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

## TL;DR

This study uses metagenomics to explore the genome and microbiome of a marine protist called Incisomonas marina, revealing its metabolic needs and unexpected traits.

## Contribution

The study provides the most complete genome of a MAST-3 stramenopile and identifies unexpected metabolic features like DSYB gene presence and B-vitamin dependencies.

## Key findings

- The genome of Incisomonas marina is 68 Mbp and 93% complete, the most complete MAST genome to date.
- I. marina is predicted to be auxotrophic for several B vitamins and lacks half the protein amino acid biosynthesis pathways.
- The DSYB gene for dimethylsulphoniopropionate biosynthesis was unexpectedly found in I. marina, a trait previously unknown in heterotrophic stramenopiles.

## Abstract

Protists are polyphyletic single-celled eukaryotes that underpin global ecosystem functioning, particularly in the oceans. Most remain uncultured, limiting the investigation of their physiology and cell biology. MArine STramenopiles (MASTs) are heterotrophic protists that, although related to well-characterized photosynthetic diatoms and parasitic oomycetes, are poorly studied. The Nanomonadea (MAST-3) species Incisomonas marina has 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 of I. marina was retrieved to an estimated completeness of 93%, representing the most complete MAST genome so far. We also characterized the diversity of, and assembled genomes for, 23 co-cultured bacteria. Auxotrophy of I. marina for B vitamins (B1, B2, B6, B7 and B12), but not vitamins C, B3, B5 and B9, was predicted. Several bacteria also lacked complete B-vitamin biosynthesis pathways, suggesting that vitamins and/or their precursors are exchanged in the consortium. Moreover, I. marina lacked the ability to synthesize 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 gene DSYB for dimethylsulphoniopropionate biosynthesis. Biosynthesis of this important stress protectant and bacterial chemoattractant is typically found in photosynthetic eukaryotes and has not been identified before 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.