Selection of marine bacterial consortia efficient at degrading chitin leads to the discovery of new potential chitin degraders.

Chitin degradation is a keystone process in the oceans, mediated by marine microorganisms with the help of several enzymes, mostly chitinases. Sediment, seawater, and filter-feeding marine invertebrates, such as sponges, are known to harbor chitin-degrading bacteria and are presumably hotspots for chitin turnover. Here, we employed an artificial selection process involving enrichment cultures derived from microbial communities associated with the marine sponge Hymeniacidon perlevis, its surrounding seawater and sediment, to select bacterial consortia capable of degrading raw chitin. Throughout the artificial selection process, chitin degradation rates and the taxonomic composition of the four successive enrichment cultures were followed. To the best of our knowledge, chitin degradation was characterized for the first time using size exclusion chromatography, which revealed significant shifts in the numbered average chitin molecular weight, strongly suggesting the involvement of endo-chitinases in the breakdown of the chitin polymer during the enrichment process. Concomitantly with chitin degradation, the enrichment cultures exhibited a decrease in alpha diversity compared with the environmental samples. Notably, some of the dominant taxa in the enriched communities, such as Motilimonas, Arcobacter, and Halarcobacter, were previously unknown to be involved in chitin degradation. In particular, the analysis of published genomes of these genera suggests a pivotal role of Motilimonas in the hydrolytic cleavage of chitin. This study provides context to the microbiome of the marine sponge Hymeniacidon perlevis in light of its environmental surroundings and opens new ground to the future discovery and characterization of novel enzymes of marine origin involved in chitin degradation processes.IMPORTANCEChitin is the second most abundant biopolymer on Earth after cellulose, and the most abundant in the marine environment. At present, industrial processes for the conversion of seafood waste into chitin, chitosan, and chitooligosaccharide (COS) rely on the use of high amounts of concentrated acids or strong alkali at high temperature. Developing bio-based methods to transform available chitin into valuable compounds, such as chitosan and COS, holds promise in promoting a more sustainable, circular bioeconomy. By employing an artificial selection procedure based on chitin as a sole C and N source, we discovered microorganisms so-far unknown to metabolize chitin in the rare microbial biosphere of several marine biotopes. This finding represents a first important step on the path towards characterizing and exploiting potentially novel enzymes of marine origin with biotechnological interest, since products of chitin degradation may find applications across several sectors, such as agriculture, pharmacy, and waste management.

Aquimarina aquimarini sp. nov. and Aquimarina spinulae sp. nov., novel bacterial species with versatile natural product biosynthesis potential isolated from marine sponges.

This study describes two Gram-negative, flexirubin-producing, biofilm-forming, motile-by-gliding and rod-shaped bacteria, isolated from the marine sponges Ircinia variabilis and Sarcotragus spinosulus collected off the coast of Algarve, Portugal. Both strains, designated Aq135T and Aq349T, were classified into the genus Aquimarina by means of 16S rRNA gene sequencing. We then performed phylogenetic, phylogenomic and biochemical analyses to determine whether these strains represent novel Aquimarina species. Whereas the closest 16S rRNA gene relatives to strain Aq135T were Aquimarina macrocephali JAMB N27T (97.8 %) and Aquimarina sediminis w01T (97.1 %), strain Aq349T was more closely related to Aquimarina megaterium XH134T (99.2 %) and Aquimarina atlantica 22II-S11-z7T (98.1 %). Both strains showed genome-wide average nucleotide identity scores below the species level cut-off (95 %) with all Aquimarina type strains with publicly available genomes, including their closest relatives. Digital DNA-DNA hybridization further suggested a novel species status for both strains since values lower than 70 % hybridization level with other Aquimarina type strains were obtained. Strains Aq135T and Aq349T grew from 4 to 30°C and with between 1-5 % (w/v) NaCl in marine broth. The most abundant fatty acids were iso-C17 : 03-OH and iso-C15 : 0 and the only respiratory quinone was MK-6. Strain Aq135T was catalase-positive and ß-galactosidase-negative, while Aq349T was catalase-negative and ß-galactosidase-positive. These strains hold unique sets of secondary metabolite biosynthetic gene clusters and are known to produce the peptide antibiotics aquimarins (Aq135T) and the trans-AT polyketide cuniculene (Aq349T), respectively. Based on the polyphasic approach employed in this study, we propose the novel species names Aquimarina aquimarini sp. nov. (type strain Aq135T=DSM 115833T=UCCCB 169T=ATCC TSD-360T) and Aquimarina spinulae sp. nov. (type strain Aq349T=DSM 115834T=UCCCB 170T=ATCC TSD-361T).

Natural product biosynthetic potential reflects macroevolutionary diversification within a widely distributed bacterial taxon.

IMPORTANCE: This is the most comprehensive study performed thus far on the biosynthetic potential within the Flavobacteriaceae family. Our findings reveal intertwined taxonomic and natural product biosynthesis diversification within the family. We posit that the carbohydrate, peptide, and secondary metabolism triad synergistically shaped the evolution of this keystone bacterial taxon, acting as major forces underpinning the broad host range and opportunistic-to-pathogenic behavior encompassed by species in the family. This study further breaks new ground for future research on select Flavobacteriaceae spp. as reservoirs of novel drug leads.

Widespread occurrence of chitinase-encoding genes suggests the Endozoicomonadaceae family as a key player in chitin processing in the marine benthos.

Chitin is the most abundant natural polymer in the oceans, where it is primarily recycled by chitin-degrading microorganisms. Endozoicomonadaceae (Oceanospirillales) bacteria are prominent symbionts of sessile marine animals, particularly corals, and presumably contribute to nutrient cycling in their hosts. To reveal the chitinolytic potential of this iconic, animal-dwelling bacterial family, we examined 42 publicly available genomes of cultured and uncultured Endozoicomonadaceae strains for the presence of chitinase-encoding genes. Thirty-two of 42 Endozoicomonadaceae genomes harbored endo-chitinase- (EC 3.2.1.14), 25 had exo-chitinase- (EC 3.2.1.52) and 23 polysaccharide deacetylase-encoding genes. Chitinases were present in cultured and uncultured Endozoicomonadaceae lineages associated with diverse marine animals, including the three formally described genera Endozoicomonas, Paraendozoicomonas and Kistimonas, the new genus Candidatus Gorgonimonas, and other, yet unclassified, groups of the family. Most endo-chitinases belonged to the glycoside hydrolase family GH18 but five GH19 endo-chitinases were also present. Many endo-chitinases harbored an active site and a signal peptide domain, indicating the enzymes are likely functional and exported to the extracellular environment where endo-chitinases usually act. Phylogenetic analysis revealed clade-specific diversification of endo-chitinases across the family. The presence of multiple, distinct endo-chitinases on the genomes of several Endozoicomonadaceae species hints at functional variation to secure effective chitin processing in diverse micro-niches and changing environmental conditions. We demonstrate that endo-chitinases and other genes involved in chitin degradation are widespread in the Endozoicomonadaceae family and posit that these symbionts play important roles in chitin turnover in filter- and suspension-feeding animals and in benthic, marine ecosystems at large.

The AnimalAssociatedMetagenomeDB reveals a bias towards livestock and developed countries and blind spots in functional-potential studies of animal-associated microbiomes.

BACKGROUND: Metagenomic data can shed light on animal-microbiome relationships and the functional potential of these communities. Over the past years, the generation of metagenomics data has increased exponentially, and so has the availability and reusability of data present in public repositories. However, identifying which datasets and associated metadata are available is not straightforward. We created the Animal-Associated Metagenome Metadata Database (AnimalAssociatedMetagenomeDB - AAMDB) to facilitate the identification and reuse of publicly available non-human, animal-associated metagenomic data, and metadata. Further, we used the AAMDB to (i) annotate common and scientific names of the species; (ii) determine the fraction of vertebrates and invertebrates; (iii) study their biogeography; and (iv) specify whether the animals were wild, pets, livestock or used for medical research. RESULTS: We manually selected metagenomes associated with non-human animals from SRA and MG-RAST.  Next, we standardized and curated 51 metadata attributes (e.g., host, compartment, geographic coordinates, and country). The AAMDB version 1.0 contains 10,885 metagenomes associated with 165 different species from 65 different countries. From the collected metagenomes, 51.1% were recovered from animals associated with medical research or grown for human consumption (i.e., mice, rats, cattle, pigs, and poultry). Further, we observed an over-representation of animals collected in temperate regions (89.2%) and a lower representation of samples from the polar zones, with only 11 samples in total. The most common genus among invertebrate animals was Trichocerca (rotifers). CONCLUSION: Our work may guide host species selection in novel animal-associated metagenome research, especially in biodiversity and conservation studies. The data available in our database will allow scientists to perform meta-analyses and test new hypotheses (e.g., host-specificity, strain heterogeneity, and biogeography of animal-associated metagenomes), leveraging existing data. The AAMDB WebApp is a user-friendly interface that is publicly available at https://webapp.ufz.de/aamdb/ .

Exploring the Potential Molecular Mechanisms of Interactions between a Probiotic Consortium and Its Coral Host.

Beneficial microorganisms for corals (BMCs) have been demonstrated to be effective probiotics to alleviate bleaching and mitigate coral mortality in vivo. The selection of putative BMCs is traditionally performed manually, using an array of biochemical and molecular tests for putative BMC traits. We present a comprehensive genetic survey of BMC traits using a genome-based framework for the identification of alternative mechanisms that can be used for future in silico selection of BMC strains. We identify exclusive BMC traits associated with specific strains and propose new BMC mechanisms, such as the synthesis of glycine betaine and ectoines. Our roadmap facilitates the selection of BMC strains while increasing the array of genetic targets that can be included in the selection of putative BMC strains to be tested as coral probiotics. IMPORTANCE Probiotics are currently the main hope as a potential medicine for corals, organisms that are considered the marine "canaries of the coal mine" and that are threatened with extinction. Our experiments have proved the concept that probiotics mitigate coral bleaching and can also prevent coral mortality. Here, we present a comprehensive genetic survey of probiotic traits using a genome-based framework. The main outcomes are a roadmap that facilitates the selection of coral probiotic strains while increasing the array of mechanisms that can be included in the selection of coral probiotics.

MarineMetagenomeDB: a public repository for curated and standardized metadata for marine metagenomes.

BACKGROUND: Metagenomics is an expanding field within microbial ecology, microbiology, and related disciplines. The number of metagenomes deposited in major public repositories such as Sequence Read Archive (SRA) and Metagenomic Rapid Annotations using Subsystems Technology (MG-RAST) is rising exponentially. However, data mining and interpretation can be challenging due to mis-annotated and misleading metadata entries. In this study, we describe the Marine Metagenome Metadata Database (MarineMetagenomeDB) to help researchers identify marine metagenomes of interest for re-analysis and meta-analysis. To this end, we have manually curated the associated metadata of several thousands of microbial metagenomes currently deposited at SRA and MG-RAST. RESULTS: In total, 125 terms were curated according to 17 different classes (e.g., biome, material, oceanic zone, geographic feature and oceanographic phenomena). Other standardized features include sample attributes (e.g., salinity, depth), sample location (e.g., latitude, longitude), and sequencing features (e.g., sequencing platform, sequence count). MarineMetagenomeDB version 1.0 contains 11,449 marine metagenomes from SRA and MG-RAST distributed across all oceans and several seas. Most samples were sequenced using Illumina sequencing technology (84.33%). More than 55% of the samples were collected from the Pacific and the Atlantic Oceans. About 40% of the samples had their biomes assigned as 'ocean'. The 'Quick Search' and 'Advanced Search' tabs allow users to use different filters to select samples of interest dynamically in the web app. The interactive map allows the visualization of samples based on their location on the world map. The web app is also equipped with a novel download tool (on both Windows and Linux operating systems), that allows easy download of raw sequence data of selected samples from their respective repositories. As a use case, we demonstrated how to use the MarineMetagenomeDB web app to select estuarine metagenomes for potential large-scale microbial biogeography studies. CONCLUSION: The MarineMetagenomeDB is a powerful resource for non-bioinformaticians to find marine metagenome samples with curated metadata and stimulate meta-studies involving marine microbiomes. Our user-friendly web app is publicly available at https://webapp.ufz.de/marmdb/ .

Genome Sequencing Suggests Diverse Secondary Metabolism in Coral-Associated Aquimarina megaterium.

We report here the genome sequences of three Aquimarina megaterium strains isolated from the octocoral Eunicella labiata. We reveal a coding potential for versatile carbon metabolism and biosynthesis of natural products belonging to the polyketide, nonribosomal peptide, and terpene compound classes.

Metagenomics-resolved genomics provides novel insights into chitin turnover, metabolic specialization, and niche partitioning in the octocoral microbiome.

BACKGROUND: The role of bacterial symbionts that populate octocorals (Cnidaria, Octocorallia) is still poorly understood. To shed light on their metabolic capacities, we examined 66 high-quality metagenome-assembled genomes (MAGs) spanning 30 prokaryotic species, retrieved from microbial metagenomes of three octocoral species and seawater. RESULTS: Symbionts of healthy octocorals were affiliated with the taxa Endozoicomonadaceae, Candidatus Thioglobaceae, Metamycoplasmataceae, unclassified Pseudomonadales, Rhodobacteraceae, unclassified Alphaproteobacteria and Ca. Rhabdochlamydiaceae. Phylogenomics inference revealed that the Endozoicomonadaceae symbionts uncovered here represent two species of a novel genus unique to temperate octocorals, here denoted Ca. Gorgonimonas eunicellae and Ca. Gorgonimonas leptogorgiae. Their genomes revealed metabolic capacities to thrive under suboxic conditions and high gene copy numbers of serine-threonine protein kinases, type 3-secretion system, type-4 pili, and ankyrin-repeat proteins, suggesting excellent capabilities to colonize, aggregate, and persist inside their host. Contrarily, MAGs obtained from seawater frequently lacked symbiosis-related genes. All Endozoicomonadaceae symbionts harbored endo-chitinase and chitin-binging protein-encoding genes, indicating that they can hydrolyze the most abundant polysaccharide in the oceans. Other symbionts, including Metamycoplasmataceae and Ca. Thioglobaceae, may assimilate the smaller chitin oligosaccharides resulting from chitin breakdown and engage in chitin deacetylation, respectively, suggesting possibilities for substrate cross-feeding and a role for the coral microbiome in overall chitin turnover. We also observed sharp differences in secondary metabolite production potential between symbiotic lineages. Specific Proteobacteria taxa may specialize in chemical defense and guard other symbionts, including Endozoicomonadaceae, which lack such capacity. CONCLUSION: This is the first study to recover MAGs from dominant symbionts of octocorals, including those of so-far unculturable Endozoicomonadaceae, Ca. Thioglobaceae and Metamycoplasmataceae symbionts. We identify a thus-far unanticipated, global role for Endozoicomonadaceae symbionts of corals in the processing of chitin, the most abundant natural polysaccharide in the oceans and major component of the natural zoo- and phytoplankton feed of octocorals. We conclude that niche partitioning, metabolic specialization, and adaptation to low oxygen conditions among prokaryotic symbionts likely contribute to the plasticity and adaptability of the octocoral holobiont in changing marine environments. These findings bear implications not only for our understanding of symbiotic relationships in the marine realm but also for the functioning of benthic ecosystems at large. Video Abstract.

Assessing the genomic composition, putative ecological relevance and biotechnological potential of plasmids from sponge bacterial symbionts.

Plasmid-mediated transfer of genes can have direct consequences in several biological processes within sponge microbial communities. However, very few studies have attempted genomic and functional characterization of plasmids from marine host-associated microbial communities in general and those of sponges in particular. In the present study, we used an endogenous plasmid isolation method to obtain plasmids from bacterial symbionts of the marine sponges Stylissa carteri and Paratetilla sp. and investigated the genomic composition, putative ecological relevance and biotechnological potential of these plasmids. In total, we isolated and characterized three complete plasmids, three plasmid prophages and one incomplete plasmid. Our results highlight the importance of plasmids to transfer relevant genetic traits putatively involved in microbial symbiont adaptation and host-microbe and microbe-microbe interactions. For example, putative genes involved in bacterial response to chemical stress, competition, metabolic versatility and mediation of bacterial colonization and pathogenicity were detected. Genes coding for enzymes and toxins of biotechnological potential were also detected. Most plasmid prophage coding sequences were, however, hypothetical proteins with unknown functions. Overall, this study highlights the ecological relevance of plasmids in the marine sponge microbiome and provides evidence that plasmids of sponge bacterial symbionts may represent an untapped resource of genes of biotechnological interest.