Metagenomic and chemical characterization of soil cobalamin production

Abstract

Cobalamin (vitamin B₁₂) is an essential enzyme cofactor for most branches of life. Despite the potential importance of this cofactor for soil microbial communities, the producers and consumers of cobalamin in terrestrial environments are still unknown. Here we provide the first metagenome-based assessment of soil cobalamin-producing bacteria and archaea, quantifying and classifying genes encoding proteins for cobalamin biosynthesis, transport, remodeling, and dependency in 155 soil metagenomes with profile hidden Markov models. We also measured several forms of cobalamin (CN-, Me-, OH-, Ado-B₁₂) and the cobalamin lower ligand (5,6-dimethylbenzimidazole; DMB) in 40 diverse soil samples. Metagenomic analysis revealed that less than 10% of soil bacteria and archaea encode the genetic potential for de novo synthesis of this important enzyme cofactor. Predominant soil cobalamin producers were associated with the Proteobacteria, Actinobacteria, Firmicutes, Nitrospirae, and Thaumarchaeota. In contrast, a much larger proportion of abundant soil genera lacked cobalamin synthesis genes and instead were associated with gene sequences encoding cobalamin transport and cobalamin-dependent enzymes. The enrichment of DMB and corresponding DMB synthesis genes, relative to corrin ring synthesis genes, suggests an important role for cobalamin remodelers in terrestrial habitats. Together, our results indicate that microbial cobalamin production and repair serve as keystone functions that are significantly correlated with microbial community size, diversity, and biogeochemistry of terrestrial ecosystems.

Fig. 1

Cobalamin biosynthesis and transport pathways, separating anaerobic and aerobic pathways with early and late incorporation of cobalt, respectively. Adapted from Doxey et al. [15] and Fang et al. [86]. Sequence homology and functional equivalency between aerobic and anaerobic pathway enzymes are indicated by horizontal arrows. Colors denote the groups of profile HMMs (see TIGR or PFAM HMM numbers adjacent to corresponding pathway steps) that contribute to various stages of cobalamin synthesis and/or salvage transport

Fig. 2

Taxonomic composition among all 155 soil metagenomes for a all 12 cobalamin biosynthesis genes, b corrin ring biosynthesis genes, c final synthesis and repair genes, and d the DMB synthesis and activation genes. Only the top five phyla are shown individually, other phyla are collectively denoted as “others”

Fig. 3

Proportion of genera encoding three cobalamin-dependent genes (i.e., mutA, metH, and rsmB) and those encoding the complete cobalamin biosynthesis pathway. Relative abundances were calculated as ratios to total rpoB gene abundances across all 155 soil metagenomes

Fig. 4

Relationship between genes coding for cobalamin transport (btuB) and synthesis enzymes (cob/cbi/bluB) across 155 soil metagenomes. Color denotes the number of genera occurring with a given cob/cbi/bluB and btuB gene relative abundance. a–c represent a genus from the rare biosphere (rpoB gene relative abundances were below 0.1%), d–f intermediate abundance taxa (rpoB gene relative abundance between 0.1 and 5%), and g–i dominant taxa (rpoB gene relative abundance over 5%). Mutual exclusion between the cobalamin transporter encoding gene (btuB) and cobalamin synthesis enzyme genes (cob/cbi/bluB) were tested with 10⁵ permutations, and significant (p < 0.05) for each gene group and either rare biosphere or intermediate abundance taxa. From top to bottom: Group A, corrin ring biosynthesis; Group B, final synthesis and repair; Group C, 5,6-dimethylbenzimidazole (DMB) synthesis

Fig. 5

Concentration of total cobalamin (sum of both water-leachable and non-water-leachable cobalamin) and 5,6-dimethylbenzimidazole (DMB) measured in soils collected from the Canadian MetaMicroBiome Library (CM²BL) [82], the rare Charitable Research Reserve [83], and the Craibstone pH plots [84]. Water-leachable cobalamin and DMB only accounted for a small portion of total cobalamin and DMB. 1AT: Arctic Tundra; 2ATN: Arctic Tundra; 4TS: Tar sand; 5BF: Boreal coniferous forest; 6TD: Temperate deciduous forest; 7TR: temperate rain forest; 8NP: Northern peatlands; 9WLM: Wetland soil; 10AS: Agricultural soil (soy); 11AW: Agricultural soil (wheat); 13CO: Compost. AA: rare active agricultural site; D03: rare decommissioned agricultural (since 2003) site; D07: rare decommissioned agricultural (since 2007) site; D10: rare decommissioned agricultural (since 2010) site; CA: rare forest site; HB: rare forest site; IW: rare forest site. A full list of the samples and their details can be found in Supplementary material S2.xlsx

Fig. 6

Measured total cobalamin (sum of both water-leachable and non-water-leachable cobalamin) and 5,6-dimethylbenzimidazole (DMB) concentrations in 40 soil samples (a), and cobalamin biosynthesis gene summary for 155 soil metagenomes (b). Cobalamin-producing genes in b were grouped as: Group A, corrin ring biosynthesis; Group B, final synthesis and repair; Group C, 5,6-dimethylbenzimidazole (DMB) synthesis. The 155 soil metagenomes are independent from the 40 soil samples used for cobalamin measurement. The center line in the box plot represents the median value