VB12Path for Accurate Metagenomic Profiling of Microbially Driven Cobalamin Synthesis Pathways

Abstract

Cobalamin (vitamin B12; VB₁₂) is an indispensable nutrient for all living entities in the Earth's biosphere and plays a vital role in both natural and host environments. Currently in the metagenomic era, gene families of interest are extracted and analyzed based on functional profiles by searching shotgun metagenomes against public databases. However, critical issues exist in applying public databases for specific processes such as VB₁₂ biosynthesis pathways. We developed a curated functional gene database termed VB₁₂Path for accurate metagenomic profiling of VB₁₂ biosynthesis gene families of microbial communities in complex environments. VB₁₂Path contains a total of 60 VB₁₂ synthesis gene families, 287,731 sequences, and 21,154 homology groups, and it aims to provide accurate functional and taxonomic profiles of VB₁₂ synthesis pathways for shotgun metagenomes and minimize false-positive assignments. VB₁₂Path was applied to characterize cobalamin biosynthesis gene families in human intestines and marine environments. The results demonstrated that ocean and human intestine had dramatically different VB₁₂ synthesis processes and that gene families belonging to salvage and remodeling pathway dominated human intestine but were lowest in the ocean ecosystem. VB₁₂Path is expected to be a useful tool to study cobalamin biosynthesis processes via shotgun metagenome sequencing in both environmental and human microbiome research. IMPORTANCE Vitamin B12 (VB₁₂) is an indispensable nutrient for all living entities in the world but can only be synthesized by a small subset of prokaryotes. Therefore, this small subset of prokaryotes controls ecosystem stability and host health to some extent. However, critical accuracy and comprehensiveness issues exist in applying public databases to profile VB₁₂ synthetic gene families and taxonomic groups in complex metagenomes. In this study, we developed a curated functional gene database termed VB₁₂Path for accurate metagenomic profiling of VB₁₂ communities in complex environments. VB₁₂Path is expected to serve as a valuable tool to uncover the hidden microbial communities producing this precious nutrient on Earth.

Keywords: cobalamin synthesis; database; functional gene; shotgun metagenome.

FIG 1

VB₁₂ synthesis gene (sub)families in VB₁₂Path and other orthologous databases. (A) The number of gene (sub)families or orthologous groups responsible for VB₁₂ biosynthesis detected in different databases. (B) The accuracy of gene families defined in orthologous databases. Accuracy was measured by the percentage of sequences belonging to the selected VB₁₂ synthesis gene (sub)families in corresponding orthology groups in public databases. Gene families appearing in multiple pathways are marked with an asterisk. Although separately plotted, some gene families may belong to a single orthologous group in public orthology databases.

FIG 2

Situation of VB₁₂ synthesis pathways in sequenced microbial genomes. (A) The current sequenced microbial genomes indicate that the cobalamin synthesis pathway is mainly contributed by bacterial genes. The taxonomic composition and relative abundance distribution of bacterial (B) and archaeal (C) gene families in different VB₁₂ synthesis processes at the phylum level are also displayed.

FIG 3

Application of VB₁₂Path to characterize cobalamin synthesis gene (sub)family in marine and human intestinal environments. Principal coordinate analysis (PCoA) clustering of ocean and gut examples based on Bray-Curtis distances of (A) taxonomic composition and (B) functional genes composition. A clear separation of human intestine and ocean samples could be observed for both taxonomic and functional gene compositions.

FIG 4

Composition and relative abundance of microbial groups at family level and gene family level in ocean (A and B) and human intestine samples (C and D). The left panels represent taxonomic compositions at family level, while the right panels represent functional gene families. Different colors refer to different species groups and gene families. Color legends are not shown in the figure because the main purpose of this figure is to describe the high variation of taxonomic compositions but stable functional gene compositions of VB₁₂ synthesis pathways among different samples.

FIG 5

Workflow summary for constructing VB₁₂Path. Multiple steps were included in the framework, including seed database construction, core database construction, database merging, and full database construction. A set of PERL scripts are also provided to generate taxonomic and functional gene profiles from shotgun metagenomes.