Unique genomic features of crAss-like phages, the dominant component of the human gut virome

Abstract

CrAssphage is the most abundant virus identified in the human gut virome and the founding member of a large group of bacteriophages that infect bacteria of the phylum Bacteroidetes and have been discovered by metagenomics of both animal-associated and environmental habitats. By analysis of circular contigs from human gut microbiomes, we identified nearly 600 genomes of crAss-like phages. Phylogenetic analysis of conserved genes demonstrates the monophyly of crAss-like phages, which can be expected to become a new order of viruses, and of 5 distinct branches, likely, families within that order. Two of these putative families have not been identified previously. The phages in one of these groups have large genomes (145-192 kilobases) and contain an unprecedented high density of self-splicing introns and inteins. Many crAss-like phages encode suppressor tRNAs that enable readthrough of UGA or UAG stop-codons, mostly, in late phage genes, which could represent a distinct anti-defense strategy. Another putative anti-defense mechanism that might target an unknown defense system in Bacteroidetes inhibiting phage DNA replication involves multiple switches of the phage DNA polymerase type between A and B families. Thus, comparative genomic analysis of the expanded assemblage of crAss-like phages reveals several unusual features of genome architecture and expression as well as phage biology that were not apparent from the previous crAssphage analyses.

Probing the “Dark Matter” of the Human Gut Phageome: Culture Assisted Metagenomics Enables Rapid Discovery and Host-Linking for Novel Bacteriophages

“…Samples with high proportion of viral DNA in them tended to consist of one or very few viral contigs suggesting successful enrichment ( Figure 2 ). The taxonomic range of recovered viral contigs was very wide and included prokaryotic viruses in the families Inoviridae , Microviridae , Siphoviridae , Podoviridae , Myoviridae , members of provisional group of crAss-like phages [proposed order Crassvirales ( Koonin and Yutin, 2020 ; Yutin et al., 2020 )] as well as different eukaryotic DNA and RNA viruses ( Figure 2 ). Viral community composition in enriched viral supernatants reflected the composition of original faecal virome pools, with exception of some samples where successful enrichment was evident.…”

“…Some select examples of the identified phages include complete circular genomes of (i), a 45.3 kb Bacteroides uniformis DSM6597 phage ( Siphoviridae , Figure 3 ) previously observed in human gut metagenomes; (ii), a 46.4 kb temperate Siphoviridae phage infecting Anaerostipes hadrus APC942/1 ( Figure 3 ); (iii), a 168.6 kb E. coli phage ( Figure 3 ), 97% identical to previously isolated phage vB_EcoM-fHoEco02 [genus T4virus , family Myoviridae , ( Kiljunen et al, 2018 )]; (iv), a 40.3 kb Siphoviridae prophage spontaneously inducing from the chromosome of Subdoligranulum sp. APC924/74 [ Figure 3 ; ( Fitzgerald et al., 2018 )]; and (v), a 104.9 kb crAss-like phage ( Figure 3 ), belonging to subfamily Deltacrassvirinae , apparently using a non-standard genetic code [suppression of UAG codon is required to translate most of the genes ( Guerin et al., 2018 ; Yutin et al., 2020 )] and enriching in the presence of Prevotella stercorea DSM 18206. Another crAss-like phage replicating on Bacteroides intestinalis APC919/174 and identified as part of this study was previously reported ( Shkoporov et al., 2018a ).…”

“…Two out of the 56 genomes belong to the provisional group of crAss-like phages, highly abundant in the human gut and associated with the phylum Bacteroidetes. One of the detected genomes belongs to the phage which infects P. stercorea and apparently uses a non-standard genetic code with suppression of UAG stop codon throughout its genome ( Yutin et al., 2020 ). This phage belongs to a previously proposed genus VIII of subfamily Deltacrassvirinae of crAss-like phages, a subgroup which is highly abundant in non-Western human populations with high faecal Prevotella counts ( Gorvitovskaia et al., 2016 ; Guerin et al., 2018 ).…”

“…Contigs (length >1 kb) from all enrichment samples were then pooled together and demultiplexed by picking the longest representatives for each group of contigs with >90% sequence identity and >90% of sequence overlap. Viral contigs were identified using matches to NCBI viral RefSeq database, human gut virome database ( Gregory et al., 2020 ), crAss-like phage genome database ( Guerin et al., 2018 ; Yutin et al., 2020 ), IMG/VR viral metagenomics database ( Paez-Espino et al., 2017 ) as well as using VirSorter algorithm ( Roux et al., 2015a ) and presence of three conserved prokaryotic viral protein orthologues (pVOGs) per 10 kb of contig length ( Grazziotin et al., 2017 ). Filtered reads were aligned back to the common demultiplexed database of contigs, to quantify presence of various contigs in the enrichment samples.…”

Long-term persistence of crAss-like phage crAss001 is associated with phase variation inBacteroides intestinalis

“…The crAss-like bacteriophages are a recently described family of dsDNA tailed bacterial viruses of the order Caudovirales that are predicted to infect bacteria of the phylum Bacteroidetes (Yutin et al, 2020). They are present in variety of host-associated, aquatic and terrestrial habitats, but are especially predominant in the human gut (Honap et al, 2020;Koonin and Yutin, 2020;Yutin et al, 2018).…”