Infection strategy and biogeography distinguish cosmopolitan groups of marine jumbo bacteriophages

Detection and validation of high-quality jumbo phage bins

Due to the large size of jumbo bacteriophage genomes, it is likely that they are present in multiple distinct contigs in metagenomic datasets and therefore require binning to recover high-quality metagenome-assembled genomes (MAGs) [28]. This has been shown for large DNA viruses that infect eukaryotes, where several recent studies have successfully employed binning approaches to recover viral MAGs [2, 3, 30]. Here, we used the same 1545 high-quality metagenomic assemblies [31] used in a recent study to recover giant viruses of eukaryotes [3], but we modified the bioinformatic pipeline to identify bins of jumbo bacteriophages. These metagenomes were compiled by Parks et al. [31] and included available metagenomes on the NCBI’s Short Read Archive by December 31, 2015 (see Parks et al. [31]). This dataset includes a wide variety of marine metagenomes (n = 469) including many non-Tara metagenomes (n = 165). We focused our benchmarking and distribution analyses on Tara data [29] because of the well-curated metadata and size fractions in this dataset. We first binned the contigs from these assemblies with MetaBat2 [32], which groups contigs together based on similar nucleotide composition and coverage profiles, and focused on bins of at least 200 kilobases in total length. We subsequently identified bins composed of bacteriophage contigs through analysis with VirSorter2 [33], VIBRANT [34], and CheckV [35] (see Methods for details).

The occurrence of multiple copies of highly conserved marker genes is typically used to assess the level of contamination present in metagenome-derived genomes of bacteria and archaea [36]. Because bacteriophage lack these marker genes [37], we developed alternative strategies to assess possible contamination in our jumbo phage bins. Firstly, we refined the set of bins by retaining those with no more than 5 contigs that were each at least 5 kilobases in length to reduce the possibility that spurious contigs were put together. Secondly, we assessed the possibility that two strains of smaller phages with similar nucleotide composition may be binned together by aligning the contigs in a bin to each other. Bins that had contigs with high sequence similarity across the majority of their lengths were discarded (Supplementary Fig. 1). Thirdly, we discarded bins if their contigs exhibited aberrant co-abundance profiles in different metagenomes (see Supplementary Methods). To generate these co-abundance profiles, we mapped reads from 225 marine metagenomes provided by Tara Oceans [29] onto the bins. Coverage variation between contigs was benchmarked based on read-mapping results from artificially-fragmented reference genomes present in the samples (See Methods for details). Only bins with coverage variation below our empirically-derived threshold were retained. Using this stringent filtering, we identified 85 bins belonging to jumbo bacteriophages. These bins ranged in length from 202 kbp to 498 kbp, and 31 consisted of a single contig, while 54 consisted of 2–5 contigs (Supplementary Fig. 2).

To assess global diversity patterns of jumbo bacteriophages, we combined these jumbo phage bins together with a compiled database of previously-identified jumbo phages that included all complete jumbo Caudovirales genomes on RefSeq (downloaded July 5th, 2020), the INPHARED database [14], a recent survey of cultivated jumbo phages [6], the Al-Shayeb et al. study [4], and marine jumbo phage contigs from metagenomic surveys of GOV 2.0 [26] (60 jumbo phages), ALOHA 2.0 [38] (8 jumbo phages), and one megaphage MAG recovered from datasets of the English Channel [39]. Ultimately, we arrived at a set of 244 jumbo phages, including the 85 bins, that were present in at least one Tara Oceans sample (min. 20% genome covered, see Methods) or deriving from a marine dataset (i.e. ALOHA, GOV 2.0) which we analyzed further in this study and refer to as marine jumbo phages. Statistics on genomic features can be found in Supplementary Dataset 1.

Marine jumbo phages belong to distinct groups with diverse infection strategies

Because bacteriophages lack high-resolution, universal marker genes for classification, such as 16S rRNA in bacteria, phages are often grouped by gene content [40, 41]. Here, we generated a bipartite network that included the 85 bins of jumbo phages with a dataset of available Caudovirales complete genomes in RefSeq (3012 genomes; downloaded July 5th, 2020) and the full set of reference jumbo phages described above. To construct the bipartite network, we compared proteins encoded in all the phage genomes to the VOG database, and each genome was linked to VOG hits that were present (Fig. 1, Supplementary Dataset 2, see Methods for details). To identify groups of phage genomes with similar VOG profiles, we employed a spinglass community detection algorithm [42] to generate genome clusters. Similar methods have recently been used to analyze evolutionary relationships in other dsDNA viruses [41]. The marine jumbo phages of this study clustered into five groups that included both jumbo and non-jumbo phage genomes (Fig. 2a). We refer to these five clusters as Phage Genome Clusters (PGCs): PGC_A, PGC_B, PGC_C, PGC_D, and PGC_E. These PGCs included cultured phages and metagenome-derived jumbo phages found in various environments (i.e. aquatic, engineered) and isolated on a diversity of hosts (i.e. Firmicutes, Proteobacteria, Bacteroidetes) (Fig. 2b, c). Of the marine jumbo phages, 135 belonged to PGC_A, 11 to PGC_B, 90 to PGC_C, 7 to PGC_D, and 1 to PGC_E (Fig. 1b). In addition to this network-based analysis, we also examined phylogenies of the major capsid protein (MCP) and the terminase large subunit (TerL) encoded by the marine jumbo phages and the same reference phage set examined in the network (Fig. 1c, d). With the exception of PGC_A, the marine jumbo phages that belong to the same PGC appeared more closely related to each other than those belonging to different clusters. The polyphyletic placement of jumbo phage PGCs in these marker gene phylogenies is consistent with the view that genome gigantism evolved multiple times, independently within the Caudovirales [6].

We then compared functional content encoded by the marine jumbo phages in the PGCs to identify functional differences that distinguish these groups. PGC_E was excluded from this analysis because this genome cluster contained only a single jumbo phage. Collectively, most genes of the marine jumbo phages could not be assigned a function (mean: 86.60%, std dev: 7.01%; Supplementary Dataset 3), which is common with environmental viruses [43, 44]. Genes with known functions primarily belonged to functional categories related to viral replication machinery, such as information processing and virion structure (Fig. 3a), and these genes drove the variation between the genome clusters of marine jumbo phages (Fig. 3b). A recent comparative genomic analysis of cultivated jumbo phages was able to identify three types of jumbo phages that are defined by different infection strategies and host interactions (referred to as Groups 1–3) [6]. We cross-referenced our PGCs and found that PGCs B, C, and D of this study corresponded to Groups 1, 2, and 3, respectively, suggesting that these genome clusters contain phages with distinct infection and replication strategies. PGC_A corresponded to multiple groups, indicating that this genome cluster contains a particularly broad diversity of phages.

The second largest phage cluster with marine jumbo phages, PGC_B, consists of 238 phages (11 (4.6%) marine jumbo phages, including 10 bins generated here), and included cultured phages of Group 1, which is typified by Pseudomonas aeruginosa phage PhiKZ. Supporting this correspondence with Group 1, all marine jumbo phages of PGC_B encoded the same distinct replication and transcription machinery, including a divergent family B DNA polymerase and a multi-subunit RNA polymerase (Fig. 3b, Supplementary Dataset 3). These marine jumbo phages also encoded a PhiKZ internal head protein, and they uniquely encoded shell and tubulin homologs which has recently been found in PhiKZ phages to assist in the formation of a nucleus-like compartment during infection that protects the replicating phage from host defenses [18, 19]. Although we could not confidently predict hosts for the 11 metagenomic marine jumbo phages in this PGC_B (Supplementary Dataset 1), the cultured phages of this genome cluster infect pathogenic bacteria belonging to the phyla Proteobacteria (178 phages) and Firmicutes (6 phages) (Fig. 2c), implicating a potential host range for marine jumbo phages in PGC_B.

The next largest phage genome cluster, PGC_C, comprised of 156 phages total (90 marine jumbo phages (57.7%); 4 bins generated from this study) and included reference jumbo phages in Group 2 (31, 19.9%) which are typified by Alphaproteobacteria and Cyanobacteria phages. Likewise, the host range of other cultured phages in PGC_C support the Group 2 correspondence, either infecting Cyanobacteria (139 phages) or Proteobacteria (4 phages) (Fig. 2c). Furthermore, all 3 marine metagenomic phages in PGC_C for which hosts could be predicted were matched to Cyanobacteria hosts (Supplementary Dataset 1). Functional annotations of PGC_C marine jumbo phages revealed nearly all encode a family B DNA polymerase (97.8% of phages) and the photosystem II D2 protein (PF00124, VOG04549) characteristic of cyanophages (90% of phages) (Fig. 3b). This PGC included the reference Prochlorococcus phage P-TIM68 (NC_028955.1), which encodes components of both photosystem I and II as a mechanism to enhance cyclic electron flow during infection [45]. This suggests that an enhanced complement of genes used to manipulate host physiology during infection may be a driver of large genome sizes in this group. Additionally, most of the PGC_C marine jumbo phages encoded lipopolysaccharide biosynthesis proteins (76%), which have been found in cyanophage genomes that may induce a “pseudolysogeny” state, when infected host cells are dormant, by changing the surface of the host cell and preventing additional phage infections [6] (Supplementary Dataset 3). Taken together, most marine jumbo phages of PGC_C likely follow host interactions of jumbo cyanophages, such as potentially manipulating host metabolism by encoding their own photosynthetic genes and potentially inducing a pseudolysogenic state.

Finally, phages of PGC_D totaled at 47 phages, of which 7 were marine jumbo phages generated in this study (14.9%). This group included Group 3 jumbo phages (15, 31.9%), which is primarily distinguished by encoding a T7-type DNA polymerase but is not typified by a particular phage type or host range. Supporting this grouping, all marine jumbo phages in this study encoded a T7 DNA polymerase which belongs to family A DNA polymerases (Fig. 3b, Supplementary Dataset 3). Most of the other genes distinctively encoded by the marine jumbo phages in this group included structural genes related to T7 (T7 baseplate, T7 capsid proteins), a eukaryotic DNA topoisomerase I catalytic core (PF01028), and DNA structural modification genes (MmcB-like DNA repair protein, DNA gyrase B). Hosts of metagenomic marine jumbo phages in PGC_D could not be predicted (Supplementary Dataset 1); however, cultured Group 3 jumbo phages in PGC_D all infect Proteobacteria, primarily Enterobacteria and other pathogens.

The largest of the phage genome clusters, PGC_A, contained 469 phages, including 135 marine jumbo phages (63 bins from this study). This genome cluster contained the largest jumbo phages, such as Bacillus phage G (498 kb) and the marine megaphage Mar_Mega_1 (656 kb) recently recovered from the English Channel [39]. Unlike other PGCs, PGC_A contained mostly metagenomic phages (401, 85%, Fig. 2b, c). Considering PGC_A contains the largest jumbo phages (Figs. 1b, 2a), the vast genetic diversity in this PGC might explain why few genes were found to distinguish this group. Of the genes unique to PGC_A, only one was present in at least half of the phages (51.9%), which was a Bacterial DNA polymerase III alpha NTPase domain (PF07733). The host ranges of cultured phages from this PGC further reflect the large diversity of this group and included a variety of phyla and genera that can perform complex metabolisms or lifestyles, such as the nitrogen-fixing Cyanobacteria of the Nodularia genus isolated from the Baltic Sea (accessions NC_048756.1 and NC_048757.1) and the Bacteroidetes bacteria Rhodothermus isolated from a hot spring in Iceland (NC_004735.1) [46]. Because this group contains an abundance of metagenome-derived genomes that encode mostly proteins with no VOG annotation (Supplementary Dataset 2), it is possible that it includes several distinct lineages that could not be distinguished using the community detection algorithm of the bipartite network analysis.

Relative abundance of jumbo bacteriophages across size fractions

To explore the distribution of the marine jumbo phages in the ocean, we first examined the size fractions in which the jumbo phages were most prevalent. To remove redundancy for the purposes of read mapping, we examined the 244 jumbo phages at the population-level (>80% genes shared with >95% average nucleotide identity [24]), corresponding to 142 populations (11 unique to this study, corresponding to 47 bins). We then mapped Tara Oceans metagenomes onto the 142 jumbo phage populations, and 102 of these populations could be detected [min. 20% of genome covered], resulting in 74 populations in PGC_A, 2 in PGC_B, 22 in PGC_C, 3 in PGC_D, and 1 in PGC_E. Out of the 225 Tara Oceans metagenomes examined, 213 (94.6%) contained at least one jumbo phage population (median: 7, Supplementary Dataset 4). Jumbo phages were more frequently detected in samples below 0.22 µm (<−0.22 µm, 0.1–0.22 µm) than those above 0.22 µm (0.45–0.8 µm, 0.22–0.45 µm, 0.22–1.6 µm, 0.22–3 µm) (Fig. 4a). All samples in the <−0.22 µm fraction and the 0.1–0.22 µm fraction had at least one jumbo phage present, while the larger fractions ranged from 89 to 97%. Interestingly, we detected 34 populations (33.3%) exclusive to samples above 0.22 µm, compared to only one population (0.98%) exclusive to samples below 0.22 µm. A similar disparity in virus detection between size fractions has been reported for large eukaryotic viruses, where roughly 41% of phylotypes were present in the 0.22–3 µm size fraction but absent in fractions below 0.22 µm [9]. In contrast to this study, where certain viral groups were more prevalent in larger size fractions than smaller, a jumbo phage’s PGC membership or genome size generally did not affect its probability of detection at different size fractions (Supplementary Figs. 3 and 4).

We also compared jumbo phage diversity (defined as population richness), relative abundance (calculated in reads per kilobase per million (RPKM)), and community composition between the size fractions (based on Bray–Curtis distance matrices). Collectively, samples of the size fractions below 0.22 µm were significantly more diverse (p value <0.0001, Wilcox test) and had significantly higher relative abundances (p value < 0.0001, Wilcox test) of jumbo phages relative to the size fractions above 0.22 µm. Despite these differences in diversity and relative abundances, jumbo phage community composition did not significantly differ between the >0.22 and <0.22 µm size fractions when comparing samples based on presence/absence data (p value = 0.1082, ANOSIM, presence/absence Bray–Curtis distance matrix, Fig. 4d), but did differ when using relative abundance data (p value = 0.0001, ANOSIM, RPKM Bray–Curtis distance matrix, Fig. 4e).

To directly test the effect of the 0.22 µm size fraction cut-off on jumbo phage recovery, we examined a subset of the samples that were co-collected at the same station and depth for the fractions below 0.22 µm (<−0.22 or 0.1–0.22) and above 0.22 µm (0.22–1.6 µm or 0.22–3 µm). The number of detected jumbo phage populations was significantly higher in samples below 0.22 µm than above 0.22 µm (p value = 0.000138, Wilcox test, Supplementary Fig. 5a). The relative abundance of jumbo phages was also significantly higher in size fractions below 0.22 µm than above 0.22 µm (p value = 0.00001, Wilcox test, Supplementary Fig. 5b). Likewise, community composition significantly differed between samples above and below 0.22 µm (p value = 0.0001, ANOSIM, presence/absence Bray–Curtis distance matrix, Supplementary Fig. 5c, d). Taken together, these findings suggest that using size fractions below 0.22 µm to analyze phages enhances the signal of jumbo phage sequences, relative to samples of larger size fractions, likely due to cellular sequences present in the larger sizes. Notwithstanding, roughly 33% of jumbo phages in this study were exclusive to size fractions above 0.22 µm, indicating that analyzing a range of size fractions is necessary for a more synoptic view of jumbo phages in the environment.

Biogeography of jumbo bacteriophages in the global ocean

Jumbo phage populations varied by depth in different PGCs

Jumbo phage populations in this study varied in their distribution (Supplementary Fig. 6) but were collectively found in all three depths (Surface (SRF), Deep Chlorophyll Maximum (DCM), Mesopelagic (MES)) examined (Fig. 5, Supplementary Fig. 7; Supplementary Dataset 4). Although jumbo phages were more prevalent in samples of the viral size fractions (<−0.22 or 0.1–0.22 µm), we focused biogeographic analyses on the 0.22–1.6 or 0.22–3 µm size fractions because the most sites were available for these samples, thereby enabling comparisons between depths and biomes. When applicable, analyses were also completed with viral fraction samples, and results are deposited at the end of the Supplement (Supplementary Figs. 16–24). Jumbo phage communities differed significantly between depths (p value = 0.0001, ANOSIM based on presence/absence and RPKM Bray–Curtis distance matrices, Supplementary Fig. 8), consistent with the dramatic transition in community composition that occurs from surface waters to below the deep chlorophyll maximum [38, 47]. Specifically, the diversity of jumbo phages across depths varied by genome cluster (Fig. 5d, Supplementary Fig. 10), with PGC_A and PGC_C exhibiting higher prevalence in the epipelagic (SRF and DCM). Although PGC_B and PGC_D had too few populations detected to generalize for these clusters (2 and 3, respectively), our results for these phages showed that PGC_B and PGC_D were typically less prevalent in SRF samples compared to DCM and MES samples. PGC_C is typified by cyanophages, providing a clear reason why this phage group is enriched in surface waters. Conversely, PGC_B is typified by Pseudomonas aeruginosa PhiKZ phages, suggesting these PGC_B marine jumbo phages may be infecting heterotrophic bacteria lineages that are less prevalent in surface waters. Overall relative abundance and diversity of jumbo phages in this study were significantly higher in the epipelagic zone (Supplementary Fig. 8), partly because most of these phages are in PGC_A. These collective and PGC-specific patterns held when examining only those samples that were co-collected at all three depths (Fig. 5c, Supplementary Figs. 8, 9, 10). This general pattern therefore reflects what has been found in previous studies on the depth distribution of viruses and viral protein clusters, where more were unique in the euphotic (i.e. epipelagic) than aphotic depths [9, 26, 48, 49], although this contrasts what has recently been found in the Pacific Ocean, where overall viral diversity increased in the mesopelagic [38].

Jumbo phage biogeography across biomes

Collectively, jumbo phages could be found in all three Longhurst biomes (Coastal, Westerlies, Trades) (Supplementary Fig. 11), and jumbo phage communities in this study significantly differed in composition between the biomes (p value < 0.05, ANOSIM, presence/absence Bray–Curtis distance matrix, Supplementary Fig. 11c). However, no biome appeared to be a particular hotspot for jumbo phages, as they were not significantly more diverse in any biome (Supplementary Fig. 11a). When looking at depths separately, the relative abundance of jumbo phages in SRF samples was significantly higher in Coastal samples (Supplementary Fig. 12b), but no clear biome was a hotspot for phages in DCM and MES samples (Supplementary Fig. 11). Likewise, jumbo phage community composition only differed between samples in the SRF and DCM, but not in MES samples (p value < 0.05, ANOSIM, presence/absence Bray–Curtis distance matrices). Upon examining jumbo phages by group, jumbo phages from PGCs A, C, and D could be detected in all biomes, while PGB_B phages could not be detected in Westerlies samples (Supplementary Fig. 13). Similar to their collective results, no PGC was enriched in a single biome (Supplementary Fig. 13). A recent global study on marine viruses has found that viral diversity is better explained by ecological zones defined by physicochemical factors like temperature, rather than by Longhurst biomes defined by patterns of chlorophyll a concentrations [26], suggesting that Longhurst biomes may not be good predictors of viral diversity in general.

Jumbo phage populations ranged in endemicity

Fifteen populations (14.7%) were detected in only one Tara station, and six (5.6%) were present in over half of the stations  ≥34 (Fig. 5c). Both the more endemic populations and the more prevalent jumbo phages belonged to PGCs A and C. PGC_A and PGC_C contained the most populations, which likely explains the wide range of endemicity of phages in these clusters (Supplementary Figs. 14, 15). Moreover, the cyanobacterial hosts that are known for many of the jumbo phages in PGC_C are widespread in the ocean, which may also explain the prevalence of this group of phages. In general, the heterogeneous distribution and abundance of these jumbo phages is consistent with the seed bank hypothesis, which postulates that viruses are passively dispersed throughout the ocean and viral community structure is shaped by local selective forces [24, 50]. This framework has previously been used to explain why phage distributions range from extremely cosmopolitan to extremely rare, which is a pattern that also appears to hold for jumbo bacteriophages.

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