CeMbio - The Caenorhabditis elegans Microbiome Resource

Abstract

The study of microbiomes by sequencing has revealed a plethora of correlations between microbial community composition and various life-history characteristics of the corresponding host species. However, inferring causation from correlation is often hampered by the sheer compositional complexity of microbiomes, even in simple organisms. Synthetic communities offer an effective approach to infer cause-effect relationships in host-microbiome systems. Yet the available communities suffer from several drawbacks, such as artificial (thus non-natural) choice of microbes, microbe-host mismatch (e.g., human microbes in gnotobiotic mice), or hosts lacking genetic tractability. Here we introduce CeMbio, a simplified natural Caenorhabditis elegans microbiota derived from our previous meta-analysis of the natural microbiome of this nematode. The CeMbio resource is amenable to all strengths of the C. elegans model system, strains included are readily culturable, they all colonize the worm gut individually, and comprise a robust community that distinctly affects nematode life-history. Several tools have additionally been developed for the CeMbio strains, including diagnostic PCR primers, completely sequenced genomes, and metabolic network models. With CeMbio, we provide a versatile resource and toolbox for the in-depth dissection of naturally relevant host-microbiome interactions in C. elegans.

Keywords: C. elegans; Host-microbe interactions; Metabolic networks; Microbiome resource; Synthetic communities.

Figure 1

The CeMbio strains. (A) The CeMbio strains (blue) were selected based on a comparison of 510 cultured C. elegans microbiome bacteria with the 12 most common OTUs inferred by repeating our previous meta-analysis [purple, (Zhang et al. 2017)] with only the natural worm samples. The tree is based on a maximum-likelihood analysis using a TIM3e+R4 model and 10000 bootstraps. Nodes with bootstrap support >75% are denoted with a red dot. Some branches include several highly similar OTUs, as indicated (e.g., +6 more). (B) Fluorescence in situ hybridization of C. elegans N2 colonized with the CeMbio strains [red, general bacterial probe EUB338; blue, DAPI].

Figure 2

Colonization levels of C. elegans gut by each CeMbio strain alone. Colony forming units (CFUs) of each CeMbio strain in C. elegans gut (N2) were measured at 72 h or 120 h post L1 larvae. At least six biological replicates were performed for each condition. These results are from colonization experiment 1.

Figure 3

Colonization of N2 and CB4856 C. elegans strains by the CeMbio community. (A) Proportion of reads in the initial community assembly used as inoculum for the lawns. (B) Proportion of reads in the C. elegans strains N2 and CB4856 and the corresponding lawn samples. The two Enterobacteriaceae CEent1 and JUb66 share a similar 16S rRNA sequence and the V4 PCR primers used in this 16S amplicon sequencing experiment do not discriminate between the two 16S rRNA sequences over this region. (C) Colony forming units (CFUs) of the CeMbio community isolated from N2 and CB4856 nematodes. (D) Mean observed number of CeMbio members (top) and Inverse Simpson Index (bottom) with standard deviation, indicating richness and diversity of the bacterial communities in N2 and CB4856 worms. (E) Principle coordinate analysis of Bray-Curtis dissimilarities of the microbial communities of nematode and lawn samples with an ellipse representing the 95% confidence interval of the nematode samples. These results are from colonization experiment 2.

Figure 4

Colonization of C. elegans gut by the CeMbio community under different plating conditions. (A) Proportion of reads in the initial community assembly used as inoculum for the lawns. (B) Proportion of reads in NGM worm and lawn samples. (C) Proportion of reads in PFM worm and lawn samples. (D) Mean observed number of CeMbio members (top) and Inverse Simpson Index (bottom) with standard deviation indicating richness and diversity of the communities. Stars indicate significant differences in alpha diversity (P < 0.005). (E) Principle coordinate analysis of Bray-Curtis dissimilarities of the microbial communities in nematode and lawn samples with ellipses representing the 95% confidence intervals of the nematode (dashed) and lawn (solid) samples. (F) Colony forming units (CFUs) of the CeMbio community in single nematodes. These results are from colonization experiment 3.

Figure 5

Effect of the CeMbio community and individual bacteria on C. elegans growth rates. (A) Developmental speed, represented by the number of adults counted on an hourly basis, when N2 and CB4856 nematodes are raised on the CeMbio mixture or the individual bacteria. Continuous lines indicate CeMbio bacteria or mixture; the dotted line the E.coli OP50 control. Each combination of nematode and bacteria was performed in duplicate. (B) Developmental timing snapshot at 52 h post L1. The black dotted line represents the median number of adult worms on E. coli at that time point where roughly 50% of the N2 population reached adulthood (n = 50-100 animal/replicate).

Figure 6

Comparison of metabolic pathways between the 12 CeMbio strains. (A) Distribution of unique and shared metabolic pathways across the 12 CeMbio members. Pathways are categorized from the most commonly found (present in 10 to 12 genomes) to unique pathways (present in 1 to 3 genomes). (B) Principal component analysis of the metabolic profiles of the 12 CeMbio members. (C) Summary of carbon source utilization for each CeMbio strain as inferred from the genome-scale metabolic models, additionally trained with Biolog EcoPlate plate data, given as a binary response (circle, ability to utilize the indicated carbon source; no circle, inability to utilize it). The carbon source utilization by the whole CeMbio community is highlighted in yellow.