Expanding the Marine Virosphere Using Metagenomics

<div><p>Viruses infecting prokaryotic cells (phages) are the most abundant entities of the biosphere and contain a largely uncharted wealth of genomic diversity. They play a critical role in the biology of their hosts and in ecosystem functioning at large. The classical approaches studying phages require isolation from a pure culture of the host. Direct sequencing approaches have been hampered by the small amounts of phage DNA present in most natural habitats and the difficulty in applying meta-omic approaches, such as annotation of small reads and assembly. Serendipitously, it has been discovered that cellular metagenomes of highly productive ocean waters (the deep chlorophyll maximum) contain significant amounts of viral DNA derived from cells undergoing the lytic cycle. We have taken advantage of this phenomenon to retrieve metagenomic fosmids containing viral DNA from a Mediterranean deep chlorophyll maximum sample. This method allowed description of complete genomes of 208 new marine phages. The diversity of these genomes was remarkable, contributing 21 genomic groups of tailed bacteriophages of which 10 are completely new. Sequence based methods have allowed host assignment to many of them. These predicted hosts represent a wide variety of important marine prokaryotic microbes like members of SAR11 and SAR116 clades, <i>Cyanobacteria</i> and also the newly described low GC <i>Actinobacteria</i>. A metavirome constructed from the same habitat showed that many of the new phage genomes were abundantly represented. Furthermore, other available metaviromes also indicated that some of the new phages are globally distributed in low to medium latitude ocean waters. The availability of many genomes from the same sample allows a direct approach to viral population genomics confirming the remarkable mosaicism of phage genomes.</p></div

General features of CGR groups and putative host assignment.

<p>CGR (Complete Genome Representative): contig representing a complete phage genome of a cluster of highly related contigs (>95% identity and 20% coverage in nucleotide sequence). The putative host taxon assigned to one or more CGRs in a group is shown. The last column shows the evidence for host assignment in brackets next to the host name. SS: putative host inferred by the high sequence similarity to known phages; AMG: Auxiliary metabolic gene; INT: putative host inferred by an exact match of a putative phage site-specific attachment site (attP) in an integrase carrying CGR to a host tRNA (host site-specific attachment site attB). The asterisk (*) in G13 indicates that host prediction was performed using a GF (genome fragment of an incomplete phage genome) and not a CGR.</p

Novel Autographivirinae phages.

<p>G7, G8 and G9 belonging to the subfamily Autographivirinae are compared to each other, and to the closest related reference phage genomes (boxed). CGR names are abbreviated, e.g. S45-C4 for uvMED-CGR-U-MedDCM-OCT-S45-C4 (for the complete names see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003987#pgen.1003987.s001" target="_blank">Data S1</a>). Size and GC% of the CGR/phage genomes are also indicated. Selected genes are uniformly colored and labeled. AA (amino acids) and NT (nucleotides) labels to the right indicate if the genome comparisons were made using TBLASTX or BLASTN. A color scale for the %identity (protein or nucleotide) is shown on the bottom right side. A 5 Kb length scale is also shown (bottom right). Some gene clusters are shown displaced and underlined in the graphic indicating that they have been moved to improve comparison across all genomes.</p

Genomic comparison of novel, complete phage genomes (CGRs) with known tailed phages.

<p>An all-vs-all comparison of several reference tailed bacteriophage genomes with the 208 CGRs identified in this study was achieved by a clustering based on a sequence similarity derived metric (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003987#s4" target="_blank">Materials and Methods</a> for details). Branches are colored according to phage family classification (See color key top left). Branches representing unclassified phages are shown in black. The ICTV (International Committee on Taxonomy of Viruses) nomenclature of several phages is also shown for reference. In addition, color dots indicate positions of phages infecting important marine microbes (color key at the bottom). The CGRs in this study are represented by blue diamonds at the tip of the branches, and the CGR groups are highlighted in grey and labeled (G1–G21). For those groups where host prediction was possible for one or more CGRs, a taxonomic rank of the host and the organisms supporting the prediction, and the nature of the evidence supporting the assignment (<i>SS</i>: sequence similarity, <i>INT</i>: integrase/att) are shown. The asterisk (<b>*</b>) in G13 indicates that host prediction was performed using an incomplete genome and not a CGR.</p

Comparative fragment recruitment of CGRs, GFs contigs (genome fragments of incomplete phage genomes) and representative cultivated phage genomes.

<p>Number of reads recruited by each, expressed as RPKG (Reads per Kb per Gb) from several metaviromes and metagenomes (color coded). Only hits that had >95% identity, minimum length of 50 bp and e-value<1e-5 were considered in this analysis. The names of the fosmid contigs are abbreviated as before. The reference genome names are highlighted in color according to their family (<i>blue</i>, Podoviridae; <i>green</i>, Siphoviridae and <i>red</i>, Myoviridae). Blue diamonds on top of the bars mark the contigs identified in this study with different colors (<i>cyan</i>, CGRs; <i>purple</i>, GFs) and the predicted (or known) host is also indicated when possible. Inset is a magnified view of the lesser recruiting fragments.</p

Terminase phylogeny.

<p>Maximum-likelihood tree of known representative terminase genes along with all identified terminases in the uncultured MedDCM viral contigs (uvMED) and putative prophage terminases (401 uvMED, 631 in total). Branches in the tree are colored according to the following codes: <i>black</i>, sequences from cultured phages; <i>blue</i>, putative prophage derived sequences; <i>red</i>, uncultured phage terminases obtained in this study. Known terminase types are labeled in bold. The microbes from which the putative prophage terminases were identified are marked by colored circles in the tree and a key is provided below. Bootstrap support of nodes is indicated on selected nodes. The terminase of pelagiphage HTVC010P is also labeled.</p

The hybrid NRPS-PKS cluster in <i>A</i><i>. macleodii</i>.

<p>Linear representation of the plasmid pAMDE1. Scale shown in the figure is in Kilobases. GC content and GC skew are shown. The high GC region corresponding to the cluster is shown in red. Genes are color coded and a legend is provided below.</p

Gene expression of the NRPS-PKS cluster.

<p>The graph shows the estimated gene expression using RPKM values associated with the NRPS-PKS cluster of pAMDE1. Genes are colored according to the inferred function. The numbers below some of the box arrows correspond to ORF numbers for that particular gene.</p

Phleomycin resistance in <i>Alteromonas</i><i>macleodii</i> strains.

<p>A) Phleomycin assay of AltDE (DE, lacking the NRPS-PKS cluster) and AltDE1 (DE1, harboring the NRPS-PKS cluster in the plasmid pAMDE1) on marine agar plates. B) Phleomycin assay of strains U7 and U8 (both harbor the NRPS-PKS cluster in their chromosome) and UM7 and U4 (both harbor the NRPS-PKS cluster in their plasmids).</p

Comparison of the protein domains of pAMDE1 to bleomycin related clusters.

<p>NRPS genes are represented in red and the PKS gene in blue. Within the arrows the names of the ORF in pAMDE1 and the homologs in the bleomycin or the other clusters are indicated. The protein domains are shown inside the boxes. In addition, functional classification is shown for condensation domains (C) and the substrate amino acid for each adenylation domain (A). The modules (NRPS-0 to NRPS-9) described for the bleomycin compounds are represented by the black lines above the domains. Light blue arrows indicate putative missing domains in pAMDE1 in comparison to bleomycin. AT and KR domains are also shown in light blue. Blm genes: bleomycin, zbm genes: zorbamycin, tlm genes: tallyzomycin. LCL: catalyzes peptide bond formation between 2 L-amino acids, CYC: heterocyclization domain, modAA: modify the incorporated amino acids, unc: unclassified. A non-functional A-domain is marked with a red-cross.</p