In-Situ Biofloc Affects the Core Prokaryotes Community Composition in Gut and Enhances Growth of Nile Tilapia (Oreochromis niloticus).

Biofloc technology is commonly applied in intensive tilapia (Oreochromis niloticus) culture to maintain water quality, supply the fish with extra protein, and improve fish growth. However, the effect of dietary supplementation of processed biofloc on the gut prokaryotic (bacteria and archaea) community composition of tilapia is not well understood. In this study one recirculating aquaculture system was used to test how biofloc, including in-situ biofloc, dietary supplementation of ex-situ live or dead biofloc, influence fish gut prokaryotic community composition and growth performance in comparison to a biofloc-free control treatment. A core gut prokaryotic community was identified among all treatments by analyzing the temporal variations in gut prokaryotes. In-situ produced biofloc significantly increased the prokaryotic diversity in the gut by reducing the relative abundance of dominant Cetobacterium and increasing the relative abundance of potentially beneficial bacteria. The in-situ biofloc delivered a unique prokaryotic community in fish gut, while dietary supplementation of tilapias with 5% and 10% processed biofloc (live or dead) only changed the relative abundance of minor prokaryotic taxa outside the gut core microbiota. The modulatory effect of in-situ biofloc on tilapia gut microbiota was associated with the distinct microbial community in the biofloc water and undisturbed biofloc. The growth-promoting effect on tilapia was only detected in the in-situ biofloc treatment, while dietary supplementation of processed biofloc had no effect on fish growth performance as compared to the control treatment.

Bacterial diversity in different outdoor pilot plant photobioreactor types during production of the microalga Nannochloropsis sp. CCAP211/78.

As large-scale outdoor production cannot be done in complete containment, cultures are (more) open for bacteria, which may affect the productivity and stability of the algae production process. We investigated the bacterial diversity in two indoor reactors and four pilot-scale outdoor reactors for the production of Nannochloropsis sp. CCAP211/78 spanning four months of operation from July to October. Illumina sequencing of 16S rRNA gene amplicons demonstrated that a wide variety of bacteria were present in all reactor types, with predominance of Bacteroidetes and Alphaproteobacteria. Bacterial communities were significantly different between all reactor types (except between the horizontal tubular reactor and the vertical tubular reactor) and also between runs in each reactor. Bacteria common to the majority of samples included one member of the Saprospiraceae family and one of the NS11-12_marine group (both Bacteroidetes). Hierarchical clustering analysis revealed two phases during the cultivation period separated by a major shift in bacterial community composition in the horizontal tubular reactor, the vertical tubular reactor and the raceway pond with a strong decrease of the Saprospiraceae and NS11-12_marine group that initially dominated the bacterial communities. Furthermore, we observed a less consistent pattern of bacterial taxa appearing in different reactors and runs, most of which belonging to the classes Deltaproteobacteria and Flavobacteriia. In addition, canonical correspondence analysis showed that the bacterial community composition was significantly correlated with the nitrate concentration. This study contributes to our understanding of bacterial diversity and composition in different types of outdoor reactors exposed to a range of dynamic biotic and abiotic factors. Key points • Reactor types had significantly different bacterial communities except HT and VT • The inoculum source and physiochemical factors together affect bacterial community • The bacterial family Saprospiraceae is positively correlated to microalgal growth.

Sponge holobionts shift their prokaryotic communities and antimicrobial activity from shallow to lower mesophotic depths.

In this study, we used 16S rRNA gene amplicon sequencing to investigate prokaryotic community composition of the Caribbean sponges Xestospongia muta and Agelas sventres from three depth ranges: < 30 m (shallow), 30-60 m (upper mesophotic), and 60-90 m (lower mesophotic). The prokaryotic community in shallow samples of X. muta was enriched in Cyanobacteria, Chloroflexota, and Crenarchaeota compared to samples from mesophotic depths, while mesophotic samples of X. muta were enriched in Acidobacteriota. For A. sventres, relative abundance of Acidobacteriota, Chloroflexota, and Gammaproteobacteria was higher in shallow samples, while Proteobacteria and Crenarchaeota were enriched in mesophotic A. sventres samples. Antimicrobial activity was evaluated by screening crude extracts of sponges against a set of Gram-positive and Gram-negative bacteria, a yeast, and an oomycete. Antibacterial activities from crude extracts of shallow sponge individuals were generally higher than observed from mesophotic individuals, that showed limited or no antibacterial activities. Conversely, the highest anti-oomycete activity was found from crude extracts of X. muta individuals from lower mesophotic depth, but without a clear pattern across the depth gradient. These results indicate that sponge-associated prokaryotic communities and the antimicrobial activity of sponges change within species across a depth gradient from shallow to mesophotic depth.

Diversity of Bacterial Secondary Metabolite Biosynthetic Gene Clusters in Three Vietnamese Sponges.

Recent reviews have reinforced sponge-associated bacteria as a valuable source of structurally diverse secondary metabolites with potent biological properties, which makes these microbial communities promising sources of new drug candidates. However, the overall diversity of secondary metabolite biosynthetic potential present in bacteria is difficult to access due to the fact that the majority of bacteria are not readily cultured in the laboratory. Thus, use of cultivation-independent approaches may allow accessing "silent" and "cryptic" secondary metabolite biosynthetic gene clusters present in bacteria that cannot yet be cultured. In the present study, we investigated the diversity of secondary metabolite biosynthetic gene clusters (BGCs) in metagenomes of bacterial communities associated with three sponge species: Clathria reinwardti, Rhabdastrella globostellata, and Spheciospongia sp. The results reveal that the three metagenomes contain a high number of predicted BGCs, ranging from 282 to 463 BGCs per metagenome. The types of BGCs were diverse and represented 12 different cluster types. Clusters predicted to encode fatty acid synthases and polyketide synthases (PKS) were the most dominant BGC types, followed by clusters encoding synthesis of terpenes and bacteriocins. Based on BGC sequence similarity analysis, 363 gene cluster families (GCFs) were identified. Interestingly, no GCFs were assigned to pathways responsible for the production of known compounds, implying that the clusters detected might be responsible for production of several novel compounds. The KS gene sequences from PKS clusters were used to predict the taxonomic origin of the clusters involved. The KS sequences were related to 12 bacterial phyla with Actinobacteria, Proteobacteria, and Firmicutes as the most predominant. At the genus level, the KSs were most related to those found in the genera Mycolicibacterium, Mycobacterium, Burkholderia, and Streptomyces. Phylogenetic analysis of KS sequences resulted in detection of two known 'sponge-specific' BGCs, i.e., SupA and SwfA, as well as a new 'sponge-specific' cluster related to fatty acid synthesis in the phylum Candidatus Poribacteria and composed only by KS sequences of the three sponge-associated bacterial communities assessed here.

Diversity and Antimicrobial Activity of Vietnamese Sponge-Associated Bacteria.

This study aimed to assess the diversity and antimicrobial activity of cultivable bacteria associated with Vietnamese sponges. In total, 460 bacterial isolates were obtained from 18 marine sponges. Of these, 58.3% belonged to Proteobacteria, 16.5% to Actinobacteria, 18.0% to Firmicutes, and 7.2% to Bacteroidetes. At the genus level, isolated strains belonged to 55 genera, of which several genera, such as Bacillus, Pseudovibrio, Ruegeria, Vibrio, and Streptomyces, were the most predominant. Culture media influenced the cultivable bacterial composition, whereas, from different sponge species, similar cultivable bacteria were recovered. Interestingly, there was little overlap of bacterial composition associated with sponges when the taxa isolated were compared to cultivation-independent data. Subsequent antimicrobial assays showed that 90 isolated strains exhibited antimicrobial activity against at least one of seven indicator microorganisms. From the culture broth of the isolated strain with the strongest activity (Bacillus sp. M1_CRV_171), four secondary metabolites were isolated and identified, including cyclo(L-Pro-L-Tyr) (1), macrolactin A (2), macrolactin H (3), and 15,17-epoxy-16-hydroxy macrolactin A (4). Of these, compounds 2-4 exhibited antimicrobial activity against a broad spectrum of reference microorganisms.

Bioactivity Screening and Gene-Trait Matching across Marine Sponge-Associated Bacteria.

Marine sponges harbor diverse microbial communities that represent a significant source of natural products. In the present study, extracts of 21 sponge-associated bacteria were screened for their antimicrobial and anticancer activity, and their genomes were mined for secondary metabolite biosynthetic gene clusters (BGCs). Phylogenetic analysis assigned the strains to four major phyla in the sponge microbiome, namely Proteobacteria, Actinobacteria, Bacteroidetes, and Firmicutes. Bioassays identified one extract with anti-methicillin-resistant Staphylococcus aureus (MRSA) activity, and more than 70% of the total extracts had a moderate to high cytotoxicity. The most active extracts were derived from the Proteobacteria and Actinobacteria, prominent for producing bioactive substances. The strong bioactivity potential of the aforementioned strains was also evident in the abundance of BGCs, which encoded mainly beta-lactones, bacteriocins, non-ribosomal peptide synthetases (NRPS), terpenes, and siderophores. Gene-trait matching was performed for the most active strains, aiming at linking their biosynthetic potential with the experimental results. Genetic associations were established for the anti-MRSA and cytotoxic phenotypes based on the similarity of the detected BGCs with BGCs encoding natural products with known bioactivity. Overall, our study highlights the significance of combining in vitro and in silico approaches in the search of novel natural products of pharmaceutical interest.

Comparative genomic analysis of Flavobacteriaceae: insights into carbohydrate metabolism, gliding motility and secondary metabolite biosynthesis.

BACKGROUND: Members of the bacterial family Flavobacteriaceae are widely distributed in the marine environment and often found associated with algae, fish, detritus or marine invertebrates. Yet, little is known about the characteristics that drive their ubiquity in diverse ecological niches. Here, we provide an overview of functional traits common to taxonomically diverse members of the family Flavobacteriaceae from different environmental sources, with a focus on the Marine clade. We include seven newly sequenced marine sponge-derived strains that were also tested for gliding motility and antimicrobial activity. RESULTS: Comparative genomics revealed that genome similarities appeared to be correlated to 16S rRNA gene- and genome-based phylogeny, while differences were mostly associated with nutrient acquisition, such as carbohydrate metabolism and gliding motility. The high frequency and diversity of genes encoding polymer-degrading enzymes, often arranged in polysaccharide utilization loci (PULs), support the capacity of marine Flavobacteriaceae to utilize diverse carbon sources. Homologs of gliding proteins were widespread among all studied Flavobacteriaceae in contrast to members of other phyla, highlighting the particular presence of this feature within the Bacteroidetes. Notably, not all bacteria predicted to glide formed spreading colonies. Genome mining uncovered a diverse secondary metabolite biosynthesis arsenal of Flavobacteriaceae with high prevalence of gene clusters encoding pathways for the production of antimicrobial, antioxidant and cytotoxic compounds. Antimicrobial activity tests showed, however, that the phenotype differed from the genome-derived predictions for the seven tested strains. CONCLUSIONS: Our study elucidates the functional repertoire of marine Flavobacteriaceae and highlights the need to combine genomic and experimental data while using the appropriate stimuli to unlock their uncharted metabolic potential.

Marine Rare Actinomycetes: A Promising Source of Structurally Diverse and Unique Novel Natural Products.

Rare actinomycetes are prolific in the marine environment; however, knowledge about their diversity, distribution and biochemistry is limited. Marine rare actinomycetes represent a rather untapped source of chemically diverse secondary metabolites and novel bioactive compounds. In this review, we aim to summarize the present knowledge on the isolation, diversity, distribution and natural product discovery of marine rare actinomycetes reported from mid-2013 to 2017. A total of 97 new species, representing 9 novel genera and belonging to 27 families of marine rare actinomycetes have been reported, with the highest numbers of novel isolates from the families Pseudonocardiaceae, Demequinaceae, Micromonosporaceae and Nocardioidaceae. Additionally, this study reviewed 167 new bioactive compounds produced by 58 different rare actinomycete species representing 24 genera. Most of the compounds produced by the marine rare actinomycetes present antibacterial, antifungal, antiparasitic, anticancer or antimalarial activities. The highest numbers of natural products were derived from the genera Nocardiopsis, Micromonospora, Salinispora and Pseudonocardia. Members of the genus Micromonospora were revealed to be the richest source of chemically diverse and unique bioactive natural products.

Cultivation of Sponge-Associated Bacteria from Agelas sventres and Xestospongia muta Collected from Different Depths.

Sponge-associated bacteria have been mostly cultured from shallow water (≤30 m) sponges, whereas only few studies targeted specimens from below 30 m. This study assessed the cultivability of bacteria from two marine sponges Xestospongia muta and Agelas sventres collected from shallow (<30 m), upper mesophotic (30-60 m), and lower mesophotic (60-90 m) reefs. Sponge-associated bacteria were cultivated on six different media, and replicate plates were used to pick individual colonies or to recover the entire biomass. Prokaryotic community analysis was conducted using Illumina MiSeq sequencing of 16S rRNA gene amplicons. A total of 144 bacterial isolates were picked following a colony morphology coding scheme and subsequently identified by 16S rRNA gene sequence analysis. Sponge individuals at each depth-range harboured specific cultivable bacteria that were not retrieved from specimens collected at other depths. However, there were substantial differences in the number of colonies obtained for replicate sponges of the same species. In addition, source of inoculum and cultivation medium had more impact on the cultured prokaryotic community than sample collection depth. This suggests that the "plate count anomaly" is larger than differences in sponge-associated prokaryotic community composition related to depth.

The multi-omics promise in context: from sequence to microbial isolate.

The numbers and diversity of microbes in ecosystems within and around us is unmatched, yet most of these microorganisms remain recalcitrant to in vitro cultivation. Various high-throughput molecular techniques, collectively termed multi-omics, provide insights into the genomic structure and metabolic potential as well as activity of complex microbial communities. Nonetheless, pure or defined cultures are needed to (1) decipher microbial physiology and thus test multi-omics-based ecological hypotheses, (2) curate and improve database annotations and (3) realize novel applications in biotechnology. Cultivation thus provides context. In turn, we here argue that multi-omics information awaits integration into the development of novel cultivation strategies. This can build the foundation for a new era of omics information-guided microbial cultivation technology and reduce the inherent trial-and-error search space. This review discusses how information that can be extracted from multi-omics data can be applied for the cultivation of hitherto uncultured microorganisms. Furthermore, we summarize groundbreaking studies that successfully translated information derived from multi-omics into specific media formulations, screening techniques and selective enrichments in order to obtain novel targeted microbial isolates. By integrating these examples, we conclude with a proposed workflow to facilitate future omics-aided cultivation strategies that are inspired by the microbial complexity of the environment.

Geochemical Parameters and Reductive Dechlorination Determine Aerobic Cometabolic vs Aerobic Metabolic Vinyl Chloride Biodegradation at Oxic/Anoxic Interface of Hyporheic Zones.

Hyporheic zones mediate vinyl chloride (VC) biodegradation in groundwater discharging into surface waters. At the oxic/anoxic interface (OAI) of hyporheic zones subjected to redox oscillations, VC is degraded via coexisting aerobic ethenotrophic and anaerobic reductive dechlorination pathways. However, the identity of aerobic VC degradation pathways (cometabolic vs metabolic) and their interactions with reductive dechlorination in relation to riverbed sediment geochemistry remain ill-defined. We addressed this using microcosms containing OAI sediments incubated under fluctuating oxic/anoxic atmosphere. Under oxic atmosphere, aerobic metabolic VC oxidation was absent in sediments with high total organic carbon (TOC) and VC was reductively dechlorinated to ethene. Ethene was oxidized by ethenotrophs that can degrade VC cometabolically. Contrastingly, VC was metabolically oxidized by ethenotrophs in low-TOC sediments with low reductive dechlorination potential. Accordingly, enrichment and isolation of metabolic VC-oxidizing ethenotrophs was successful only from the low-TOC sediment. Sequence analysis of etnE genes from the microcosms as well phylogenetic typing of the isolates showed that ethenotrophs in the sediments were facultative anaerobic Proteobacteria capable of coping with OAI-associated redox fluctuations. Our results suggest that local sediment heterogeneity supports/selects divergent VC degradation processes at the OAI and that high reductive dechlorination potential suppresses development of aerobic metabolic VC oxidation potential.

Bioprospecting Sponge-Associated Microbes for Antimicrobial Compounds.

Sponges are the most prolific marine organisms with respect to their arsenal of bioactive compounds including antimicrobials. However, the majority of these substances are probably not produced by the sponge itself, but rather by bacteria or fungi that are associated with their host. This review for the first time provides a comprehensive overview of antimicrobial compounds that are known to be produced by sponge-associated microbes. We discuss the current state-of-the-art by grouping the bioactive compounds produced by sponge-associated microorganisms in four categories: antiviral, antibacterial, antifungal and antiprotozoal compounds. Based on in vitro activity tests, identified targets of potent antimicrobial substances derived from sponge-associated microbes include: human immunodeficiency virus 1 (HIV-1) (2-undecyl-4-quinolone, sorbicillactone A and chartarutine B); influenza A (H1N1) virus (truncateol M); nosocomial Gram positive bacteria (thiopeptide YM-266183, YM-266184, mayamycin and kocurin); Escherichia coli (sydonic acid), Chlamydia trachomatis (naphthacene glycoside SF2446A2); Plasmodium spp. (manzamine A and quinolone 1); Leishmania donovani (manzamine A and valinomycin); Trypanosoma brucei (valinomycin and staurosporine); Candida albicans and dermatophytic fungi (saadamycin, 5,7-dimethoxy-4-p-methoxylphenylcoumarin and YM-202204). Thirty-five bacterial and 12 fungal genera associated with sponges that produce antimicrobials were identified, with Streptomyces, Pseudovibrio, Bacillus, Aspergillus and Penicillium as the prominent producers of antimicrobial compounds. Furthemore culture-independent approaches to more comprehensively exploit the genetic richness of antimicrobial compound-producing pathways from sponge-associated bacteria are addressed.

Similar sponge-associated bacteria can be acquired via both vertical and horizontal transmission.

Marine sponges host diverse communities of microorganisms that are often vertically transmitted from mother to oocyte or embryo. Horizontal transmission has often been proposed to co-occur in marine sponges, but the mechanism is poorly understood. To assess the impact of the mode of transmission on the microbial assemblages of sponges, we analysed the microbiota in sympatric sponges that have previously been reported to acquire bacteria via either vertical (Corticium candelabrum and Crambe crambe) or horizontal transmission (Petrosia ficiformis). The comparative study was performed by polymerase chain reaction-denaturing gradient gel electrophoresis and pyrosequencing of barcoded PCR-amplified 16S rRNA gene fragments. We found that P. ficiformis and C. candelabrum each harbour their own species-specific bacteria, but they are similar to other high-microbial-abundance sponges, while the low-microbial-abundance sponge C. crambe hosts microbiota of a very different phylogenetic signature. In addition, nearly 50% of the reads obtained from P. ficiformis were most closely related to bacteria that were previously reported to be vertically transmitted in other sponges and comprised vertical-horizontal transmission phylogenetic clusters (VHT clusters). Therefore, our results provide evidence for the hypothesis that similar sponge-associated bacteria can be acquired via both vertical and horizontal transmission.

Candidatus Nemesobacterales is a sponge-specific clade of the candidate phylum Desulfobacterota adapted to a symbiotic lifestyle.

Members of the candidate phylum Dadabacteria, recently reassigned to the phylum Candidatus Desulfobacterota, are cosmopolitan in the marine environment found both free-living and associated with hosts that are mainly marine sponges. Yet, these microorganisms are poorly characterized, with no cultured representatives and an ambiguous phylogenetic position in the tree of life. Here, we performed genome-centric metagenomics to elucidate their phylogenomic placement and predict the metabolism of the sponge-associated members of this lineage. Rank-based phylogenomics revealed several new species and a novel family (Candidatus Spongomicrobiaceae) within a sponge-specific order, named here Candidatus Nemesobacterales. Metabolic reconstruction suggests that Ca. Nemesobacterales are aerobic heterotrophs, capable of synthesizing most amino acids, vitamins and cofactors and degrading complex carbohydrates. We also report functional divergence between sponge- and seawater-associated metagenome-assembled genomes. Niche-specific adaptations to the sponge holobiont were evident from significantly enriched genes involved in defense mechanisms against foreign DNA and environmental stressors, host-symbiont interactions and secondary metabolite production. Fluorescence in situ hybridization gave a first glimpse of the morphology and lifestyle of a member of Ca. Desulfobacterota. Candidatus Nemesobacterales spp. were found both inside sponge cells centred around sponge nuclei and in the mesohyl of the sponge Geodia barretti. This study sheds light on the enigmatic group Ca. Nemesobacterales and their functional characteristics that reflect a symbiotic lifestyle.

First continuous marine sponge cell line established.

The potential of sponge-derived chemicals for pharmaceutical applications remains largely unexploited due to limited available biomass. Although many have attempted to culture marine sponge cells in vitro to create a scalable production platform for such biopharmaceuticals, these efforts have been mostly unsuccessful. We recently showed that Geodia barretti sponge cells could divide rapidly in M1 medium. In this study we established the first continuous marine sponge cell line, originating from G. barretti. G. barretti cells cultured in OpM1 medium, a modification of M1, grew more rapidly and to a higher density than in M1. Cells in OpM1 reached 1.74 population doublings after 30 min, more than twofold higher than the already rapid growth rate of 0.74 population doublings in 30 min in M1. The maximum number of population doublings increased from 5 doublings in M1 to at least 98 doublings in OpM1. Subcultured cells could be cryopreserved and used to inoculate new cultures. With these results, we have overcome a major obstacle that has blocked the path to producing biopharmaceuticals with sponge cells at industrial scale for decades.

Omics and imaging combinatorial approach reveals butyrate-induced inflammatory effects in the zebrafish gut.

BACKGROUND: Prebiotic feed additives aim to improve gut health by influencing the microbiota and the gut barrier. Most studies on feed additives concentrate on one or two (monodisciplinary) outcome parameters, such as immunity, growth, microbiota or intestinal architecture. A combinatorial and comprehensive approach to disclose the complex and multifaceted effects of feed additives is needed to understand their underlying mechanisms before making health benefit claims. Here, we used juvenile zebrafish as a model species to study effects of feed additives by integrating gut microbiota composition data and host gut transcriptomics with high-throughput quantitative histological analysis. Zebrafish received either control, sodium butyrate or saponin-supplemented feed. Butyrate-derived components such as butyric acid or sodium butyrate have been widely used in animal feeds due to their immunostimulant properties, thereby supporting intestinal health. Soy saponin is an antinutritional factor from soybean meal that promotes inflammation due to its amphipathic nature. RESULTS: We observed distinct microbial profiles associated with each diet, discovering that butyrate (and saponin to a lesser extent) affected gut microbial composition by reducing the degree of community-structure (co-occurrence network analysis) compared to controls. Analogously, butyrate and saponin supplementation impacted the transcription of numerous canonical pathways compared to control-fed fish. For example, both butyrate and saponin increased the expression of genes associated with immune response and inflammatory response, as well as oxidoreductase activity, compared to controls. Furthermore, butyrate decreased the expression of genes associated with histone modification, mitotic processes and G-coupled receptor activity. High-throughput quantitative histological analysis depicted an increase of eosinophils and rodlet cells in the gut tissue of fish receiving butyrate after one week of feeding and a depletion of mucus-producing cells after 3 weeks of feeding this diet. Combination of all datasets indicated that in juvenile zebrafish, butyrate supplementation increases the immune and the inflammatory response to a greater extent than the established inflammation-inducing anti-nutritional factor saponin. Such comprehensive analysis was supplemented by in vivo imaging of neutrophil and macrophage transgenic reporter zebrafish (mpeg1:mCherry/mpx:eGFPi114) larvae. Upon exposure to butyrate and saponin, these larvae displayed a dose-dependent increase of neutrophils and macrophages in the gut area. CONCLUSION: The omics and imaging combinatorial approach provided an integrated evaluation of the effect of butyrate on fish gut health and unraveled inflammatory-like features not previously reported that question the usage of butyrate supplementation to enhance fish gut health under basal conditions. The zebrafish model, due to its unique advantages, provides researchers with an invaluable tool to investigate effects of feed components on fish gut health throughout life.

Distribution and diversity of 'Tectomicrobia', a deep-branching uncultivated bacterial lineage harboring rich producers of bioactive metabolites.

Genomic and functional analyses of bacterial sponge symbionts belonging to the uncultivated candidate genus 'Entotheonella' has revealed them as the prolific producers of bioactive compounds previously identified from their invertebrate hosts. These studies also suggested 'Entotheonella' as the first members of a new candidate phylum, 'Tectomicrobia'. Here we analyzed the phylogenetic structure and environmental distribution of this as-yet sparsely populated phylum-like lineage. The data show that 'Entotheonella' and other 'Tectomicrobia' are not restricted to marine habitats but widely distributed among terrestrial locations. The inferred phylogenetic trees suggest several intra-phylum lineages with diverse lifestyles. Of these, the previously described 'Entotheonella' lineage can be more accurately divided into at least three different candidate genera with the terrestrial 'Candidatus Prasianella', the largely terrestrial 'Candidatus Allonella', the 'Candidatus Thalassonella' comprising sponge-associated members, and the more widely distributed 'Candidatus Entotheonella'. Genomic characterization of 'Thalassonella' members from a range of sponge hosts did not suggest a role as providers of natural products, despite high genomic similarity to 'Entotheonella' regarding primary metabolism and implied lifestyle. In contrast, the analysis revealed a correlation between the revised 'Entotheonella' 16S rRNA gene phylogeny and a specific association with sponges and their natural products. This feature might serve as a discovery method to accelerate the identification of new chemically rich 'Entotheonella' variants, and led to the identification of the first 'Entotheonella' symbiont in a non-tetractinellid sponge, Psammocinia sp., indicating a wide host distribution of 'Entotheonella'-based chemical symbiosis.

Cultivation of sponges, sponge cells and symbionts: achievements and future prospects.

Marine sponges are a rich source of bioactive compounds with pharmaceutical potential. Since biological production is one option to supply materials for early drug development, the main challenge is to establish generic techniques for small-scale production of marine organisms. We analysed the state of the art for cultivation of whole sponges, sponge cells and sponge symbionts. To date, cultivation of whole sponges has been most successful in situ; however, optimal conditions are species specific. The establishment of sponge cell lines has been limited by the inability to obtain an axenic inoculum as well as the lack of knowledge on nutritional requirements in vitro. Approaches to overcome these bottlenecks, including transformation of sponge cells and using media based on yolk, are elaborated. Although a number of bioactive metabolite-producing microorganisms have been isolated from sponges, and it has been suggested that the source of most sponge-derived bioactive compounds is microbial symbionts, cultivation of sponge-specific microorganisms has had limited success. The current genomics revolution provides novel approaches to cultivate these microorganisms.

Genomic diversity and biosynthetic capabilities of sponge-associated chlamydiae.

Sponge microbiomes contribute to host health, nutrition, and defense through the production of secondary metabolites. Chlamydiae, a phylum of obligate intracellular bacteria ranging from animal pathogens to endosymbionts of microbial eukaryotes, are frequently found associated with sponges. However, sponge-associated chlamydial diversity has not yet been investigated at the genomic level and host interactions thus far remain unexplored. Here, we sequenced the microbiomes of three sponge species and found high, though variable, Chlamydiae relative abundances of up to 18.7% of bacteria. Using genome-resolved metagenomics 18 high-quality sponge-associated chlamydial genomes were reconstructed, covering four chlamydial families. Among these, Candidatus Sororchlamydiaceae shares a common ancestor with Chlamydiaceae animal pathogens, suggesting long-term co-evolution with animals. Based on gene content, sponge-associated chlamydiae resemble members from the same family more than sponge-associated chlamydiae of other families, and have greater metabolic versatility than known chlamydial animal pathogens. Sponge-associated chlamydiae are also enriched in genes for degrading diverse compounds found in sponges. Unexpectedly, we identified widespread genetic potential for secondary metabolite biosynthesis across Chlamydiae, which may represent an unexplored source of novel natural products. This finding suggests that Chlamydiae members may partake in defensive symbioses and that secondary metabolites play a wider role in mediating intracellular interactions. Furthermore, sponge-associated chlamydiae relatives were found in other marine invertebrates, pointing towards wider impacts of the Chlamydiae phylum on marine ecosystems.

Comparative Metagenomic Analysis of Biosynthetic Diversity across Sponge Microbiomes Highlights Metabolic Novelty, Conservation, and Diversification.

Marine sponges and their microbial symbiotic communities are rich sources of diverse natural products (NPs) that often display biological activity, yet little is known about the global distribution of NPs and the symbionts that produce them. Since the majority of sponge symbionts remain uncultured, it is a challenge to characterize their NP biosynthetic pathways, assess their prevalence within the holobiont, and measure the diversity of NP biosynthetic gene clusters (BGCs) across sponge taxa and environments. Here, we explore the microbial biosynthetic landscapes of three high-microbial-abundance (HMA) sponges from the Atlantic Ocean and the Mediterranean Sea. This data set reveals striking novelty, with <1% of the recovered gene cluster families (GCFs) showing similarity to any characterized BGC. When zooming in on the microbial communities of each sponge, we observed higher variability of specialized metabolic and taxonomic profiles between sponge species than within species. Nonetheless, we identified conservation of GCFs, with 20% of sponge GCFs being shared between at least two sponge species and a GCF core comprised of 6% of GCFs shared across all species. Within this functional core, we identified a set of widespread and diverse GCFs encoding nonribosomal peptide synthetases that are potentially involved in the production of diversified ether lipids, as well as GCFs putatively encoding the production of highly modified proteusins. The present work contributes to the small, yet growing body of data characterizing NP landscapes of marine sponge symbionts and to the cryptic biosynthetic potential contained in this environmental niche. IMPORTANCE Marine sponges and their microbial symbiotic communities are a rich source of diverse natural products (NPs). However, little is known about the sponge NP global distribution landscape and the symbionts that produce them. Here, we make use of recently developed tools to perform untargeted mining and comparative analysis of sponge microbiome metagenomes of three sponge species in the first study considering replicate metagenomes of multiple sponge species. We present an overview of the biosynthetic diversity across these sponge holobionts, which displays extreme biosynthetic novelty. We report not only the conservation of biosynthetic and taxonomic diversity but also a core of conserved specialized metabolic pathways. Finally, we highlight several novel GCFs with unknown ecological function, and observe particularly high biosynthetic potential in Acidobacteriota and Latescibacteria symbionts. This study paves the way toward a better understanding of the marine sponge holobionts' biosynthetic potential and the functional and ecological role of sponge microbiomes.