ABSTRACT
We report the genome sequences of eight bacteriophages isolated using Gordonia rubripertincta NRRL B-16540-SEA. Based on gene content similarity to phages in the Actinobacteriophage database, six of the phages are assigned to phage cluster CT while two are assigned to cluster DJ.
KEYWORDS: actinobacteriophages
ANNOUNCEMENT
The Science Education Alliance Phage Hunters Advancing Genomics and Evolutionary Science (SEA-PHAGES) project studies the genetic diversity and evolution of actinobacteriophages (1). Here, we report the identification and characterization of eight phages isolated using Gordonia rubripertincta NRRL B-16540-SEA, using standard methods (2). Briefly, soil samples collected from various sites in Omaha and Lincoln, NE, and Conway, SC (Table 1) were each washed with peptone-yeast extract-calcium medium, and the wash was filtered through a 0.22 µm filter. Filtrates were plated as top agar overlays with G. rubripertincta either immediately (direct isolation) or after inoculation with G. rubripertincta and incubated with shaking at 30°C for 2 days (enriched isolation). The direct isolation samples yielded phages BigChungus, Chikenjars, Dre3, Feastonyeet, and Gibbous, while enriched samples yielded phages Mayweather, Nithya, and Vine. All phages were plaque-purified through multiple rounds of plating. Negative-stain transmission electron microscopy revealed a siphovirus morphology for the phages (Mayweather was not imaged).
TABLE 1.
Characteristics of genomes of eight phages isolated using host G. rubripertincta NRRL B-16540 SEA
| Phage (cluster; year isolated) | GPS coordinates of soil collection sites | No. of sequencing reads | Approximate shotgun coverage (X) | Genome length (bp) | GC content (%) | 3′ Overhang | No. of CDS a | CDS assigned function |
|---|---|---|---|---|---|---|---|---|
| BigChungus (CT; 2019) | 41.2097 N, 96.2161 W | 575,645 | 1724 | 47,166 | 60.7 | CGGCGGTAGGCTT | 69 | 33 |
| Dre3 (CT; 2018) | 40.8228 N, 96.6958 W | 111,427 | 383 | 45,810 | 60.5 | CGGTAGGCAT | 68 | 33 |
| Feastonyeet (CT; 2019) | 41.2097 N, 96.2161 W | 439,254 | 1310 | 47,166 | 60.7 | CGGCGGTAGGCTT | 69 | 33 |
| Gibbous (CT; 2018) | 40.8228 N, 96.6958 W | 253,287 | 782 | 45,810 | 60.5 | CGGTAGGCAT | 68 | 33 |
| Mayweather (CT; 2018) | 33.903299 N, 79.062125 W | 320,874 | 937 | 48,382 | 60.6 | CGGCGGTAGGCTT | 74 | 35 |
| Vine (CT; 2020) | 40.8203 N, 96.6967 W | 67,933 | 202 | 48,092 | 60.4 | CGGTAGGCTT | 74 | 35 |
| Chikenjars (DJ; 2018) | 40.828025 N, 96.693583 W | 320,431 | 735 | 61,544 | 51.3 | CGCCGCTCT | 92 | 20 |
| Nithya (DJ; 2020) | 40.8195 N, 96.6845 W | 159,586 | 374 | 61,092 | 51.3 | CGCCGCTCT | 91 | 27 |
CDS, coding DNA sequences.
Genomic DNA was extracted from lysates with the Wizard DNA Clean-Up System (Promega), prepared for sequencing using the NEB Ultra II Library kit, and sequenced using an Illumina MiSeq (v3 reagents) to yield 150 bp single-end reads with indicated coverage (Table 1). Reads were assembled and checked for completeness using Newbler v2.9 and Consed v29.0, respectively (3 – 5). Table 1 provides genome lengths, GC%, and genome termini, determined by comparison to similar phages with known ends and confirmed by read start buildups (5). Based on gene content similarity of at least 35% to phages in the Actinobacteriophage database, phages were assigned to either phage cluster CT or DJ (6). Cluster CT phages BigChungus and Feastonyeet, which were isolated from different soil samples collected in the same location, differ only by 2 bp. Dre3 and Gibbous, similarly isolated, also differ by 2 bp.
Annotation of phage genomes was accomplished with DNA Master (7) (Version 5.23.3 or later), NCBI BLASTp (8), HHPred (9), GeneMark v2.5p (10), Glimmer v3.02 (11), Phamerator (12), SOSUI v1.11 (13), TMHMM v2.0 (14), and databases PhagesdDB (15) and Protein Data Bank (16). All software programs were run using default parameters.
For all six cluster CT phages, predicted L-Ala-D-Glu peptidase and glycosyl hydrolase domains of lysin A, often a single multi-domain protein in actinobacteriophages, are encoded by separate but adjacent genes. These are located on the left arm of the genomes and transcribed rightward. A predicted lysin B is encoded among DNA metabolism genes on the right arm of the genome where most genes are transcribed leftward. The predicted tail assembly chaperone proteins were annotated with a (–1) translational frameshift conserved in mycobacteriophage (17, 18). For the Cluster DJ phages, functional assignments were made for 20 of 92 genes for Chikenjars and 27 of 91 genes for Nithya. These include two major tail proteins, and a major capsid and maturation protease fusion protein. None of the eight phages encode identifiable tRNA genes.
ACKNOWLEDGMENTS
Student Contributors from the SEA-PHAGES courses at the University of Nebraska-Lincoln (School of Biological Sciences; 2018–2020) were MacKenzie J. Asche, Lindsay R. Birge, Layton D. Bivans, Seth M. Blakestad, Renee E. Box, Emma K. Chesley, Jane E. Frank, Kiyara R. Grosz, Austin D. Herold, Grace E. Hilfiker, Sarah Z. Hoagland, Justin Hultquist, Noah C. Lawrence, Nicholas R. Lee, Jace K. Miller, Andrew J. Mize, Phoebe B. Peña, Ethan P. Ramsey, Grant T. Schneider, and Chanasei M. Ziemann. Student Contributors from the SEA-PHAGES courses at the Department of Biology of Coastal Carolina University (2018) were Takiah C. Anderson, Victoria G. Ballou, Viktoria K. Berkey, Krystal R. Bonaccorso, Laura B. Busby, Devon A. Carey, Tyler Cutaia, James Dalenburg, Elmer N. Diaz-Ramirez, Kenard J. Holmes, Shebbena T. McGrew, Da'Wayne P. Meares, Robert E. Mordente, Paige A. Pietrzak, Mya Roblee, Olivia A. Shirley, Zahra Slimani, Alysia M. Smith, Shionnah D. Wallace, and Ymani S. Wright.
We thank members of the Howard Hughes Medical Institute SEA-PHAGES Program for wet lab and bioinformatics training, reviewers of annotation files (Susan Gurney, University of St. Andrews; Karen Klyczek, University of Wisconsin-River Falls; Sara Tolsma, Northwestern College), members of the University of Pittsburgh SEA-PHAGES team for guidance (Graham F. Hatfull, Deborah Jacobs-Sera, Welkin H. Pope) and genome sequencing, assembly, and deposition of raw reads into the Sequence Read Archive (Rebecca A. Garlena and Daniel A. Russell). This announcement was prepared in part to fulfill the Contract for Honors Credit undertaken by X.F.R. for BIOS137 Discovery Research: Virus Genome Analyses, an undergraduate course at UNL.
Contributor Information
Catherine P. Chia, Email: cchia1@unl.edu.
John J. Dennehy, Department of Biology, Queens College, Queens, New York, USA
DATA AVAILABILITY
Under BioProject PRJNA488469, the following are the sequence reads in the sequence read archive (SRA) and Accession numbers: BigChungus (SRX11422988; GenBank MT776810), Chikenjars (SRX11422995; GenBank MN204501), Dre3 (SRX11422996; GenBank MW507135), Feastonyeet (SRX11422997; GenBank MT776808), Gibbous (SRX11422998; GenBank MN310549), Mayweather (SRX11555142; GenBank MN062716) Nithya (SRX11422991; GenBank MZ388556) and Vine (SRX11422992; GenBank MZ622167).
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Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Data Availability Statement
Under BioProject PRJNA488469, the following are the sequence reads in the sequence read archive (SRA) and Accession numbers: BigChungus (SRX11422988; GenBank MT776810), Chikenjars (SRX11422995; GenBank MN204501), Dre3 (SRX11422996; GenBank MW507135), Feastonyeet (SRX11422997; GenBank MT776808), Gibbous (SRX11422998; GenBank MN310549), Mayweather (SRX11555142; GenBank MN062716) Nithya (SRX11422991; GenBank MZ388556) and Vine (SRX11422992; GenBank MZ622167).