Identification of Huge Phages from Wastewater Metagenomes

Abstract

Huge phages have genomes larger than 200 kilobases, which are particularly interesting for their genetic inventory and evolution. We screened 165 wastewater metagenomes for the presence of viral sequences. After identifying over 600 potential huge phage genomes, we reduced the dataset using manual curation by excluding viral contigs that did not contain viral protein-coding genes or consisted of concatemers of several small phage genomes. This dataset showed seven fully annotated huge phage genomes. The phages grouped into distinct phylogenetic clades, likely forming new genera and families. A phylogenomic analysis between our huge phages and phages with smaller genomes, i.e., less than 200 kb, supported the hypothesis that huge phages have undergone convergent evolution. The genomes contained typical phage protein-coding genes, sequential gene cassettes for metabolic pathways, and complete inventories of tRNA genes covering all standard and rare amino acids. Our study showed a pipeline for huge phage analyses that may lead to new enzymes for therapeutic or biotechnological applications.

Keywords: huge phage; metagenome; viral metagenomics; virus genome annotation; virus phylogeny; wastewater.

Figure 1

Phylogenomic GBDP tree inferred using the formula D0 (nucleotide) and yielding average support of 64%. The numbers near branches are GBDP pseudo-bootstrap support values from 100 replications. The branch lengths of the resulting VICTOR tree are scaled in terms of the respective distance formula used. The OPTSIL clustering [76] yielded 81 species clusters and 60 genus clusters. The number of clusters determined at the family level was fifteen. The geometrical shapes and colours represent different taxa. Studied phages are shown in red.

Figure 2

Phylogenetic relationship of identified huge phage major capsid proteins. Maximum likelihood phylogeny based on the amino acid sequences of the major capsid proteins was performed in iqtree with the best model option. Confidence tests were performed with 1000 bootstrap replicates. Virus names are shown, and GenBank accession numbers are given in parentheses. Some clades were collapsed for clarity (A–G). Coloured backgrounds represent the environment from which the sequences were identified (see legend).