Genome Sequences of Nine Erwinia amylovora Bacteriophages

At an estimated total number of 10³¹, phages are by far the most abundant biological entity on the planet (1–7). They dramatically influence the evolution of bacteria by their ability to infect and kill their hosts and to transfer genetic material. Erwinia amylovora is a rod-shaped facultative anaerobic member of the Enterobacteriaceae bacterial family, which includes many well-characterized Gram-negative plant and animal pathogens, such as Salmonella spp., Escherichia coli, and Klebsiella spp. As the causative agent of fire blight, Erwinia amylovora infects members of the Rosaceae plant family, causing diseased areas to appear burnt (8–10). The isolation and characterization of phages that infect E. amylovora may aid in our understanding of these bacteria and provide potential treatment for this devastating agricultural disease. Herein, we announce the genome sequences of nine E. amylovora bacteriophages, vB_EamM_Asesino, vB_EamM_Alexandra, vB_EamM_Bosolaphorus, vB_EamM_Desertfox, vB_EamM_MadMel, vB_EamM_Mortimer, vB_EamP_Pavtok, vB_EamM_SunLIRen, and vB_EamM_Wellington.

Phages were isolated from apple trees along the Wasatch Front in Utah that appeared to harbor fire blight infection. Phages were plaque purified through a minimum of three passages after amplification via enrichment culture (11). All nine phages reported in this announcement infect the Erwinia amylovora ATCC 29780 strain, as indicated by plaque assays, and their characteristics are summarized in Table 1. Genomic DNA was extracted (Phage DNA isolation kit; Norgen Biotek), a library was made using the Illumina TruSeq DNA Nano kit, and sample genomes were sequenced by Illumina HiSeq 2500 sequencing (250-bp paired end) and assembled with Geneious (12) version 8.1 using de novo assembly with medium-low sensitivity and various percentages of data. All phages circularized upon assembly and were annotated using DNA Master (http://cobamide2.bio.pitt.edu/computer.htm), giving preference for calls that gave full coding potential coverage.

The nine phages were grouped into five distinct clusters by genomic dot plot and average nucleotide identity analyses, as previously described (11), with the first three groups containing jumbo Myoviridae. The first jumbo group included four myoviruses, vB_EamM_Bosolaphorus, vB_EamM_Desertfox, vB_EamM_MadMel, and vB_EamM_Mortimer, which are similar to previously published Erwinia phage Ea35-70 (13), as well as other phages we have isolated (14). The second group included two jumbo myoviruses, vB_EamM_Asesino and vB_EamM_Wellington, with similarity to the well-characterized Salmonella SPN3US phage (15) and related phages. The third is a single jumbo myovirus, EamM_Alexandra, which has similarity to previously published Erwinia phages EamM_Yoloswag (14) and EamM_Y3 (16). Podovirus vB_EamP_Pavtok and myovirus vB_EamM_SunLIRen are similar to Erwinia phages PEp14 and phiEa21-4 (17), respectively. The three jumbo myovirus groups package DNA by headful packaging (14) based on homology to phage phiKZ terminase (18), and their bp 1 was chosen by alignment to their phage family. PhageTerm (19) was used to determine the packaging strategy of SunLIRen and Pavtok. SunLIRen appeared to have headful packaging, and its bp 1 was assigned based on homology alignment to Erwinia phage phiEa21-4, while the packaging strategy of Pavtok is unknown, and its bp 1 was assigned due to homology to PEp14.

We thank the Howard Hughes Medical Institute Science Education Alliance–Phage Hunters Advancing Genomics and Evolutionary Science (SEA-PHAGES) for phage analysis training. In addition, we thank Ed Wilcox (BYU DNA Sequencing Center) and Michael Standing (BYU Microscopy Lab).

This work was graciously funded by a USDA grant (to D.A.B., University of Arizona) and the Department of Microbiology and Molecular Biology and the College of Life Sciences at Brigham Young University, as well as a private donor.