Uneven distribution of cobamide biosynthesis and dependence in bacteria predicted by comparative genomics

Abstract

The vitamin B₁₂ family of cofactors known as cobamides are essential for a variety of microbial metabolisms. We used comparative genomics of 11,000 bacterial species to analyze the extent and distribution of cobamide production and use across bacteria. We find that 86% of bacteria in this data set have at least one of 15 cobamide-dependent enzyme families, but only 37% are predicted to synthesize cobamides de novo. The distribution of cobamide biosynthesis and use vary at the phylum level. While 57% of Actinobacteria are predicted to biosynthesize cobamides, only 0.6% of Bacteroidetes have the complete pathway, yet 96% of species in this phylum have cobamide-dependent enzymes. The form of cobamide produced by the bacteria could be predicted for 58% of cobamide-producing species, based on the presence of signature lower ligand biosynthesis and attachment genes. Our predictions also revealed that 17% of bacteria have partial biosynthetic pathways, yet have the potential to salvage cobamide precursors. Bacteria with a partial cobamide biosynthesis pathway include those in a newly defined, experimentally verified category of bacteria lacking the first step in the biosynthesis pathway. These predictions highlight the importance of cobamide and cobamide precursor salvaging as examples of nutritional dependencies in bacteria.

Fig. 1

Functions carried out by cobamide-dependent processes. Reactions carried out by cobamide-dependent enzymes are shown on the left side of the arrows and cobamide-independent alternative processes, if known, on the right. Annotations or query genes used for searching for each function are listed in Supplementary Table 4. Tetrahydrofolate (THF), radical S-adenosylmethionine (rSAM)

Fig. 2

Cobamide biosynthesis and structure. a The cobamide biosynthesis pathway is shown with each enzymatic step indicated by a white box labeled with the gene names and functional annotation. Subsections of the pathway and salvaging and remodeling pathways are bracketed or boxed with labels in bold. Orthologous enzymes that carry out similar reactions in aerobic and anaerobic corrin ring biosynthesis are indicated by dashed lines. b Structure of cobalamin. The upper ligand R can be a 5'-deoxyadenosyl or methyl group. Classes of possible lower ligand structures are also shown. Benzimidazoles: R₁, R₂ = H, OH, CH₃, OCH₃. Purines: R₁ = H, CH₃, NH₂; R₂ = H, NH₂, OH, O. Phenolics: R = H, CH₃.

Fig. 3

Cobamide dependence in bacteria. a Histogram of the number of cobamide-dependent enzyme families (shown in Fig. 1, Supplementary Table 4) per genome in the complete filtered data set and the four most abundant phyla in the data set. The numbers are given for bars with values less than 1%. The inset lists the mean, standard deviation (St. Dev.), median, and mode of cobamide-dependent enzyme families for each phylum. b Rank abundance of cobamide-dependent enzyme families in the filtered data set and the four most abundant phyla. The inset shows an expanded view of the nine less abundant functions. c Abundance of five cobamide-dependent processes and cobamide-independent alternatives in the complete filtered data set. Genomes with only the cobamide-dependent, only the cobamide-independent, or both pathways are shown for each process

Fig. 4

Predicted cobamide biosynthesis phenotypes in the complete filtered data set and the four most abundant phyla in the data set. Genomes were classified into predicted cobamide biosynthesis phenotypes based on the criteria listed in Supplementary Table 7. The “Partial biosynthesis” category includes cobinamide (Cbi) salvagers and tetrapyrrole precursor salvagers. The “Uses cobamides” category is defined as having one or more of the cobamide-dependent enzyme families shown in Fig. 1. The numbers are given for bars that are not visible

Fig. 5

Lower ligand structure predictions. a, b Proportion of genomes containing the indicated lower ligand structure determinants (inner circle), α-ribazole salvaging gene (inner ring), and corrinoid remodeling gene (outer ring) in the complete filtered data set separated by cobamide producer category (a) and in cobamide producers separated by phylum (b). c. The anaerobic benzimidazole biosynthesis pathway is shown with the functions that catalyze each step above the arrows. The genes required to produce each benzimidazole are shown below each structure, with the number of genomes in the complete filtered data set containing each combination of genes in parentheses. The sets of bza genes that do not have a predicted structure are listed on the right. Aminoimidazole ribotide (AIR), 5-hydroxybenzimidazole (5-OHBza), 5-methoxybenzimidazole (5-OMeBza), 5-methoxy-6-methylbenzimidazole (5-OMe-6-MeBza)

Fig. 6

Characterization of putative tetrapyrrole precursor salvagers a Steps in cobamide biosynthesis. The enzymes that catalyze each step are indicated to the right of each arrow. The number of genomes in the complete filtered data set in each precursor salvage category is on the left. Two genomes had cobamide biosynthesis pathways inconsistent with simple auxotrophy (*). Specific tetrapyrrole precursor salvager genomes are listed in Supplementary Table 10. b HPLC analysis of corrinoid extracts from Clostridium scindens, Clostridium sporogenes, and Treponema primitia grown with and without added ALA. A cyanocobalamin standard (200 pmoles) is shown for comparison. Asterisks denote peaks with ultraviolet–visible (UV–Vis) spectra consistent with that of a corrinoid. c T. primitia ZAS-2 growth in 4YACo medium with and without added cyanocobalamin (37 nM) or ALA (1 mM). Each point represents the average of three biological replicates. Error bars are the standard deviation