ABSTRACT
Erwinia amylovora is the causal agent of fire blight, a devastating disease affecting some plants of the Rosaceae family. We isolated bacteriophages from samples collected from infected apple and pear trees along the Wasatch Front in Utah. We announce 19 high-quality complete genome sequences of E. amylovora bacteriophages.
GENOME ANNOUNCEMENT
Erwinia amylovora is a Gram-negative facultative anaerobic rod-shaped bacterium and the causative agent of fire blight (1), a disease that affects some members of the plant family Rosaceae and causes the infected areas of the plant to appear burnt (2, 3). E. amylovora is a member of the Enterobacteriaceae family, which includes many well-characterized pathogenic bacteria such as Salmonella enterica and Escherichia coli. Thus, understanding the evolution of this plant pathogen and the bacteriophages that infect it may provide insight into the evolution of the Enterobacteriaceae family, including other pathogenic strains.
Herein, we announce the genome sequences of 19 novel E. amylovora bacteriophages, vB_EamP_Frozen, vB_EamP_Gutmeister, vB_EamP_Rexella, vB_EamM_Deimos-Minion, vB_EamM_RAY, vB_EamM_Simmy50, vB_EamM_Special G, vB_EamM_Caitlin, vB_EamM_ChrisDB, vB_EamM_EarlPhillipIV, vB_EamM_Huxley, vB_EamM_Kwan, vB_EamM_Machina, vB_EamM_Parshik, vB_EamM_Phobos, vB_EamM_Stratton, vB_EamM_Joad, vB_EamM_RisingSun, and vB_EamM_Yoloswag. Samples were collected from apple and pear trees bearing symptoms of fire blight infection that were found along the Wasatch Front of Utah. Phages were amplified via enrichment culture of these samples, and resulting phages were then plaque purified by a minimum of three passages. All phages reported in this announcement infect the Erwinia amylovora ATCC 29780 strain.
Genomic DNA was extracted using the Phage DNA isolation kit (Norgen Biotek Corporation) and sequenced using 454 pyrosequencing (454 Life Sciences, Roche Diagnostics) or Illumina HiSeq 2500 sequencing (Illumina, 250-bp reads). Contigs were assembled using Newbler version 2.9 (Roche Diagnostics, Branford, CT) and Consed (4) for 454 pyrosequencing reads or Geneious version R8 (5) for Illumina reads. Assembled genomes were annotated using DNA Master (6) and other programs as described previously (7, 8).
The 19 phages fell into five distinct clusters according to genomic analysis. The first group included the jumbo myoviruses vB_EamM_Deimos-Minion, vB_EamM_RAY, vB_EamM_Simmy50, and vB_EamM_Special G, which share a minimum of 97.2% average nucleotide identity to one another. The second group included two jumbo myoviruses, vB_EamM_RisingSun and vB_EamM_Joad, which differ by only two putative gene products. The third group included diverse jumbo myoviruses vB_EamM_Caitlin, vB_EamM_ChrisDB, vB_EamM_EarlPhillipIV, vB_EamM_Huxley, vB_EamM_Kwan, vB_EamM_Machina, vB_EamM_Parshik, vB_EamM_Phobos, and vB_EamM_Stratton, which share a minimum of 50.5% average nucleotide identity. An additional jumbo myovirus, vB_EamM_Yoloswag, did not have any close phage relatives. Podovirus phages vB_EamP_Frozen, vB_EamP_Gutmeister, and vB_EamP_Rexella share at least 97.2% average nucleotide identity. The four jumbo myovirus groups package DNA by headful packaging based on homology of their putative terminase genes to the phiKZ terminase (9). Three of these genomically permuted myovirus groups were assigned their base pair (bp) 1 by alignment to previously published genomes by use of BLASTN (10) and Gepard (11) (Ea35-70 for the Deimos-Minion group [12], EL [13, 14] for the RisingSun group, and SPN3US [15] for the Caitlin group). vB_EamM_Yoloswag shared very little DNA homology with any other phage; therefore, its bp 1 was assigned to position its putative terminase at the beginning of the genome. The podovirus group genomes were assigned bp 1 by their relation to N4, in terms of both terminase similarity and whole-genome alignment, suggesting they have small terminal repeats.
Accession number(s).
GenBank accession numbers for the 19 Erwinia bacteriophages are listed in Table 1.
TABLE 1 .
Properties of 19 novel Erwinia amylovora bacteriophage genomes
| Phage name | GenBank accession no. |
Sequencing type |
Minimum–maximum fold coverage (avg read depth) |
Genome length (bp) |
No. of ORFsa |
No. of tRNAsb |
G+C content (%) |
|---|---|---|---|---|---|---|---|
| vB_EamP_Gutmeister | KX098391 | Illumina | 423–2,415 (662) | 71,173 | 84 | 8 | 46.9 |
| vB_EamP_Frozen | KX098389 | 454 | 79–1,779 (862) | 75,147 | 92 | 8 | 46.9 |
| vB_EamP_Rexella | KX098390 | 454 | 69–1,780 (885) | 75,448 | 92 | 7 | 46.9 |
| vB_EamM_Deimos-Minion | KU886225 | 454 | 61–1,780 (873) | 273,501 | 326 | NA | 49.9 |
| vB_EamM_RAY | KU886224 | Illumina | 335–910 (677) | 271,182 | 319 | 1 | 49.9 |
| vB_EamM_Special G | KU886222 | 454 | 19–1,779 (874) | 273,224 | 324 | NA | 49.8 |
| vB_EamM_Simmy50 | KU886223 | Illumina | 150–831 (282) | 271,088 | 322 | 1 | 49.9 |
| vB_EamM_Caitlin | KX397365 | Illumina | 84–249 (174) | 241,147 | 271 | 7 | 52.2 |
| vB_EamM_ChrisDB | KX397366 | 454 | 66–1,780 (874) | 244,840 | 277 | 11 | 49.4 |
| vB_EamM_EarlPhillipIV | KX397367 | Illumina | 75–243 (164) | 223,935 | 241 | NA | 50.6 |
| vB_EamM_Huxley | KX397368 | 454 | 75–1,779 (880) | 240,761 | 271 | 9 | 51.1 |
| vB_EamM_Kwan | KX397369 | Illumina | 192–554 (362) | 246,390 | 285 | 8 | 52.1 |
| vB_EamM_Machina | KX397370 | 454 | 65–1,780 (879) | 241,654 | 272 | 9 | 51.0 |
| vB_EamM_Parshik | KX397371 | 454 | 64–1,779 (880) | 241,050 | 271 | 10 | 51.0 |
| vB_EamM_Phobos | KX397372 | 454 | 59–1,779 (873) | 229,501 | 247 | NA | 49.1 |
| vB_EamM_Stratton | KX397373 | 454 | 64–1,779 (874) | 243,953 | 276 | 12 | 51.3 |
| vB_EamM_Yoloswag | KY448244 | Illumina | 5–265 (99.5) | 259,700 | 334 | NA | 46.91 |
| vB_EamM_RisingSun | MF459646 | Illumina | 50–293 (138.6) | 235,108 | 243 | NA | 48.32 |
| vB_EamM_Joad | MF459647 | Illumina | 232–1,065 (522.2) | 235,374 | 245 | NA | 48.29 |
ORFs, open reading frames.
NA, no tRNAS were identified.
ACKNOWLEDGMENTS
We extend a special thanks to the Howard Hughes Medical Institute Science Education Alliance–Phage Hunters Advancing Genomics and Evolutionary Science (SEA-PHAGES) for support and training on phage analysis. We appreciate the help of Ed Wilcox (BYU DNA Sequencing Center), Michael Standing (BYU Microscopy Lab), Alisa L. Buchanan, Brett Buhler, and Brianna R. Keele.
This work was graciously funded by the Department of Microbiology and Molecular Biology and the College of Life Sciences at Brigham Young University, as well as by a private donor.
J.H.G. is in the process of submitting a patent for using Erwinia phages for the treatment of fire blight. J.H.G., S.H., and D.P.B. have a license agreement with a company for distribution of Erwinia phages.
Footnotes
Citation Esplin IND, Berg JA, Sharma R, Allen RC, Arens DK, Ashcroft CR, Bairett SR, Beatty NJ, Bickmore M, Bloomfield TJ, Brady TS, Bybee RN, Carter JL, Choi MC, Duncan S, Fajardo CP, Foy BB, Fuhriman DA, Gibby PD, Grossarth SE, Harbaugh K, Harris N, Hilton JA, Hurst E, Hyde JR, Ingersoll K, Jacobson CM, James BD, Jarvis TM, Jaen-Anieves D, Jensen GL, Knabe BK, Kruger JL, Merrill BD, Pape JA, Payne Anderson AM, Payne DE, Peck MD, Pollock SV, Putnam MJ, Ransom EK, Ririe DB, Robinson DM, Rogers SL, Russell KA, Schoenhals JE, Shurtleff CA, Simister AR, Smith HG, Stephenson MB, Staley LA, Stettler JM, Stratton ML, Tateoka OB, Tatlow PJ, Taylor AS, Thompson SE, Townsend MH, Thurgood TL, Usher BK, Whitley KV, Ward AT, Ward MEH, Webb CJ, Wienclaw TM, Williamson TL, Wells MJ, Wright CK, Breakwell DP, Hope S, Grose JH. 2017. Genome sequences of 19 novel Erwinia amylovora bacteriophages. Genome Announc 5:e00931-17. https://doi.org/10.1128/genomeA.00931-17.
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