Isolation and characterization of Septuagintavirus; a novel clade of Escherichia coli phages within the subfamily Vequintavirinae

Abstract

Escherichia coli is a commensal inhabitant of the mammalian gut microbiota, frequently associated with various gastrointestinal diseases. There is increasing interest in comprehending the variety of bacteriophages (phages) that target this bacterium, as such insights could pave the way for their potential use in therapeutic applications. Here, we report the isolation and characterization of four newly identified E. coli infecting tailed phages (W70, A7-1, A5-4, and A73) that were found to constitute a novel genus, Septuagintavirus, within the subfamily Vequintavirinae. Genomes of these phages ranged from 137 kbp to 145 kbp, with a GC content of 41 mol%. They possess a maximum nucleotide similarity of 30% with phages of the closest phylogenetic genus, Certrevirus, while displaying limited homology to other genera of the Vequintavirinae family. Host range analysis showed that these phages have limited activity against a panel of E. coli strains, infecting 6 out of 16 tested isolates, regardless of their phylotype. Electrospray ionization-tandem mass spectrometry (ESI-MS/MS) was performed on the virion of phage W70, allowing the identification of 28 structural proteins, 19 of which were shared with phages of other genera of Vequintavirinae family. The greatest diversity was identified with proteins forming tail fiber structures, likely indicating the adaptation of virions of each phage genus of this subfamily for the recognition of their target receptor on host cells. The findings of this study provide greater insights into the phages of the subfamily Vequintavirinae, contributing to the pool of knowledge currently known about these phages.

Importance: Escherichia coli is a well-known bacterium that inhabits diverse ecological niches, including the mammalian gut microbiota. Certain strains are associated with gastrointestinal diseases, and there is a growing interest in using bacteriophages, viruses that infect bacteria, to combat bacterial infections. Here, we describe the isolation and characterization of four novel E. coli bacteriophages that constitute a new genus, Septuagintavirus, within the subfamily Vequintavirinae. We conducted mass spectrometry on virions of a representative phage of this novel clade and compared it to other phages within the subfamily. Our analysis shows that virion structure is highly conserved among all phages, except for proteins related to tail fiber structures implicated in the host range. These findings provide greater insights into the phages of the subfamily Vequintavirinae, contributing to the existing pool of knowledge about these phages.

Keywords: Escherichia coli; Vequintavirinae; bacteriophage; characterization; isolation; mass-spectrometry; virion.

Fig 1

Bacteriophage kill-curves of E. coli K12 MG-1655 in response to infection by novel isolated E. coli phages. The host was individually infected with the four phages: (A) A73, (B) W70, (C) A5-4, and (D) A7-1 at different MOIs: 10 (red), 1 (green), 0.1 (purple), 0.01 (blue), and 0.001 (orange). Growth control (only bacteria) is represented with black color. All samples were analyzed in triplicate. Results are expressed as mean values ± SD.

Fig 2

Transmission electron micrographs of Escherichia phage W70 stained with 2% (wt/vol) uranyl acetate. (A) Uncontracted phage W70 virion particle and (B) two particles with empty capsids and contracted tail sheath. The scale bar represents 100 nm.

Fig 3

Heatmap showing the percentage of nucleotide similarity of Escherichia phages isolated in this study and other closer phylogenetic members of the subfamily Vequintavirinae as calculated with VIRIDIC (29).

Fig 4

Circular genome map of the E. coli phage W70 serving as a representative of the novel isolated E. coli phages. The innermost ring represents the GC skew (red for the positive strand and blue for the negative strand) while the central ring (black) shows the GC content. BLAST alignment was performed for sequencing similarity comparison versus A54 phage (orange ring), A7-1 phage (dark green ring), and A73 phage (purple ring). The outermost circle displays the coding genes (CDS) for W70 phage with predicted functions as labels. The coloration of the CDS corresponds to their general functions as indicated in the legend. Genes whose function could not be determined are colored gray and remain unlabeled.

Fig 5

Amino acid VICTOR-generated phylogenomic Genome-BLAST Distance Phylogeny (GBDP) tree inferred using the formula D4 and yielding an average support of 86% (30). The phylogram includes the Escherichia phages isolated in this study and other members of the subfamily Vequintavirinae. The genus (if allocated) of phages in the analysis is illustrated. Branch support was inferred from 100 pseudo-bootstrap replicates.

Fig 6

Genome map comparison of the genomes of Escherichia phages isolated in this study and other members of the subfamily Vequintavirinae employing tBLASTx with easyfig (31). The genome maps display arrows indicating the locations and orientation of ORFs among different phage genomes. The large terminase was set as the first gene among all genomes.