Isolation and complete genome sequence of Citrobacter bacteriophage Ursula

Phage isolation and cultivation

The bacteriophage Ursula was isolated during efforts to sample diverse bacteriophages from aquatic environments, specifically targeting Citrobacter spp. strains known to cause infections in African sharptooth catfish (Clarias gariepinus) in Russian aquaculture facilities. On September 14, 2021, a water sample was collected from the Norishka Stream in Mikhailovsky Park, Moscow, Russia. To isolate the phage, the following enrichment process was used:

Initial Preparation: 0.5 liters of unsterilized water were added to a 2-liter sterile plastic bottle.

Medium Addition: 50 milliliters of sterile 10× LB medium were mixed into the water (the 1× medium contains: 10 g Bacto Tryptone (Amresco, Am-J859-0.25), 5 g yeast extract (Amresco, J850-500G), 10 g NaCl (Amresco, J869-500G), and distilled water up to 1 liter).

Bacterial Culture Introduction: 0.5 milliliters of an overnight liquid culture of Citrobacter spp. CF69 strain were added to the enriched mixture.

Incubation: The enriched culture was incubated at 37°C with shaking at 240 rpm.

After incubation, 1 milliliter of the enriched culture was centrifuged at 12,000 rpm using a tabletop microcentrifuge. The resulting supernatant was filtered through a 0.22-micron syringe membrane filter (Millex-GP, Millipore) and then plated onto a lawn of Citrobacter spp. CF69 strain using the standard double-layer method.⁶

Phage plaques were formed after an overnight incubation at 37 °C. The phage was purified using two consecutive single-plaque isolations. To obtain a high-titer lysate, five 90 mm Petri dishes were filled with solid LB medium (15 g of Bacto Agar (BD214030, BD Difco) per 1 l). Without drying the plates, they were overlaid with 5 ml of soft LB agar (6 g Bacto Agar (BD214030, BD Difco) per 1 l) per plate. Soft agar was inoculated with 300 μl of a 4h log-phase liquid culture of the host strain and approximately 1×10⁵ PFU of the phage per plate. The plates were incubated overnight at 37 °C. To extract the phage, the soft agar layer was gently destroyed with a spreader, transferred into 50 ml plastic centrifuge tubes, and layered with 10 ml of LB medium. Chloroform (Fisher Scientific, C298-4) (100 μL) was added to each tube, followed by vigorous vortexing for 1 min, and allowed to stand at room temperature for 2 h. After incubation, the agar fragments and bacterial cells were pelleted by centrifugation at 10 000 g for 5 min, and the supernatant was collected and centrifuged again under the same conditions. The phage clarified stock was pelleted in Beckman Ti45 angle rotor (75 000 g, 1 h, 20 °C), resuspended in saline and layered on sucrose step gradient in 5 ml tubes (60%-50%-40%-30%-20% sucrose w/w, 800 μL each). Tubes were centrifuged in Beckman SW55Ti swing bucket rotor (75 000 g, 1 h, 20 °C) and a compact opalescent band between 40% and 50% layers was removed, corresponding to intact phage particles.⁷ The purified phage sample was used for DNA extraction and transmission electron microscopy (TEM), as previously described.⁸ Briefly, a drop of purified phage preparation was adsorbed on a carbon-coated TEM support grids, negatively contrasted with 1% uranyl acetate in methanol (Reachim, USSR), dried and studied in Jeol JEM-1400Flash electron microscope Jeol Ltd., Japan).

DNA extraction, sequencing, assembly and annotation

To extract phage DNA, the high-titer phage stock (about 10¹¹ pfu/ml) was incubated with DNase (Thermo Scientific, EN0521) at a concentration of 0.01 mg/ml for 30 minutes at room temperature to destroy extraneous DNA traces. Subsequently, the phage particles were collected via ultracentrifugation in an angle rotor at room temperature (Beckman 45Ti, 1 hour, 75,000 g). Genomic DNA was extracted from pelleted bacteriophage using CTAB (cetyltrimethylammonium bromide) extraction as previously described.⁹ The quality and quantity of the DNA were assessed using agarose gel electrophoresis and a Qubit dsDNA HS fluorometric assay (Qubit, USA). Libraries of phage genomic DNA were prepared and sequenced using an Ion Proton sequencer (Applied Biosystems, Foster City, CA, USA) with standard reagents according to the manufacturer’s protocol. Raw sequencing reads were pooled and filtered using the error-correction tool Pollux (RRID:SCR_026200). Final contigs were assembled using Newbler version 3.0 (RRID:SCR_011916) (Roche Diagnostics, USA), yielding a single contig representing the phage genome, consisting of 183,411 base pairs with an average coverage of 200 bp.

Annotation was performed using Prokka¹⁰ (RRID:SCR_014732) and Pharokka¹¹ (RRID:SCR_026017)-Phold (RRID:SCR_026016)-Phynteny (RRID:SCR_026015) pipeline with subsequent manual curation. Potential open reading frames (ORFs) were initially detected using GeneMarkS (https://genemark.bme.gatech.edu/genemarks.cgi) (RRID:SCR_011930) and subsequently analyzed using HMMER¹² (RRID:SCR_005305), HHPRED¹³ (RRID:SCR_010276) (MPI Bioinformatics Toolkit), NCBI BLAST¹⁴ (RRID:SCR_004870), and tRNAscan-SE¹⁵ (RRID:SCR_008637).

Ethical considerations

No animal research was conducted in this work. Ethical approval and consent were not required.