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.

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Reviewer #1 (Comments for the Author): The authors describe a new clade of Ecoli phages and named it Septuagintavirus within the subfamily, Vequintavirinae.They isolated four novel Ecoli phages (W70, A7-1, A5-4, and A73) that infect E.coli K12 MG1655.This study and discovery of novel phages offers an alternative treatment for GI and other diseases in which E. coli maybe involved.With the emergence of antibiotic resistance strains.E.coli phages offer the option of using phages as therapeutic agents.The isolation of lytic phages, which the above-mentioned phages are, are an important step in that direction.The phages were isolated from wastewater and animal feces and produced clear plaques.The phages also showed some lytic activity against other E. coli strains.The authors did a lysis profile of all four phages and identified them to be myovirus based on electron microscopy.The genomes of phages ranged from 137,323 and 145, 365 bp and showed a 90% homology amongst them.Genome annotation assigned functions to about 23% of the genes.Using phylogenetic analysis VICTOR, it was deduced that these phages form a distinct clade within Vequentevirinae and are placed close to Certrevirus genus.The different classes of genes and their assigned functions were discussed; these include structural proteins (proteome analysis was done by SDS PAGE and mass spectrometry), DNA replication, cell wall degrading enzymes and cell lysis.The authors conclude by stating that these phages are close relatives of Certevirus.The resemblance of tail fibers between Septuagintavirus and Certevirus suggest a shared utilization of cell surface receptors.Was curious to know if these phages would be effective against other bacterial stains that are part of the human microbiome.
Reviewer #2 (Comments for the Author): The author isolated four E.coli phages, analyzed their host ranges, structural proteins, and morphologies, and concluded that these phages belong to a novel genus, Septuagintavirus, based on genome analysis.Main document: 1.Figure S1 only displays plaques produced by phage W70.Why are there no images of plaques from the other three phages?The document mentions only spot assays for these phages without showcasing their individual plaque morphologies.2.Lines 122-123 state, "lower MOIs appeared to result in less efficient recovery than higher MOIs."What could be the underlying reason for this observation?3.Are there electron microscope images available for the other three phages?4.What are the pH and temperature sensitivities of the other three phages?5.Line 134 mentions, "with a shared nucleotide similarity between genomes exceeding 90%."What is the exact nucleotide similarity?Additionally, which phage in the NCBI database shares the highest homology with these four phages?6.Lines 155-156: Among the Certrevirus phages, which one shares the highest homology with W70?What is the nucleotide similarity?7.The author concludes that the four phages belong to a distinct subfamily, Vequintavirinae, solely based on VICTOR analysis.Is there any other evidence to support this classification?

Comments and Suggestions for the authors:
The authors describe a new clade of Ecoli phages and named it Septuagintavirus within the subfamily, Vequintavirinae.They isolated four novel Ecoli phages (W70, A7-1, A5-4, and A73) that infect E.coli K12 MG1655.This study and discovery of novel phages offers an alternative treatment for GI and other diseases in which E. coli maybe involved.With the emergence of antibiotic resistance strains.E.coli phages offer the option of using phages as therapeutic agents.The isolation of lytic phages, which the above-mentioned phages are, are an important step in that direction.The phages were isolated from wastewater and animal feces and produced clear plaques.The phages also showed some lytic activity against other E. coli strains.The authors did a lysis profile of all four phages and identified them to be myovirus based on electron microscopy.The genomes of phages ranged from 137,323 and 145, 365 bp and showed a 90% homology amongst them.Genome annotation assigned functions to about 23% of the genes.Using phylogenetic analysis VICTOR, it was deduced that these phages form a distinct clade within Vequentevirinae and are placed close to Certrevirus genus.
The different classes of genes and their assigned functions were discussed; these include structural proteins (proteome analysis was done by SDS PAGE and mass spectrometry), DNA replication, cell wall degrading enzymes and cell lysis.