The ultrasmall ocean microbiome: a reservoir of microbial diversity and nitrogen fixation.

Marine microbiology has long considered ultrasmall (<0.2 {micro}m) size fractions as virus-dominated, yet emerging evidence suggests potential prokaryotic activity with implications for global biogeochemical cycles. Here, we present the first comprehensive genome-resolved survey of this fraction, built on 4,058 metagenome-assembled genomes (MAGs) representing 1,152 species across Bacteria (90.7%) and Archaea (9.3%), dominated by Pseudomonadota, Bacteroidota, and Nanoarchaeota. Over 490 non-redundant MAGs occurred exclusively in the ultrasmall fraction, with 66.8% representing novel taxa, particularly abundant in Arctic surface waters. Beyond known ultrasmall organisms (e.g., DPANN archaea and Patescibacteria), which show expected genomic and metabolic reduction, functional annotation revealed diverse metabolic potential across nitrogen, sulfur, and carbon cycling. Focusing on nitrogen fixation, we identified 13 non-cyanobacterial diazotroph species with complete nitrogenase operons spanning four bacterial classes. These resolved into two ecotypes: cosmopolitan low-abundance taxa, and Arctic ultrasmall-restricted strains reaching up to 16.7% relative abundance. Two lineages (50-400-T64 and Novosphingobium) showed ultrasmall exclusive bipolar distributions across Arctic and Southern Oceans. Beyond nitrogen fixation, these diazotrophs encoded pathways for denitrification, DNRA, sulfur oxidation, and carbon fixation. Our findings reveal an overlooked reservoir of microbial diversity including diazotrophs in the ultrasmall ocean microbiome, with significant implications for polar nitrogen budgets and global biogeochemical models.