Genome sequence of PSonyx, a singleton bacteriophage infecting Corynebacterium glutamicum

ABSTRACT PSonyx is a newly isolated phage that infects Corynebacterium glutamicum. This siphovirus was isolated from a French pond in the south of Paris by students from Paris-Saclay University. Its 80,277-bp singleton genome carries 136 protein-coding genes and 5 tRNAs.

Here, we report the complete genome of PSonyx, a bacteriophage that was isolated from pond water collected in Massy (48.72538N, 2.27359 E), France.The sample was first passed through a 0.22-µm filter and then supplemented with LB (5 g/L NaCl; 5 g/L yeast extract; 10 g/L tryptone), 1 mM CaCl 2 , and C. glutamicum strain MB001, a derivative of the reference strain ATCC 13032 in which the prophages CGP1, CGP2, and CGP3 were deleted (8).After incubation for 24 h at 30°C, the filtered supernatant produced plaques when plated in the double agar layer in the presence of MB001 and incubated for 24 h at 30°C.The phage was purified through three rounds of plating using the procedures outlined in the SEA-PHAGES discovery guide (https://seaphagesphage discoveryguide.helpdocsonline.com/).PSonyx showed clear plaques of approximately 1.5 mm diameter with irregular edges (Fig. 1).Transmission electron microscopy revealed a capsid with a diameter of 80 (±6) nm and a flexible tail with a length of 320 (±11) nm (n = 30) (Fig. 1).
Phage DNA was extracted from a high titer lysate using the PCI/SDS protocol (https:// phagesdb.org/media/workflow/protocols/pdfs/PCI_SDS_DNA_Extraction_2.2013.pdf).A library was prepared using the NEB Ultra II kit and sequenced using an Illumina Miseq instrument (v3 reagents).De novo assembly of 133,928 single-end 150-bp reads (approxi mate coverage of 791) was performed with Newbler v2.9 as previously described (9), resulting in an 80,277-bp genome sequence, with a 53% G + C content and circularly permuted termini.Consed v29 (10) was used to check the completeness, accuracy, and termini.
The left third of the genome contains 30 genes that are transcribed forward and are likely involved in virion assembly and lysis.The encoded proteins include tail assembly chaperones using programmed translational frameshift, an endolysin, and a holin.The middle third of the genome is transcribed in reverse orientation.Although it contains 65% of the genes, most of them lack any known homologs, except for tRNAs and genes responsible for DNA replication/modifications. Finally, the last 19 genes are forward transcribed and include two intriguing long ORFs encoding proteins of 1,263 and 1,116 residues, respectively and both without homologs.The PSonyx genome does not share sufficient sequence homology with other genomes in the actinobacteriophage database and was therefore classified as a singleton.

ACKNOWLEDGMENTS
Seed funding was provided by the "Appel à projet pour la pédagogie" from the Ecole Universitaire de Premier Cycle of Paris-Saclay.The present work has benefited from the platform of Cryo-EM of I2BC supported by IBiSA and by French Infrastructure for Integrated Structural Biology (FRISBI) ANR-10-INBS-05, as well as Pascale Boulanger.
We are grateful to Audrey Guitton for her help with the budgeting and ordering when the teaching unit was created.We thank Billy Biederman, Véronique Delesalle, Deborah

FIG 1
FIG 1 Characterization of siphovirus PSonyx.(A) Clear plaques observed in the LB agar overlay supplemented with C. glutamicum strain MB001 and 1 mM CaCl 2 after 24 h at 30°C.(B) Negative staining of PSonyx phages with 2% wt/vol uranyl acetate observed with a Tecnai Spirit microscope operated at 100 kV (TFS) and equipped with a K2 Base 4kx4k direct electron detection camera (Ametek/Gatan).The magnification used was 19,800× with a pixel size of 2.5 Å at the level of the specimen.Bar: 100 nm.