Metagenomics of the MAST-3 stramenopile,Incisomonas,and its associated microbiome reveals unexpected metabolic attributes and extensive nutrient dependencies

Protists are polyphyletic singled-celled eukaryotes that underpin global ecosystem functioning, particularly in the oceans. Most remain uncultured, limiting investigation of their physiology and cell biology.<underline>MA</underline>rine<underline>ST</underline>ramenopiles (MASTs) are heterotrophic protists that, although related to well-characterised photosynthetic diatoms and parasitic oomycetes, are poorly studied. The Nanomonadea (MAST-3) speciesIncisomonas marinahas been maintained in co-culture with a bacterial consortium, offering opportunities to investigate the metabolic attributes and nutritional dependencies of the community. Employing a metagenomics approach, the 68 Mbp haploid genome ofI. marinawas retrieved to an estimated completeness of 93%, representing the most complete MAST genome so far. We also characterised the diversity of, and assembled genomes for, 23 co-cultured bacteria. Auxotrophy ofI. marinafor B vitamins (B₁, B₂, B₆, B₇and B₁₂), but not vitamins C, B₃, B₅and B₉was predicted. Several bacteria also lacked complete B-vitamin biosynthesis pathways, suggesting that vitamins and/or their precursors are exchanged in the consortium. Moreover,I. marinalacked the ability to synthesise half the protein amino acids, although genes encoding the complete urea cycle were identified, like diatoms; this may play a role in recycling organic nitrogen compounds. Unexpectedly, we also identified the geneDSYBfor dimethylsulphoniopropionate (DMSP) biosynthesis. Biosynthesis of this important stress-protectant and bacterial chemoattractant is typically found in photosynthetic eukaryotes and has not before been identified in heterotrophic stramenopiles. Together, our study reveals the metabolic attributes of a hitherto understudied organism, advancing knowledge of the evolution and adaptations of the stramenopiles and informing future culturing efforts.