Open access
Bacteriophages
Announcement
19 September 2022

Annotation of the Complete Genome Sequences of Bacteriophages Sara and Birdfeeder

ABSTRACT

Sara is a siphovirus with a linear 17,362bp genome containing 25 genes. Birdfeeder is a podovirus with a circularly permuted 53,897bp genome containing 52 genes. Sara and Birdfeeder were isolated from environmental samples in Plattsburgh, NY, USA and Forest Hill, MD, USA, respectively, using Microbacterium foliorum NRRL B-24224.

ANNOUNCEMENT

Characterizing bacteriophages improves understanding of the most plentiful biological constructs on earth and their human health applications (1, 2). As Microbacterium have been associated with plant drought resistance and meat spoilage, phages infecting this genus are of interest for the agricultural and food industries (3). Here, we describe two bacteriophages, Sara and Birdfeeder, that infect soil bacterium M. foliorum.
Phage isolation followed standard procedures (4). Sara was isolated from soil collected near the Saranac River in Plattsburg, New York, (Global Positioning System [GPS] coordinates 44.69° N, 73.46° W), whereas Birdfeeder was isolated from soil collected underneath a bird feeder in Forest Hill, Maryland, (GPS coordinates 39.559167° N, 76.423611° W). Soil samples were washed with peptone/yeast/calcium (PYCa) liquid media and bacteriophages extracted through a 0.22-μm filter. Filtrate was mixed with soft agar containing M. foliorum NRRL B-24224, overlaid on PYCa agar, and incubated at 20°C for 48 h. Sara produced small, clear pinpoint plaques while Birdfeeder produced large, haloed plaques. Both phages were purified with at least three rounds of plating.
DNA was extracted from high-titer lysates using the Promega Wizard DNA cleanup kit and prepared for sequencing using the NEBNext UltraII FS kit. DNA was sequenced using Illumina MiSeq (v3 reagents), generating 375,319 and 131,281 150-bp unpaired reads for Sara and Birdfeeder, respectively. Raw reads were trimmed and assembled using Newbler v2.9 with default parameters, yielding a single contig for each phage genome; Consed v29 used used to check genomes for completeness and accuracy and to determine phage termini (5, 6). Genome characteristics are provided in Table 1. Based on gene content similarity of 35% of higher to phages in the Actinobacteriophage database, Sara and Birdfeeder are assigned to phage clusters EE and EK, respectively (7, 8). The GC content of the genomes are consistent with other members of their respective subclusters; additionally, the GC content for Sara is similar to that of the host bacteria, M. foliorum (68.7%) (3).
TABLE 1
TABLE 1 Genome characteristics for sara and birdfeeder genomes
PhageSubclusterAvg sequence coverage (x)Genome sizeGenome terminus arrangementGC contentNo. of protein coding genes
SaraEE308417,362bpLinear, with 3’ single-stranded overhangs (5′-CCCGCCCCA-3′)68.5%25
BirdfeederEK34953,897bpCircular60.0%52
Phage genomes were auto-annotated using DNAmaster v5.23.6 (http://cobamide2.bio.pitt.edu) embedded with GeneMark v2.5 (9) and GLIMMER v3.02 (10), with start sites then refined using Phage Evidence Collection and Annotation Network (PECAAN) (http://discover.kbrinsgd.org), Phamerator v454 (11) and Starterator v1.0.1 (https://seaphages.org/software). A total of 52 and 25 protein-coding genes were identified in Birdfeeder and Sara, respectively (Table 1). Using Aragorn v1.2.41 (12) and tRNA-SE v2.0 (13), no tRNAs were detected in either phage genome. Putative gene functions were determined using BLAST v2.11.0 (14) and HHPred v2.0 (15). All software used default parameters.
For Sara, structure and assembly genes span the first two-thirds of the genome. With the exception of three genes (SEA_SARA_20 to SEA_SARA_22) encoding DNA-binding proteins, all genes for Sara are transcribed rightwards. In contrast, the first third of the Birdfeeder genome encodes for several DNA metabolism genes are transcribed leftwards, followed by all rightwards-transcribed genes that include structure and assembly genes and a gene that is notably 13,479 bp-long. No immunity repressor or integrase functions could be identified for either phage. For Sara, this is consistent with its clear plaque morphology and with phages in cluster EE consisting of lytic siphoviruses. The lifecycle of phages in cluster EK is unknown.

Data availability.

GenBank Accession and Sequence Read Archive (SRA) numbers are ON260812 & SRX14485116, respectively (Sara) and ON456346 & SRX14989441, respectively (Birdfeeder).

ACKNOWLEDGMENTS

This study was kindly supported by the HHMI-sponsored SEA-PHAGES program, and the Idaho State University Department of Biological Sciences.
We thank Mallory Brownell, Temitayo Adegbite, and colleagues at State University of New York-Plattsburgh for the discovery and isolation of Sara, and C.J. Jednorski, E.N. Walters, J.R. Lebrasseur, and colleagues at Harford Community College for the discovery and isolation of Birdfeeder.

REFERENCES

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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.
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Russell DA. 2018. Sequencing, assembling, and finishing complete bacteriophage genomes. Methods Mol Biol 1681:109–125.
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Delcher AL, Bratke KA, Powers EC, Salzberg SL. 2007. Identifying bacterial genes and endosymbiont DNA with Glimmer. Bioinformatics 23:673–679.
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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.
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Laslett D, Canback B. 2004. ARAGORN, a program to detect tRNA genes and tmRNA genes in nucleotide sequences. Nucleic Acids Res 32:11–16.
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Information & Contributors

Information

Published In

cover image Microbiology Resource Announcements
Microbiology Resource Announcements
Volume 11Number 1020 October 2022
eLocator: e00780-22
Editor: John J. Dennehy, Queens College CUNY
PubMed: 36121218

History

Received: 2 August 2022
Accepted: 7 September 2022
Published online: 19 September 2022

Contributors

Authors

Benjamin M. Adams
Department of Biological Sciences, Idaho State University, Pocatello, Idaho, USA
Joseph B. Adams
Department of Biological Sciences, Idaho State University, Pocatello, Idaho, USA
Reganne L. Brewster
Department of Biological Sciences, Idaho State University, Pocatello, Idaho, USA
Michael S. Cutler
Department of Biological Sciences, Idaho State University, Pocatello, Idaho, USA
Anthony E. Davis
Department of Biological Sciences, Idaho State University, Pocatello, Idaho, USA
Abby H. Gallegos
Department of Biological Sciences, Idaho State University, Pocatello, Idaho, USA
Jeremy S. Hernandez
Department of Biological Sciences, Idaho State University, Pocatello, Idaho, USA
Luke H. May
Department of Biological Sciences, Idaho State University, Pocatello, Idaho, USA
Emma G. Montoya
Department of Biological Sciences, Idaho State University, Pocatello, Idaho, USA
Andrew T. Reagan
Department of Biological Sciences, Idaho State University, Pocatello, Idaho, USA
Jack F. Shurley
Department of Biological Sciences, Idaho State University, Pocatello, Idaho, USA
Anna S. Grinath
Department of Biological Sciences, Idaho State University, Pocatello, Idaho, USA
Department of Biological Sciences, Idaho State University, Pocatello, Idaho, USA

Editor

John J. Dennehy
Editor
Queens College CUNY

Notes

The authors declare no conflict of interest.

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