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 phageome 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 3,738 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.

Prediction of prophages and their host ranges in pathogenic and commensal Neisseria species

“…Elucidating the host range of phages is critical for understanding how they influence their microbial hosts, including their role in mobilizing genetic material (12). CRISPR targeting data are frequently used to predict the bacterial hosts of phages (35–39). Here, our findings that prophages are extensively targeted by spacers encoded by other species suggest that phages may be shared between different Neisseria species.…”

UG/Abi: a highly diverse family of prokaryotic reverse transcriptases associated with defense functions

“…To verify the presence of UG27 in viral genomes, we expanded the number of UG27 systems by searching on IMG/VR, GPD, and mMGE databases (44,51,53) obtaining 1447 dereplicated genomes harboring the complete system (781 of which also encode a TerL homolog, Supplementary Figure S3). In an attempt to assign taxonomy to these viral sequences or test whether they belong to a known family, we compared them against reference databases and a compendium of new phage families (54–56) using ANI (average nucleotide identity) and gene-content network information (Methods). At the ANI level, some UG27-containing viral genomes share similarities with recently proposed orders of bacterial viruses, including Crassvirales and Friedlandervirales (55) and viruses from the proposed Quimbyviridae family (54) (Supplementary Table S5).…”

“…Only genomes harboring RT, cluster 337, and cluster 346 were further considered, which rendered 3861 predicted viral genomes. Genomes were dereplicated at 95% sequence identity using dRep with default parameters as previously described (54), resulting in 1447 distinct genomes. In addition, dereplicated viral genomes were searched for Large Terminases (TerL) as a viral marker using the PFAM profiles PF03354, PF04466, PF03237, and PF05876 and a profile built from an alignment of TerL derived from a recent phylogenetic analysis (54).…”

“…Genomes were dereplicated at 95% sequence identity using dRep with default parameters as previously described (54), resulting in 1447 distinct genomes. In addition, dereplicated viral genomes were searched for Large Terminases (TerL) as a viral marker using the PFAM profiles PF03354, PF04466, PF03237, and PF05876 and a profile built from an alignment of TerL derived from a recent phylogenetic analysis (54). To identify these putative viral sequences and assign taxonomy, whole-genome comparisons and gene-content networks were made using novel phage families (54–56) as references.…”

“…In addition, dereplicated viral genomes were searched for Large Terminases (TerL) as a viral marker using the PFAM profiles PF03354, PF04466, PF03237, and PF05876 and a profile built from an alignment of TerL derived from a recent phylogenetic analysis (54). To identify these putative viral sequences and assign taxonomy, whole-genome comparisons and gene-content networks were made using novel phage families (54–56) as references. For whole-genome comparisons, fastANI (57) with parameter –fragLen 500 and default parameters were used.…”