Endozoicomonadaceae symbiont in gills of Acesta clam encodes genes for essential nutrients and polysaccharide degradation.

Gammaproteobacteria from the family Endozoicomonadaceae have emerged as widespread associates of dense marine animal communities. Their abundance in coral reefs involves symbiotic relationships and possibly host nutrition. We explored functions encoded in the genome of an uncultured Endozoicomonadaceae 'Candidatus Acestibacter aggregatus' that lives inside gill cells of large Acesta excavata clams in deep-water coral reefs off mid-Norway. The dominance and deep branching lineage of this symbiont was confirmed using 16S rRNA gene sequencing and phylogenomic analysis from shotgun sequencing data. The 4.5 Mb genome binned in this study has a low GC content of 35% and is enriched in transposon and chaperone gene annotations indicating ongoing adaptation. Genes encoding functions potentially involved with the symbiosis include ankyrins, repeat in toxins, secretion and nutritional systems. Complete pathways were identified for the synthesis of eleven amino acids and six B-vitamins. A minimal chitinolytic machinery was indicated from a glycosyl hydrolase GH18 and a lytic polysaccharide monooxygenase LPMO10. Expression of the latter was confirmed using proteomics. Signal peptides for secretion were identified for six polysaccharide degrading enzymes, ten proteases and three lipases. Our results suggest a nutritional symbiosis fuelled by enzymatic products from extracellular degradation processes.

Diversity of deep-water coral-associated bacteria and comparison across depth gradients.

Environmental conditions influence species composition, including the microbial communities that associate with benthic organisms such as corals. In this study we identified and compared bacteria that associate with three common deep-water corals, Lophelia pertusa, Madrepora oculata and Paragorgia arborea, from a reef habitat on the mid-Norwegian shelf. The 16S rRNA gene amplicon sequencing data obtained revealed that >50% of sequences were represented by only five operational taxonomic units. Three were host-specific and unclassified below class level, belonging to Alphaproteobacteria with affiliation to members of the Rhizobiales order (L. pertusa), Flavobacteria affiliated with members of the Elisabethkingia genus (M. oculata) and Mollicutes sequences affiliated with the Mycoplasma genus (P. arborea). In addition, gammaproteobacterial sequences within the genera Sulfitobacter and Oleispira were found across all three deep-water coral taxa. Although highly abundant in the coral microbiomes, these sequences accounted for <0.1% of the surrounding bacterioplankton, supporting specific relationships. We combined this information with previous studies, undertaking a meta-data analysis of 165 widespread samples across coral hosts and habitats. Patterns in bacterial diversity indicated enrichment of distinct uncultured species in coral microbiomes that differed among deep (>200 m), mesophotic (30-200 m) and shallow (<30 m) reefs.

Norwegian deep-water coral reefs: cultivation and molecular analysis of planktonic microbial communities.

Deep-sea coral reefs do not receive sunlight and depend on plankton. Little is known about the plankton composition at such reefs, even though they constitute habitats for many invertebrates and fish. We investigated plankton communities from three reefs at 260-350 m depth at hydrocarbon fields off the mid-Norwegian coast using a combination of cultivation and small subunit (SSU) rRNA gene and transcript sequencing. Eight months incubations of a reef water sample with minimal medium, supplemented with carbon dioxide and gaseous alkanes at in situ-like conditions, enabled isolation of mostly Alphaproteobacteria (Sulfitobacter, Loktanella), Gammaproteobacteria (Colwellia) and Flavobacteria (Polaribacter). The relative abundance of isolates in the original sample ranged from ∼ 0.01% to 0.80%. Comparisons of bacterial SSU sequences from filtered plankton of reef and non-reef control samples indicated high abundance and metabolic activity of primarily Alphaproteobacteria (SAR11 Ia), Gammaproteobacteria (ARCTIC96BD-19), but also of Deltaproteobacteria (Nitrospina, SAR324). Eukaryote SSU sequences indicated metabolically active microalgae and animals, including codfish, at the reef sites. The plankton community composition varied between reefs and differed between DNA and RNA assessments. Over 5000 operational taxonomic units were detected, some indicators of reef sites (e.g. Flavobacteria, Cercozoa, Demospongiae) and some more active at reef sites (e.g. Gammaproteobacteria, Ciliophora, Copepoda).

High diversity of microplankton surrounds deep-water coral reef in the Norwegian Sea.

Coral reefs that exist in the depths of the oceans are surrounded by Eukarya, Archaea and bacterial communities that may play an important role in the nutrition and health of the reef. The first interdomain community structure of planktonic organisms in seawater from a deep-water coral reef is described. Community profiling and analysis of ribosomal RNA gene sequences from a coral reef system at 350 m depth in the Norwegian Sea revealed a rich diversity of Eukarya and Bacteria and a moderate diversity of Archaea. Most sequences affiliated with marine microplankton from deep-sea to cold-surface regions, with many sequences being similar to those described in studies of mesopelagic and oxygen minimum zones. Dominant phylotypes belonged to the Alveolata (group I, II, dinoflagellates), Stramenopiles (silicoflagellates), Alphaproteobacteria (Pelagibacter ubique), Gammaproteobacteria (ARCTIC96BD-19), Bacteroidetes (Flavobacteria) and mesophilic Crenarchaeota (Nitrosopumilus maritimus). Several rare and novel members of the community fell into distinct phylogenetic groups. The inferred function of dominant community members suggested autotrophs that utilise light, ammonium or sulphide, and lifestyles based on host associations. The high diversity reflected a microplankton community structure, which is significantly different from that of microplankton collected at the same depth at a pelagic station away from reefs.

Intracellular Oceanospirillales bacteria inhabit gills of Acesta bivalves.

A novel bacterium was discovered in the gills of the large bivalve Acesta excavata (Limidae) from coral reefs on the northeast Atlantic margin near the shelf break of the fishing ground Haltenbanken of Norway, and confirmed present in A. excavata from a rock-wall in the Trondheimsfjord. Purified gill DNA contained one dominant bacterial rRNA operon as indicated from analysis of broad range bacterial PCR amplicons in denaturant gradient gels, in clone libraries and by direct sequencing. The sequences originated from an unknown member of the order Oceanospirillales and its 16S rRNA gene fell within a clade of strictly marine invertebrate-associated Gammaproteobacteria. Visual inspection by fluorescent in situ hybridization and transmission electron microscopy indicated a pleomorphic bacterium with no visible cell wall, located in aggregates inside vacuoles scattered within the gill cells cytoplasm. Intracellular Oceanospirillales exist in bathymodiolin mussels (parasites), Osedax worms and whiteflies (symbionts). This bacterium apparently lives in a specific association with the Acesta.

Methane assimilation and trophic interactions with marine Methylomicrobium in deep-water coral reef sediment off the coast of Norway.

Deep-water coral reefs are seafloor environments with diverse biological communities surrounded by cold permanent darkness. Sources of energy and carbon for the nourishment of these reefs are presently unclear. We investigated one aspect of the food web using DNA stable-isotope probing (DNA-SIP). Sediment from beneath a Lophelia pertusa reef off the coast of Norway was incubated until assimilation of 5 micromol 13CH4 g(-1) wet weight occurred. Extracted DNA was separated into 'light' and 'heavy' fractions for analysis of labelling. Bacterial community fingerprinting of PCR-amplified 16S rRNA gene fragments revealed two predominant 13C-specific bands. Sequencing of these bands indicated that carbon from 13CH4 had been assimilated by a Methylomicrobium and an uncultivated member of the Gammaproteobacteria. Cloning and sequencing of 16S rRNA genes from the heavy DNA, in addition to genes encoding particulate methane monooxygenase and methanol dehydrogenase, all linked Methylomicrobium with methane metabolism. Putative cross-feeders were affiliated with Methylophaga (Gammaproteobacteria), Hyphomicrobium (Alphaproteobacteria) and previously unrecognized methylotrophs of the Gammaproteobacteria, Alphaproteobacteria, Deferribacteres and Bacteroidetes. This first marine methane SIP study provides evidence for the presence of methylotrophs that participate in sediment food webs associated with deep-water coral reefs.