A cryptic plasmid is among the most numerous genetic elements in the human gut

Abstract

Plasmids are extrachromosomal genetic elements that often encode fitness-enhancing features. However, many bacteria carry "cryptic" plasmids that do not confer clear beneficial functions. We identified one such cryptic plasmid, pBI143, which is ubiquitous across industrialized gut microbiomes and is 14 times as numerous as crAssphage, currently established as the most abundant extrachromosomal genetic element in the human gut. The majority of mutations in pBI143 accumulate in specific positions across thousands of metagenomes, indicating strong purifying selection. pBI143 is monoclonal in most individuals, likely due to the priority effect of the version first acquired, often from one's mother. pBI143 can transfer between Bacteroidales, and although it does not appear to impact bacterial host fitness in vivo, it can transiently acquire additional genetic content. We identified important practical applications of pBI143, including its use in identifying human fecal contamination and its potential as an alternative approach to track human colonic inflammatory states.

Keywords: Bacteroides; cryptic; horizontal gene transfer; human gut microbiome; inflammatory bowel disease; metagenomics; mobile genetic element; plasmid; sewage.

Figure 1. pBI143 prevalence and abundance in globally distributed human populations.

(A) Plasmid maps of the three distinct versions of pBI143, which differ primarily in the repA gene. IR = inverted repeat. The repA genes are colored according to Version 1 (blue), Version 2 (red) and Version 3 (green). (B) Read recruitment results from 4,516 metagenomes originating from 23 globally representative countries and mapped to pBI143. Top: The percentage of reads in each metagenome that mapped to pBI143 normalized by number of reads in the metagenome. Bottom: The proportion of individuals in a country that have pBI143 in their gut. Each red dot represents an individual metagenome. (C) Countries that are represented in our collection of 4,516 global adult gut metagenomes. Each country’s pie chart is colored based on the version(s) of pBI143 that is most prevalent in that country (Version 1 = blue, Version 2 = red, Version 3 = green). Each country is colored based on the proportion of Version 1, 2 or 3 present in the population, or gray if fewer than 20% of individuals carry pBI143. Pie charts show the proportions of pBI143 versions in all individuals that carry it within a country. See also Table S1, Figure S1.

Figure 2.. Presence or absence of pBI143 in non-human gut environments.

(A) Survey of pBI143 across more than 100,000 metagenomes from diverse environments. (B) Copy number of pBI143 compared to two established human fecal markers Bacteroides or Lachnospiraceae, as measured by qPCR. Zero, trace, moderate, high and sewage categories and sample order designations are determined based on pBI143 copy number relative to the established markers. See also Table S1, S2, Figure S2, S3.

Figure 3.. The mutational landscape of pBI143 in sewage and the human gut.

(A) The proportion of SNVs across 4,516 human gut metagenomes that are present in the same location (match) or different locations (do not match) as variation in one of the versions of pBI143 (turquoise). Each point is a single metagenome. (B) The proportion of SNVs across 68 sewage gut metagenomes that are present in the same location (match) or different locations (do not match) as variation in one of the versions of pBI143 (pink). (C) Non-consensus SNVs present in 4,516 human gut metagenomes and 68 sewage metagenomes. (D) AlphaFold 2 predicted structure of the catalytic domain of MobA with single amino acid variants from all 4,516 human gut metagenomes superimposed as ball- and-stick residues. oriT DNA (gray) and a Mn2+ ion marking the active site (purple) were modeled based on 4lvi.pdb . The size of the ball-and-stick spheres indicate the proportion of samples carrying variation in that position (the larger the sphere, the more prevalent the variation at the residue) and the color is in CPK format. The color of the ribbon diagram indicates the pLDDT from AlphaFold 2 with red = very high (> 90 pLDDT) and orange = confident (80 pLDDT). See also Table S3, S4.

Figure 4.. Phylogeny of pBI143 in human donors versus the phylogeny of bacterial isolates recovered from the same individuals.

pBI143 (left) and bacterial host (right) genome phylogenies. The pBI143 phylogeny was constructed using the MobA and RepA genes; the bacterial phylogeny was constructed using 38 ribosomal proteins (see Methods). Blue alluvial plots are isolates with Version 1 pBI143 and red alluvial plots are isolates with Version 2 pBI143. No isolates had the rarer Version 3. See also Table S1, S4.

Figure 5.. Transfer and maintenance of pBI143.

(A) The network shows the degree of similarity between pBI143 SNVs across 154 mother and infant metagenomes from Finland, Italy, Sweden and the USA. Each node is an individual metagenome and nodes are colored based on family grouping. The surrounding coverage plots (colored) are visual representations of SNV patterns present in the indicated metagenomes. Nodes labeled with an “M” are mothers; nodes with no labels are infants. (B) Representative coverage plots showing different coverage patterns (maintained, two versions or wilt) observed in plasmids transferred from mothers to infants. See also Table S5, Figure S5.

Figure 6.. The relationship between pBI143 and its bacterial hosts.

(A) The average coverage of pBI143 and the corresponding coverage of predicted host genomes (Bacteroides, Parabacteroides and Phocaeicola) in 4,516 metagenomes. (B) Competition experiments in gnotobiotic mice between B. fragilis with and without pBI143. The proportion of pBI143-carrying cells in male (M) and female (F) mice in the initial inoculum, at Day 14, Day 28 and Day 40. (C) Four examples of pBI143 assembled from metagenomes that carry additional cargo genes. Gray genes are the canonical repA and mobA genes of naive pBI143; lilac genes are additional cargo. See also Table S2, S6, Figure S6.

Figure 7.. pBI143 copy number increases in stressful environments.

(A) Copy number of pBI143 in B. fragilis cultures with increasing exposure to oxygen. Top: B. fragilis 214. Bottom: B. fragilis RI6. Arrows indicate the time point at which the culture was returned to the anaerobic chamber. The control cultures (gray) were never exposed to oxygen. Opaque lines are the mean of 5 replicates (translucent lines). (B) Host-specific approximate copy number ratio (ACNR) of pBI143 in healthy individuals (gray) versus those with IBD (purple). See also Table S2, S7.