Deciphering a marine bone degrading microbiome reveals a complex community effort

The marine bone biome is a complex assemblage of macro- and microorganisms, however the enzymatic repertoire to access bone-derived nutrients remains unknown. The bone matrix is a composite material made up mainly of organic collagen and inorganic hydroxyapatite. We conducted field experiments to study microbial assemblages that can use organic bone components as nutrient source. Bovine and turkey bones were deposited at 69 m depth in a Norwegian fjord (Byfjorden, Bergen). Metagenomic sequence analysis was used to assess the functional potential of microbial assemblages from bone surface and the bone eating worm Osedax mucofloris, which is a frequent colonizer of whale falls and known to degrade bone. The bone microbiome displayed a surprising taxonomic diversity revealed by the examination of 59 high-quality metagenome assembled genomes from at least 23 bacterial families. Over 700 genes encoding enzymes from twelve relevant enzymatic families pertaining to collagenases, peptidases, glycosidases putatively involved in bone degradation were identified. Metagenome assembled genomes (MAGs) of the class Bacteroidia contained the most diverse gene repertoires. We postulate that demineralization of inorganic bone components is achieved by a timely succession of a closed sulfur biogeochemical cycle between sulfur-oxidizing and sulfur-reducing bacteria, causing a drop in pH and subsequent enzymatic processing of organic components in the bone surface communities. An unusually large and novel collagen utilization gene cluster was retrieved from one genome belonging to the gammaproteobacterial genus Colwellia. Importance Bones are an underexploited, yet potentially profitable feedstock for biotechnological advances and value chains, due to the sheer amounts of residues produced by the modern meat and poultry processing industry. In this metagenomic study we decipher the microbial pathways and enzymes that we postulate to be involved in bone degradation marine environment. We herein demonstrate the interplay between different bacterial community members, each supplying different enzymatic functions with the potential to cover an array of reactions relating to the degradation of bone matrix components. We identify and describe a novel gene cluster for collagen utilization, which is a key function in this unique environment. We propose that the interplay between the different microbial taxa is necessary to achieve the complex task of bone degradation in the marine environment.

Two distinct bacterial endosymbiont genomes belonging to the order Oceanospirillales have metagenome is made up of 7% to 22% Oceanospirillales affiliated reads, whereas the bone 3 2 0 surface metagenome only contains 1% to 4% reads of this order according to the performed 3 2 1 Kaiju analysis. This relative difference confirms that the methodological approach to minimize 3 2 2 cross-contamination was successful and that the OB-MAGs affiliated to Oceanospirilalles likely Oceanospirillales were identified in the Osedax-associated metagenome, belonging to the genera Neptunomonas (OB1) and Amphritea (OB2). Both genera are known to have an aerobic 3 2 6 organotrophic metabolism and also able to thrive as free-living bacteria (70). In fact, the scarce 3 2 7 representation of Oceanospirillales in the bone surface has been reported before (30, 31) and we cannot rule out that Oceanospirillales preferentially colonize bone surfaces in earlier or later 3 3 0 stages, their preference for a symbiont life supports the notion of a casual and facultative 3 3 1 association to Osedax worms, triggered by the common benefit from a sudden nutrient bonanza 3 3 2 as previously hypothesized (18,21). Free-living microbial communities, must deal with similar challenges as the Osedax holobiont to 3 3 6 access the nutrient-rich, collagen-made organic bone matrix and eventually the lipid-rich bone consortia may be a beneficial strategy for achieving this task. We hypothesize that sulfur-driven geomicrobiology (sulfate/thiosulfate/tetrathionate reduction and sulfide/sulfur/thiosulfate 3 4 0 oxidation) is the major responsible factor for bone dissolution in the marine environment by free- organic matrix to enzymatic degradation (30, 31). Besides thiotrophy, that seems to be a major 3 5 6 acid-producing mechanism in the microbial community, other mechanisms might also contribute 3 5 7 significantly. In this respect, a number of carbonic anhydrases (CA) were annotated, normally 3 5 8 housekeeping genes involved in internal pH homeostasis and other processes (76), but known to 3 5 9 play a role in environmental acidification by Osedax (15). Here, the CAs were found to contain 3 6 0 signal peptides for extracellular export (19 out of 94) and therefore could also be involved in α -CA family is only found in gram negative bacteria, which is also the case here, and it is 3 6 4 evolutionarily the youngest of the three bacterial CA families (49).

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Besides a large number of SOB, eight MAGs related to SRB were identified that are affiliated to 3 6 6 the families Desulfobulbaceae (also SOB), Desulfobacteraceae, Geopsychrobacteraceae and 3 6 7 Desulfovibrionacaeae. Moreover, they are prevalently associated with the free-living community 3 6 8 attached to the bone surface in this study. Sulfate, tetrathionate or thiosulfate can serve as process. The generated sulfide is known to quickly react with iron, blackening the bone surfaces polypeptides like elastin (84), spongin (85) and others. The recently described Marinifilaceae to the bone-surface associated microbiome, we hypothesize that Bacteroidia play a pivotal and 3 9 7 specialized role in the free-living community via the degradation of specific organic bone 3 9 8 components.

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Differential microbial colonization of the spongy cancellous bone tissue over the cortical 4 0 0 compact bone has also been observed in the terrestrial environment and has been related to easier 4 0 1 access to the red marrow (88) although a priming effect linked to the differential composition of  proteinogenic compounds within bone, as they are able to degrade collagen, the main source of kind of functional condensation for collagen utilization has not been described before in 4 2 8 Colwellia or elsewhere. Interestingly, Colwellia bacteria are also one partner in a dual carboxylate to alpha-ketoglutarate semialdehyde).

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Bone degradation -a complex microbial community effort 4 4 7 The marine bone microbiome is a complex assemblage of various bacterial classes that requires Bone material after manual meat deboning was kindly provided by a local slaughterhouse DNA was quantified and quality controlled using a NanoDrop2000 (ThermoFisher Scientific) Illumina raw reads were quality trimmed and adapters were removed with Trimmomatic version   identification, coupled with an estimate of relative evolutionary divergence (RED) to their next 5 2 2 common ancestor. RED is a normalization method to assign taxonomic ranks according to MAGs were predicted with Prodigal version 2.6.3 (100). Predicted ORFs were annotated using MAGs were annotated and metabolic models were calculated using the RAST (rapid annotation Traitar (40). MEBS is a software package used here to detect genes related to sulfur metabolism, Heatmapper (110). Gene cluster maps were drawn with Gene Graphics (111). Signal peptides 5 4 3 were predicted with SignalP-5.0 server using nucleotide sequences to predict the presence of 5 4 4 Sec/SPI, Tat/SPI and Sec/SPII signal peptides in a given sequence (112).

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Enzyme profiling 5 4 6 Based on the organic composition of bone matrix, we hypothesized twelve enzyme families to be 5 4 7 necessary for its degradation. Accordingly, the following enzymes were selected for in-depth reference database for each of these families was generated using the NCBI repository, based on 5 5 5 sequences from 287 M9 collagenases, 4453 S1 peptidases, 3237 S8/S53 peptidases, 3653 U32 option and a bitscore threshold of 100. MAGs were here reanalyzed with GTDB-Tk and the generated HMM profiles as previously Tara Oceans MAGs were generated with a quality threshold of >70% completion, therefore the to avoid bias due to the higher threshold used for functional analysis in other parts of this 5 7 5 manuscript.

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Data availability 5 7 7 The raw sequencing reads have been deposited in the sequence read archive (SRA) of NCBI The 59 high-quality MAGs analyzed in this study were deposited in the NCBI database as well, Osedax MAGs 1-15 SAMN16086371-SAMN16086385).  To the memory of Prof. Hans Tore Rapp and his effort on characterizing Osedax mucofloris. We nov., isolated from a marine sponge, and emended description of the genus Amphritea. Peninsula. Polar Research 38. broad-spectrum activity from the ulvan utilisation loci of Formosa agariphila KMM  112:93-100.    Microbiol 56:719-34.  resolved metagenomic data analysis. Microbiome 6:158. Bioinformatics 11:119.