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Genome Sequences
bacteriophagesBacteriophagesGenome characterization of BI2 subcluster Streptomyces scabiei bacteriophages GoblinVoyage and Doxi13
Jin Hanna 1 Conceptualization Data curation Formal analysis Investigation Methodology Validation Visualization Writing – original draft Writing – review and editing
Chana Nihal K. 1 Conceptualization Data curation Formal analysis Methodology Project administration Validation Visualization Writing – original draft Writing – review and editing
Tang Annie L. 1 Conceptualization Data curation Formal analysis Methodology Project administration Validation Visualization Writing – original draft Writing – review and editing
Kaur Paramjit 1 Conceptualization Data curation Formal analysis Methodology Project administration Validation Visualization Writing – original draft Writing – review and editing
Lamichhane Brishti 1 Conceptualization Data curation Formal analysis Methodology Project administration Validation Visualization Writing – original draft Writing – review and editing
Leung Sze Ching 1 Conceptualization Data curation Formal analysis Methodology Project administration Validation Visualization Writing – original draft Writing – review and editing
Scheiderer Diane 1 Conceptualization Data curation Formal analysis Methodology Project administration Validation Visualization Writing – original draft Writing – review and editing
Sivaprakasam Vighnesh V. 1 Conceptualization Data curation Formal analysis Methodology Project administration Validation Visualization Writing – original draft Writing – review and editing
Marcelino Dannah T. 1 Conceptualization Data curation Formal analysis Methodology Project administration Validation Visualization Writing – original draft Writing – review and editing
Hull Gregory J. 1 Conceptualization Data curation Formal analysis Methodology Project administration Validation Visualization Writing – original draft Writing – review and editing
Kamara Toma M. 1 Conceptualization Data curation Formal analysis Methodology Project administration Visualization Writing – original draft Writing – review and editing
2023 UMBC Phage Hunters 1
STEM BUILD at UMBC Cohort 7 2
Guimaro Maria C. 2 Funding acquisition Project administration Resources Supervision Writing – review and editing
https://orcid.org/0000-0001-9166-9137
Caruso Steven M. 1 Conceptualization Data curation Formal analysis Funding acquisition Investigation Methodology Project administration Resources Software Supervision Validation Visualization Writing – review and editing scaruso@umbc.edu

1 Department of Biological Sciences, University of Maryland Baltimore County , Baltimore, Maryland, USA
2 College of Natural and Mathematical Sciences, University of Maryland Baltimore County , Baltimore, Maryland, USA
Editor Dennehy John J. Department of Biology, Queens College , Queens, New York, USA

Address correspondence to Steven M. Caruso, scaruso@umbc.edu
The authors declare no conflict of interest.

9 2024
20 8 2024
20 8 2024
13 9 e00581-2431 5 2024
03 7 2024
Copyright © 2024 Jin et al.
2024
Jin et al.
https://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license.

ABSTRACT

We present the bacteriophages GoblinVoyage and Doxi13, siphoviruses isolated on Streptomyces scabiei RL-34. They belong to the BI2 cluster and have genomes consisting of 60.9% GC content with identical 3’ end sticky overhangs. The genome lengths of GoblinVoyage and Doxi13 are 43,540 bp and 43,696 bp, respectively.

KEYWORDS

bacteriophage
Streptomyces
plant pathogen
phytopathogens
soil microbiology
bacteriophage genetics
actinomycetes
genomes
HHS | NIH | National Institute of General Medical Sciences (NIGMS) TL4GM118989, UL1GM118988, RL5GM118987 Guimaro Maria C. Caruso Steven M. cover-dateSeptember 2024
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pmcANNOUNCEMENT

Streptomyces scabiei is a Gram-positive phytopathogen, primarily responsible for the common potato scab disease (1). Genomic characterization of bacteriophages that infect S. scabiei reveals roles that they may serve to address the deleterious effects of the infection.

Samples of bacteriophages GoblinVoyage and Doxi13 were collected from soils using S. scabiei RL-34 as a host (Table 1). All protocols were derived from the Science Education Alliance-Phage Hunters Advancing Genomics and Evolutionary Science (SEA-PHAGES) Phage Discovery Guide (2). Soil samples were combined with phage buffer (10 mM [Tris pH 7.5], 10 mM MgSO4, 68 mM NaCl, and 1 mM CaCl2) and passed through a 0.22-µm filter. Then, 500 µL of the filtrate was added to 250 µL of a 48-hour S. scabiei culture, incubated for 10 minutes, combined with 3 mL of tryptic soy soft agar (BD), then plated onto nutrient agar plates (BD Difco) with Streptomyces phage supplement (10 mM MgCl2, 8 mM Ca(NO3)2, 0.5% glucose) and incubated for 24–48 hours at 30°C. Phages underwent a minimum of three rounds of plaque purification. In short, picked plaques were serially diluted tenfold and used for plaque assays on the host. The fresh lysate was harvested from plates containing near-confluent lysis for imaging and DNA isolation. Negative-stained transmission electron microscopy revealed siphoviral morphotypes for both GoblinVoyage and Doxi13 (Fig. 1A). Both phages demonstrated a similar plaque morphology (Fig. 1B). Neither phage produced discernable lysogens when tested by spotting dilutions of the lysate on S. mirabilis and incubating for 7 days at 30°C, suggesting they followed a lytic infection cycle.

Fig 1 Morphological and functional characterization of BI2 phages GoblinVoyage and Doxi13. (A) Representative transmission electron microscopy images of GoblinVoyage and Doxi13. The fresh lysate sample was stained with 2% uranyl acetate. TEM imaging revealed GoblinVoyage and Doxi13 are siphoviruses with flexible and noncontractile tails. GoblinVoyage (left) has a capsid diameter of 58 nm (n = 3, SD = 2.5 nm) and tail length 261 nm (n = 3, SD = 1.53). Doxi13 (right) has a capsid diameter of 52 nm (n = 3, SD = 1.5) and tail length 248 nm (n = 3, SD = 12.56). All measurements were taken using ImageJ v1.54i (3). (B) Plaque morphology of GoblinVoyage and Doxi13 (left to right). Both phages showed turbid plaques with an average diameter of 1.10 mm (n = 30, SD = 0.337) and 1.60 mm (n = 16, SD = 0.46), respectively. Doxi13 plaques were observed in S. mirabilis. Plaques were measured after approximately 24 to 48 hours of incubation at 30°C. All measurements were taken using ImageJ v1.54i (3). (C) Comparison of GoblinVoyage and Doxi13 genome size and GoblinVoyage to other BI subcluster phages. From top to bottom: GoblinVoyage (BI2), Doxi13 (BI2), TonyStarch (BI1), LibertyBell (BI3), and SendItCS (BI4). All phages are compared to GoblinVoyage from approximately 21,500 bp. All phages selected are representative or the only member of their subcluster. Phages from subclusters BI5, BI6, and BI7 show similar results and are not included. Image produced using Phamerator vActino_Draft 558 (4).

TABLE 1 Properties of Streptomyces bacteriophages GoblinVoyage and Doxi13

Phage characteristics	GoblinVoyage	Doxi13	
Isolation host	Streptomyces scabiei RL-34	Streptomyces scabiei RL-34	
Sample type	Dry, dusty soil	Dry, sandy soil	
Sample location [GPS]	Golansville, Virginia, USA
[37.980611 N, 77.491896 W]	Laurel, Maryland, USA
[39.15222 N, 76.96722 W]	
Approximate shotgun coverage	1,166 bp	10,298 bp	
Total number of reads	357,901	3,008,314	
Approximate fold coverage	1,233 x	10,327 x	
Genome length	43,540 bp	43,696 bp	
GC% content of phage	60.9%	60.9%	
Genome end type	3' sticky overhangs	3' sticky overhangs	
Character of genome ends	5′-CGCCGCCCT-3′	5′-CGCCGCCCT-3′	
Identified protein coding genes (assigned a function)	59 (29)	60 (21)	
Identified tRNA encoding genes	0	0	

Phage DNA was extracted from the freshly prepared crude lysate using the Promega Wizard DNA cleanup kit and sequenced at the Pittsburgh Bacteriophage Institute using the NEB Ultra II Library Kit and an Illumina MiSeq with v3 reagents with 150-bp single-end raw reads. The sequencing results are described in Table 1. Newbler v2.9 (5) and Consed v29 (6) were used for assembly, quality verification, and end determination as described (7).

Genome annotation was completed using DNA Master v5.23.6 (8) with internal Glimmer v3.02 (9) and GeneMarkS v2.5 (10, 11) programs. GeneMark.hmm v2.5p (10, 11) was used to confirm these annotations with S. scabiei 87–22 as the species. Putative functions were assigned by assessing amino acid sequence homology using NCBI BLASTp v2.15.0 (12) and Conserved Domain Database v3.19 (13), structural homology with HHpred v57c87 (minimum probability: 90%) (14, 15), and synteny via Phamerator vActino_Draft 558 (4). Default parameters were used for all software, unless otherwise specified. The phages were assigned to cluster BI2 in the Actinobacteriophage Database (PhagesDB) based on gene content as described (16, 17) and the genus Scapunavirus (18). Genomic characterization and annotation results are reported in Table 1.

Annotations of GoblinVoyage and Doxi13 failed to identify genes consistent with a temperate phage, agreeing with the earlier virulent designation and classification of the cluster in PhagesDB. Genomic comparison of phages from BI subclusters shows that BI2 phages have smaller genomes, differing by approximately 10 kbp, including multiple small genes in the center of the genome (Fig. 1C).

ACKNOWLEDGMENTS

This work was supported by the UMBC Department of Biological Sciences and the Howard Hughes Medical Institute SEA-PHAGES program. We thank Becca Elrod and Jessica Schafer for isolating Doxi13. We are grateful to Ralph Murphy for preparing the necessary equipment and materials and to Dr. Tagide deCarvalho at Keith R. Porter Imaging Facility for conducting the transmission electron microscopy of bacteriophages. We extend our gratitude to the Pittsburgh Bacteriophage Genome Center for sequencing and assembling the genomes of the GoblinVoyage and Doxi13. This research was supported by the National Institute of General Medical Sciences of the National Institutes of Health under grants TL4GM118989, UL1GM118988, and RL5GM118987. The content is solely the responsibility of the authors and does not necessarily represent the ofﬁcial views of the National Institutes of Health.

Members of the 2023–2024 UMBC Phage Hunters class are listed at https://phages.umbc.edu/home/class-lists/2023-24/. Members of STEM BUILD at UMBC Cohort 7 are listed at https://phages.umbc.edu/cohort-7-summer-2022/.

DATA AVAILABILITY

GoblinVoyage is available at GenBank with accession number PP725412 and at SRA with accession number SRX24123906. Doxi13 is available at GenBank with accession number ON970617 and at Sequence Read Archive (SRA) with accession number SRX20165790.
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