A Novel, Highly Related Jumbo Family of Bacteriophages That Were Isolated Against Erwinia

Abstract

Erwinia amylovora is a plant pathogen from the Erwiniaceae family and a causative agent of the devastating agricultural disease fire blight. Here we characterize eight lytic bacteriophages of E. amylovora that we isolated from the Wasatch front (Utah, United States) that are highly similar to vB_EamM_Ea35-70 which was isolated in Ontario, Canada. With the genome size ranging from 271 to 275 kb, this is a novel jumbo family of bacteriophages. These jumbo bacteriophages were further characterized through genomic and proteomic comparison, mass spectrometry, host range and burst size. Their proteomes are highly unstudied, with over 200 putative proteins with no known homologs. The production of 27 of these putative proteins was confirmed by mass spectrometry analysis. These bacteriophages appear to be most similar to bacteriophages that infect Pseudomonas and Ralstonia rather than Enterobacteriales bacteria by protein similarity, however, we were only able to detect infection of Erwinia and the closely related strains of Pantoea.

Keywords: Agrican357virus; Erwinia; Pantoea; burst size; genome; jumbo bacteriophage; novel; proteome.

FIGURE 1

Electron microscopy STEM images of Deimos-Minion, Special G, RAY, Simmy50, Bosolaphorus, Desertfox, MadMel, and Mortimer revealed Myoviruses with long contractile tails.

FIGURE 2

Growth Curve of Deimos-Minion with host E. amylovora ATCC 29780 by plaque assays shows first burst at ∼4 h and second burst at ∼6 h.

FIGURE 3

Whole-genome nucleotide (A) and protein terminase (B) or major capsid protein (C) dot plot analysis reveals a fairly isolated cluster of bacteriophages that includes Deimos-Minion, Special G, RAY, Simmy50, Bosolaphorus, Desertfox, MadMel, Mortimer and Ea35-70. Dot plots were constructed using Gepard.

FIGURE 4

Phylogenetic analyses of phage terminase proteins supports the relationships depicted by dot plot analysis of the Agrican357virus bacteriophages. The evolutionary history was inferred using the Maximum Likelihood method and Poisson correction model in MEGA X. Branches corresponding to partitions reproduced in less than 50% bootstrap replicates are collapsed where 100 was set to be initial bootstrapping value.

FIGURE 5

Whole genome Phamerator map of E. amylovora bacteriophages illustrates the high similarity of bacteriophages Mortimer, MadMel, Desertfox, Bosolaphorus, Deimos-Minion, Special G, RAY, Simmy50, and Ea35-70. Bacteriophages were mapped using Phamerator and arranged based on highest protein similarity. Violet shading between genomes indicates genome nucleotide homology (with standard e-value cutoff of 1.00E-04) and the ruler indicates genome base pairs, while white spaces indicate areas without significant nucleotide similarity. Boxes above and below the genome ruler indicate ORFs going in the forward and reverse direction, respectively. They are labeled with predicted function, occasionally numbered, and colored to indicate protein homologs between the bacteriophages.

FIGURE 6

Protein-conservation analysis displayed by Splitstree of the Agrican357virus genus with related jumbo Myoviridae bacteriophages reveals Agrican357virus as a distant evolutionary group.

FIGURE 7

Halo formation on P. vagans by RAY (A) and bio-film degradation activity of gp76 on P. vagans (B).