Heterotrophic bacterial diazotrophs are more abundant than their cyanobacterial counterparts in metagenomes covering most of the sunlit ocean

Abstract

Biological nitrogen fixation contributes significantly to marine primary productivity. The current view depicts few cyanobacterial diazotrophs as the main marine nitrogen fixers. Here, we used 891 Tara Oceans metagenomes derived from surface waters of five oceans and two seas to generate a manually curated genomic database corresponding to free-living, filamentous, colony-forming, particle-attached, and symbiotic bacterial and archaeal populations. The database provides the genomic content of eight cyanobacterial diazotrophs including a newly discovered population related to known heterocystous symbionts of diatoms, as well as 40 heterotrophic bacterial diazotrophs that considerably expand the known diversity of abundant marine nitrogen fixers. These 48 populations encapsulate 92% of metagenomic signal for known nifH genes in the sunlit ocean, suggesting that the genomic characterization of the most abundant marine diazotrophs may be nearing completion. Newly identified heterotrophic bacterial diazotrophs are widespread, express their nifH genes in situ, and also occur in large planktonic size fractions where they might form aggregates that provide the low-oxygen microenvironments required for nitrogen fixation. Critically, we found heterotrophic bacterial diazotrophs to be more abundant than cyanobacterial diazotrophs in most metagenomes from the open oceans and seas, emphasizing the importance of a wide range of heterotrophic populations in the marine nitrogen balance.

Fig. 1. The phylogeny of 48 marine bacterial diazotrophs.

Top panel displays a phylogenomic tree of the 48 diazotroph MAGs using 37 gene markers and visualized with anvi’o [43]. Additional layers of information display the length of MAGs alongside environmental signal computed using genome-wide metagenomic read recruitments across 937 metagenomes, and nifH primer compatibilities (only full length and non-fragmented nifH genes were considered). For each MAG, the “maximal percent of mapped reads” layer displays the percent of mapped reads corresponding to the sample for which this metric was the highest among all 937 metagenomes. Thus, this sample is MAG dependent. In contrast, the “relative abundance” layers display for each MAG the average number of mapped reads across samples corresponding to the same size fraction. Bottom panel displays the ratio of cumulative genome-scale mean coverage between eight cyanobacterial diazotrophs (green) and 40 HBDs (red) across 385 metagenomes we organized into five size fractions.

Fig. 2. Functional lifestyle of marine diazotrophs.

The figure displays a heatmap of the completeness of 322 functional modules across the 48 diazotrophic MAGs. Clustering of MAGs and modules is based on completeness values (Euclidean distance and ward linkage) and the data were visualized using anvi’o [43]. The cosmopolitan score corresponds to the number of stations in which a given MAG was detected (cut-off: >25% of the MAG is covered by metagenomic reads).

Fig. 3. Oceanic stations with highest metagenomic signal for diazotrophs.

The world map provides coordinates for 15 Tara Oceans metagenomes (10 stations) displaying cumulative genomic coverage >100X for MAGs affiliated to diazotrophic Trichodesmium, UCYN-A or the HBDs. The bottom panel summarizes multi-omic signal (including at the level of nifH genes) statistics for those 15 metagenomes.

Fig. 4. Detection of nifH genes across marine metagenomes and metatranscriptomes.

The figure displays the proportion of metagenomic and metatranscriptomic reads mapping onto nifH genes as a function of ranges in two size fractions. Target genes correspond to the extended nifH gene database of 328 sequences including 280 orphan genes. The mapped samples (781 metagenomes and 520 metatranscriptomes) correspond to the surface and deep chlorophyll maximum layers of all oceans and two seas. For each size fraction range, the number of cumulated mapped reads represents each diazotrophic lineage (seven categories) across all samples. Results are displayed in relative proportion. The >0.8 μm size fraction range includes up to five size fractions: 0.8–5 μm, 5–20 μm, 20–180 μm, 180–2000 μm, and 0.8–2000 μm.