Analysis of metagenome-assembled viral genomes from the human gut reveals diverse putative CrAss-like phages with unique genomic features

Abstract

CrAssphage is the most abundant human-associated virus and the founding member of a large group of bacteriophages, discovered in animal-associated and environmental metagenomes, that infect bacteria of the phylum Bacteroidetes. We analyze 4907 Circular Metagenome Assembled Genomes (cMAGs) of putative viruses from human gut microbiomes and identify nearly 600 genomes of crAss-like phages that account for nearly 87% of the DNA reads mapped to these cMAGs. Phylogenetic analysis of conserved genes demonstrates the monophyly of crAss-like phages, a putative virus order, and of 5 branches, potential families within that order, two of which have not been identified previously. The phage genomes in one of these families are almost twofold larger than the crAssphage genome (145-192 kilobases), with high density of self-splicing introns and inteins. Many crAss-like phages encode suppressor tRNAs that enable read-through of UGA or UAG stop-codons, mostly, in late phage genes. A distinct feature of the crAss-like phages is the recurrent switch of the phage DNA polymerase type between A and B families. Thus, comparative genomic analysis of the expanded assemblage of crAss-like phages reveals aspects of genome architecture and expression as well as phage biology that were not apparent from the previous work on phage genomics.

Fig. 1. The crAss-like phage assemblage.

a Phylogenetic tree of the TerL protein of crAss-like phages. CrAss-like groups are marked with red color. Previously analyzed crAss-like sequences are highlighted in yellow. Cultured phages are marked with bold font. Numbers in green circles indicate genomes for which the gene conservation pattern is shown in c. b Some key genome features of CrAss-like cMAGs. In parentheses are numbers on distinct genomes (sharing <90% of similarity at the DNA level). c Pattern of gene conservation in crAss-like phages. TerL terminase large subunit, portal portal protein, gene77 putative structural protein (gene 77), MCP major capsid protein, gene75 putative structural protein (gene 75), gene74 putative structural protein (gene 74), gene73 putative structural protein (gene 73), IHF_54 IHF subunit (gene 54), IHF_53 IHF subunit (gene 53), Ttub Tail tubular protein (P22 gp4-like), Tstab tail stabilization protein (P22 gp10-like), gene49 uncharacterized protein (gene 49), gene86 putative structural protein (gene 86), PDDEXK PD-(D/E)XK family nuclease, DnaB phage replicative helicase, DnaB family, primase DnaG family primase, SNF2 SNF2 helicase, SF1 SF1 helicase, ATP_43b AAA domain ATPase, PolB DNA polymerase family B, PolA DNA polymerase family A, Thy1 thymidylate synthase, dUTP dUTPase, UDG Uracil-DNA glycosylase, MPP metallophosphatase, Rep_Org replisome organizer protein, RNR ribonucleotide reductase, phage_O bacteriophage replication protein O (gene10 of IAS virus), gene48b phage endonuclease I, dNK deoxynucleotide monophosphate kinase.

Fig. 2. Genome organizations of representative crAss-like phages.

The circular genomes were linearized by breaking the circle after the transcription gene block. Conserved proteins are abbreviated as in Fig. 1c; RNApB’ marks the conserved DxDxD motif of the large RNAP subunit homologous to the bacterial β’ subunit.

Fig. 3. Transcription gene block in crAss-like phages.

The predicted genes are shown by empty block arrows, and recognized conserved domains are indicated by colored rectangles. Vertical bars indicate in-frame stop codons. LAGLIDADG is intron-encoded endonuclease maturase.

Fig. 4. Rarefaction analysis of the crAss-like phage pangenome.

The results of 1001 rarefaction runs for 110 representative genomes are shown. The black line shows the median, and the gray lines show the 5 and 95 percentiles.

Fig. 5. The crAss-like phages dominate the human gut virome.

The schematic shows the fractions of sequence reads mapped to different groups of virus genomes. The total represents 49,540,005 reads mapped to virus sequences.

Fig. 6. DNA polymerase switches in the evolution of crAss-like phages.

The left panel shows a schematic phylogenetic tree of TerL for the entire crAss-like phage assemblage, with the branches collapsed into triangles. The right panels show the expanded beta and zeta branches. The portions of the triangles (left panel) and the tree leaves (right panels) are colored by the DNAP type, red for PolA and purple for PolB.

Fig. 7. Examples of stop codon reassignments, intein, and self-splicing intron insertions in conserved genes of crAss-like phages.

Horizontal lines denote co-directed reading frames in the nucleotide sequence; short vertical bars indicate standard stop codons (red: TAG, green: TGA, blue: TAA); colored shapes indicate domains or domain fragments, mapped to the nucleotide sequence.