Thousands of previously unknown phages discovered in whole-community human gut metagenomes

Abstract

Background: Double-stranded DNA bacteriophages (dsDNA phages) play pivotal roles in structuring human gut microbiomes; yet, the gut virome is far from being fully characterized, and additional groups of phages, including highly abundant ones, continue to be discovered by metagenome mining. A multilevel framework for taxonomic classification of viruses was recently adopted, facilitating the classification of phages into evolutionary informative taxonomic units based on hallmark genes. Together with advanced approaches for sequence assembly and powerful methods of sequence analysis, this revised framework offers the opportunity to discover and classify unknown phage taxa in the human gut.

Results: A search of human gut metagenomes for circular contigs encoding phage hallmark genes resulted in the identification of 3738 apparently complete phage genomes that represent 451 putative genera. Several of these phage genera are only distantly related to previously identified phages and are likely to found new families. Two of the candidate families, "Flandersviridae" and "Quimbyviridae", include some of the most common and abundant members of the human gut virome that infect Bacteroides, Parabacteroides, and Prevotella. The third proposed family, "Gratiaviridae," consists of less abundant phages that are distantly related to the families Autographiviridae, Drexlerviridae, and Chaseviridae. Analysis of CRISPR spacers indicates that phages of all three putative families infect bacteria of the phylum Bacteroidetes. Comparative genomic analysis of the three candidate phage families revealed features without precedent in phage genomes. Some "Quimbyviridae" phages possess Diversity-Generating Retroelements (DGRs) that generate hypervariable target genes nested within defense-related genes, whereas the previously known targets of phage-encoded DGRs are structural genes. Several "Flandersviridae" phages encode enzymes of the isoprenoid pathway, a lipid biosynthesis pathway that so far has not been known to be manipulated by phages. The "Gratiaviridae" phages encode a HipA-family protein kinase and glycosyltransferase, suggesting these phages modify the host cell wall, preventing superinfection by other phages. Hundreds of phages in these three and other families are shown to encode catalases and iron-sequestering enzymes that can be predicted to enhance cellular tolerance to reactive oxygen species.

Conclusions: Analysis of phage genomes identified in whole-community human gut metagenomes resulted in the delineation of at least three new candidate families of Caudovirales and revealed diverse putative mechanisms underlying phage-host interactions in the human gut. Addition of these phylogenetically classified, diverse, and distinct phages to public databases will facilitate taxonomic decomposition and functional characterization of human gut viromes. Video abstract.

Fig. 1

Three candidate families of Caudovirales phages discovered in human gut metagenomes. a Phylogenetic tree of the large terminase subunit encoded by Caudovirales phage genomes in GenBank (n = 3931) and in gut metagenomes (n = 1298). Branches are colored according to the current ICTV families, except for the Myoviridae, Podoviridae, or Siphoviridae, which are in orange. The outermost ring indicates the location of candidate families proposed in this study: 1, “Quimbyviridae” phages; 2, “Flandersviridae” phages; 3, “Gratiaviridae” phages (see main text). b Gene sharing network of the Urovicota phages. Phage genomes identified in human gut metagenomes (blue nodes) were compared to phages in the GenBank database (colored as in Figure 1, with the addition of the crAss-like phages in brown and the new Caudovirales families proposed in this study in black). c Abundance of phages across human gut viromes. The x-axis depicts the fractional abundance of a given phage averaged across all viromes (n = 1241); the y-axis is the fraction of viromes that a given phage recruits at least one read. Each phage genome (n = 7888 total) is colored at the taxonomic level of order (c) or family (Uroviricota families only) (d)

Fig. 2

Phylogenetic tree of the large terminase subunit and genome maps of Quimby-like phages. a Individual genome maps of Quimby-like phages and ICTV classified phages are shown to the right of each branch. The ORFs are colored according to function: large terminase subunit (red), structural components (blue), DNA replication and repair (orange), lysogeny (pink), general function (green), and unknown (grey). b Expansion of four Quimby-like phages and a single gut phage genome from an adjacent branch (“group 4986”). The diversity-generating retroelement and hypervariable ORFs are highlighted with a dashed box and asterisk. The nucleotide scales differ between individual genome maps in both panels

Fig. 3

Phylogenetic tree of the large terminase subunit and complete genome maps for “Flandersviridae.” a Genome maps of members of the “Flandersviridae and selected ICTV-classified phages were constructed and colored as in Fig 3. b Genome maps of three genera from the “Flandersviridae” family. The dashed box highlights the insertion of licD- and ispD-family enzymes in the replication module of one “Flandersviridae” phage

Fig. 4

Phylogenetic tree of the large terminase subunit and genome maps of the “Gratiaviridae” phages. a Genome maps of ICTV-classified phages were constructed and colored as in Fig. 3. b Genome maps of four genera from the Gratiaviridae family. The dashed box highlights a HipA-family kinase domain-containing protein, AAA-family ATPase, and glycosyltransferase (see main text)