Genome sequence and characterization of Streptomyces phages Vanseggelen and Verabelle, representing two new species within the genus Camvirus

Bacteriophage isolation

Streptomyces phages Vanseggelen and Verabelle were isolated from a soil sample obtained from the National Park Zuid-Kennemerland in the Netherlands (N52° 23′ 31″, E4° 34′ 49″) using Streptomyces coelicolor as the host. Details on isolation procedures are reported previously in detail12. The collected phage stock solutions were stored at 4 °C.

Host range analysis

The host range of Vanseggelen and Verabelle was determined by using serial dilutions on double agar overlay plates. The phage stock solutions were serial diluted with Difco Nutrient Broth (DNB) (BD biosciences) supplemented with 4 mM Ca(NO3)2 and 0.5% w/v glucose. Droplets of 3 µl of the phage dilutions were spotted in duplicate on a bacterial lawn of nine different Streptomyces strains and one Kitasatospora strain from the Microbial Biotechnology (MBT) collection at Leiden University13. Streptomyces strains MBT13, MBT61 and MBT86 are new species and have not been taxonomically classified yet. The plates were incubated at 30 °C for at least 24 h before lysis was observed, after which the presence of a lysis zone was scored as a positive result. The efficiency of plating (EOP) for a given strain was calculated relative to the host strain S. coelicolor, only when distinct clear individual plaques were observed.

Morphology analysis

The plaque morphology of Vanseggelen and Verabelle was determined by using serial dilutions on double agar overlay plates on the host S. coelicolor.

Representative images of the phages were made using transmission electron microscopy (TEM). For a single grid preparation, 3 µl of the phage lysates (Vanseggelen = 106 PFU/ml and Verabelle = 107 PFU/ml) was placed on a glow-discharged 200 mesh carbon coated copper grid (EMS) and allowed to set for 30 s before excess sample solution was removed by filter paper. The phages were stained with 2% uranyl acetate for 45 s after which excess liquid was removed and the samples were air-dried for an additional 30 min. The grids were observed using a single tilt specimen holder inside a 120 kV Talos L120C TEM with a Lab6 electron source and Ceta detector at the Netherlands Center for Nanoscopy (NeCEN, Leiden).

Stability analysis

A one-step growth curve analysis was performed in triplicate to determine the latent period and burst size of both phages14. Since Streptomyces phage adsorption was found to be maximal for germlings15, 108 spores ml−1 of S. coelicolor were allowed to germinate in 10 ml DNB medium at 30 °C while shaking at 200 rpm. After approximately 5 h, the culture was infected with Vanseggelen or Verabelle at Multiplicity of Infection (MOI) of 0.01 and 100 µl was sampled at 10 min intervals up to 180 min. The samples were filtered with a 0.20 µm filter, serial diluted with DNB medium and immediately plated on double agar overlay plates. The burst size was calculated using the following formula:

$$ \begin{aligned} & {\text{Burst}}\;{\text{size}} = {\text{average}}\;{\text{of}}\;{\text{free}}\;{\text{phages}}\;{\text{after}}\;{\text{burst}}\left( {{\text{T}} = {15}0\;{\text{to}}\;{\text{T}} = {18}0} \right) \\ & – {\text{average}}\;{\text{of}}\;{\text{free}}\;{\text{phages}}\;{\text{before}}\;{\text{burst}}\left( {{\text{T}} = {1}0\;{\text{to}}\;{\text{T}} = {14}0} \right)/{\text{number}}\;{\text{of}}\;{\text{phages}}\;{\text{at}}\;{\text{T}} = 0 \\ \end{aligned} $$

The thermal stability of Vanseggelen and Verabelle was determined by adding 100 µl of phage suspension (Vanseggelen = 105 PFU/ml and Verabelle = 107 PFU/ml) to 900 µl DNB medium and incubating the phages at 25, 30, 37, 45, 55 and 65 °C. The samples were filtered with a 0.20 µm filter after 1 h of incubation. A serial dilution was made with DNB medium, which was immediately spotted on double agar overlay plates to determine the phage titers. To assess viability at common storage temperatures, the phages were incubated in DNB medium without glycerol at -80, -20 and 4 °C for seven days. To determine pH stability, 100 µl of the phage suspensions was added to 900 µl DNB medium that was pH adjusted using 1 M HCl or 1 M NaOH. The samples were incubated at 30 °C for 1 h before filtration. A serial dilution was made with DNB medium, which was immediately spotted on double agar overlay plates to determine the phage titer. All the phage stability measurements were performed in triplicate. All statistical analyses were performed with a Student’s t-test.

DNA extraction and phylogenetic analysis

DNA isolation, whole genome sequencing and de novo assembly of Vanseggelen and Verabelle were performed by the Institute Pasteur using Illumina NovaSeq PE150 sequencing (Paris, France). The complete genome sequences of Vanseggelen and Verabelle were deposited at GenBank under accession number OQ970438 and OQ970439, respectively. Linear maps of the genomes were constructed by Geneious Prime 2022.1.1 ( and genomes were de novo assembled using SPAdes v3.15.5 and aligned starting by the phage terminase16.

A viral proteomic phylogenetic tree and a genomic alignment based on the whole genome sequences of Streptomyces phages Alsaber, Amela, Endor1, Endor2, Hydra, Indigo, Joe, Pablito, phiCAM, Saftant, Sitrop, Verse and Yosif were constructed using ViPtree: the Viral Proteomic Tree version 3.517. The genome sequences of these phages were acquired from the National Center for Biotechnology Information (NCBI). Pairwise genome comparisons of phages within the Camvirus genus were visualized and performed with clinker18. A heatmap to determine intergenomic relatedness was constructed with the use of VIRIDIC19.


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