Characterization and Genome Structure of Virulent Phage EspM4VN to Control Enterobacter sp. M4 Isolated From Plant Soft Rot

Abstract

Enterobacter sp. M4 and other bacterial strains were isolated from plant soft rot disease. Virulent phages such as EspM4VN isolated from soil are trending biological controls for plant disease. This phage has an icosahedral head (100 nm in diameter), a neck, and a contractile sheath (100 nm long and 18 nm wide). It belongs to the Ackermannviridae family and resembles Shigella phage Ag3 and Dickeya phages JA15 and XF4. We report herein that EspM4VN was stable from 10°C to 50°C and pH 4 to 10 but deactivated at 70°C and pH 3 and 12. This phage formed clear plaques only on Enterobacter sp. M4 among tested bacterial strains. A one-step growth curve showed that the latent phase was 20 min, rise period was 10 min, and an average of 122 phage particles were released from each absorbed cell. We found the phage's genome size was 160,766 bp and that it annotated 219 open reading frames. The genome organization of EspM4VN has high similarity with the Salmonella phage SKML-39; Dickeya phages Coodle, PP35, JA15, and Limestone; and Shigella phage Ag3. The phage EspM4VN has five tRNA species, four tail-spike proteins, and a thymidylate synthase. Phylogenetic analysis based on structural proteins and enzymes indicated that EspM4VN was identified as a member of the genus Agtrevirus, subfamily Aglimvirinae, family Ackermannviridae.

Keywords: Agtrevirus; Enterobacter; genome; soft rot disease; virulent phage.

FIGURE 1

Plaques formed in the top agar layer (A), morphology of the extended tail phage EspM4VN (B), contracted tail phage EspM4VN (C), and displaying an umbrella-like structure (D). Bars indicate 3 mm (A), 100 nm (B,C), and 50 nm (D).

FIGURE 2

Thermal and pH stability of EspM4VN. Course of survival ratio (%) of EspM4VN particles during heat (A) and pH (B) treatments is plotted. The mean titer is shown from triplicate assays.

FIGURE 3

One-step growth curve of the EspM4VN.

FIGURE 4

Chemical sensitivity of EspM4VN. Phage particles were inoculated in LB containing different concentrations of Tween 20, ethanol, SDS, skim milk, and sucrose from 0.1 to 1.5?%. SM buffer (outlined bars) was used as a control. Quantification was achieved by plaque assay. The plates were incubated at 37°C for 12 h, and the phage growth was assayed by plaque-forming units (pfu). Experiments were performed in triplicate on three different occasions, and means ± SD are shown.

FIGURE 5

Genome map of EspM4VN. The outer lane represents genes in the plus strand. The next lane illustrates genes on the minus strand. The lane with black peaks and valleys indicate the GC content, while the innermost lane (green and violet) shows a GC skew analysis.

FIGURE 6

Phylogenetic trees generated based on (A) DNA polymerases, (B) DNA ligase, (C) major capsid protein, and (D) baseplate hub subunit of EspM4VN and homologous proteins from other phage members of the Ackermannviridae family. Nucleic acid sequences were compared using ClustalW, and phylogenetic trees were generated using the neighbor-joining method. Numbers in brackets show the gene ID.

FIGURE 7

Bacteriophage EspM4VN structural proteins separated in SDS-PAGE gel (A) and identification of structural proteins with ESI-MS/MS (B). (A) For SDS-PAGE, EspM4VN particles (∼10¹² pfu) were mixed with lysis buffer and then boiled for 10 min. Phage proteins were separated in a 15% acrylamide SDS-PAGE gel for approximately 19 h at 50 V at 22°C. The bands were stained with Coomassie Brilliant Blue according to the protocol provided by the manufacturer. For ESI-MS/MS analysis of phage structural proteins, protein bands obtained from SDS-PAGE were excised from the gel with a sterile scalpel.

FIGURE 8

Comparison of gene organization between tail-protein encoding genes in EspM4VN and its homologous genes. (A) Gene clusters of phage tail proteins in EspM4VN and Ackermannviridae family phages. Same-colored arrows indicate homologous genes. Blanked arrows show no homology with gene 102 in EspM4VN. Numbers indicate positions of ORFs in the genome. (B) Homologous amino acid sequence distribution of gene 101 product (gray bar). White and black bars show partial regions of phage gene products and bacterial chromosomal gene products, respectively. Numbers indicate amino acid positions in each protein. (C) Gene organization adjacent to gene 101 homologs in bacterial chromosomes. Black arrows indicate gene 101 homologous genes. Predicted gene products are shown below each arrow.