Extended Data Fig. 9. Uncultivated OTUs are depleted in numerous functions, including genes for osmotic and oxidative stress.

Genes from high-quality MAGs were functionally annotated on the basis of the KEGG and TIGRFAM databases, and the presence and absence of functions was averaged across MAGs per OTU. Functions were then compared between uncultivated OTUs (n = 271) and cultivated OTUs (n = 233) using the Ives–Garland phylogenetic logistic regression test, and P values were corrected for multiple hypothesis testing using the Benjamini–Hochberg procedure. a, The number of genes associated with cultivation status does not depend on the database used for functional annotation. b, KEGG functional annotations were compared between high-quality MAGs and reference genomes from the same species-level OTU (left) (n = 665 OTUs), and between MAGs and reference genomes from different OTUs using Pearson correlation (right) (n = 665 OTUs). MAGs and reference genomes have concordant functional annotations. In the box plots, the middle line denotes the median, the box denotes the IQR and the whiskers denote 1.5× IQR. c, Phylogenetic tree of 271 uncultivated OTUs and 233 cultivated OTUs. The inner ring indicates whether an OTU is cultivated or not. The outer ring indicates the presence or absence genes from the KEGG database. The top ten genes associated with cultivation status are shown. Of these, four are related to the maintenance of osmotic pressure (KEGG identifiers K05846, K01547, K01546 and K01548) and two (including the top hit) are related to oxidative stress (identifiers K0986 and K03386). Note that identifier K0986 (the top hit) is listed as encoding an uncharacterized protein in the KEGG database, but as encoding a peroxide stress protein in the PFam database (identifier PF03883). Organisms that lack these functions may have decreased viability during cultivation owing to oxygen exposure and osmotic stress from growth in culture medium.