 phylotypes), and 198 Gram + low GC content isolates (24 phylotypes). Notably, 11 phylotypes were < or =93% similar to the closest sequence match in the GenBank database even after sequencing a larger portion of the 16S rRNA gene (approximately 1400 bp), indicating the likely discovery of novel microbial taxa. Furthermore, previously reported "uncultured" microbes, such as sponge-specific isolates, are part of the HBMMCC. The results of this research will be available online as a searchable taxonomic database (www.hboi.edu/dbmr/dbmr_hbmmd.html).     

Stability of Sponge-Associated Bacteria over Large Seasonal Shifts in Temperature and Irradiance

Complex microbiomes reside in marine sponges and consist of diverse microbial taxa, including functional guilds that may contribute to host metabolism and coastal marine nutrient cycles. Our understanding of these symbiotic systems is based primarily on static accounts of sponge microbiota, while their temporal dynamics across seasonal cycles remain largely unknown. Here, we investigated temporal variation in bacterial symbionts of three sympatric sponges (Ircinia spp.) over 1.5 years in the northwestern (NW) Mediterranean Sea, using replicated terminal restriction fragment length polymorphism (T-RFLP) and clone library analyses of bacterial 16S rRNA gene sequences. Bacterial symbionts in Ircinia spp. exhibited host species-specific structure and remarkable stability throughout the monitoring period, despite large fluctuations in temperature and irradiance. In contrast, seawater bacteria exhibited clear seasonal shifts in community structure, indicating that different ecological constraints act on free-living and on symbiotic marine bacteria. Symbiont profiles were dominated by persistent, sponge-specific bacterial taxa, notably affiliated with phylogenetic lineages capable of photosynthesis, nitrite oxidation, and sulfate reduction. Variability in the sponge microbiota was restricted to rare symbionts and occurred most prominently in warmer seasons, coincident with elevated thermal regimes. Seasonal stability of the sponge microbiota supports the hypothesis of host-specific, stable associations between bacteria and sponges. Further, the core symbiont profiles revealed in this study provide an empirical baseline for diagnosing abnormal shifts in symbiont communities. Considering that these sponges have suffered recent, episodic mass mortalities related to thermal stresses, this study contributes to the development of model sponge-microbe symbioses for assessing the link between symbiont fluctuations and host health.     

Metagenomic Analysis Reveals Diverse Polyketide Synthase Gene Clusters in Microorganisms Associated with the Marine Sponge Discodermia dissoluta

Sponge-associated bacteria are thought to produce many novel bioactive compounds, including polyketides. PCR amplification of ketosynthase domains of type I modular polyketide synthases (PKS) from the microbial community of the marine sponge Discodermia dissoluta revealed great diversity and a novel group of sponge-specific PKS ketosynthase domains. Metagenomic libraries totaling more than four gigabases of bacterial genomes associated with this sponge were screened for type I modular PKS gene clusters. More than 90% of the clones in total sponge DNA libraries represented bacterial DNA inserts, and 0.7% harbored PKS genes. The majority of the PKS hybridizing clones carried small PKS clusters of one to three modules, although some clones encoded large multimodular PKSs (more than five modules). The most abundant large modular PKS appeared to be encoded by a bacterial symbiont that made up <1% of the sponge community. Sequencing of this PKS revealed 14 modules that, if expressed and active, is predicted to produce a multimethyl-branched fatty acid reminiscent of mycobacterial lipid components. Metagenomic libraries made from fractions enriched for unicellular or filamentous bacteria differed significantly, with the latter containing numerous nonribosomal peptide synthetase (NRPS) and mixed NRPS-PKS gene clusters. The filamentous bacterial community of D. dissoluta consists mainly of Entotheonella spp., an unculturable sponge-specific taxon previously implicated in the biosynthesis of bioactive peptides.     

Metagenomic analysis reveals diverse polyketide synthase gene clusters in microorganisms associated with the marine sponge Discodermia dissoluta

Sponge-associated bacteria are thought to produce many novel bioactive compounds, including polyketides. PCR amplification of ketosynthase domains of type I modular polyketide synthases (PKS) from the microbial community of the marine sponge Discodermia dissoluta revealed great diversity and a novel group of sponge-specific PKS ketosynthase domains. Metagenomic libraries totaling more than four gigabases of bacterial genomes associated with this sponge were screened for type I modular PKS gene clusters. More than 90% of the clones in total sponge DNA libraries represented bacterial DNA inserts, and 0.7% harbored PKS genes. The majority of the PKS hybridizing clones carried small PKS clusters of one to three modules, although some clones encoded large multimodular PKSs (more than five modules). The most abundant large modular PKS appeared to be encoded by a bacterial symbiont that made up < 1% of the sponge community. Sequencing of this PKS revealed 14 modules that, if expressed and active, is predicted to produce a multimethyl-branched fatty acid reminiscent of mycobacterial lipid components. Metagenomic libraries made from fractions enriched for unicellular or filamentous bacteria differed significantly, with the latter containing numerous nonribosomal peptide synthetase (NRPS) and mixed NRPS-PKS gene clusters. The filamentous bacterial community of D. dissoluta consists mainly of Entotheonella spp., an unculturable sponge-specific taxon previously implicated in the biosynthesis of bioactive peptides.     

Functional characterization and taxonomic classification of novel gammaproteobacterial diversity in sponges

Sponges harbour exceptionally diverse microbial communities, whose members are largely uncultured. The class Gammaproteobacteria often dominates the microbial communities of various sponge species, but most of its diversity remains functional and taxonomically uncharacterised. Here we reconstructed and characterised 32 metagenome-assembled genomes (MAGs) derived from three sponge species. These MAGs represent ten novel species and belong to seven orders, of which one is new. We propose nomenclature for all these taxa. These new species comprise sponge-specific bacteria with varying levels of host specificity. Functional gene profiling highlights significant differences in metabolic capabilities across the ten species, though each also often exhibited a large degree of metabolic diversity involving various nitrogen- and sulfur-based compounds. The genomic features of the ten species suggest they have evolved to form symbiotic interaction with their hosts or are well-adapted to survive within the sponge environment. These Gammaproteobacteria are proposed to scavenge substrates from the host environment, including metabolites or cellular components of the sponge. Their diverse metabolic capabilities may allow for efficient cycling of organic matter in the sponge environment, potentially to the benefit of the host and other symbionts.