Larger Than Life: Isolation and Genomic Characterization of a Jumbo Phage That Infects the Bacterial Plant Pathogen, Agrobacterium tumefaciens

Abstract

Agrobacterium tumefaciens is a plant pathogen that causes crown gall disease, leading to the damage of agriculturally-important crops. As part of an effort to discover new phages that can potentially be used as biocontrol agents to prevent crown gall disease, we isolated and characterized phage Atu_ph07 from Sawyer Creek in Springfield, MO, using the virulent Agrobacterium tumefaciens strain C58 as a host. After surveying its host range, we found that Atu_ph07 exclusively infects Agrobacterium tumefaciens. Time-lapse microscopy of A. tumefaciens cells subjected to infection at a multiplicity of infection (MOI) of 10 with Atu_ph07 reveals that lysis occurs within 3 h. Transmission electron microscopy (TEM) of virions shows that Atu_ph07 has a typical Myoviridae morphology with an icosahedral head, long tail, and tail fibers. The sequenced genome of Atu_ph07 is 490 kbp, defining it as a jumbo phage. The Atu_ph07 genome contains 714 open reading frames (ORFs), including 390 ORFs with no discernable homologs in other lineages (ORFans), 214 predicted conserved hypothetical proteins with no assigned function, and 110 predicted proteins with a functional annotation based on similarity to conserved proteins. The proteins with predicted functional annotations share sequence similarity with proteins from bacteriophages and bacteria. The functionally annotated genes are predicted to encode DNA replication proteins, structural proteins, lysis proteins, proteins involved in nucleotide metabolism, and tRNAs. Characterization of the gene products reveals that Atu_ph07 encodes homologs of 16 T4 core proteins and is closely related to Rak2-like phages. Using ESI-MS/MS, the majority of predicted structural proteins could be experimentally confirmed and 112 additional virion-associated proteins were identified. The genomic characterization of Atu_ph07 suggests that this phage is lytic and the dynamics of Atu_ph07 interaction with its host indicate that this phage may be suitable for inclusion in a phage cocktail to be used as a biocontrol agent.

Keywords: Agrobacterium; Atu_ph07; biocontrol; genomics; jumbo bacteriophage; mass spectrometry.

Figure 1

Characterization of Atu_ph07. (A) Atu_ph07 forms small plaques on a lawn of A. tumefaciens C58 on 0.3% soft agar (left) and larger plaques on 0.15% soft agar (right). (B) Growth curve of A. tumefaciens infected with Atu_ph07 at different MOIs. (C) Time-lapse microscopy of A. tumefaciens cells infected with Atu_ph07 at an MOI of 10. (D) TEM image of Atu_ph07 shows the phage is in the family Myoviridae. Inset of a single phage particle at higher magnification is shown to emphasize the presence of tail fibers and whiskers.

Figure 2

Host range of Atu_ph07. Phylogenetic tree of Alphaproteobacteria tested for Atu_ph07 susceptibility in this study was constructed using 16S rRNA sequences. Agrobacterium strains are represented in purple, Rhizobium strains in pink, Sinorhizobium strains in brown, Caulobacter in orange, and the outgroup, Escherichia coli, is in blue. Agrobacteria biovars and genomospecies are indicated. Phage susceptibility (green) or resistance (red) for each strain is indicated.

Figure 3

The annotated genome of Atu_ph07. ORFs are represented by functional categories in corresponding colors. Regions shaded in beige represent putative structural protein clusters as identified using ESI-MS/MS analysis. Proteins detected by ESI-MS/MS analysis are indicated with an asterisk (*) below the corresponding ORF.

Figure 4

Phylogenetic comparison of Atu_ph07 and related phages. Phylogenetic trees of phages based on alignments of the (A) major capsid protein, (B) large terminase, and (C) portal vertex protein. Jumbo phages are labeled in blue and Atu_ph07 is indicated with bold font. Bootstrap values of 100 replicates are indicated. Rak2-like phages are indicated by a green line.

Figure 5

Similarity of annotated gene products in Atu_ph07 and related phages. Heat map displaying Atu_ph07 gene products compared with homologs in 12 related phages, including members of the Rak2-like phages (indicated with a green line). Intensity of the red color indicates the degree of similarity among homologs. Gray boxes indicate that a homolog with an E-value smaller than 1e-03 was not detected (ND). Gene products are organized by functional category and Atu_ph07 gp numbers are indicated.

Figure 6

tRNAs are encoded in the Atu_ph07 genome. Graphical representation of codon bias of phage Atu_ph07 and its host A. tumefaciens strain C58. Data points represent the usage of each codon in the Atu_ph07 and A. tumefaciens genomes. Red points represent codons only found in A. tumefaciens, purple points represent codons only found in Atu_ph07, and blue points represent codons found in both genomes. Gray lines outline the region in which codon usage in both genomes is similar.

Figure 7

SDS-PAGE of Atu_ph07 structural proteins as identified by ESI-MS/MS. Phage proteins were separated by size and fragments covering the full lane of the gel were excised for proteomic analysis. Numbers at the right of the gel indicate the position of fragments which were excised from the gel. Proteins identified in each fragment are listed. Bold font indicates validation of annotated structural proteins.