Resolving the structure of phage–bacteria interactions in the context of natural diversity
SamplingEnvironmental samplingSamples were collected from the littoral marine zone at Canoe Cove, Nahant, Massachusetts, USA, on 22 August (ordinal day 222), 18 September (261) and 13 October (286) 2010, during the course of the three month Nahant Collection Time Series sampling11.Bacterial isolation and characterizationBacterial isolationBacterial strains were isolated from water samples using a fractionation-based approach7 as previously described19,20. In brief, seawater was passed first through a 63um plankton net and then sequentially through 5um (Whatman 111113 or Sterlitech PCT5047100), 1um (Whatman 111110 or Sterlitech PCT1047100), and 0.2um (Whatman 111106) hydrophilic polycarbonate filters; material recovered on the filters was resuspended by shaking for 20 min; dilution series of resuspended cells were filtered onto 0.2um polyethersulfone filters (Pall 66234) in a carrier solution of artificial seawater (40 g Sigma Sea Salts, S9883; 0.2um filtered), and filters placed directly onto Vibrio-selective MTCBS plates (BD Difco TCBS Agar 265020, supplemented with with 10 g NaCl per liter to 2% final w/v). Colonies (96) from each of three replicates of each size fraction were selected from the dilution plates with the fewest numbers of colonies (1,152 isolates per isolation day). Colonies were purified by serial passage, first onto TSB-II (Difco Tryptic Soy Broth, 1.5% BD Difco Bacto Agar 214010, amended with 15 g NaCl to 2% w/v), second onto MTCBS, finally onto TSB-II again. Colonies were inoculated into 1 ml of 2216 Marine Broth (BD Difco 279110) in 96-well 2 ml culture blocks and allowed to grow, shaking at room temperature, for 48 h. Glycerol stocks were prepared by combining 100 ul of culture with 100 ul of 50% glycerol (BDH 1172-4LP) in 96-well microtiter plates and sealed with adhesive aluminum foil for preservation at −80 °C.Bacterial hsp60 gene sequencingTo obtain hsp60 gene sequences for isolates, Lyse and Go (LNG) (Pierce, Thermo Scientific 78882) treatments of subsamples of the same overnights cultures used in the bait assay (described below) were used directly as template in PCR amplification reactions. PCR reactions were prepared in 30 ul volumes, as follows: 1 ul LNG template, 3 ul 10x buffer, 3 ul 2 mM dNTPs, 3 ul 2um hsp60-F primer, 3 ul 2um hsp60-R primer, 0.3 ul NEB Taq, 16.7 ul PCR-grade HOH; with hsp60-F (H279) primer sequence: 5′-GAA TTC GAI III GCI GGI GAY GGI ACI ACI AC-3′, and hsp60-R (H280) primer sequence: 5′-CGC GGG ATC CYK IYK ITC ICC RAA ICC IGG IGC YTT-3′61 (Supplementary Table 1). PCR thermocycling conditions were as follows: initial denaturation at 94 °C for 2 min; 35 cycles of 94 °C for 1 min, 37 C for 1 min, 72 °C for 1 min; final annealing at 72 °C for 6 min; hold at 10 °C. PCR products were cleaned up by isopropyl alcohol (IPA) precipitation, as follows: addition of 100 ul 75% IPA to 30 ul PCR reaction product, gentle inversion mixing followed by 25 min incubation at RT, 30 min centrifugation at 2800 rcf, addition of 50 ul 70% IPA with gentle inversion wash, centrifugation at 2000 rcf, inversion on paper towels to remove IPA, 10 min centrifugation at 700 rcf, air drying in PCR hood for 30 min, resuspension in 30 ul PCR HOH. PCR products were Sanger sequenced (Genewiz, Inc.) using hsp60-R primer, as follows: 5 ul of 5 um hsp60-R primer, 7 ul nuclease free water, 3 ul DNA template. For a subset of strains hsp60 sequences were obtained from subsequently determined whole-genome sequences. Hsp60 sequences were aligned to the hsp60 sequence previously published for Vibrio 1S_84 and trimmed to 422 bases using Geneious (https://www.geneious.com/). Accession numbers for these 1287 strains are provided in Supplementary Data 1, where they are identified as baxSet1287.Bacterial hsp60 phylogeniesA phylogenetic tree of relationships among bacterial isolates screened in the bait assay (described below) was produced based on a 422 bp fragment of the hsp60 gene, derived either from Sanger or whole genome sequences; with E. coli K12 serving as the outgroup. Sequences from each of the three days of isolation were aligned using muscle v.3.8.3162 with default settings (muscle -in $seqsALL -out $seqsALL.muscleAln), and a single tree including all 1287 sequences from all the days was generated using FastTree v.2.1.863 (FastTree -gtr -gamma -nt -spr 4 -slow $seqsALL.muscleAln.fasttree). For presentation in Fig. 1 three sub-trees including only nodes from each day were produced using PareTree v.1.0.264 (java -jar PareTree1.0.2.jar -t O -del notDay222.txt -f $seqsALL.$round.muscleAln.fasttree.DAY222). Trees were visualized using iTOL65 and painted with metadata for each of the strains, including: sensitivity to killing in agar overlay by co-occurring phage predators collected on the same day and, for the subset of strains that were genome sequenced and also included in the host range matrix, the bacterial species, based on concatenated ribosomal protein analysis using RiboTree66 as described below. Isolation days for each of the strains included in these analyses are provided in Supplementary Data 1, where these strains are identified as baxSet1287.Bacterial genome sequencing and assignment to populationsTo assign genome-sequenced bacterial isolates used in the host range assay to species, we use the RiboTree tool66 to produce a phylogeny based on concatenated single copy ribosomal proteins as in23. We include strains of previously described Vibrionaceae in preliminary analyses as reference strains and assign species names to new isolates based on clustering with named representatives, as well as provide placeholder names for newly identified clades with no previously described representatives. Trees were visualized using iTOL65 and the representation including only those strains included in the host range assay is shown in Supplementary Fig. 1; population assignments and accession numbers for this set of 294 genomes, which also includes a small number of previously isolated bacterial strains that were included in the host range assay (described below), are provided in Supplementary Data 1, where they are identified as baxSet294.Viral isolation and characterizationWe have previously described features of the viruses of the Nahant Collection20, as well as approaches used for the standardization of their genome assemblies19, additional details are provided below.Viral sample collectionThe iron chloride flocculation approach was used to generate 1000-fold concentrated viral samples from 0.2 um-filtered seawater, as follows. For each isolation day, triplicate 4 L seawater samples were filtered through 0.2 um polyethersulfone cartridge filters (Millipore, Sterivex, SVGP0150) into collection bottles, spiked with 400 uL of FeCl3 solution (10 gL−1 Fe; as 4.83 g FeCl3•6H2O (Mallinckrodt 5029) into 100 ml H2O), and allowed to incubate at room temperature for at least 1 h. Virus-containing flocs were then recovered from the sample by filtration onto 90 mm 0.2 um polycarbonate filters (Millipore, Isopore, GTTP09030) under gentle vacuum in a 90 mm glass cup-frit system (e.g Kontes funnel 953755-0090, fritted base 953752-0090, and clamp 953753-0090); once liquid was fully passed, the funnel was removed and, with the vacuum pump left on, the filters were folded into quarters, removed from the fritted base, and inserted into a 7 ml borosilicate glass vial. A volume of 4 ml of oxalate-EDTA solution (prepared from stock solution as 10 ml 2 M Mg2EDTA (J.T. Baker, JTL701-5), 10 ml 2.5 M Tris-HCl (Promega PAH5123), 25 ml 1 M oxalic acid (Mallinckrodt 2752); adjusted to pH 6 with 10 M NaOH (J.T. Baker, 3722-01); final volume 100 ml; used within 7 days of preparation and maintained at room temperature in the dark) was added to the vial and the sample allowed to dissolve at room temperature for at least 30 min before transfer to storage at 4 °C. A reagent used in this original formulation (JT-Baker 7501 Mg2EDTA) is no longer available and an updated recipe is provided elsewhere67.Bait assay and associated viral plaque archivalIn order to obtain estimates of co-occurring phage predator loads at bacterial strain level resolution, and generate plaques from which to isolate phages, we exposed 1440 purified bacterial isolates to phage concentrates from their same day of isolation (1334 yielded lawns sufficient to evaluate for plaques, and hsp60 sequences could be determined for 1287 of these). Bacterial strains screened included 480 isolates from each ordinal day, representing 120 strains from each of 4 size-fractionation classes (0.2 um, 1.0 um, 5.0 um, 63 um) details of isolation origin are provided for each strain in Supplementary Data 1, and description of naming conventions is as previously described19. For the bait assay each strain was mixed in agar overlay with seawater concentrates containing viruses (15 ul concentrate, equivalent to 15 ml unconcentrated seawater assuming 100% recovery efficiency; derived from pooling of three replicate virus concentrates from each day). We note that recoveries were not tested for individual samples and that previous tests14 of recovery efficiency have shown that resuspension of iron flocculates in oxalate solution yields initial recoveries of approximately 50% (49 ± 3% and 55 ± 11% for a marine sipho- and myo-virus respectively, at 24 h post re-suspension) and shows low decay rate over time (47 ± 5% and 73 ± 16% for a marine sipho- and myo-virus respectively, at 38 days post re-suspension). All of our assays were performed approximately 8–9 months post-sampling from oxalate concentrates stored at 4 °C. Agar overlays were performed based on the previously described Tube-free method13, as follows. Bacterial strains were prepared for agar overlay plating by streaking out from glycerol stocks onto 2216MB agar plates with 1.5% agar (Difco, BD Bacto, 214010), and allowed to grow for 2 days at room temperature. Strains were then inoculated into 1 ml 2216MB in a 96-well culture block and incubated 24 h at room temperature shaking at 275 rpm on a VWR DS500E orbital shaker. Immediately prior to use in direct plating the OD600 was measured in 96-well microtiter plates and subsamples were taken for Lyse and Go (LNG) processing for DNA (10 ul culture, 10 ul LNG). Phage concentrates were prepared for plating by pooling 1.2 ml from each of the concentrate replicates into a 7 ml borosilicate scintillation vial. Cultures were transferred from overnight culture blocks to 96-well PCR plates in 100 ul volume and 15 ul of pooled phage concentrate was added to cultures one row at a time, with each row plated in agar overlay before adding phage concentrate to the next row of bacterial cultures. Mixed samples of 100 ul bacterial overnight culture and 15 ul pooled phage concentrate were transferred to the surface of bottom agar plates (2216MB, 1% agar, 5% glycerol, 125 ml L−1 of chitin supplement [40 g L−1 coarsely ground chitin, autoclaved, 0.2 um filtered]). A 2.5 ml volume of 52 °C molten top agar (2216MB, 0.4% agar, 5% glycerol BDH 1172-4LP) was added to the surface of the bottom agar and swirled around to incorporate and evenly disperse the mixed bacterial and phage sample into an agar overlay lawn. Agar overlay lawns were held at room temperature for 14–16 days and observed for plaque formation. Glycerol was incorporated into this assay to facilitate detection of plaques68. Chitin supplement was incorporated into this assay to facilitate detection of phages interacting with receptors upregulated in response to chitin degradation products. A variety of preliminary tests exploring potential optimizations to agar compositions for direct plating indicated that the addition of chitin did not negatively impact recovery of plaques with control phage strains tested. After approximately 2 weeks, plaques on agar overlay lawns were cataloged and described with respect to plaque morphology and plaques were picked for storage based on the previously described Archiving Plaques method13, as follows. All plaques were archived from plates containing less than 25 plaques, on plates with larger numbers of plaques a random subsample of plaques from each distinct morphology were archived. A polypropylene 96-well PCR plate was filled with 200 ul aliquots of 0.2 um filtered 2216MB, agar plugs were collected from plates using a 1 ml barrier pipette tip and ejected into the 2216MB, skipping one well between each sample to minimize potential for cross-contamination, for a final count of 48 phage plugs per plate. Plaque plugs were soaked at 4 °C for several hours to allow elution of phage particles into the media. After soaking, 96-well plates were centrifuged at 2,000 rcf for 3 min before proceeding to the next step. Plug soaks were then processed for two independent storage treatments. For storage at 4 °C, plates were processed by transferring 150 ul of eluate from each well to a 0.2 um filtration plate (Millipore, Multiscreen HTS GV 0.22um Filter Plate MSGVS22) and gently filtered under vacuum to remove bacteria, the cell-free filtrates containing eluted phage particles from each plaque plug were stored at 4 °C. For storage at −20 °C, 50 ul of 50% glycerol was added to the residual ~50 ul of the plug elution, often still containing the agar plug. In this way all plaques were characterized and many plaques from each strain were archived in two independent sets of conditions. Total plaque counts for all strains included in the bait assay are represented in Fig. 1, and provided in Supplementary Data 1, where they are identified as baxSet1287. Notes on limitations to the assay: Water temperatures on each of the three isolations days were 13.8 °C, 16.3 °C, and 14.2 °C, for days 222, 261, and 286; as bait assays were performed at room temperature (approximately 22 °C) some phages requiring lower temperatures may not have yielded plaques. The majority of plates were evaluated for plaque formation twice, on day 1 and day 13, thus any plaques appearing after day 1 and disappearing before day 13 – for example as a result of overgrowth of lysogens—are likely to have been missed in these assays.Viral purificationA subset of plaques archived during the bait assay was selected for phage purification, genome sequencing, and host range characterization. This subset included single randomly-selected representatives from each plaque-positive bacterial strain. Minor details of the purification and lysate preparation varied across samples but were largely as follows. Phages were purified from inocula derived primarily from −20 °C plaque archives, and secondarily from 4 °C archives when primary attempts with −20 °C stocks failed to produce plaques. Three serial passages were performed using Molten Streaking for Singles13 method. Agar overlay lawns for passages were prepared by aliquoting 100 ul of host overnight culture (4 ml 2216MB, colony inoculum from streak on 2216MB with 1.5% Bacto Agar, shaken overnight at RT at 250 rpm on VWR DS500E orbital shaker) onto a standard size bottom agar plate and adding 2.5 ml of molten 52 °C top agar as in the bait assay, swirling to disperse the host into the top agar and form a lawn, and streaking-in phage with a toothpick either from the plaque archive or directly from well-separated plaques in overlays from the previous step in serial purification. Following plaque formation on the third serial passage plate plaque plugs were picked using barrier tip 1 ml pipettes and ejected into 250 ul of 2216MB to elute overnight at 4 °C. Plaque eluates were spiked with 20 ul of host culture and grown with shaking for several hours to generate a primary small-scale lysate. Small scale primary lysates were centrifuged to pellet cells and titered by drop spot assay to estimate optimal inoculum volume to achieve confluent lysis in a 150 mm agar overlay plate lysate. Plate lysates were generated by mixing 250 ul of overnight host culture with primary lysate and plating in 7.5 ml agar overlay. After development of confluent lysis of lawns as compared against negative control without phage addition, the lysates were harvested by addition of 25 ml of 2216MB, shredding of the agar overlay with a dowel, and collection of the broth and top agar. Freshly harvested lysates were stored at 4 °C overnight for elution of phage particles, the following day lysates were centrifuged at 5,000 rcf for 20 min and the supernatant filtered through a 0.2 um Sterivex filter into a 50 ml tube and stored at 4 °C.Viral genome sequencingSequencing of Nahant Collection viruses was described in previous work19, and was performed as follows. For DNA extraction approximately 18 ml of phage lysate was concentrated using a 30 kD centrifugal filtration device (Millipore, Amicon Ultra Centrifugal Filters, Ultracel 30 K, UFC903024) and washed with 1:100 2216MB to reduce salt concentrations inhibitory to downstream nuclease treatments. Concentrates were brought to approximately 500 ul using 1:100 diluted 2216MB and then treated with DNase I and RNase A (Qiagen RNase A 100 mg mL−1) for 65 min at 37 °C to digest unencapsidated nucleic acids. Nuclease treated concentrates were extracted using an SDS, KOAc, phenol-chloroform extraction and resuspended in EB Buffer (Qiagen 19086) for storage at 20 °C. Phage genomic DNA was sheared by sonication in preparation for genome library preparation. DNA concentrations of extracts were determined using PicoGreen (Invitrogen, Quant-iT PicoGreen dsDNA Reagent and Kits P7589) in a 96-well format and samples brought to 5 ug in 100 ul final volume of PCR-grade water diluent for sonication. Samples were sonicated in batches of 6 for 6 cycles of 5 min each, at an interval of 30 s on/off on the Low Intensity setting of the Biogenode Bioruptor to enrich for a fragment size of ~300 bp. Illumina constructs were prepared from sheared DNA as follows: end repair of sheared DNA (NEB, Quick Blunting Kit, E1201L), 0.72×/0.21× dSPRI (AMPure XP SPRI Beads) size selection to enrich for ~300 bp sized fragments, ligation (NEB, Quick Ligation Kit, M2200L) of Illumina adapters and unique pairs of forward and reverse barcodes for each sample, SPRI (AMPure XP SPRI Beads) clean up, nick translation (NEB, Bst DNA polymerase, M0275L), and final SPRI (AMPure XP SPRI Beads) clean up (Rodrigue et al., 2010). Constructs were enriched by PCR using PE primers following qPCR-based normalization of template concentrations. Enrichment PCRs were prepared in octuplicate 25 ul volumes, with the recipe: 1 ul Illumina construct template, 5 ul 5x Phusion polymerase buffer (NEB, 5X Phusion HF Reaction Buffer, B0518S), 0.5 ul 10 mM dNTPs (NEB, dNTP Mix (1 mM; 0.5 ml), N1201AA), 0.25 ul 40 uM IGA-PCR-PE-F primer, 0.25 ul 40 uM IGA-PCR-PE-R primer, 0.25 ul Phusion polymerase (NEB, Phusion High Fidelity DNA Pol, M0530L), 17.75 ul PCR-grade water. PCR thermocycling conditions were as follows: initial denaturation at 98 °C for 20 sec; batch dependent number of cycles (range of 12–28) of 98 °C for 15 sec, 60 °C for 20 see, 72 °C for 20 sec; final annealing at 72 °C for 5 min; hold at 10 °C. For each sample 8 replicate enrichment PCR reactions were pooled and purified by 0.8x SPRI beads (AMPure XP) clean up. Each sample was then checked by Bioanalyzer (2100 expert High Sensitivity DNA Assay) to confirm the presence of a unimodal distribution of fragments with a peak between 350–500 bp. Sequencing of phage genomes was distributed over 4 paired-end sequencing runs as follows: HiSeq library of 18 samples pooled with 18 external samples, 3 MiSeq libraries each containing ~100 multiplexed phage genomes. Accession numbers for all sequenced phage genomes are provided in Supplementary Data 1, where they are identified as phageSet283; the subset of phages used in the majority of analyses in this work are identified as phageSet248 and exclude non-independent isolates derived from the same plaque, as well as well as identical phages isolated from multiple independent plaques from the same host strain in the bait assay.Viral protein clusteringTo characterize and annotate groups of proteins in assembled viral genomes in the Nahant Collection19, proteins were clustered using MMseqs2 v. 2.2339469 with default parameter settings, the 21,937 proteins reported in the GenBank files associated with each of the 283 Nahant Collection phage genomes were clustered into 5,929 clusters including 2,978 singletons. MMseqs2 cluster assignments for each protein sequence are provided in Supplementary Data 6.Viral protein cluster annotationAll proteins were annotated using InterProScan70 v.5.39–77.0; eggNOG-mapper71,72 v.2 using both automated and viral HMM selection options; Meta-iPVP73; and with best matches to 9518 Viral Orthologous Groups74 HMM profiles (obtained at http://dmk-brain.ecn.uiowa.edu/pVOGs/downloads.html); search was performed with hmmer, requiring a bitscore of 50 or greater (highest e-value 5.80E-13), as follows: hmmsearch -o $out_dir/$hmm_group.$hmmfile.$prots_short_name.hmm.out -tblout $out_dir/$hmm_group.$hmmfile.$prots_short_name.hmm.tbl.out -noali -T 50 $hmmfile $prots_dir/$prots_file. Annotations for viral protein clusters are provided in Supplementary Data 6.Receptor binding proteins (RBPs) were annotated as follows. RBPs were defined here to include both globular and fibrous host interacting proteins and general protein annotations were reviewed for similarity to known phage receptor binding proteins and supplemented with Phyre275, HHpred, and literature review76. Annotated RBPs were mapped onto phage genome diagrams and additional RBPs were annotated based on gene order conservation with phages in the same genus for which RBPs were already identified; annotated RBPs were then used to iteratively search against all Nahant Collection phage proteins using the jackhmmer search tool in the HMMER77 v.3.2.1 package (jackhmmer -cpu 16 -N 3 -E 0.00001 -incE 0.01 -incdomE 0.01 -o $run.$1.vs.$2.jackhmmer.iters-$iters.out -tblout $run.$1.vs.$2.jackhmmer.iters-$iters.tbl.out -domtblout $run.$1.vs.$2.jackhmmer.iters-$iters.dom.tbl.out $queryFASTAS $subjectFASTAS) and new hits were manually reviewed. All annotations were performed on a protein-cluster level and annotations of proteins and protein clusters as “adsorption – RBP” are indicated in Supplementary Data 6.Recombinases were annotated as follows: Homologs of single strand annealing protein recombinases in the Rad52, Rad51 and Gp2.5 superfamilies in the Nahant Collection phages were identified as described below. First, iterative HMM searches were performed against the Nahant Collection phage proteins using as seeds 194 recombinases identified in Lopes et al.44 (excluding RecET fusion protein YP_512292.1; http://biodev.extra.cea.fr/virfam/table.aspx), these represent 6 families of SSAP recombinases (UvsX, Sak4, Sak, RedB, ERF, and Gp2.5); searches were performed using the jackhmmer function of HMMER v.3.1.2 (jackhmmer -cpu 16 -N 5 -E 0.00001 -incE 0.01 -incdomE 0.01 -o $run.$1.vs.$2.jackhmmer.out -tblout $run.$1.vs.$2.jackhmmer.tbl.out -domtblout $run.$1.vs.$2.jackhmmer.dom.tbl.out $queryFASTAS $subjectFASTAS) – this yielded 156 proteins. Second, all hits were plotted onto genome diagrams for all phages in the collection and additional candidate recombinases identified based on gene neighborhood comparisons (Supplementary Data 9) – this step identified 4 additional protein clusters (mmseqs 297, 149, 2211, and 600), totaling 224 proteins. Third, all proteins clusters were curated by manual review of annotations made using InterProScan70, EggNOG-mapper71, and Phyre275 (annotations provided in Supplementary Data 6) to identify potential false positives (none identified), and references to recombinases in annotations. Where these annotation methods did not provide additional support, sequences were evaluated for additional support using HHpred78 (hhsearch -cpu 8 -i../results/full.a3m -d /cluster/toolkit/production/databases/hh-suite/mmcif70/pdb70 -o../results/2058109.hhr -oa3m../results/2058109.a3m -p 20 -Z 250 -loc -z 1 -b 1 -B 250 -ssm 2 -sc 1 -seq 1 -dbstrlen 10000 -norealign -maxres 32000 -contxt /cluster/toolkit/production/bioprogs/tools/hh-suite-build-new/data/context_data.crf) as implemented on the MPI Bioinformatics Toolkit webserver (mmseq 2896 and 5138 both gave >99% probability hits to DNA repair protein Rad52 with PDB ID 5JRB_G), or JackHMMER (-E 1 -domE 1 -incE 0.01 -incdomE 0.03 -mx BLOSUM62 -pextend 0.4 -popen 0.02 -seqdb uniprotkb) as implemented on the EMBL-EBI webserver (mmseq 2990 showed hits to diverse RedB family RecT-like sequences at e-value ≤1e-05). Following this third step, there were 3 protein clusters for which support was limited, these were included in the final dataset as putative SSAP recombinases but are highlighted here. Protein cluster mmseq 297 (present in 21 phages in 6 genera): was always encoded by genes adjacent to genes in protein cluster mmseq 3923, which was itself a recombinase associated exonuclease that was found either adjacent to mmseq 297 or to the well-supported putative SSAP recombinase mmseq 3721 (sometimes separated by one gene from mmseq 3721). Protein cluster mmseq 600 (present in 2 phages in 2 genera): was encoded adjacent to a protein cluster annotated as a recombination associated exonuclease; iterative HHMER searches of a mmseq 600 cluster representative (AUR82881.1) against Viruses in UniProtKB using jackhmmer yielded hits to proteins in mmseq 297 in iteration 3. Protein cluster mmseq 2990 (present in 1 phage): was encoded adjacent to two small proteins encoding putative recombination associated exonucleases and was in the same genomic position relative to neighboring genes as putative recombinases in related phages in the genus. Finally, all putative SSAP recombinase genes were assigned to a recombinase family by clustering based on 2 iterations of all-by-all HMM jackhmmer sequence similarity searches of all candidates and the reference seed set of Lopes44 (jackhmmer -cpu 16 -N 2 -E 0.00001 -incE 0.01 -incdomE 0.01 -o $run.$1.vs.$2.jackhmmer.out -tblout $run.$1.vs.$2.jackhmmer.tbl.out -domtblout $run.$1.vs.$2.jackhmmer.dom.tbl.out $queryFASTAS $subjectFASTAS); similarities were were visualized using Cytoscape v.3.3.0 using the “Edge-weighted Spring Embedded Layout” based on jackhmmer score, clusters were identified using the ClusterMaker2 v.1.2.1 Cytoscape plugin with the MCL cluster option and all settings at default and Granularity=2.5. Proteins in 3 mmseq clusters (149, 297, 600) did not fall into MCL clusters with recombinases from the annotated seed set and therefore are described as “unknown” rather than being assigned to a family of recombinases. All final assignments of genes to a recombinase superfamily and family, as well as all associated annotations, are provided in Supplementary Data 6 (sheet A.prots_overview column anno_Recombinase_manual). Additional details regarding seed sequences and MCL cluster assignments associated with recombinase analyses are provided in Supplementary Data 7 which contains a main descriptor sheet (00.readme), an overview of the 224 Nahant phages with recombinases (sheet 01.NahantPhageRecombinases_224), a table of InterPro domains associated with each of the reference and Nahant recombinases, with specific mmseqs and MCL clusters (sheet 02.IPR_annos_Lopes+Nahant), a list of all references used (sheet 03.List1_LOPES_ALL.noETfusion), the output of the iterative jackhmmer search with seeds against all Nahant Collection proteins (sheet 04.List1_vs_NahantProts), the output of the all-by-all jackhmmer search for 194 references and 224 putative Nahant recombinases (sheet 05.Lopes+Nahant224_v_self2iter), and information on the assignment of all Nahant and reference proteins to MCL clusters as shown in Fig. 6 (06.Recombinase_assign_by_MCL).All proteins were assigned to one of three broad categories – structural, other (non-structural), or no prediction – based on manual review of annotations derived from: NCBI product ID, Virfam21, PhANNs79, pVOGs74, eggNOG-mapper72, Phyre275, the MPI Bioinformatics implementation of HHpred78, and targeted annotations of predicted receptor binding proteins and recombinases (see descriptions for targeted annotations in Methods, above). Protein clusters (mmseq groups) were reviewed for conflicting calls and ultimately all proteins within each protein cluster (mmseqsID) were assigned to a single category. All assignments, and annotations on which they were based, are provided in Supplementary Data 6.The approach for assigning annotations to these broad categories was as follows: Step 1) All genes identified as putative recombinases through targeted annotations were assigned as “other”. Step 2) All genes identified as putative receptor binding proteins through targeted annotations were assigned as “structural”. Step 3) Genes not assigned to a category in steps 1 and 2, and which were identified by Virfam as “head-neck-tail” associated were assigned as follows: Genes annotated by Virfam as a terminase (TerL) were assigned as “other”; genes annotated by Virfam as a major capsid protein (MCP), portal (portal), adaptor (Ad1, Ad2, Ad3), head-closure (Hc1, Hc2, Hc3), tail completion (Tc1, Tc2), major tail protein (MTP), neck (Ne), or sheath (sheath) were assigned as “structural”. Step 4) Genes not assigned to a category in steps 1–3, were assigned as “structural” or “other” (non-structural) if identified as such by PhANNs with a confidence of ≥95%. Cases where conflicting annotations were observed between PhANNs and other annotations were flagged for review in subsequent steps. Step 5) Genes with annotations of VOG0263 (DNA transfer protein); terminal protein, any reference to internal virion protein, DNA circularization protein, and MuF-like proteins were assigned as “other”; in the case of conflict the Step 5 annotation superseded the prior annotations. Step 6) Genes with annotation as a terminase (large subunit, small subunit, and unspecified) by any of the tools (requiring ≥ 90% confidence if based on Phyre2) were assigned as “other”. Step 7) All genes lacking support across annotations were assigned as “no prediction”, high confidence Phyre2 predictions qualitatively judged as inappropriate were disregarded. Step 8) Genes flagged in Step 4 were reviewed and assigned as “structural” when containing any structural related genes (i.e. those listed in Step 3 and any others identifiable as structural based on words in the annotations and consensus across tools, e.g. containing the word baseplate, capsid, coat, head, spike, tail, whisker, fibritin). Additional targeted annotation by HHpred was used to facilitate assignment to “structural” (known structural proteins as described for Step 3 and in the aforementioned list), “other” (non-structural), “no prediction” (e.g. no assignable function based on available annotations and a PhANNs confidence of More
