---
id: "edwards-2021-joy99"
title: "Complete Genome Sequence of Mycobacteriophage Joy99"
authors:
  - "Faith Cox"
  - "Josh Katuri"
  - "Megan Adams"
  - "Danielle Bachhofer"
  - "Ashleigh Cooper"
  - "Jessica Doty"
  - "Miranda Fuentes"
  - "Leeila Hanson"
  - "Travis Miller"
  - "Jonathan Musgrave"
  - "Aleksey Palumbo"
  - "Camille Trautman"
  - "Julie Edwards"
  - "Dustin Edwards"
venue: "Microbiology Resource Announcements"
year: 2021
date: "2021-08-12"
doi: "10.1128/mra.00556-21"
url: "/research/publications/10-1128-mra-00556-21/"
pdf: "/research/publications/10-1128-mra-00556-21/dustin-edwards-10-1128-mra-00556-21.pdf"
pmc: "https://pmc.ncbi.nlm.nih.gov/articles/PMC8359790/"
openAccess: true
license: "cc-by"
accessions:
  - "genbank:MH536822"
  - "sra:SRX4721439"
citedBy: 1
citedBySource: "OpenAlex, read 2026-09-12"
---
# Complete Genome Sequence of Mycobacteriophage Joy99

Genome of phage Joy99, annotated by Bluff Dale High School students; 59,837 bp, 97 genes.

## Abstract

Joy99 is a siphoviral mycobacteriophage with a 59,837-base pair double-stranded DNA genome and is predicted to contain 97 protein-coding genes and a single tRNA gene. Joy99 was isolated in Saint Louis, MO, and annotated by students at Bluff Dale High School in community engagement with Tarleton State University.

## Full text

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Complete Genome Sequence of Mycobacteriophage Joy99
Faith Cox,a Josh Katuri,a Megan Adams,a Danielle Bachhofer,a Ashleigh Cooper,a Jessica Doty,a Miranda Fuentes,a Leeila Hanson,a
Travis Miller,a Jonathan Musgrave,a Aleksey Palumbo,a Camille Trautman,a Julie Edwards,a Dustin Edwardsa
aDepartment of Biological Sciences, Tarleton State University, Stephenville, Texas, USA
ABSTRACT Joy99 is a siphoviral mycobacteriophage with a 59,837-base pair double-
stranded DNA genome and is predicted to contain 97 protein-coding genes and a single
tRNA gene. Joy99 was isolated in Saint Louis, MO, and annotated by students at Bluff Dale
High School in community engagement with Tarleton State University.
In 2019, 10 million Mycobacterium tuberculosis infections occurred worldwide (1). An
estimated half million infections were rifampin-resistant tuberculosis, and 206,030 were
multidrug-resistant tuberculosis (MDR-TB), a 10% increase from 2018 (1). Characterized bacte-
riophages could offer an alternative treatment option for MDR-TB (2, 3). Mycobacteriophage
Joy99 was discovered in soil from a municipal mulch pile in Saint Louis, MO (global positioning
system [GPS] coordinates 38.664567 N, 90.3179 W) (4, 5). Soil samples were incubated in 7H9
liquid medium at 37°C for 2 h before the supernatant was centrifuged, filtered through a
0.22-
mm filter, and incubated with Mycobacterium smegmatis mc2 155 at 37°C on Luria agar
plates. Joy99 was isolated by two rounds of picking a single, well-separated plaque, fol-
lowed by diluting the bacteriophage sample in a 10-fold dilution series and plating it with M.
smegmatis mc2 155 (6).
Joy99 plaques formed a three-ring morphology with a clear center spot, thin middle
ring, and turbid outer ring. Negative-staining transmission electron microscopy with 1% uranyl
acetate on a carbon type B 300-mesh grid showed Siphoviridae morphology (4). ImageJ
v1.53h (7) measured a 75-nm-wide isometric capsid and 180-nm-long tail (Fig. 1). High-titer
lysate was obtained by flooding “webbed” plates, as described in the Phage Discovery Guide,
and DNA extraction was performed at Washington University using the Promega Wizard DNA
clean-up system (8). Sequencing libraries were prepared from genomic DNA using a 454 DNA
Citation Cox F, Katuri J, Adams M, Bachhofer D,
Cooper A, Doty J, Fuentes M, Hanson L, Miller T,
Musgrave J, Palumbo A, Trautman C, Edwards J,
Edwards D. 2021. Complete genome sequence
of mycobacteriophage Joy99. Microbiol Resour
Announc 10:e00556-21. https://doi.org/10
.1128/MRA.00556-21.
Editor Kenneth M. Stedman, Portland State
University
Copyright © 2021 Cox et al. This is an open-
access article distributed under the terms of
the Creative Commons Attribution 4.0
International license.
Address correspondence to Dustin Edwards,
dcedwards@tarleton.edu.
Received 29 May 2021
Accepted 22 July 2021
Published 12 August 2021
FIG 1 Transmission electron microscopy (TEM) of mycobacteriophage Joy99. High-titer lysate on a
carbon type B 300-mesh grid was negatively stained with 1% uranyl acetate. A TEM micrograph
of mycobacteriophage Joy99 shows an approximately 75-nm-diameter nonenveloped icosahedral capsid
and a 180-nm flexible noncontractile tail.
Volume 10 Issue 32 e00556-21 mra.asm.org 1
GENOME SEQUENCES
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library prep kit and sequenced with a Roche 454 GS FLX instrument at the Pittsburgh
Bacteriophage Institute to approximately 157-fold coverage from 17,587 total single-end reads
with an average read length of 165 bases (9). The single-bacteriophage contig assembly was
performed using Newbler v2.7, and the assembly was checked for completeness, accuracy,
and genome termini using Consed v22.0 software (9, 10). Joy99 has a linear double-stranded
DNA genome that is 59,837 bp long with 66.6% G1C content and an 11-base 39 sticky over-
hang composed of 59-CTCGTAGGCAT-39.
Whole-genome nucleotide alignment using BLASTn (https://blast.ncbi.nlm.nih.gov/)
indicated that Joy99 has nucleotide sequence identity of more than 99% with other
subcluster K1 bacteriophages (Table 1) (11). Initial auto-annotations were generated
using Glimmer v3.02 (12) and GeneMark v2.5p (13) and were revised manually using DNA
Master v5.23.2 (http://phagesdb.org/DNAMaster/) and PECAAN (https://pecaan.kbrinsgd.org/).
Gene functions were assigned using HHpred v3.0beta (14, 15) and BLASTp (11). The tRNA
was characterized using ARAGORN v1.2.38 (16) and tRNAscan-SE v2.0 (17). All tools were run
with default parameters. The Joy99 genome contains 97 predicted protein-coding genes
and a single tRNA gene for tryptophan; 14.3% of K1 bacteriophages encode a tRNA. The
start codon usage is 58.7% AUG, 40.2% GUG, and 0.1% UUG. Like other K1 bacteriophage
genomes, Joy99 contains genes for virion assembly and structure, lysis proteins, host inte-
gration and excision proteins, DNA primase, RusA-like resolvase, and RtcB-like ligase.
Data availability. The Joy99 genome sequence is available under GenBank accession
number MH536822. The raw reads are available under SRA accession number SRX4721439.
ACKNOWLEDGMENTS
Support for this research was provided by Tarleton State University College of Science
and Technology and by the Howard Hughes Medical Institute Science Education Alliance-
Phage Hunters Advancing Genomics and Evolutionary Science (SEA-PHAGES) research
and education program.
Mycobacteriophage Joy99 was originally isolated by Ho Yee Joyce Fung and Suchita
Rastogi at Washington University in Saint Louis, MO, USA. We thank Graham Hatfull, Welkin
Pope, Deborah Jacobs-Sera, Daniel Russell, Rebecca Garlena, and Sally Molloy for their
technical support during the sequencing and annotation of this genome. We also thank the
students at Bluff Dale High School, Texas, that participated in the annotation of this genome
as part of a Tarleton State University science outreach program.
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TABLE 1 Characteristics of similar bacteriophages with M. smegmatis as a hosta
Name GenBank accession no. Genome size (bp) GC content (%) No. of ORFsb No. of tRNAs Identity to Joy99 (%)
Joy99 MH536822 59,837 66.6 97 1
BellaDonna MH697578 59,708 66.6 97 1 99.46
Dalmuri MH727544 59,708 66.5 97 1 99.45
Inky MN369746 59,708 66.5 96 1 99.44
Jekyll MF140412 59,708 66.6 97 1 99.44
CREW KY380102 59,707 66.6 95 1 99.44
a Similarity defined as an identity of .95%.
b ORFs, open reading frames.
Cox et al.
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