DOI: 10.5061/dryad.z8w9ghx7s
title: Resurrection of a global, metagenomically defined gokushovirus

description: Gokushoviruses are single-stranded, circular DNA bacteriophages found in
 metagenomic datasets from diverse ecosystems wordwide, including human gut
 microbiomes. Despite their ubiquity and abundance, little is known about
 their biology or host range: isolates are exceedingly rare, known only
 from three obligate intracellular bacterial genera. By synthesizing
 circularized phage genomes from prophages embedded in diverse enteric
 bacteria, we produced gokushoviruses in an experimentally tractable model
 system, allowing us to investigate their features and biology. We
 demonstrate that virions can reliably infect and lysogenize hosts by
 hijacking a conserved chromosome-dimer resolution system. Sequence motifs
 required for lysogeny are detectable in other metagenomically defined
 gokushoviruses; however, we show that even partial motifs enable phages to
 persist cytoplasmically without leading to collapse of their host culture.
 This ability to employ multiple, disparate survival strategies is likely
 key to the long-term persistence and global distribution of
 Gokushovirinae.
description: <b>Detection and phylogeny
 of gokushovirus prophages and their hosts.</b><b><i>
 </i></b>Using blastp<i>,</i> we queried the NCBI
 nr database (April 2019) with the <i>Chlamydia</i> Phage 4
 major capsid protein VP1 (NCBI Gene ID 3703676) and downloaded the
 complete genomes of 95 strains within the
 <i>Enterobacteriaceae</i> containing sequences returning
 E-values less than 0.0001. Chromosome contigs containing the VP1 gene were
 visually inspected in Geneious R9 (www.geneious.com) for the presence of
 prophage insertion boundaries by searching for identical 17-bp sequences
 within 5-kb regions upstream and downstream of the VP1 gene. Prophage
 genes were annotated with GLIMMER3 (Delcher et al., 2007) using default
 settings, specifying a minimum gene length of 110 bp and a maximum overlap
 of 50 bp. Initial alignments of prophage regions (ranging in size
 from 4047 to 4692 bp) were made with ClustalO 1.2.4 using standard
 settings, and were refined manually to accommodate hypervariable
 regions and the phage insertion sites at the 3’ and 5’ ends of the
 alignment. Average nucleotide identity at each position in the
 alignment was calculated and visualized using Geneious R9.
 Maximum likelihood phylogenetic trees of enterobacterial prophages were
 generated with RAxML 8.0.26 (Stamatakis, 2014) using the GTR+GAMMA
 substitution model and 100 fast-bootstrap replicates and visualized with
 FigTree 1.4.3 To evaluate the distribution of
 prophage hosts within the broad diversity of <i>E. coli</i> at
 large, we produced core genome alignments of prophage hosts and
 representative genomes from the <i>Escherichia coli</i>
 reference (ECOR) collection (Ochman and Selander, 1984)
 based on protein families satisfying a 30% amino-acid identity cutoff
 (USEARCH 11, Edgar, 2010), which were aligned in MUSCLE 3.8.31 (Edgar,
 2004) as implemented in the BPGA 1.3 pipeline (Chaudhari, 2016). The
 maximum likelihood phylogenetic tree of core genome alignments was built
 with IQTree 1.6.2 (Nguyen, 2015), using the JTT
 substitution model (Jones et al., 1992) and 100 bootstrap
 replicates. <b>Phylogenetic
 analysis of
 Gokushovirinae</b><b><i>.</i></b><i>
 </i>We downloaded a total of 1284 metagenome-assembled genomes
 (MAGs) of microviruses, which were then reannotated in GLIMMER3 (Delcher
 et al., 2007) using default settings, with a minimum gene length of 110 bp
 and a maximum overlap of 50 bp. We recovered homologues to the conserved
 major capsid protein VP1 and replication initiation protein VP4 in the set
 of metagenome-assembled microviruses using PSI-BLAST searches and querying
 with VP1 and VP4 proteins from detected enterobacterial gokushoviruses,
 gokushovirus genomes of <i>Chlamydia</i>,
 <i>Spiroplasma</i> and <i>Bdellvibrio</i>, and
 <i>Bullavirinae</i> phage <i>phiX</i>174.
 After individual protein alignments using Clustal Omega 1.2.4
 (standard settings), we concatenated the VP1 and VP4 alignments, and
 removed all sites with &gt;10% gaps to decrease the amount
 of spuriously aligned sites using Geneious R9. The initial phylogenetic
 tree of all microviruses was built with IQTree 1.6.2 using the LG+F+R10
 substitution model as determined by ModelFinder (Kalyaanamoorthy et al.,
 2017), and branch support was tested using 1000 ultra-fast bootstrap
 replicates (Hoang et al., 2018) and 1000 SH-aLRT tests. Collapsing all
 branches with &lt;95% bootstrap support and &lt;80% SH-aLRT
 support yielded a single, well-supported clade containing all known
 <i>Gokushovirinae</i>, and all subsequent alignments and
 phylogenetic trees were refined by including only those genomes
 represented in this clade, with branch support assessed with 100 bootstrap
 replicates. <b>Recovering
 prophages from metagenomes</b>. To assemble prophages from
 metagenomic datasets, we downloaded SRA files from
 BioProjects PRJEB29491 (viral human, Moreno-Gallego et
 al., 2019), PRJNA362629 (cellular bovine, unpublished), PRJNA290380
 (cellular human, Kostic et al., 2015), PRJNA352475 (cellular human,
 Ferretti et al. 2018), PRJEB6456 (cellular human, Bäckhed et al., 2015),
 PRJNA385126 (viral human, Stockdale et al., 2018),PRJEB7774 (cellular
 human, Feng et al., 2016), and PRJNA545408 (human viral, Shkoporov et al.,
 2019). We performed initial trimming and quality filtering with BBDuk
 (Bushnell, 2014a) with options ktrim=r k=23 mink=11 hdist=1 tbe tbo. Reads
 having a minimum nucleotide sequence identity of 50% to sequences of
 enterobacterial prophages, as determined by BBMap (Bushnell, 2014b), were
 assembled into contigs using MEGAHIT 1.1.3 (Li et al., 2015) implemented
 with default settings, and those contigs &gt;1000 bp were retained.