Skip to main content
  • ASM
    • Antimicrobial Agents and Chemotherapy
    • Applied and Environmental Microbiology
    • Clinical Microbiology Reviews
    • Clinical and Vaccine Immunology
    • EcoSal Plus
    • Eukaryotic Cell
    • Microbiology Resource Announcements
    • Infection and Immunity
    • Journal of Bacteriology
    • Journal of Clinical Microbiology
    • Journal of Microbiology & Biology Education
    • Journal of Virology
    • mBio
    • Microbiology and Molecular Biology Reviews
    • Microbiology Spectrum
    • Molecular and Cellular Biology
    • mSphere
    • mSystems
  • Log in
  • My alerts
  • My Cart

Main menu

  • Home
  • Articles
    • Latest Articles
    • Archive
  • Types of Resources
    • Amplicon Sequence Collections
    • Culture Collections/Mutant Libraries
    • Databases and Software
    • Omics Data Sets
    • Other Genetic Resources
    • Genome Sequences
  • For Authors
    • Getting Started
    • Submit a Manuscript
    • Scope
    • Editorial Policy
    • Submission, Review, & Publication Processes
    • Organization and Format
    • Errata, Author Corrections, Retractions
    • Illustrations and Tables
    • Nomenclature
    • Publication Fees
    • Ethics Resources and Policies
  • About the Journal
    • About MRA
    • Editor in Chief
    • Board of Editors
    • For Reviewers
    • For the Media
    • For Librarians
    • For Advertisers
    • Alerts
    • RSS
    • FAQ
  • ASM
    • Antimicrobial Agents and Chemotherapy
    • Applied and Environmental Microbiology
    • Clinical Microbiology Reviews
    • Clinical and Vaccine Immunology
    • EcoSal Plus
    • Eukaryotic Cell
    • Microbiology Resource Announcements
    • Infection and Immunity
    • Journal of Bacteriology
    • Journal of Clinical Microbiology
    • Journal of Microbiology & Biology Education
    • Journal of Virology
    • mBio
    • Microbiology and Molecular Biology Reviews
    • Microbiology Spectrum
    • Molecular and Cellular Biology
    • mSphere
    • mSystems

User menu

  • Log in
  • My alerts
  • My Cart

Search

  • Advanced search
Microbiology Resource Announcements
publisher-logosite-logo

Advanced Search

  • Home
  • Articles
    • Latest Articles
    • Archive
  • Types of Resources
    • Amplicon Sequence Collections
    • Culture Collections/Mutant Libraries
    • Databases and Software
    • Omics Data Sets
    • Other Genetic Resources
    • Genome Sequences
  • For Authors
    • Getting Started
    • Submit a Manuscript
    • Scope
    • Editorial Policy
    • Submission, Review, & Publication Processes
    • Organization and Format
    • Errata, Author Corrections, Retractions
    • Illustrations and Tables
    • Nomenclature
    • Publication Fees
    • Ethics Resources and Policies
  • About the Journal
    • About MRA
    • Editor in Chief
    • Board of Editors
    • For Reviewers
    • For the Media
    • For Librarians
    • For Advertisers
    • Alerts
    • RSS
    • FAQ
Genome Sequences

Complete Genome Sequences of Escherichia coli Phages vB_EcoM-EP75 and vB_EcoP-EP335

Joël van Mierlo, Steven Hagens, Sander Witte, Silvia Klamert, Leoni van de Straat, Lars Fieseler
John J. Dennehy, Editor
Joël van Mierlo
aMicreos Food Safety, Wageningen, The Netherlands
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Steven Hagens
aMicreos Food Safety, Wageningen, The Netherlands
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Sander Witte
aMicreos Food Safety, Wageningen, The Netherlands
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Silvia Klamert
aMicreos Food Safety, Wageningen, The Netherlands
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Leoni van de Straat
aMicreos Food Safety, Wageningen, The Netherlands
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Lars Fieseler
bFood Microbiology Research Group, Institute of Food and Beverage Innovation, Zurich University of Applied Sciences (ZHAW), Wädenswil, Switzerland
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
John J. Dennehy
Queens College
Roles: Editor
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
DOI: 10.1128/MRA.00078-19
  • Article
  • Info & Metrics
  • PDF
Loading

ABSTRACT

Phages vB_EcoM-EP75 (EP75) and vB_EcoP-EP335 (EP335) specifically infect Shiga toxin (Stx)-producing Escherichia coli (STEC) O157 strains. EP75 has a genome size of 158,143 bp and belongs to the genus Vi1virus. The genome size of EP335 is 76,622 bp, and it belongs to the genus Phieco32virus.

ANNOUNCEMENT

Shiga toxin (Stx)-producing Escherichia coli (STEC) strains are a tremendous threat to human health, as they cause severe infections and kidney failure (1). Some antibiotics cannot be applied to control the disease either because the bacteria are resistant to the antibiotic or because the expression of Shiga toxins is upregulated after antibiotic exposure (1). To control these pathogenic bacteria, bacteriophage treatment represents one of the alternatives to antibiotics (1). Importantly, treatment using bacteriophages does not seem to induce expression of Shiga toxins (2). Although more research is needed before bacteriophages can be applied in human therapy, the application of bacteriophages in food is already industry practice. This creates the opportunity to exploit bacteriophages as an intervention against STEC in food. To serve this purpose, we have set out to isolate and sequence STEC O157-specific bacteriophages.

STEC O157-specific phages vB_EcoM-EP75 (EP75) and vB_EcoP-EP335 (EP335) were isolated from sewage water from The Netherlands using the LB soft agar overlay method (3) and E. coli O157 NC13127 as an isolation strain. Plates were incubated overnight at 37°C (EP75) or 20°C (EP335). Single plaques were picked using a Pasteur pipette and transferred to 300 µl of sodium chloride-magnesium sulfate (SM) buffer (100 mM NaCl, 8 mM MgSO4, 50 mM Tris-HCl [pH 7.4]), followed by a 2-h incubation at 30°C. After centrifugation, the supernatant was filter sterilized (0.2-µm membrane filter) and used in two consecutive rounds of plaque isolation to obtain a pure phage solution. Filter-sterilized lysates (0.2-µm membrane filter) of EP75 and EP335 were precipitated using polyethylene glycol (10% [wt/vol] PEG 8000, 1 M NaCl; 4°C, overnight) and pelletized (10 min, 10,000 × g), after which the pellet was resuspended in 5 ml of SM buffer. Phages were purified using CsCl gradient centrifugation (4).

Phage DNA was extracted using the Quick-DNA viral kit (Zymo Research, Irvine, CA) according to the manufacturer’s protocol. Library preparation was performed using the Nextera XT library preparation kit (Illumina, San Diego, CA) according to the manufacturer’s instructions. The resulting paired-end sequence reads were generated using an Illumina HiSeq 2500 system. Sequencing of the EP75 genome revealed read lengths of >50 nucleotides and a total of 3,491,911 reads, with an average quality (Phred) score of 37.82. Sequencing of the EP335 genome revealed read lengths of >50 nucleotides, a total of 589,508 reads, and an average quality (Phred) score of 38.78. For read quality control, FASTQ sequence files were generated using bcl2fastq2 version 2.18. Initial quality assessment was based on data passing Illumina Chastity filtering. Subsequently, reads containing the PhiX control signal were removed using an in-house filtering protocol. In addition, reads containing adapters were clipped. The second quality assessment was based on the remaining reads, using the FASTQC version 0.11.5 quality control tool. Reads of the EP75 genome were assembled into 8 contigs using SPAdes version 3.10 (5). For the EP335 genome, reads were assembled into 15 contigs. BLASTN 2.8.1+ (default settings) was used to search for the closest matches for all contigs. Contigs were placed into scaffolds using the SSPACE Premium Scaffolder version 2.3 (6). Contigs with more than 50% coverage and 80% sequence identity to non-phage-related sequences were discarded for genome analysis. The average coverages of the assembled genomes were 5,129 (EP75) and 1,664 (EP335) reads per nucleotide.

Coding sequences (CDS) were annotated using Rapid Annotations using Subsystems Technology (RAST) 2.0 (7) and BLAST (8) comparisons with the nonredundant GenBank database. ARAGORN version 1.2.38 (9) and tRNAscan-SE version 2.0 (10) identified tRNA sequences. Overall nucleotide sequence identities were analyzed using EMBOSS Stretcher (11) and BLAST2seq.

The EP75 genome is 158,143 bp long and likely circularly permuted (12). The G+C content is 44.6%. Of the 209 CDS annotated, 57 were assigned a putative function. Five tRNA sequences were found. EP75 shares a nucleotide identity of 96.59% with Escherichia phage PhaxI (query coverage, 91%; GenBank accession number JN673056), 92.8% with Salmonella phage Marshall (query coverage, 86%; accession number KF669653), and 91.92% with Salmonella phage Maynard (query coverage, 85%; accession number KF669654), placing EP75 into the family Ackermannviridae, subfamily Cvivirinae, and genus Vi1virus. With nucleotide identities ranging from 91.92 to 95.69% and query coverages ranging from 85 to 90%, EP75 also exhibited homologies to Escherichia phage ECML-4 (GenBank accession number JX128257), Salmonella phage 38 (accession number KR296692), and Escherichia virus CBA120 (accession number JN593240).

The double-stranded linear DNA of EP335 is 76,622 bp long with a G+C content of 42.5% and contains 125 annotated CDS, 20 with assigned putative functions, and a tRNAArg sequence. Due to the nucleotide identity of 96.73% with Escherichia phage KBNP1711 (query coverage, 88%; GenBank accession number KF981730) and 85.47% with Escherichia phage NJ01 (query coverage, 69%; accession number JX867715), EP335 can be assigned to the family Podoviridae, genus Phieco32virus. Compared to other phages, EP335 exhibited homology to Escherichia phage 172-1 (accession number KP308307), Enterobacteria phage phiEco32 (accession number EU330206), and phage vB_EcoP_SU10 (accession number KM044272), with nucleotide identities of ca. 85% and query coverages ranging from 65 to 70%.

Data availability.The annotated sequences of the two E. coli phage genomes were deposited at GenBank under the accession numbers MG748547 (vB_EcoM-EP75) and MG748548 (vB_EcoP-EP335). The raw data are available under BioProject and SRA accession numbers PRJNA517780 and SRP182885, respectively.

ACKNOWLEDGMENTS

J.V.M. and S.H. designed the experiments; S.W., L.V.D.S., and S.K. performed the experiments, and L.F., J.V.M., and S.H. wrote the paper.

We declare no conflicts of interest.

FOOTNOTES

    • Received 1 February 2019.
    • Accepted 22 March 2019.
    • Published 18 April 2019.
  • Copyright © 2019 van Mierlo et al.

This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license.

REFERENCES

  1. 1.↵
    1. Mir RA,
    2. Kudva IT
    . 2019. Antibiotic-resistant Shiga toxin-producing Escherichia coli: an overview of prevalence and intervention strategies. Zoonoses Public Health 66:1–13. doi:10.1111/zph.12533.
    OpenUrlCrossRef
  2. 2.↵
    1. Howard-Varona C,
    2. Vik DR,
    3. Solonenko NE,
    4. Li Y-F,
    5. Gazitua MC,
    6. Chittick L,
    7. Samiec JK,
    8. Jensen AE,
    9. Anderson P,
    10. Howard-Varona A,
    11. Kinkhabwala AA,
    12. Abedon ST,
    13. Sullivan MB
    . 2018. Fighting fire with fire: phage potential for the treatment of E. coli O157 infection. Antibiotics (Basel) 7:101. doi:10.3390/antibiotics7040101.
    OpenUrlCrossRef
  3. 3.↵
    1. Adams MH
    . 1959. Bacteriophages. Interscience Publishers, Inc., New York, NY.
  4. 4.↵
    1. Sambrook J,
    2. Russell DW
    . 2001. Molecular cloning, 3rd ed, vol 1. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
  5. 5.↵
    1. Bankevich A,
    2. Nurk S,
    3. Antipov D,
    4. Gurevich AA,
    5. Dvorkin M,
    6. Kulikov AS,
    7. Lesin VM,
    8. Nikolenko SI,
    9. Pham S,
    10. Prjibelski AD,
    11. Pyshkin AV,
    12. Sirotkin AV,
    13. Vyahhi N,
    14. Tesler G,
    15. Alekseyev MA,
    16. Pevzner PA
    . 2012. SPAdes: a new genome assembly algorithm and its applications to single-cell sequencing. J Comput Biol 19:455–477. doi:10.1089/cmb.2012.0021.
    OpenUrlCrossRefPubMed
  6. 6.↵
    1. Boetzer M,
    2. Pirovano W
    . 2014. SSPACE-LongRead: scaffolding bacterial draft genomes using long read sequence information. BMC Bioinformatics 15:211. doi:10.1186/1471-2105-15-211.
    OpenUrlCrossRefPubMed
  7. 7.↵
    1. Aziz RK,
    2. Bartels D,
    3. Best AA,
    4. DeJongh M,
    5. Disz T,
    6. Edwards RA,
    7. Formsma K,
    8. Gerdes S,
    9. Glass EM,
    10. Kubal M,
    11. Meyer F,
    12. Olsen GJ,
    13. Olson R,
    14. Osterman AL,
    15. Overbeek RA,
    16. McNeil LK,
    17. Paarmann D,
    18. Paczian T,
    19. Parrello B,
    20. Pusch GD,
    21. Reich C,
    22. Stevens R,
    23. Vassieva O,
    24. Vonstein V,
    25. Wilke A,
    26. Zagnitko O
    . 2008. The RAST server: Rapid Annotations using Subsystems Technology. BMC Genomics 9:75. doi:10.1186/1471-2164-9-75.
    OpenUrlCrossRefPubMed
  8. 8.↵
    1. Altschul SF,
    2. Gish W,
    3. Miller W,
    4. Myers EW,
    5. Lipman DJ
    . 1990. Basic local alignment search tool. J Mol Biol 215:403–410. doi:10.1016/S0022-2836(05)80360-2.
    OpenUrlCrossRefPubMedWeb of Science
  9. 9.↵
    1. Laslett D,
    2. Canback B
    . 2004. ARAGORN, a program to detect tRNA genes and tmRNA genes in nucleotide sequences. Nucleic Acids Res 32:11–16. doi:10.1093/nar/gkh152.
    OpenUrlCrossRefPubMedWeb of Science
  10. 10.↵
    1. Lowe TM,
    2. Chan PP
    . 2016. tRNAscan-SE on-line: integrating search and context for analysis of transfer RNA genes. Nucleic Acids Res 44:W54–W57. doi:10.1093/nar/gkw413.
    OpenUrlCrossRefPubMed
  11. 11.↵
    1. Chojnacki S,
    2. Cowley A,
    3. Lee J,
    4. Foix A,
    5. Lopez R
    . 2017. Programmatic access to bioinformatics tools from EMBL-EBI update: 2017. Nucleic Acids Res 45:W550–W553. doi:10.1093/nar/gkx273.
    OpenUrlCrossRefPubMed
  12. 12.↵
    1. Kutter EM,
    2. Skutt-Kakaria K,
    3. Blasdel B,
    4. El-Shibiny A,
    5. Castano A,
    6. Bryan D,
    7. Kropinski AM,
    8. Villegas A,
    9. Ackermann H-W,
    10. Toribio AL,
    11. Pickard D,
    12. Anany H,
    13. Callaway T,
    14. Brabban AD
    . 2011. Characterization of a ViI-like phage specific to Escherichia coli O157:H7. Virol J 8:430–444. doi:10.1186/1743-422X-8-430.
    OpenUrlCrossRefPubMed
PreviousNext
Back to top
Download PDF
Citation Tools
Complete Genome Sequences of Escherichia coli Phages vB_EcoM-EP75 and vB_EcoP-EP335
Joël van Mierlo, Steven Hagens, Sander Witte, Silvia Klamert, Leoni van de Straat, Lars Fieseler
Microbiology Resource Announcements Apr 2019, 8 (16) e00078-19; DOI: 10.1128/MRA.00078-19

Citation Manager Formats

  • BibTeX
  • Bookends
  • EasyBib
  • EndNote (tagged)
  • EndNote 8 (xml)
  • Medlars
  • Mendeley
  • Papers
  • RefWorks Tagged
  • Ref Manager
  • RIS
  • Zotero
Print
Alerts
Sign In to Email Alerts with your Email Address
Email

Thank you for sharing this Microbiology Resource Announcements article.

NOTE: We request your email address only to inform the recipient that it was you who recommended this article, and that it is not junk mail. We do not retain these email addresses.

Enter multiple addresses on separate lines or separate them with commas.
Complete Genome Sequences of Escherichia coli Phages vB_EcoM-EP75 and vB_EcoP-EP335
(Your Name) has forwarded a page to you from Microbiology Resource Announcements
(Your Name) thought you would be interested in this article in Microbiology Resource Announcements.
CAPTCHA
This question is for testing whether or not you are a human visitor and to prevent automated spam submissions.
Share
Complete Genome Sequences of Escherichia coli Phages vB_EcoM-EP75 and vB_EcoP-EP335
Joël van Mierlo, Steven Hagens, Sander Witte, Silvia Klamert, Leoni van de Straat, Lars Fieseler
Microbiology Resource Announcements Apr 2019, 8 (16) e00078-19; DOI: 10.1128/MRA.00078-19
del.icio.us logo Digg logo Reddit logo Twitter logo CiteULike logo Facebook logo Google logo Mendeley logo
  • Top
  • Article
    • ABSTRACT
    • ANNOUNCEMENT
    • ACKNOWLEDGMENTS
    • FOOTNOTES
    • REFERENCES
  • Info & Metrics
  • PDF

Related Articles

Cited By...

About

  • About MRA
  • Editor in Chief
  • Board of Editors
  • Policies
  • For Reviewers
  • For the Media
  • For Librarians
  • For Advertisers
  • Alerts
  • RSS
  • FAQ
  • Permissions
  • Journal Announcements

Authors

  • Getting Started
  • Submit a Manuscript
  • Author Warranty
  • Ethics
  • Contact Us
  • ASM Author Center

Follow #MRAJournal

@ASMicrobiology

       

ASM Journals

ASM journals are the most prominent publications in the field, delivering up-to-date and authoritative coverage of both basic and clinical microbiology.

About ASM | Contact Us | Press Room

 

ASM is a member of

Scientific Society Publisher Alliance

 

American Society for Microbiology
1752 N St. NW
Washington, DC 20036
Phone: (202) 737-3600

Copyright © 2021 American Society for Microbiology | Privacy Policy | Website feedback

Online ISSN: 2576-098X