Article Search
닫기

Microbiology and Biotechnology Letters

Genome Report(Note)

View PDF

Genome Report  |  Genome Report

Microbiol. Biotechnol. Lett. 2023; 51(4): 562-565

https://doi.org/10.48022/mbl.2311.11017

Received: November 27, 2023; Revised: December 24, 2023; Accepted: December 26, 2023

Complete Genome Sequence of Escherichia coli - Specific Phage KFS-EC1 Isolated from a Slaughterhouse

Su-Hyeon Kim and Mi-Kyung Park*

School of Food Science and Biotechnology, and Food and Bio-industry Research Institute, Kyungpook National University, Daegu 41566, Republic of Korea

Correspondence to :
Mi-Kyung Park,            parkmik@knu.ac.kr

Escherichia coli-specific phage, KFS-EC1, was isolated and purified from a slaughterhouse. The complete genome of the phage was obtained using Illumina MiSeq platforms. Its assembled genome consisted of a single chromosome of 164,715 bp with a GC content of 40.5%. The phage genome contained 170 hypothetical and 101 functional ORFs, and exhibited orthologous average nucleotide identity values of >95% with other E. coli phages belonging to the family Straboviridae. Additionally, phylogenetic analysis revealed that KFS-EC1 was finally classified into the family Straboviridae of the genus Caudoviricetes. The genome has been deposited in GenBank under the accession number NC_055757.1.

Keywords: Bacteriophage, Straboviridae, Escherichia coli, whole genome sequencing

Bacteriophages (phages) have garnered interest as potent biocontrol agents against Escherichia coli due to their ubiquitous existence, specificity, bactericidal effect, and safety [1]. Recent advancements in sequencing and bioinformatics technologies have expanded an understanding of phage diversity and enabled in-depth analysis of novel genes and differences in the genomes among the reported phages [2]. Along with this trend, the International Committee on Taxonomy of Viruses (ICTV) amended and ratified phage taxonomy by transitioning from morphology-based to genome-based classification in 2022. The significant alterations in this update involved the elimination of morphology-driven families, such as Myoviridae, Siphoviridae, and Podoviridae, and the replacement of the Caudovirales order to the class Caudoviricetes for categorizing all tailed phages possessing icosahedral capsid and a double-stranded DNA genome [3]. These paradigm shifts have necessitated the re-evaluation of previously classified phages. Thus, this study presents a complete genome sequence and revised taxonomic identification of E. coli-specific phage KFSEC1, aligning it with the updated ICTV standards.

KFS-EC1 was isolated and purified from slaughterhouse wastewater in Daegu, Korea [4]. For phage isolation, 1% (v/v) host culture of E. coli ATCC 43895 was incubated with 225 ml of tryptic soy broth (TSB, Difco Laboratories, USA) and 25 ml of the sample at 37℃ for 16 h. After centrifugation at 2,400 ×g for 20 min and filtration of the supernatant using a 0.22-μm pore size filter (Advantec MFS Inc., USA), the presence of phage in the filtrate was confirmed via plaque assay. This involved placing a mixture of host and phage suspension on the surface of pre-solidified tryptic soy agar (TSA) plates and incubating them at 37℃ for 16 h. The isolated phage was propagated by incubation with 1% (v/v) host culture in TA broth (8 g/l nutrient broth, 5 g/l NaCl, 0.2 g/l MgSO4, 0.05 g/l MnSO4, and 0.15 g/l CaCl2) at 37℃ for 2 h, followed by adding phage solution and reincubation at 37℃ for 2 h. After centrifugation of the mixture at 6,000 ×g for 20 min, supernatant was filtrated using a 0.22-μm filter. This propagation was repeated with increasing volumes of host culture and TA broth. Afterward, phage purification was performed by precipitation using 10% (w/v) polyethylene glycol 6000 (Sigma-Aldrich Co., USA) and CsCl-gradient ultracentrifugation at 39,000 ×g for 2 h. Finally, the obtained bluish band was dialyzed using sodium chloride-magnesium sulfate buffer (50 mM/L Tris-HCl, 100 mM NaCl, and 10 mM MgSO4, pH 7.5). The purified phage (KFS-EC1) was stored in a glass vial at 4℃ for further analyses.

The genomic DNA of KFS-EC1 was extracted using a Phage DNA Isolation Kit (Norgen Biotek Co., Canada) according to the manufacturer’s instructions. Quantification of the DNA was carried out using a Qubit 3.0 fluorometer (Thermo Fisher Scientific, USA). Subsequently, whole genome sequencing of the phage was performed using the Illumina Miseq platform with paired-end reads of 2 × 150 bp size. Error correction of the generated raw reads was conducted by quality filtering of the Illumina raw reads. The reads that had Phred-score of Q30 and above were collected and assembled using the SPAdes genome assembler (Illumina Inc., USA). The complete genome of KFS-EC1 was annotated using the Rapid Annotations Systems Technology (RAST) server [5] and BLASTP, and the genes linked to lysogenic properties were confirmed using the PHASTER’s database (https://phaster.ca/). After categorizing of the annotated open reading frames (ORFs), a genome map of KFS-EC1 was generated using Geneious software (v. 11.1.5, Biomatters Ltd., New Zealand). In addition, the genome of KFS-EC1 was compared with that of similar E. colispecific phages listed in the National Center for Biotechnology Information (NCBI) database, and their average nucleotide identity (ANI) values were determined using OrthoANI Tool (v. 0.93) [6]. Phylogenetic analysis of KFS-EC1 was then performed using the Virus Classification and Tree Building Online Resource [7].

The genetic characteristics of KFS-EC1 are shown in Table 1. The genome of KFS-EC1 consisted of one contig with a genome size of 164,715 bp and a GC content of 40.5%. In addition, its genome was predicted to comprise 170 hypothetical ORFs and 101 functional ORFs, and no tRNA was detected. Although some lytic phages have tRNAs for supporting protein synthesis, but their presence is not universal across all phages. This is because tRNA belongs to the accessory genome that is not an essential component [8]. Furthermore, the functional ORFs were categorized into four groups, including nucleotide metabolism, phage structure and packaging, host lysis, and additional functions (Fig. 1). Expressly, 42 genes related to nucleotide metabolism were confirmed, such as nucleotide replication, DNA repair, and its regulation, which encoded RNA polymerase, DNA polymerase, DNA ligase, DNA helicase, endonuclease, etc. Furthermore, 38 genes involved in the phage structure and packaging were found, such as capsid protein, baseplate and wedge proteins, phage tail, tail fibers, phage neck, phage prohead assembly and capsid scaffolding proteins, and phage tail assembly and scaffolding protein. The main components associated with host lysis (marked as pink arrows, Fig. 1) were also predicted to encode endolysin (9,050 bp−10,849 bp), phage spanin (73,706 bp−74,029 bp), holin (99,392−100,018), and peptidoglycan hydrolase (162,495 bp−162,884 bp). Endolysins degrade the peptidoglycan layer of the cell wall, whereas holins and spanins disrupt the inner membrane and outer membrane of the host, respectively [9], facilitating effective lysis of the host during phages’ lytic cycle. In addition, phage-encoded depolymerases, including hydrolases and lyases, can support the control of the host and its biofilm by cleaving the O-glycosidic bonds of bacterial polysaccharides and then degrading polymers [10]. More importantly, KFS-EC1 did not contain genes associated with prophages compared to the PHASTER’s database (20 December 2023). Overall, these genetic analyses confirmed that KFS-EC1 can control E. coli by expressing lytic action rather than lysogenic activity.

Table 1 . Comparison of genetic characteristics between KFS-EC1 and other similar E. coli-specific phages.

PhageGenome size (bp)GC content (%)tRNAPredicted ORFQuery coverage (%)Identity (%)ANI (%)Accession number
KFS-EC1164,71540.5ND271This studyThis studyThis studyNC_055757.1
vB_Eco_TB34165,22040.5ND28995.098.696.6OX_001802.1
JEP8165,29540.5ND27295.098.195.7MT_764208.1
W115163,99740.5ND26894.098.496.5ON_286974.1
vB_EcoM-pEE20166,42240.4ND27395.097.096.7OP_114734.1
vB_EcoM_PHB13165,64140.4ND27495.094.596.3MK_573636.1

ORF, open reading frame; ANI, average nucleotide identity; ND, not detected.


Figure 1.Genome map of KFS-EC1.

Among E. coli phages listed in the NCBI database, the most closely related phages to KFS-EC1 were selected, including vB_Eco_TB34, JEP8, W115, vB_EcoM-pEE20, and vB_EcoM_PHB13, to compare their genetic characteristics. Notably, KFS-EC1 exhibited orthologous ANI values of >95% with all selected phages belonging to the family Straboviridae (Table 1). In addition, the phylogenetic analysis of KFS-EC1 (Fig. 2) was performed with ten Straboviridae phages, three Kyanoviridae phages, one Herelleviridae phage, one Autographiviridae phage, and one Drexleviridae phage to re-classify KFS-EC1 aligning with the updated ICTV standards. The phylogenetic analysis (Fig. 2) exhibited that KFS-EC1 was clustered in the same branch with ten phages within the family Straboviridae, indicating a homologous relationship at the nucleotide level. The abolished family Myoviridae has recently been amended into two families, Straboviridae and Kyanoviridae, by significant reevaluation [3]. The Straboviridae family consists of 35 genera, including newly created 11 genera, that exhibit a T4-like morphology possessing an icosahedral head and a contractile tail that terminates in a baseplate with tail fibers. It is consistent with the results of genome annotation that has tail fibers and base plate, as well as the morphology of KFS-EC with contractile tail observed in the previous study [5]. Consequently, our findings finally re-classified KFS-EC1 as a member of the family Straboviridae, unlike its previous classification into the family Myoviridae [5]. This re-identification significantly can contribute to refining the evolutionary context within the recently revised taxonomy of the genus Caudoviricetes.

Figure 2.Phylogenetic tree between KFS-EC1 and other related taxa within the genus Caudoviricetes.
The complete genome of KFS-EC1 has been deposited in the Gen-Bank database under an accession number NC_055757.1.

This research was supported by Cooperative Research Program for Agriculture Science and Technology Development (Project No. PJ01572601) funded by Rural Development Administration, Korea.

The authors have no financial conflicts of interest to declare.

  1. Kim S-H, Lee H, Park M-K. 2023. Isolation, characterization, and application of a novel, lytic phage vB_SalA_KFSST3 with depolymerase for the control of Salmonella and its biofilm on cantaloupe under cold temperature. Food Res. Int. 172: 113062.
    Pubmed CrossRef
  2. Oliveira H, Domingues R, Evans B, Sutton JM, Adriaenssens EM, Turner D. 2022. Genomic diversity of bacteriophages infecting the genus acinetobacter. Viruses 14: 181.
    Pubmed KoreaMed CrossRef
  3. Turner D, Shkoporov AN, Lood C, Millard AD, Dutilh BE, Alfenas-Zerbini P, et al. 2023. Abolishment of morphology-based taxa and change to binomial species names: 2022 taxonomy update of the ICTV bacterial viruses subcommittee. Arch. Virol. 168: 74.
    Pubmed KoreaMed CrossRef
  4. Lee C, Choi IY, Park DH, Park MK. 2020. Isolation and characterization of a novel Escherichia coli O157:H7-specific phage as a biocontrol agent. J. Environ. Health Sci. Eng. 18: 189-199.
    Pubmed KoreaMed CrossRef
  5. Aziz RK, Bartels D, Best AA, DeJongh M, Disz T, Edwards RA, et al. 2008. The RAST Server: rapid annotations using subsystems technology. BMC Genomics 9: 75.
    Pubmed KoreaMed CrossRef
  6. Lee I, Kim YO, Park S-C, Chun J. 2016. OrthoANI: an improved algorithm and software for calculating average nucleotide identity. Int. J. Syst. Evol. Microbiol. 66: 1100-1103.
    Pubmed CrossRef
  7. Meier-Kolthoff JP, Göker M. 2017. VICTOR: genome-based phylogeny and classification of prokaryotic viruses. Bioinformatics 33: 3396-3404.
    Pubmed KoreaMed CrossRef
  8. Bailly-Bechet M, Vergassola M, Rocha E. 2007. Causes for the intriguing presence of tRNAs in phages. Genome Res. 17: 1486-1495.
    Pubmed KoreaMed CrossRef
  9. Abeysekera GS, Love MJ, Manners SH, Billington C, Dobson RC. 2022. Bacteriophage-encoded lethal membrane disruptors: Advances in understanding and potential applications. Front. Microbiol. 13: 1044143.
    Pubmed KoreaMed CrossRef
  10. Knecht LE, Veljkovic M, Fieseler L. 2020. Diversity and function of phage encoded depolymerases. Front. Microbiol. 10: 2949.
    Pubmed KoreaMed CrossRef

Starts of Metrics

Share this article on :

Related articles in MBL

Most Searched Keywords ?

What is Most Searched Keywords?

  • It is most registrated keyword in articles at this journal during for 2 years.