Aquatic metagenomes implicate Thaumarchaeota in global cobalamin production

Abstract

Cobalamin (vitamin B12) is a complex metabolite and essential cofactor required by many branches of life, including most eukaryotic phytoplankton. Algae and other cobalamin auxotrophs rely on environmental cobalamin supplied from a relatively small set of cobalamin-producing prokaryotic taxa. Although several Bacteria have been implicated in cobalamin biosynthesis and associated with algal symbiosis, the involvement of Archaea in cobalamin production is poorly understood, especially with respect to the Thaumarchaeota. Based on the detection of cobalamin synthesis genes in available thaumarchaeotal genomes, we hypothesized that Thaumarchaeota, which are ubiquitous and abundant in aquatic environments, have an important role in cobalamin biosynthesis within global aquatic ecosystems. To test this hypothesis, we examined cobalamin synthesis genes across sequenced thaumarchaeotal genomes and 430 metagenomes from a diverse range of marine, freshwater and hypersaline environments. Our analysis demonstrates that all available thaumarchaeotal genomes possess cobalamin synthesis genes, predominantly from the anaerobic pathway, suggesting widespread genetic capacity for cobalamin synthesis. Furthermore, although bacterial cobalamin genes dominated most surface marine metagenomes, thaumarchaeotal cobalamin genes dominated metagenomes from polar marine environments, increased with depth in marine water columns, and displayed seasonality, with increased winter abundance observed in time-series datasets (e.g., L4 surface water in the English Channel). Our results also suggest niche partitioning between thaumarchaeotal and cyanobacterial ribosomal and cobalamin synthesis genes across all metagenomic datasets analyzed. These results provide strong evidence for specific biogeographical distributions of thaumarchaeotal cobalamin genes, expanding our understanding of the global biogeochemical roles played by Thaumarchaeota in aquatic environments.

Figure 1

Identification of the cobalamin synthesis pathway and associated gene clusters in available thaumarchaeotal genomes. The pathway in (a) is adapted from the studies by Moore et al. (2013) and Raux et al. (1999). Horizontal arrows indicate homology between aerobic and anaerobic pathway enzymes. Grey enzyme names were not detected in thaumarchaeotal genomes. *CbiJ is also not present in other known archaeal cobalamin producers and therefore should not be considered an essential gene for this pathway in Thaumarchaeota. (b) A six-member cobalamin synthesis gene cluster with conserved synteny across several thaumarchaeotal genomes. The cluster encodes enzymes (highlighted by blue boxes in (a)) in the upper pathway, and includes three enzymes (bolded) specific to the anaerobic pathway. (c) A four-member gene cluster possessing enzymes (highlighted by green boxes in (a)) corresponding to the final steps of cobalamin synthesis. The apparent missing cobD gene in AR1 may be a genome annotation error given that it appears to be in the genome but may be truncated (data not shown).

Figure 2

Survey of cobalamin synthesis genes across aquatic metagenomes. (a) The full set of cob/cbi genes and subset of eleven representative marker genes (black) used in the screen and their abundance (total number of detected genes). (b) Taxonomic breakdown of all detected cob/cbi genes across 430 aquatic metagenomes. (c) Distributions of thaumarchaeotal, cyanobacterial and proteobacterial cobalamin gene contributions (% of total cob/cbi genes) for 28 different studies. (d) Clustering and heatmap visualization of taxonomic composition of cob/cbi genes for the 430 metagenomes. The color scale reflects the proportion of cob/cbi genes in each sample affiliated with a particular taxonomic group. More information on each sample can be found in Supplementary Table S1. (e) Global distribution of cobalamin synthesis genes. To emphasize Thaumarchaeota-rich metagenomes, data points with higher thaumarchaeotal contributions have been slightly enlarged and overlayed preferentially in cases where there are overlapping data points. The Thaumarchaeota-rich Arctic metagenome described in the text is indicated by an asterisk.

Figure 3

Phylum and metadata correlations. (a) Correlation matrix for all species-metadata correlations with r>0.25. Correlations were measured between numerical metadata from all 430 metagenome samples and the per-taxon proportion of cobalamin genes. Correlations between taxonomic proportions were also measured to identify potential species–species associations. (b) Left: proportion of cobalamin synthesis genes vs depth for three phyla (computed using all 430 metagenome samples). Middle: proportion of cobalamin synthesis genes vs different time points from the 2008–2009 L4 station time series. Right: per-gene taxonomic proportions of cobalamin genes for an Arctic metagenome. Thaumarchaeota (red), Cyanobacteria (green), Proteobacteria (blue), Unclassified (dark grey), Bacteroidetes/Chlorobi (purple), Actinobacteria (black), Firmicutes (orange), and Other (light gray). Bar widths are proportional to the square root of the sample sizes. (c) Phyla-vs-Phyla contributions of cobalamin synthesis genes across all 430 metagenome samples. All correlations exhibit a negative linear relationship, except for Thaumarchaeota-vs-Cyanobacteria, which exhibits an either-or relationship, suggestive of competitive or niche exclusion. (d) Correlation analysis for the MI_LOCO metagenome. The strongest correlating variables with thaumarchaeotal cob/cbi gene contribution are shown on the left, and plots for the top two positively (nitrate concentration and sample depth) and negatively correlated (ammonium and bacterial cell concentration) variables are shown on the right.