Media and culture conditions
All bacterial culturing steps, with or without phages, were undertaken at room temperature (~ 22 °C) using R2A agar and R2B broth (Alpha Biosciences Inc., Baltimore, Maryland, USA) as growth media. Liquid cultures were grown with agitation at 200 rpm. Between uses, bacterial isolates were stored on R2A agar at 4 °C or as cryostocks at − 80 °C (~ 20% glycerol). Purified phage suspensions were stored at 4 °C between uses. The buffer solution (SM buffer) for phage resuspension contained 100 mM NaCl, 8 mM MgSO4 and 50 mM Tris–Cl (pH 7.5) in autoclaved Milli-Q water.
Bacterial isolation from sampled groundwater
Groundwater from 11 wells at the Skelstofte test sites (Vedby, Denmark, 54°52′34.1ʺN 11°16′18.7ʺE) was sampled at depths of between 7 and 12 m below the surface. These test sites are used to study bioremediation and monitoring strategies of groundwater contamination24. Briefly, to obtain water samples representative of the reservoir, wells were purged of ~ 13–22 times their volume (well ø = 4 mm) using a peristaltic pump with a 0.2 L min−1 flow rate to avoid disruption of the natural water movement, and stable values were observed for O2, pH, conductivity, temperature and other redox parameters before sampling (individual parameters varied between wells) (data not shown). Subsequently, 1-L samples were extracted into sterile glass bottles and stored at 4 °C until use. From the samples, heterotrophic bacteria were isolated on R2A agar (Alpha Biosciences Inc., Baltimore, Maryland, USA), which gave a collection of 84 isolates representing diverse colony morphotypes.
In brief, sample bottles with groundwater were shaken thoroughly and 100 µL of each well sample was plated and left to grow for 4–14 days. Colony growth from samples varied greatly and library building was based on acquiring all the colony morphotypes throughout the incubation period. Where possible, several colonies of a distinct morphotype (up to a maximum of eight) were picked. To obtain pure isolates, colonies were re-streaked three times.
Phage screening, isolation and amplification
Phage activity was detected in a pre-screening of phage enrichment cultures containing sample water, concentrated media and the relevant isolate. Screening hits, indicated by clear or turbid zones in the top agar layer as a result of plausible phage activity, were then followed by a new enrichment to verify phage activity and subsequently obtain pure phage isolates.
The initial enrichment and pre-screening were performed by mixing 2.5 mL 2 × R2B, 2.5 mL sample water and then inoculating with 50 µL culture of the relevant isolate (isolated from that sample water). Following five days of growth, enrichment cultures were centrifuged at 10,000 g for three minutes and supernatant was filtered through a 0.22-µm sterile syringe PVDF membrane filter (Millex Durapore, Burlington, MA, USA). Phage activity in the filtrate from each enrichment was screened in a standard double agar overlay assay. Briefly, 10 µL of each filtered enrichment was drop-plated in 10 replicates onto a semisolid agar, R2B + 50 mM CaCl2/MgCl2 + 0.6% agarose, inoculated with 100 µL culture of relevant bacterial isolate, using R2A as the bottom agar layer. Plates were incubated at room temperature and inspected daily for one week, with any potential plaque forming noted as ‘hits’ for groundwater sample and isolate.
The follow-up enrichment cultures of phage-host ‘hits’ contained 4 mL 10 × R2B broth, 35 mL groundwater sample, 1 mL culture of relevant host and 10 mM CaCl2/MgCl2. Enrichment cultures were incubated for 24 h. Subsequently, the culture was supplemented with 1 M NaCl and incubated for 30 min. The culture was then centrifuged at 5,000 g for five minutes and the supernatant was filtered through a 0.45-µm PVDF syringe filter (Millex Durapore, Burlington, MA, USA). Then 100 µL of the filtered supernatant was used in a double agar overlay assay as described above, and individual plaques were picked and re-plated three times to obtain pure phage samples.
A high titre of both phages was obtained by polyethylene glycol (PEG) precipitation as described in9 with modifications. Briefly, 200 mL R2B was inoculated with 100 µL host culture, infected with 100 µL of pure phage lysate and left overnight. The following day, 1 M NaCl was added to cultures and left on an orbital shaker for one hour to burst bacterial cells. Cultures were then spun at 12,000 g for 10 min, supernatant was collected and PEG was added to reach 10% w/v. The mixture was left for two hours on an orbital shaker for phage-PEG adsorption. Finally, the mixture was centrifuged at 12,000 g for 10 min and the pellets with phage virions were resuspended and collected in 3–5 mL SM buffer. Following PEG precipitation, titre was determined by PFU counts by drop-plating dilution series of collected particles on an agar overlayer, as described above. A titre range between 1010–1011 PFU mL−1 was obtained. Single plaques or high-titre phage samples (1010–1011 PFU mL−1) were used in downstream analysis to characterise and determine the phage’s (i) morphology, (ii) burst size, (iii) general genome features (iv) homology with other related phages, and (v) virion proteins. For TEM imaging and peptide sequencing, phage samples were further purified by a caesium chloride gradient according to the protocol of Clokie & Kropinski25.
Host DNA extraction, sequencing and identification
DNA was extracted from 5 mL of each host culture (grown for five days) using an Ultraclean Microbial DNA Isolation Kit (Mo bio Laboratories, Carlsbad, California, USA), following the manufacturer’s protocol. DNA libraries were prepared with the Nextera XT DNA kit (Illumina, San Diego, USA) according to the manufacturer’s protocol. The prepared libraries were then sequenced in a 2 × 251 paired-end sequencing run, as part of a flow cell, using the Illumina MiSeq v2 kit (Illumina, San Diego, CA, USA). In assembling host draft genomes, CutAdapt (v1.8.3) was used to quality-trim sequence reads (bases with < q20 removed from read ends) and to remove any contaminants (primers and indexes). Shorter reads (< 50 bp) were then removed and overlapping read pairs merged using AdapterRemoval (v2.1.0)26 at default settings. Finally, the cleaned merged and unmerged reads were assembled using SPAdes (v3.6.0)27 and assemblies evaluated in QUAST (v3.1)28.
For each host, the genome sequence data were uploaded to the Type (Strain) Genome Server (TYGS), a free bioinformatics platform available at https://tygs.dsmz.de, for a whole genome-based taxonomic analysis8. Methods (and results) for strain identification are provided in the Supplementary Information.
Transmission electron microscopy of virions
The caesium chloride-purified phage sample was adsorbed to freshly prepared ultra-thin carbon film and fixed with 2% (v/v) EM-grade glutaraldehyde (20 min). Fixed samples were then negatively stained with 1% (w/v) uranyl acetate and picked up with 400-mesh copper grids (Plano, Wetzlar, Germany). Finally, prepared samples were analysed using a Tecnai 10 transmission electron microscope (Thermo Fisher, Eindhoven, the Netherlands) at an acceleration voltage of 80 kV. Micrographs were taken with a MegaView G2 CCD-camera (EMSIS, Muenster, Germany).
Phage Lana burst size
Phage latency period and burst size were determined for phage Lana in a one-step growth curve experiment as described elsewhere9. Here, the host was grown to OD600nm 0.75, corresponding to 4 × 107 CFUs mL−1. Then, 10 mL culture was infected with a 0.05 multiplicity of infection and incubated for 20 min at 200 rpm to allow phage-host adsorption. After adsorption, three aliquots of infected culture were diluted × 10,000, and PFUs in the three diluted cultures were followed over time to determine the phage latency period and phage burst size. Experimental cultures were sampled at the beginning of the experiment and then every 10 min from 90 min until the end. Average PFU numbers before and after the burst event were used to calculate the burst size. In calculating the burst size, the infection efficacy of phage Lana after the adsorption step was also considered. After adsorption, one sample from the infected culture was centrifuged at 6,000 g for five minutes, thereby separating infecting phages (pellet) and unadsorped phages (supernatant). Subsequently, three technical replicates from the resuspended pellet and the supernatant were plated and PFUs were counted. The infection efficacy of Lana was then determined to be 19.27% ± 1.04 SEM. Prior to the experiment, the study established (i) a growth curve for the host strain to determine the OD600nm—CFU relationship and (ii) an approximate phage latency period (data not shown).
Phage DNA extraction and genome sequencing
For both phages the protocol for the direct plaque sequencing (DPS) method was used as described by Kot et al.29 with the following modifications for phage Anath only: 500 µL high titre lysate was used as input (~ 2 × 1010 PFU mL−1), 10 µL (100 mg mL−1) of protein kinase K (Thermo Scientific, Waltham, USA) was used in capsid DNA release, and 10 µL was used as elution volume for purified DNA. Phage DNA libraries were prepared with the Nextera XT kit DNA kit (Illumina, San Diego, USA), using the DPS method described in Kot et al.29 for phage Lana and the manufacturer’s kit protocol for phage Anath. Prepared libraries were sequenced in a 2 × 250 paired-end sequencing run, as part of a flow cell, using the Illumina MiSeq platform (Illumina, San Diego, USA).
Phage genome assembly and annotation
Sequence reads were trimmed and assembled in the CLC Genomic Workbench 11.1.0 (CLC bio, Aarhus, Denmark) using standard settings, and assembly was cross-verified using SPAdes (version 3.13.0, using trimmed and merged reads as input running on-careful mode), as described elsewhere30. Assembled genomes were automatically annotated using the RAST online tool31 and were manually curated by cross-referencing with four other publicly available protein recognition tools: BLASTp, Pfam, HHpred and Phyre32,33,34,35. Predicted protein functions were annotated accordingly when identical functions were predicted in at least three of the five databases used.
De novo peptide sequencing—identification of structural proteins
To identify the proteins, the previous procedure for protein purification from Lavigne et al.36 was followed with minor modifications. In short, 100 µL of the phage extract was transferred to an Amicon Ultra filter unit (MWCO 30 kDa) and centrifuged at 14,000 × g for 20 min and further desalted four times with 450 µL water. The filtrate containing the phage particles (10 µL) was denaturised in 25 µL buffer consisting of 6 M urea, 5 mM dithiothreitol and 50 mM Tris–HCl (pH 8). The phage particles were destabilised by five successive freeze-thawing cycles followed by a full hour incubation at 60 °C to reduce the phage proteins. The proteins were alkylated by adding 25 µL of 100 mM iodoacetamide and 150 µL of 50 mM ammonium bicarbonate and incubated for 45 min at room temperature. Phage proteins were digested with 0.8 µg trypsin dissolved in 40 µL 50 mM ammonia bicarbonate and incubated for 24 h at 37 °C. The protein digest was diluted with 200 µL 0.1% trifluoroacetic acid (TFA) and purified by solid-phase extraction using 2 mg hydrophobic reversed phase well-plate cartridges (Thermo Fisher Scientific) preconditioned with 200 µL acetonitrile and 200 µL 0.1% TFA. The peptides were eluted from the cartridges with two times 25 µL 70% acetonitrile and diluted with 150 µL 0.1% TFA. The phage peptides were analysed using an Ultimate 3,000 RSLCnano UHPLC system hyphenated with a Q Exactive HF mass spectrometer (Thermo Fisher Scientific, Denmark). An amount of 6.4 µL of the sample was loaded on a preconcentration trap (C18 300 µm × 5 mm cartridge, Thermo Fisher Scientific) and eluted onto an analytical column (75 µm × 250 mm, 2 µm C18, Thermo Fisher Scientific) with a chromatographic triple-phasic 53 min gradient ranging from 1 to 64% mobile phase B (98% acetonitrile and 0.1% formic acid) at a 300 nL per minute flow rate. The total analysis time was 65 min and mobile phase A consisted of 2% acetonitrile and 0.1% formic acid. The high-resolution mass spectrometer was operated with positive electrospray ionisation in data-dependent mode by automatically switching between MS and MS/MS fragmentation. Based on a survey MS scan in the Orbitrap operated at a mass resolution of 120,000 at m/z 200 with a target of 3e6 ions and a maximum injection time at 50 ms, the twelve most intense peptide ions were selected for MS/MS fragmentation in subsequent scans. The selected ions were isolated (in a m/z 1.4 window) and higher-energy collision dissociation was performed at a normalised collision energy (28) and fragments recorded in centroid mode at a resolution of 60,000 (m/z 200) with a 250 ms maximum filling time and target of 1e5 ions. The high-resolution data generated were analysed in Proteome Discoverer 2.2 (Thermo Fisher Scientific) and searched against predicted phage/host proteins by the Sequest HT algorithm in an iterative processing pipeline. The search criteria were enzyme, trypsin (full); dynamic modifications, methionine oxidation and acetyl (N-terminus); precursor mass tolerance, 5 ppm; fragment mass tolerance, 20 mDa. The processed data were filtered in a Proteome Discoverer consensus workflow with the Peptide Validator algorithm (q-value < 0.01) to ensure the peptide-spectrum match had a false discovery rate under 1%. The de novo peptide sequencing identified 136 proteins in total with a false discovery rate < 1%. Individual samples contained proteins mapping to predicted proteins of both the phage and its host: 17 identified proteins in Anath/B. mycoides and 122 identified proteins in Lana/Pseudomonas sp. To avoid false positive identification, only phage proteins identified with quality scores (sequest HT score) exceeding the highest quality score of identified host proteins were regarded as virion proteins.