--- id: "edwards-2019-ryadel" title: "Complete Genome Sequence of Cluster O Mycobacterium smegmatis Bacteriophage Ryadel" authors: - "Travis Miller" - "Danielle Bachhofer" - "Ashleigh Cooper" - "Jessica Doty" - "Josh Katuri" - "Jonathan Musgrave" - "Aleksey Palumbo" - "Heidi Spann" - "Amanda Stone" - "Keith Emmert" - "Julie Edwards" - "Jesse Meik" - "James Pierce" - "Dustin Edwards" venue: "Microbiology Resource Announcements" year: 2019 date: "2019-03-28" doi: "10.1128/mra.01594-18" url: "/research/publications/10-1128-mra-01594-18/" pdf: "/research/publications/10-1128-mra-01594-18/dustin-edwards-10-1128-mra-01594-18.pdf" pmc: "https://pmc.ncbi.nlm.nih.gov/articles/PMC6439249/" openAccess: true license: "cc-by" accessions: - "genbank:MH590592" - "sra:SRX4721442" citedBy: 0 citedBySource: "OpenAlex, read 2026-09-12" --- # Complete Genome Sequence of Cluster O Mycobacterium smegmatis Bacteriophage Ryadel Genome of phage Ryadel, a cluster O phage with an elongated head; 72,658 bp, 132 genes. ## Abstract Mycobacteriophage Ryadel is a newly isolated cluster O Siphoviridae bacteriophage, characterized by an unusual prolate capsid, containing a 72,658-base-pair double-stranded DNA genome with 132 predicted protein-coding genes. Conserved among cluster O bacteriophages, the Ryadel genome contains 31 copies of a unique 17-bp sequence with dyad symmetry. ## Full text Machine-extracted from the PDF linked above. It carries the artifacts that come with reading a typeset two-column page: running heads, figure captions in the flow of the prose, and words broken across line ends. The abstract above is the registry's deposit and is the authoritative text. Complete Genome Sequence of Cluster O Mycobacterium smegmatis Bacteriophage Ryadel Travis Miller,a Danielle Bachhofer,a Ashleigh Cooper,a Jessica Doty,a Josh Katuri,a Jonathan Musgrave,a Aleksey Palumbo,a Heidi Spann,a Amanda Stone,a Keith Emmert,b Julie Edwards,a Jesse Meik,a James Pierce,a Dustin Edwardsa aDepartment of Biological Sciences, Tarleton State University, Stephenville, Texas, USA bDepartment of Mathematics, Tarleton State University, Stephenville, Texas, USA ABSTRACT Mycobacteriophage Ryadel is a newly isolated cluster O Siphoviridae bacteriophage, characterized by an unusual prolate capsid, containing a 72,658-base- pair double-stranded DNA genome with 132 predicted protein-coding genes. Con- served among cluster O bacteriophages, the Ryadel genome contains 31 copies of a unique 17-bp sequence with dyad symmetry. Mycobacteriophage Ryadel was isolated from sandy soil samples collected beneath a rotting hay bale in Stephenville, Texas (32°16=43.601N, 98°08=52.101W). Soil samples were placed in 7H9 liquid medium, and the supernatant was passed through a 0.22- m filter for direct isolation of bacteriophages. Filtered supernatant was incu- bated with Mycobacterium smegmatis mc2155 at 37°C for 48 hours and resulted in small-sized lytic plaques. Ryadel was purified by collecting virus from well-isolated plaques from the direct isolation and two successive rounds of serial dilutions. Negative-staining transmission electron microscopy (Fig. 1) of isolated mycobacterio- phage Ryadel displayed siphoviral morphology with an unusual prolate capsid that was 160 nm in length by 40 nm in width (4:1 ratio), which is characteristic of cluster O bacteriophages (1). A Promega Wizard DNA clean-up kit was used to isolate DNA from the purified bacteriophage. The Pittsburgh Bacteriophage Institute prepared a Genomic DNA se- quencing library using the NEB Ultra II kit, which was run on an Illumina MiSeq instrument with 47 other samples, yielding 172,400 single-end 150-base-pair Ryadel reads representing 325-fold genome coverage. A single bacteriophage contig was assembled from raw reads using Newbler 2.9 with default settings, and it was checked for completeness, accuracy, and genome termini using Consed 29.0 (2, 3). The virus contains a double-stranded DNA genome that is 72,658 base pairs in length with a GC content of 65.2%. The accumulation of aligned reads indicated a linear genome with a 3= single-stranded terminal overhang of 5=-GTGT-3=. NCBI BLASTn (https://blast.ncbi .nlm.nih.gov/) whole-genome alignment (4) showed 98% nucleotide identity to the cluster O mycobacteriophages Familton (GenBank accession number MG099943) and Catdawg (GenBank accession number KF017002) (5). Characteristic of cluster O bacteriophages, the Ryadel genome contains 31 copies of a unique 17-bp sequence with dyad symmetry consisting of a 7-bp inverted repeat separated by 3 bp (5=- TGTTCGGNNNCCGAACA-3=) (1). This repeat does not occur in the genomes of Myco- bacterium tuberculosis or Mycobacterium smegmatis mc2155; however, there are two copies present in the genome of Mycobacterium sp. 05-1390 (1). Glimmer v3.02 (6, 7) and Genemark v2.5p (8, 9) were used to autoannotate the genome. Manual inspection included refinement of start sites and annotation revision using Phamerator (https://phamerator.org/) (10), DNA Master v5.23.2 (http://phagesdb .org/DNAMaster/), and PECAAN (https://pecaan.kbrinsgd.org/). Mycobacteriophage Citation Miller T, Bachhofer D, Cooper A, Doty J, Katuri J, Musgrave J, Palumbo A, Spann H, Stone A, Emmert K, Edwards J, Meik J, Pierce J, Edwards D. 2019. Complete genome sequence of cluster O Mycobacterium smegmatis bacteriophage Ryadel. Microbiol Resour Announc 8:e01594-18. https://doi.org/10.1128/ MRA.01594-18. Editor Jelle Matthijnssens, KU Leuven Copyright © 2019 Miller 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 26 November 2018 Accepted 1 March 2019 Published 28 March 2019 GENOME SEQUENCES crossm Volume 8 Issue 13 e01594-18 mra.asm.org 1 Downloaded from https://journals.asm.org/journal/mra on 26 July 2026 by 156.146.253.207. Ryadel was predicted to contain 132 protein-coding genes, and no tRNA genes were identified by ARAGORN v1.2.38 (11) or tRNAscan-SE v2.0 (12). Putative functions of 34 (25.8%) of 132 predicted protein-coding genes were assigned using HHpred v3.0beta (13, 14) and NCBI BLASTp (4). Similar to other cluster O bacteriophages, the Ryadel genome is mostly arranged in three transcriptional blocks and contains nine strongly predicted SigA-like promoters (5=-TGTCAA–17 bp–TGAAT-3=) (1). Leftward-transcribed genes 1 to 13 (6.5% of genome) encode endonuclease VI and DNA methylases. Rightward-transcribed genes 14 to 76 (58.5% of genome) encode virion structural and assembly proteins, DNA primase/polymerase, HNH endonucleases, O-methyltransferase, glycosyltransferases, d-Ala-d-Ala carboxypeptidase, a lysis cassette containing lysin A and lysin B, and holin proteins. Leftward-transcribed genes 77 to 132 (35% of genome) encode DNA polymerase III sliding clamp beta, Ku-like double-stranded DNA (dsDNA) break-binding protein, and ParB-like dsDNA partitioning protein. FIG 1 Transmission electron microscopy (TEM) of mycobacteriophage Ryadel. Purified high-titer lysate was placed on a carbon type-B 300 mesh grid, stained with uranyl acetate, and imaged by a FEI Tecnai G2 Spirit BioTWIN (NL1.160G). TEM micrographs of negatively stained mycobacteriophage Ryadel showed a prolate capsid that was 160 nm in length by 40 nm in width (4:1 ratio), a morphology that corresponds to that of members of cluster O bacteriophages within the Siphoviridae family. Miller et al. Volume 8 Issue 13 e01594-18 mra.asm.org 2 Downloaded from https://journals.asm.org/journal/mra on 26 July 2026 by 156.146.253.207. Data availability. The mycobacteriophage Ryadel genome is available at GenBank under accession number MH590592. Raw reads are available in the SRA under acces- sion number SRX4721442. ACKNOWLEDGMENTS Support for this research was provided by Tarleton State University College of Science and Technology and by the Howard Hughes Medical Institute SEA-PHAGES program. 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