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A major lineage of non-tailed dsDNA viruses as unrecognized killers of marine bacteria

Abstract

The most abundant viruses on Earth are thought to be double-stranded DNA (dsDNA) viruses that infect bacteria1. However, tailed bacterial dsDNA viruses (Caudovirales), which dominate sequence and culture collections, are not representative of the environmental diversity of viruses2,3. In fact, non-tailed viruses often dominate ocean samples numerically4, raising the fundamental question of the nature of these viruses. Here we characterize a group of marine dsDNA non-tailed viruses with short 10-kb genomes isolated during a study that quantified the diversity of viruses infecting Vibrionaceae bacteria. These viruses, which we propose to name the Autolykiviridae, represent a novel family within the ancient lineage of double jelly roll (DJR) capsid viruses. Ecologically, members of the Autolykiviridae have a broad host range, killing on average 34 hosts in four Vibrio species, in contrast to tailed viruses which kill on average only two hosts in one species. Biochemical and physical characterization of autolykiviruses reveals multiple virion features that cause systematic loss of DJR viruses in sequencing and culture-based studies, and we describe simple procedural adjustments to recover them. We identify DJR viruses in the genomes of diverse major bacterial and archaeal phyla, and in marine water column and sediment metagenomes, and find that their diversity greatly exceeds the diversity that is currently captured by the three recognized families of such viruses. Overall, these data suggest that viruses of the non-tailed dsDNA DJR lineage are important but often overlooked predators of bacteria and archaea that impose fundamentally different predation and gene transfer regimes on microbial systems than on tailed viruses, which form the basis of all environmental models of bacteria–virus interactions.

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Figure 1: Autolykiviridae is a new family of non-tailed dsDNA viruses in the DJR capsid lineage.
Figure 2: Autolykiviruses dominate the lytic viral infection network of marine Vibrio.
Figure 3: Recovery of autolykiviruses is subject to multiple methodological biases.
Figure 4: DJR capsid viruses are far more diverse than the three currently recognized families, and include hosts in diverse bacterial and archaeal phyla

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Acknowledgements

We thank J. King, P. Weigele, J. Daily and J. Chodera for comments and suggestions; T. Soni and members of the Polz laboratory for assistance with sampling; S. Labrie for guidance in viral genome extractions and sequencing library preparation, and C. Haase-Pettingell for assistance with density gradients; N. Watson for electron microscopy; and R. Ratzlaff for discussions and the suggestion of electron microscopy of virus plaques in agar overlay. This work was supported by grants from the National Science Foundation OCE 1435993 to M.F.P. and L.K., the NSF GRFP to F.A.H. and the WHOI Ocean Ventures Fund to K.M.K.

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Authors and Affiliations

Authors

Contributions

K.M.K., F.A.H., L.K. and M.F.P. designed the study and planned experiments and analyses. K.M.K., L.K. and M.F.P. wrote the paper with contributions from all authors. K.M.K. conducted field sampling, isolations and experimental characterizations of lytic viruses. J.Y. conducted the statistical analyses of the viral decay experiment and wrote the scripts to visualize the infection matrix as a phylogeny-anchored network, which was based on the host ribosomal protein tree generated by P.A. W.K.C. and L.K. performed the quantification of significance of host sharing. F.A.H. performed isolation and characterization of active Vibrio DJR prophages. Bacterial genome sequencing libraries were prepared by M.B.C., assembled by P.A., and curated and annotated by P.A. and J.E. The viral genome sequencing libraries were prepared by K.M.K. and R.S.S., assembled by J.M.B. and K.M.K., and annotated and curated by J.M.B., K.M.K., J.E., W.K.C. and L.K. The viral metagenome sequencing libraries were prepared by K.M.K., and assembled and curated by P.A. and L.K. The bioinformatic analyses of microbial genomes and metagenomes for DJR capsid elements were performed by L.K. and K.M.K., and the visualization of the DJR network was performed by D.V. M.B.C. provided field and laboratory technical support throughout. Although specific contributions are highlighted for each author, all authors contributed in additional ways through contributions to figures, analyses, discussion of results and comments on the manuscript.

Corresponding authors

Correspondence to Libusha Kelly or Martin F. Polz.

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Reviewer Information Nature thanks J. Fuhrman, E. V. Koonin and the other anonymous reviewer(s) for their contribution to the peer review of this work.

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Extended data figures and tables

Extended Data Figure 1 Members of the Autolykiviridae are non-tailed viruses that may form tail tubes on contact with cells.

Thin-section electron microscopy of an agar overlay containing plaques of representative Autolykiviridae virus 1.008.O (see Methods for experimental details). a, Virus particles in contact with cell membranes are observed to occasionally possess tail-tube-like structures, whereas those not in contact with cells do not. b, Lower magnification of same field of view as a shows that tail-tube-free virions are more common than those with tail tubes. c, Lower magnification view of virion in Fig. 1b also shows that the presence of the tail tube is associated with cell contact and is not observed in nearby virions.

Extended Data Figure 2 Whole-genome alignments show that the family Autolykiviridae consists of five major sequence diversity clusters.

a, Maximum Likelihood phylogeny of whole-genome nucleotide alignments of 21 autolykiviruses. Alignments were made with Clustal Omega and the phylogenetic tree was generated with PhyML-SMS with aLRT branch supports. Scale bar, substitutions per base. b, Percentage of whole-genome nucleotide identities among 21 autolykivirus genomes on the basis of the Clustal Omega alignment. Assumptions of 50% and 95% identity for genus and species classifications83, respectively, suggest that these viruses represent two genera (groups A, B, C, D and group E) and five species. Two viruses with identical genomes were isolated at time points 39 days apart (1.048.O and 1.102.O), viruses with the same number and different letter suffixes represent lineages derived from a single plaque that gave rise to variable morphotypes during serial purification.

Extended Data Figure 3 Genomes of members of the Autolykiviridae are syntenic despite extensive diversity at the nucleotide level.

Virus genomes are grouped by nucleotide similarity (as identified in Extended Data Fig. 2). Homologous proteins were identified by performing an all-by-all BLASTp, requiring a minimum bitscore of 50, and clustering all pairs unweighted, using MCL with an inflation parameter set to 1.4 (Methods), cluster membership is identified by the label over the block arrows in the genome diagram. Protein clustering reveals that in addition to the six proteins identifiable by sequence similarity as core to all characterized autolykiviruses, additional protein clusters are shared among various subsets of the identified viral genome groups. For example, in the region of the genome to the right of the major capsid protein, 17 out of 18 viruses (genome groups A, B, C and D) share a set of seven protein clusters of unknown function (c11, c12, c13, c14, c15, c16 and c17); among these viruses, two additional proteins are shared only within subsets of the genomes (c26 in genome groups A and B; c19 in genome groups C and D).

Extended Data Figure 4 Packaging and replication protein-sequence phylogenies of autolykiviruses are incongruent with respect to other known families of non-tailed dsDNA viruses.

Autolykiviruses are most similar to the corticovirus PM2 in their major capsid protein, poorly resolved in their packaging ATPase, and most similar to the tectiviruses in their protein-primed DNA polymerase. Pairwise identities and phylogenies of the protein sequences of the DJR major capsid protein (a and b), packaging ATPase (c and d) and protein primed DNA polymerase (e and f). Members of the Tectiviridae infecting Gram-positive and Gram-negative hosts are shown separately as G+ and G−, respectively. All alignments were performed using the ETE3 Toolkit with workflow eggNOG41. All trees are maximum-likelihood trees with aLRT branch supports.

Extended Data Figure 5 Sequence-diverse autolykiviruses share extensively overlapping host ranges that include diverse hosts.

a, Pairwise coinfection significance by host count. Autolykiviruses exhibit highly significant host sharing. b, Pairwise coinfection significance compared to mean pairwise genomic similarity of the host. Autolykiviruses exhibit more significant host sharing than tailed phages of comparable host diversity. a, b, Coinfection significance as defined in Methods. c, Pairwise coinfection significance compared to viral genomic similarity measured as a fraction of shared open reading frames (ORFs). Autolykiviruses exhibit more significant host sharing than tailed viruses of comparable genomic similarity. A total of 998 reciprocal pairs of tailed viruses and 236 reciprocal pairs of autolykiviruses are shown, representing all pairs of viruses within each group (141 unique tailed, 16 unique autolykiviruses) that share at least one host.

Extended Data Figure 6 Autolykiviruses show delayed host lysis compared with other viruses.

Inverted phylogenetic tree showing the relationships among all 318 assayed bacterial strains on the basis of the concatenated alignments of the hsp60 and ribosomal protein genes, and using a partitioned model in RaxML to allow placement of 40 strains for which only the hsp60 gene sequence was available (Methods). Isolates are generally non-clonal. Leaves represent Vibrionaceae isolates and are coloured by population (Methods). Nodes represent viruses and are coloured by morphotype, as defined by major capsid protein or genome composition (Methods; non-tailed in orange, tailed in blue, unsequenced viruses in grey); edges represent infections with intensity increasing with increased time required for observation of plaques. Whereas 94% of tailed virus infections were detected within three days in host range assays, only 57% of autolykivirus infections were detected in that time, with 15% requiring more than seven days to be detected.

Extended Data Figure 7 DJR elements in Vibrionaceae include naturally excising integrated prophages and broad host-range plasmids.

Prophages of representative group 5 DJR elements (Fig. 4) naturally excise from their Vibrio hosts during growth in culture. Sequencing of nuclease-treated cell-free culture supernatants reveals sharply delineated regions of high coverage read mapping with respect to host genome background, indicating the presence of extracellular nuclease-protected prophage DNA. a, V. kanaloae 5S-149 DJR prophage. b, Vibrio 10N.286.55.C7 DJR prophage. c, Genome diagrams of the excising 5S-149 DJR prophage and the nine Vibrionaceae plasmids28 that are identified here as DJR elements show that they are syntenic and all share the DJR capsid protein, packaging ATPase and the corticovirus PM2 P17-like protein. MCL clustering of proteins on the basis of the BLASTp sequence similarity reveals that additional proteins, including integrases, repressors, peptidoglycan hydrolases and replication initiation genes, are common but not universal within these elements. d, Pairwise percentage of whole-genome nucleotide identities between 5S-149 DJR prophage and the DJR Vibrionaceae plasmids show that these elements are highly diverse at the nucleotide level and that 100% nucleotide-identical 13.6-kb plasmids are found in hosts in multiple species.

Extended Data Figure 8 Network of DJR virus capsids identified in bacterial and archaeal genomes and marine metagenomes.

Iterative HMM-based searches of marine metagenomes, on the basis of a reference panel of autolykiviruses and previously identified DJR capsid bacterial and archaeal viruses, yield approximately 15,000 proteins following stringent quality control filtering of the initial approximately 45,000 sequences that were recovered. Network visualization reflects MCL clustering of BLASTp-based similarities among sequences. a, Placement of reference panel sequences within the network. b, Characterization of proteins as DJRs on the basis of sequence- and structural-similarity-based annotation. c, Best BLASTp matches to RefSeq viruses, bitscore requirement of 50. d, Association of Tara Oceans-derived sequences to size fraction of isolation. e, Subset of sequences selected for phylogenetic analyses (Fig. 4) on the basis of membership in protein clusters strongly supported as bacterial and archaeal virus DJR capsids and requiring a length of ≥200 amino acids (Methods). We note that this selection is conservative, given the greater number and diversity of sequences recovered by our HMM-based search that passed all quality controls and show no structural- or sequence-based similarity to any other proteins, and thus were excluded from further analyses. The observed dominance of eukaryotic virus DJR capsids in this search is predicted to reflect four major aspects of our approach. First, inclusion of cellular metagenomes allows capture of large viruses such as the Mimiviridae (>400?nm), Iridoviridae (120–350?nm) and Phycodnaviridae (100–220?nm). Second, some Phycodnaviridae have been shown to encode up to eight sequence-diverse copies of their DJR major capsid gene84. Third, <0.22?μm viral metagenomes are biased against recovery of bacterial and archaeal DJR viruses, as described here. And fourth, the sequence content of HMMs using iterative searches is defined by the search space, such that if eukaryotic virus DJR capsid sequences are well represented, as they are in the larger size-fraction sequence databases used here, they will drive searches towards increased detection of similar sequences.

Extended Data Table 1 Metagenomes used in this study
Extended Data Table 2 Contigs of DJR elements

Supplementary information

Supplementary Information

This file contains Supplementary Figure 1, a comparison of tailed virus and autolykivirus genome recovery with and without protease treatment. The uncropped source gel for Fig. 3c. (PDF 288 kb)

Life Sciences Reporting Summary (PDF 74 kb)

Supplementary Data 1

This file contains accession numbers, taxonomy, and annotation of DJR capsid proteins included in the trees and network. (XLSX 781 kb)

Supplementary Data 2

This file contains accession numbers, taxonomy, and annotation of DJR capsid proteins included in the trees and network. (XLSX 6229 kb)

Supplementary Data 3

This file contains GenBank accession numbers for newly obtained sequences and previously published genomes included in Fig. 2 and Extended Data Fig. 6. (XLSX 45 kb)

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Kauffman, K., Hussain, F., Yang, J. et al. A major lineage of non-tailed dsDNA viruses as unrecognized killers of marine bacteria. Nature 554, 118–122 (2018). https://doi.org/10.1038/nature25474

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