Isolation, Characterization, and Genome Analysis of a Novel Bacteriophage, Escherichia Phage vB_EcoM-4HA13, Representing a New Phage Genus in the Novel Phage Family Chaseviridae

Abstract

Shiga toxin-producing Escherichia coli (STEC) is one of the leading causes of foodborne illnesses in North America and can lead to severe symptoms, with increased fatality risk for young children. While E. coli O157:H7 remains the dominant STEC serotype associated with foodborne outbreaks, there has been an increasing number of non-O157 STEC outbreaks in recent years. For the food industry, lytic bacteriophages offer an organic, self-limiting alternative to pathogen reduction-one that could replace or reduce the use of chemical and physical food processing methods. From EHEC-enriched sewage, we isolated a novel bacteriophage, vB_EcoM-4HA13 (4HA13). Phenotypic characterizations revealed 4HA13 to possess a myoviral morphotype, with a high specificity to non-motile O111 serotype, and a long latent period (90 min). Through genomic analyses, this 52,401-bp dsDNA phage was found to contain 81 CDS, but no detectable presence of antibiotic resistance, integrase, or virulence genes. A BLASTn search for each of the identified 81 CDS yielded homologues with low levels of similarity. Comparison of RNA polymerase and terminase large subunit amino acid sequences led to the proposal and acceptance of a new bacteriophage family, Chaseviridae, with 4HA13 representing a new species and genus. The discovery of this phage has broadened our current knowledge of bacteriophage diversity.

Keywords: Chaseviridae; Escherichia coli; STEC O111-specific bacteriophage; bacteriophage; genomics; proteomics.

Figure 1

Transmission electron micrograph of 4HA13. Phage particles show the icosahedral head and uncontracted tail. Also visible are the striations from the tail tube proteins and some tail fibers. The image was altered to include the scale bar in the same orientation as the phage particles. Scale bar = 20 nm.

Figure 2

Determination of 4HA13 virulence against E. coli O111:NM in TSB, 25 °C. (a) Growth of E. coli O111:NM treated with 4HA13 at phage-to-host ratios of 10⁻⁵ to 10² over 16 h. The curves were normalized against an assay blank average; (b) local virulence (Vi) values, as determined using the calculations set out by Storms et al., were plotted against the input ratios [22]. A curve of best fit and its polynomial equation allowed for the determination of 4HA13’s virulence index (y = Vi = 0.5) and are indicated by the red dash line.

Figure 3

Phage kinetics of 4HA13. (a) Adsorption of 4HA13 to E. coli O111:NM over 16 min; (b) one-step growth curve of 4HA13 to E. coli O111:NM. The latent period and burst are marked by orange dash lines.

Figure 4

Genome map of 4HA13 and its five closest nucleotide homologues. CDS with annotated functions are highlighted, with structural proteins in black and orientation denoted by arrow-ends. The direct terminal repeat region (3120 bp) is highlighted in grey. The intensity of color of the homologues corresponds to level of nucleotide similarity.

Figure 5

VIRIDIC heatmap of 4HA13 and its 50 closest nBlast homologues. The upper right half contains the intergenomic similarities between phage pairings, with intensity of color corresponding to level of similarity. The lower left half lists the percentage coverage of phage one, percentage alignment, and the percentage coverage of phage two.

Figure 6

Phylogenetic tree of 4HA13 and its VIRIDIC clade members. The tree was constructed using PATRIC’s PGFams using 10 genes and an allocation of 3 genome exclusions.

Figure 7

Ion chromatograms of the trypsin-treated 4HA13. Proteins were identified using LC MS/MS at a duration of 90 min. (a) Extracted ion chromatograms of the high abundance peptides of the top twelve proteins followed by the order of major capsid protein (gi|1735348871, red), tail tape measure protein (gi|1735348880, pink), tail sheath protein (gi|1735348876, orange), hypothetical protein AC4HA13_0057 (gi|1735348866, dark yellow), hypothetical protein AC4HA13_0041 (gi|1735348850, purple), tail tube initiator protein (gi|1735348882, blue), hypothetical protein AC4HA13_0070 (gi|1735348883, dark cyan), tail completion protein (gi|1735348875, oliver), baseplate wedge protein (gi|1735348886, green), tail tube protein (gi|1735348877, cyan), hypothetical protein AC4HA13_0076 (gi|1735348889, gray), and hypothetical protein AC4HA13_0049 (gi|1735348858, light gray). (b) Total ion chromatogram of a tryptic digest of 4HA13.

Figure 8

Identification of acetylated-lysine-containing peptides of 4HA13. (a) MS/MS spectrum of peptide 262–269 at m/z 449.7904 of a major capsid protein; (b) MS/MS spectrum of peptide 37–56 at m/z 1128.0223 of a tail completion protein. Symbols of b and y ions denote the fragments extended from the N-terminus and C-terminus of the peptides, respectively.