Lysogeny is prevalent and widely distributed in the murine gut microbiota

Abstract

Bacteriophages are central members and potential modulators of the gut microbiome; however, the ecological and evolutionary relationships of gut bacteria and phages are poorly understood. Here we investigated the abundance and diversity of lysogenic bacteria (lysogens) in the bacterial community of C57BL/6J mice by detecting integrated prophages in genomes reconstructed from the metagenome of commensal bacteria. For the activities of lysogens and prophages, we compared the prophage genomes with the metagenome of free phages. The majority of commensal bacteria in different taxa were identified as lysogens. More lysogens were found among Firmicutes and Proteobacteria, than among Bacteroidetes and Actinobacteria. The prophage genomes shared high sequence similarity with the metagenome of free phages, indicating that most lysogens appeared to be active, and that prophages are spontaneously induced as active phages; dietary interventions changed the composition of the induced prophages. By contrast, CRISPR-Cas systems were present in few commensal bacteria, and were rarely active against gut phages. The structure of the bacteria-phage infection networks was "nested-modular", with modularity emerging across taxonomic scales, indicating that temperate phage features have developed over a long phylogenetic timescale. We concluded that phage generalists contribute to the prevalence of lysogeny in the gut ecosystem.

Fig. 1

The distribution and abundance of commensal lysogens in the gut bacterial community. a The phylogenetic tree of 181 bacterial bins that were generated based on 43 conserved marker genes, and designated at the class taxon level by different colors. b The percentage and c abundance of lysogenic bins (n = 24) per genome completeness (>30 and >70%) and size (>1 and >2 Mb). d Bacterial taxonomic profiles of lysogenic and non-lysogenic bins (n = 24) shown at the class-level. All data are presented as the mean ± SEM

Fig. 2

The taxonomic distribution of commensal lysogens. a The percentage and b abundance of lysogenic bins (n = 24) shown by phylum-level bacterial taxa according to genome completeness (>30 and >70%) and size (>1 and >2 Mb). The number of bacterial bins per bacterial phylum is indicated in parentheses. All data are presented as the mean ± SEM

Fig. 3

High sequence similarities between the integrated prophages and the free phages. a The overall percentage of phage metagenome annotations in the reference databases shown for protein clusters (n = 24), viral contigs (n = 24) and viral clusters. b The percentage of “known” phage metagenome annotated to three reference databases (the integrated prophages, RefSeq_viral and RefSeq_proph) shown for protein clusters, viral contigs and viral clusters. c The annotation efficiency of the phage metagenome to three reference databases shown for protein clusters, viral contigs and viral clusters. All data are presented as the mean ± SEM

Fig. 4

Prophages are spontaneously induced in active commensal lysogens. a The percentage of induced prophages and active lysogenic bins estimated by mapping phage metagenomic reads to the prophage sequences. b The taxonomic profiles of lysogenic and active lysogenic bins compared at the phylum level (n = 24). The composition of induced prophages for different diets determined using c the Jaccard dissimilarity-based PCoA (Adonis; FORTH, P = 0.001; BACK, P = 0.057) and d Jaccard distance comparisons (one-way ANOVA; FORTH, P < 0.001; BACK, P = 0.006). All data are presented as the mean ± SEM. LFD low-fat diet, HFHS high-fat, high-sucrose diet, and LFPP low-fat, high-plant-polysaccharide diet

Fig. 5

The network visualization of viral clusters comprising genomic sequences of the free phages and the integrated prophages. The nodes for different bacterial bins are designated by different colors

Fig. 6

The distribution of CRISPR-Cas systems in bacterial bins. a The percentage of CRISPR arrays in bacterial bins according to the identification of lysogen and the presence of CRISPR arrays. b The percentage of bacterial bins encoding spacers homologous to the phage metagenomic sequences in CRISPR arrays. c Comparison of the spacer numbers in lysogenic (n = 32) and non-lysogenic bins (n = 13) (two-tailed Student t-test), and comparison of the prophage numbers in the presence (n = 32) and absence (n = 87) of CRISPR-Cas systems (two-tailed Student t-test). d Correlation between the number of spacers in the CRISPR arrays of bacterial bins encoding CRISPR-Cas systems and the number of prophages in lysogenic bins (n = 32, Spearman’s rank correlation, r = −0.38, P = 0.03). All data are presented as the mean ± SEM

Fig. 7

The bacteria–phage interaction networks between viral clusters and bacterial bins. The matrix is composed of virus clusters (vc; rows) and bacterial bins (columns). These bacteria–phage networks are described as a nested by NTC (N_NTC = 0.97); and b modular by lp-Brim (Q = 0.66, c = 28). The gray curve is an isocline of perfect nestedness. The different modules are highlighted in color