---
id: "edwards-2025-godfather"
title: "Complete genome sequence of bacteriophage Godfather isolated from Microbacterium foliorum"
authors:
  - "Joshua Hutchings"
  - "Lauren Bower"
  - "Ethan Collum"
  - "Timothy Hester"
  - "Melody Hunter"
  - "Chaney Kelly"
  - "Luke Reynolds"
  - "Cole Moore"
  - "Dustin Edwards"
venue: "Microbiology Resource Announcements"
year: 2025
date: "2025-02-11"
doi: "10.1128/mra.00888-24"
url: "/research/publications/10-1128-mra-00888-24/"
pdf: "/research/publications/10-1128-mra-00888-24/dustin-edwards-10-1128-mra-00888-24.pdf"
pmc: "https://pmc.ncbi.nlm.nih.gov/articles/PMC11812392/"
openAccess: true
license: "cc-by"
accessions:
  - "genbank:PP725414"
  - "sra:SRX24892095"
citedBy: 0
citedBySource: "OpenAlex, read 2026-09-12"
---
# Complete genome sequence of bacteriophage Godfather isolated from Microbacterium foliorum

Genome of phage Godfather, isolated from Stephenville soil; 17,452 bp, 24 genes, cluster EE.

## Abstract

Microbacteriophage Godfather was collected from a soil sample in Stephenville, Texas. The 17,452-bp double-stranded genome contains 24 protein-coding genes. The genome shares >99% nucleotide sequence identity with cluster EE microbacteriophages Scamander, Danno, Kojax4, and Burgy.

## 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.

| Bacteriophages | Announcement
Complete genome sequence of bacteriophage Godfather
isolated from Microbacterium foliorum
Joshua Hutchings,1 Lauren Bower,1 Ethan Collum,1 Timothy Hester,1 Melody Hunter,1 Chaney Kelly,1 Luke Reynolds,1 Cole Moore,1
Dustin Edwards1
AUTHOR AFFILIATION See affiliation list on p. 2.
ABSTRACT Microbacteriophage Godfather was collected from a soil sample in
Stephenville, Texas. The 17,452-bp double-stranded genome contains 24 protein-coding
genes. The genome shares >99% nucleotide sequence identity with cluster EE microbac
teriophages Scamander, Danno, Kojax4, and Burgy.
KEYWORDS bacteriophage genetics
Investigating the genetic diversity of bacteriophages increases our understanding of
their evolution and biological roles (1–3). We isolated the bacteriophage Godfather
using Microbacterium foliorum NRRL B-24224, which was selected as the host bacteria
for its lack of prophages and internal defense systems (4, 5). A soil sample was collected
from a flower bed in Stephenville, Texas (GPS coordinates 32.21191 N, 98.211755 W) and
incubated with peptone yeast calcium (PYCa) media at 29°C in a shaking incubator for 2
hours (6). The sample was centrifuged at 10,000 × g for 20 minutes, and the supernatant
was collected and filtered (0.22 μL pore size), inoculated with a freshly saturated M.
foliorum culture, and incubated at 29°C on a PYCa plate in a soft agar overlay (6). At 24
hours, Godfather formed 2 mm clear plaques, and the bacteriophage was isolated by two
rounds of ten-fold serial dilutions and plating before being prepared as a high-titer lysate
(6). A 300-mesh copper grid was prepared with high-titer lysate and stained with 1%
uranyl acetate. Transmission electron microscopy, using an FEI Tecnai G2 Spirit BioTWIN
(NL1.160G), showed that Godfather has siphovirus morphology (Fig. 1A). ImageJ v1.53m
(7) was used to measure an approximate tail length of 115 nm and a capsid diameter of
50 nm (n = 6).
Genomic DNA was extracted from high-titer lysate by ZnCl2 precipitation (8). A library
was prepared with the NEBNext Ultra II kit and sequenced with an Illumina MiSeq
instrument at the Pittsburgh Bacteriophage Institute (9) to 38× coverage from 30,304
single-end reads of 150 base read length. Raw reads were verified for accuracy using
Consed v29.0 (9) and assembled as a single contig using Newbler v2.9 (10). The Godfa
ther genome is 17,452 bp in length with a 9-nucleotide 3′ sticky overhang (5′-C
CCGCCCCA-3′) and contains 68.7% G+C content, similar to host M. foliorum (5).
BLASTn (11) query of the sequence with the GenBank nonredundant/nucleotide
(nr/nt) database returned nucleotide sequence identity (NSI) >99% with cluster EE
bacteriophages Scamander (NC_054431), Danno (MT316462), Kojax4 (OR159657), and
Burgy (ON755188) and 98% NSI to microbacteriophages Eightball (OK040783) and
TimoTea (MK524502) (12–15). Of interest, Godfather shared 97% NSI with cluster EE
bacteriophage Loca, which was previously isolated from a shopping cart handle at a
nearby vendor that sold commercially available bagged soil (16). Starterator v546 (http://
phages.wustl.edu/starterator/), Glimmer v3.02 (17), and GeneMark v2.5 (18) were used
for auto-annotation, followed by manual refinement with Phamerator (19), PECAAN
(https://discover.kbrinsgd.org), and DNA Master v5.23.6 (http://phagesdb.org/
February 2025 Volume 14 Issue 2 10.1128/mra.00888-24 1
Editor Kenneth M. Stedman, Portland State
University, Portland, Oregon, USA
Address correspondence to Dustin Edwards,
dcedwards@tarleton.edu.
The authors declare no conflict of interest.
See the funding table on p. 2.
Received 12 August 2024
Accepted 4 December 2024
Published 16 January 2025
Copyright © 2025 Hutchings et al. This is an
open-access article distributed under the terms of
the Creative Commons Attribution 4.0 International
license.
Downloaded from https://journals.asm.org/journal/mra on 26 July 2026 by 156.146.253.207.

DNAMaster/). TmHmm v1.0.24 (20) identified potential transmembrane helices. Consis
tent with other cluster EE bacteriophages, no tRNA genes were identified using Aragorn
v1.2.38 (21) and tRNAscan-SE v2.0 (22). Nineteen of 24 protein-coding genes were called
using queries to BLASTp v2.14.1 (11) and HHPred v.3.0beta (PDB, UniProt, Pfam-A v36,
and NCBI v3.19 databases) (23). Default settings were used for all programs. Functional
assignments include structural proteins, an Lsr2-like DNA-bridging protein, four helix-
turn-helix DNA-binding domains, and an HNH endonuclease (Fig. 1B).
ACKNOWLEDGMENTS
Support for this research was provided by the Tarleton State University College of
Science and Mathematics and the Howard Hughes Medical Institute Science Education
Alliance-Phage Hunters Advancing Genomics and Evolutionary Science (SEA-PHAGES)
research and education program. We thank Graham Hatfull, Deborah Jacobs-Sera, Daniel
Russell, Rebecca Garlena, and Phoebe Doss for their technical support during the
imaging of the bacteriophage and the sequencing and annotation of this genome.
AUTHOR AFFILIATION
1Department of Biological Sciences, Tarleton State University, Stephenville, Texas, USA
AUTHOR ORCIDs
Dustin Edwards http://orcid.org/0000-0001-6409-8041
FUNDING
Funder Grant(s) Author(s)
Tarleton State University College of Science and Mathematics Dustin Edwards
AUTHOR CONTRIBUTIONS
Joshua Hutchings, Formal analysis, Investigation, Writing – original draft | Lauren Bower,
Formal analysis, Investigation, Writing – original draft | Ethan Collum, Formal analysis,
Investigation, Writing – original draft | Timothy Hester, Formal analysis, Investigation,
Writing – original draft | Melody Hunter, Formal analysis, Investigation, Writing –
original draft | Chaney Kelly, Formal analysis, Investigation, Writing – original draft |
Luke Reynolds, Formal analysis, Investigation, Writing – original draft | Cole Moore,
FIG 1 Bacteriophage Godfather virion and genome organization. (A) A representative micrograph of bacteriophage Godfather demonstrating siphovirus
morphology with an approximate tail length of 115 nm and a capsid diameter of 50 nm. (B) Genes represented by boxes located above or below the ruler
(forward or reverse orientation) and labeled with putative functions. Auto-annotation was performed by Starterator, Glimmer, and GeneMark, followed by
manual refinement with Phamerator, PECAAN, and DNA Master. Protein-coding genes were identified by queries to BLASTp and HHPred.
Announcement Microbiology Resource Announcements
February 2025 Volume 14 Issue 2 10.1128/mra.00888-24 2
Downloaded from https://journals.asm.org/journal/mra on 26 July 2026 by 156.146.253.207.

Formal analysis, Investigation, Project administration, Supervision, Writing – original
draft, Writing – review and editing | Dustin Edwards, Conceptualization, Data curation,
Formal analysis, Funding acquisition, Investigation, Methodology, Project administration,
Resources, Supervision, Validation, Visualization, Writing – original draft, Writing – review
and editing
DATA AVAILABILITY
Godfather genome sequence is available at GenBank under the accession number
PP725414 and in the SRA under accession number SRX24892095.
REFERENCES
1. Dion MB, Oechslin F, Moineau S. 2020. Phage diversity, genomics and
phylogeny. Nat Rev Microbiol 18:125–138. https://doi.org/10.1038/
s41579-019-0311-5
2. Hatfull GF. 2020. Actinobacteriophages: genomics, dynamics, and
applications. Annu Rev Virol 7:37–61. https://doi.org/10.1146/annurev-
virology-122019-070009
3. Jansson JK, Wu R. 2023. Soil viral diversity, ecology and climate change.
Nat Rev Microbiol 21:296–311. https://doi.org/10.1038/s41579-022-
00811-z
4. Behrendt U, Ulrich A, Schumann P. 2001. Description of Microbacterium
foliorum sp. nov. and Microbacterium phyllosphaerae sp. nov., isolated
from the phyllosphere of grasses and the surface litter after mulching
the sward, and reclassification of Aureobacterium resistens (Funke et al.
1998) as Microbacterium resistens comb. nov. Int J Syst Evol Microbiol
51:1267–1276. https://doi.org/10.1099/00207713-51-4-1267
5. Russell DA, Garlena RA, Hatfull GF. 2019. Complete genome sequence of
Microbacterium foliorum NRRL B-24224, a host for bacteriophage
discovery. Microbiol Resour Announc 8:e01467-18. https://doi.org/10.
1128/MRA.01467-18
6. Poxleitner M, Pope W, Jacobs-Sera D, Sivanathan V, Hatfull G. 2018.
Phage discovery guide. Howard Hughes Medical Institute, Chevy Chase,
MD.
7. Schneider CA, Rasband WS, Eliceiri KW. 2012. NIH Image to ImageJ: 25
years of image analysis. Nat Methods 9:671–675. https://doi.org/10.
1038/nmeth.2089
8. Santos MA. 1991. An improved method for the small scale preparation of
bacteriophage DNA based on phage precipitation by zinc chloride.
Nucleic Acids Res 19:5442. https://doi.org/10.1093/nar/19.19.5442
9. Russell DA. 2018. Sequencing, assembling, and finishing complete
bacteriophage genomes. Methods Mol Biol 1681:109–125. https://doi.
org/10.1007/978-1-4939-7343-9_9
10. Margulies M, Egholm M, Altman WE, Attiya S, Bader JS, Bemben LA,
Berka J, Braverman MS, Chen Y-J, Chen Z, et al. 2005. Genome
sequencing in microfabricated high-density picolitre reactors. Nat New
Biol 437:376–380. https://doi.org/10.1038/nature03959
11. Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ. 1990. Basic local
alignment search tool. J Mol Biol 215:403–410. https://doi.org/10.1016/
S0022-2836(05)80360-2
12. Markov SA, Church JC, Lee L, Bell CM, Binkley SD, Bouma KM, Hutson KM,
Markov GS, Mason EC, Rueff GB, Sennuga TO, Simpson MH, Zimmer RJ,
Villalpando DG. 2021. Complete genome sequences of Microbacterium
bacteriophages Danno, Otwor, and Scumberland, isolated in Clarksville,
Tennessee. Microbiol Resour Announc 10:e00209-21. https://doi.org/10.
1128/MRA.00209-21
13. Van Roekel HF, Georgen JJ, Kock RA, Coleman ST. 2022. Genome
sequence and characteristics of the Microbacterium foliorum cluster EE
bacteriophage Burgy. Microbiol Resour Announc 11:e0091222. https://
doi.org/10.1128/mra.00912-22
14. Mastropaolo MD, Fallest-Strobl PC, Sequira DM, Campbell DD, Negro CJ,
Tuang KS, Sigmund-Hamre IM, Womack CL, Mansbridge T, Metzler AL,
Sasher EG, Collins C, Crowley NK, Dower VR, Bates M, Bjorkelo C, Johnson
H, Salvitti LR, Neumann University Phage Discovery Group. 2023.
Genome sequences of five Microbacterium foliorum phages, GaeCeo,
NeumannU, Eightball, Chivey, and Hiddenleaf. Microbiol Resour
Announc 12:e0110622. https://doi.org/10.1128/mra.01106-22
15. Jacobs-Sera D, Abad LA, Alvey RM, Anders KR, Aull HG, Bhalla SS, Blumer
LS, Bollivar DW, Bonilla JA, Butela KA, et al. 2020. Genomic diversity of
bacteriophages infecting Microbacterium spp. PLoS ONE 15:e0234636.
https://doi.org/10.1371/journal.pone.0234636
16. Ounsinegad A, Ashcraft M, Bliss E, Brawley D, Clements G, Densmore A,
Gastin A, Luciano M, Moore C, Munoz V, Pernarelli A, Pitner M, Velsen E,
Wiggam K, Goppert M, Edwards D. 2022. Complete genome sequence of
bacteriophage loca, isolated on a Microbacterium foliorum culture.
Microbiol Resour Announc 11:e0078322. https://doi.org/10.1128/mra.
00783-22
17. Delcher AL, Harmon D, Kasif S, White O, Salzberg SL. 1999. Improved
microbial gene identification with GLIMMER. Nucleic Acids Res 27:4636–
4641. https://doi.org/10.1093/nar/27.23.4636
18. Besemer J, Borodovsky M. 2005. GeneMark: web software for gene
finding in prokaryotes, eukaryotes and viruses. Nucleic Acids Res
33:W451–4. https://doi.org/10.1093/nar/gki487
19. Cresawn SG, Bogel M, Day N, Jacobs-Sera D, Hendrix RW, Hatfull GF.
2011. Phamerator: a bioinformatic tool for comparative bacteriophage
genomics. BMC Bioinformatics 12:395. https://doi.org/10.1186/1471-
2105-12-395
20. Möller S, Croning MD, Apweiler R. 2001. Evaluation of methods for the
prediction of membrane spanning regions. Bioinformatics 17:646–653.
https://doi.org/10.1093/bioinformatics/17.7.646
21. Laslett D, Canback B. 2004. ARAGORN, a program to detect tRNA genes
and tmRNA genes in nucleotide sequences. Nucleic Acids Res 32:11–16.
https://doi.org/10.1093/nar/gkh152
22. Lowe TM, Eddy SR. 1997. tRNAscan-SE: a program for improved
detection of transfer RNA genes in genomic sequence. Nucleic Acids Res
25:955–964. https://doi.org/10.1093/nar/25.5.955
23. Söding J, Biegert A, Lupas AN. 2005. The HHpred interactive server for
protein homology detection and structure prediction. Nucleic Acids Res
33:W244–8. https://doi.org/10.1093/nar/gki408
Announcement Microbiology Resource Announcements
February 2025 Volume 14 Issue 2 10.1128/mra.00888-24 3
Downloaded from https://journals.asm.org/journal/mra on 26 July 2026 by 156.146.253.207.