Genome sequence of cluster A6 bacteriophage Lilbunny, isolated using Mycobacterium smegmatis mc2155

TL;DR

This paper reports the genome sequence of a new bacteriophage, Lilbunny, isolated from rabbit fecal compost and infecting Mycobacterium smegmatis.

Contribution

The study provides the first genome sequence of a cluster A6 bacteriophage isolated from rabbit fecal compost.

Findings

Lilbunny's genome is 50,789 bp and contains 95 open reading frames.

Approximately 52.6% of the open reading frames encode proteins with predicted functions.

The genome includes three tRNA genes.

Abstract

Bacteriophage Lilbunny is a siphovirus infecting Mycobacterium smegmatis strain mc2155. It was isolated from compost of rabbit fecal matter. The genome of Lilbunny belongs to the A6 subcluster and is 50,789 bp, containing 95 open reading frames, 52.6% of which encode proteins with predicted functions, and three tRNA genes.

Figures

Negative-stained transmission electron micrograph of the 200 nm Lilbunny phage (500,000× magnification, 30 kV accelerating voltage, Tecnai TF-20).

Full text

ANNOUNCEMENT

The Lilbunny phage was isolated from ~100 g of rabbit feces compost in Provo, Utah, on 1 October 2023. Phages were isolated by shaking in 7H9 liquid medium, then 0.2 micron-filtering and triplicate M. smegmatis mc^2^155 plaque assays (37°C incubation for 48 hours). Plaques were clear with a ~2 mm diameter. Negative-stained transmission electron microscopy (2% uranyl-acetate) revealed a virion with siphovirus morphology and a length of ~200 nm (Fig. 1).

Negative-stained transmission electron micrograph of the 200 nm Lilbunny phage (500,000× magnification, 30 kV accelerating voltage, Tecnai TF-20).

High-titer lysate was sent to CD Genomics, where 0.5 µg DNA was extracted using the phage DNA isolation kit (Norgen) protocol prior to DNA sequencing. NEBNext Ultra DNA Library Prep Kit for Illumina (NEB, USA) following manufacturer’s recommendations and indices were used. DNA samples were sonicated to 350 bp, then end-polished, A-tailed, and ligated with the full-length adaptor for sequencing. The product was purified (AMPure XP system) and quantified with an Agilent 2100 Bioanalyzer and qPCR. In addition, 2.74 million 150 bp paired-end reads were generated using Illumina NovaSeq X PE150. TrimGalore was applied with a minimum length of 20 bases and minimum phred score of 20 (1), then de novo assembly of the 1.4 million reads using CONSED 2.9 (2) with Unicycler version 0.5.0 (3), resulting in a 50,739 base-pair genome (4,051× coverage) with 61.5% G + C content. The phage sequence, assigned to the Gladiatorvirus genus, has a 10-base 3´ sticky overhang sequence (CGGTCGGTAA; phagesdb.org) and was assigned to subcluster A6 using BLAST. Plaque morphology and similarity with other members of this cluster suggest that this phage has a temperate life cycle.

Annotation of the genome sequence used DNA Master v.5.0.2 (4), Glimmer v.3.0 (5), and GeneMark v.2.5 (6), and refined using Phamerator (7) and Starterator (https://phagesdb.org/). HHPRED (8) and BLASTP (9) searches confirmed annotations. We identified 95 protein-coding genes and three tRNA genes for aspartic acid, tryptophan, and glutamine, which were determined by Aragorn (10) and tRNAscan-SE (11). We identified open reading frames that putatively encode a holin, a DnaB-like helicase, and a metallophosphatase.