Characterization of a ViI-like phage specific to Escherichia coli O157:H7

Abstract

Phage vB_EcoM_CBA120 (CBA120), isolated against Escherichia coli O157:H7 from a cattle feedlot, is morphologically very similar to the classic phage ViI of Salmonella enterica serovar Typhi. Until recently, little was known genetically or physiologically about the ViI-like phages, and none targeting E. coli have been described in the literature. The genome of CBA120 has been fully sequenced and is highly similar to those of both ViI and the Shigella phage AG3. The core set of structural and replication-related proteins of CBA120 are homologous to those from T-even phages, but generally are more closely related to those from T4-like phages of Vibrio, Aeromonas and cyanobacteria than those of the Enterobacteriaceae. The baseplate and method of adhesion to the host are, however, very different from those of either T4 or the cyanophages. None of the outer baseplate proteins are conserved. Instead of T4's long and short tail fibers, CBA120, like ViI, encodes tail spikes related to those normally seen on podoviruses. The 158 kb genome, like that of T4, is circularly permuted and terminally redundant, but unlike T4 CBA120 does not substitute hmdCyt for cytosine in its DNA. However, in contrast to other coliphages, CBA120 and related coliphages we have isolated cannot incorporate 3H-thymidine (3H-dThd) into their DNA. Protein sequence comparisons cluster the putative "thymidylate synthase" of CBA120, ViI and AG3 much more closely with those of Delftia phage φW-14, Bacillus subtilis phage SPO1, and Pseudomonas phage YuA, all known to produce and incorporate hydroxymethyluracil (hmdUra).

Figure 1

Transmission electron micrographs of CBA120 particles stained with uranyl acetate (top) or phosphotungstic acid (bottom). Arrows indicate: A, neck with collar; B, reticular structure around the baseplate; C; tail spikes; D, tail tube after sheath contraction.

Figure 2

Effect of multiplicity of infection of phage CBA120 on host cells. 2A. Low MOI (~0.1) single-step growth curve of CBA120 infecting E. coli O157:H7 NCTC 12900. The infection graph shown is representative of three replicates. Infective centers (PFU mL^-1, O), and total viable phage (PFU mL^-1, □, as determined after the addition of chloroform). 2B. High MOI (~10) CBA120 infection of E. coli O157:H7 NCTC 12900. The infection graph shown is representative of three replicates. Infective centers (PFU mL^-1, O), phage (PFU mL^-1, □ after the addition of chloroform), optical density (600 nm, Δ) and bacterial titer/survivors (CFU mL^-1, ◇).

Figure 3

Anaerobic (MOI ~3) CBA120 infection of E. coli O157:H7 NCTC 12900 exponentially growing in TSB at 37°C under an N₂ headspace. Symbols as in Figure 2.

Figure 4

Comparison of ³H-dThd uptake in uninfected E. coli 12900 (O) and after infection with phage CBA120 (Δ) or CEV1 (□).

Figure 5

DNA-level ACT alignment of the genomes of phages ViI (top), CBA120 and AG3. Regions in red represent high DNA sequence similarity between the genomes.

Figure 6

Functional genomic map of CBA120. The "gp" (gene product) numbers refer to the corresponding gene numbers of bacteriophage T4. Red: DNA replication; yellow: nucleotide metabolism; dark blue: head-associated; turquoise: tail-associated; medium blue: orthologs seen in ViI proteomic analysis, but otherwise unidentified; grey: regulatory genes.

Figure 7

Phylogenetic tree of genes initially identified as "Thymidylate Synthase" (TS) or "Thymidylate Synthetase" by homology searches of the non-redundant NCBI protein database. The topology and branch lengths correspond to the Maximum Likelihood (ML) inference. Bootstraps appear ordered as ML, Neighbor Joining and Maximum Parsimony. Only bootstrap values greater than 50 are shown. All of the phages are members of the Myoviridae except for siphoviruses T5, infecting E. coli, YuA, infecting Pseudomonas, and JL001, targeting a marine γ-Proteobacterium. Phage SPO1 infects B. subtilis, Felix O1 infects many species of Salmonella and wV8 infects E. coli. The rest of the phages belong to the T4 superfamily, most of them infecting E. coli and some strains of Shigella. However, KVP40 infects Vibrio parahaemolyticus, 31 infects A. salmonicida, φW-14 infects D. acidovorans, and KP15 infects Klebsiella pneumoniae. Coliphage LZ2 was isolated by Sean Eddy from the Denver zoo and several segments were sequenced in the Kutter lab, in comparison with those from a number of other T4-like phages [44].