ABSTRACT
Two bacteriophages infecting Escherichia coli were isolated from activated sludge samples collected from two different domestic wastewater treatment plants in Japan. One phage is related to Kayfunavirus phages and the other to Kuravirus phages. We report the complete genome sequences of these isolates.
KEYWORDS: wastewater treatment, activated sludge
ANNOUNCEMENT
Public health concerns regarding antibiotic resistance in pathogens such as Escherichia coli have prompted the search for alternative methods to control them. Bacteriophages (phages) have received increasing attention as potential alternatives (1–3). We isolated two E. coli phages (KM01 and KM02); each phage was isolated from an activated sludge sample collected at two different domestic wastewater treatment plants in Yokohama, Japan. The phages were isolated on host E. coli strain K12 (JCM20135—from feces of a diphtheria patient). Phage isolation and genomic DNA extraction, using phenol-chloroform with zinc chloride precipitation, were conducted as previously described (4, 5). Default parameters were used for all software tools, unless otherwise specified.
DNA libraries were constructed without fragmentation or size selection using Ligation Sequencing Kit 14 (SQK-LSK114) and loaded onto R10.4.1 flow cells (FLO-MIN114) for sequencing with MinION Mk1B (Oxford Nanopore, Tokyo, Japan). Simplex and duplex basecalling used Guppy v.6.4.2 in super accurate mode. Read filtering was performed with Filtlong v.0.2.1 (https://github.com/rrwick/Filtlong). Each read had mean qualities of Q19.6 and 16.2 and mean lengths of 25.2 kb and 8.18 kb, respectively, as evaluated by NanoPlot v.1.40.0 (6). Trimming of reads was performed with Trim Galore v.0.6.7 (https://github.com/FelixKrueger/TrimGalore). The assembly process used Flye v.2.9.1 (7) in high-quality mode. Post-assembly polishing was by Medaka v.1.7.2 (https://github.com/nanoporetech/medaka) without corrections to assembly. Final assembly was screened against Y-adapter top and bottom sequences, to remove remaining Nanopore adapter sequences from ends.
To identify direct terminal repeats, we filtered the assembly using PhageTerm v.1.0.12 (8) and determined genuine genome ends. For annotation, we imported assemblies into Geneious Prime v.2025.0.3 (https://www.geneious.com/), searching for Open Reading Frames (ORFs) with Glimmer3 (9) using start codons GTG, ATG, and TTG. Gene prediction was manually performed using the NCBI Protein Blast (BLASTp) search. Table 1 shows features of genome characteristics. Characteristics of KM01 are similar to the existing Kuravirus-like group of phages (4), including genome size, number of ORFs (124), number of tRNA/tmRNA (0), and G + C content (42.2%; lower than host E. coli strain K12 [50.5%]) (https://www.ncbi.nlm.nih.gov/datasets/genome/GCF_002087985.1/). Protein similarity analysis (ViPTree) (10) indicated that KM01 belonged to subfamily Gordonclarkvirinae and genus Kuravirus. The NCBI genome BLAST also showed that this displayed similarity to Escherichia phage Kuravirus phiEc_1 (LC768462), with 98.1% sequence identity (95% query coverage). Phage KM02 has a smaller genome size with fewer ORFs and no tRNA/tmRNA, but its G + C content (49.7%) was higher than that of KM01. The ViPTree indicated that KM02 belonged to subfamily Studiervirinae and genus Kayfunavirus. NCBI genome BLAST showed the sequence displayed 95.7% sequence identity (84% query coverage) to Kayfunavirus MirsaeidiM_Esher2 (PQ464628) Escherichia phage. KM02 exhibited low query coverage against existing phages, indicating the diversity of E. coli phages.
TABLE 1.
Genometrics of E. coli phages
| Phage | Genome size (nt) | No. of ORFs | No. of tRNA /tmRNA |
G + C (%) | Mean coverage (×) | Closest relative | Query coverage (%) | Identity (%) | GenBank accession |
|---|---|---|---|---|---|---|---|---|---|
| KM01 | 75,964 | 124 | 0 | 42.2 | 91 |
Kuravirus, Escherichia phage phiEc_1 (Acc. LC768462) |
95 | 98.1 | PV018827 |
| KM02 | 40,673 | 53 | 0 | 49.7 | 95 |
Kayfunavirus, Escherichia phage MirsaeidiM_Esher2 (Acc. PQ464628) |
84 | 95.7 | PQ179042 |
ACKNOWLEDGMENTS
This work was supported by JSPS KAKENHI Grants (JP17KK0010, JP22H03771, and JP22F20714). Steven Batinovic was funded by a JSPS Postdoctoral Fellowship (P20714). The authors acknowledge the Yokohama City Government for supplying the activated sludge samples.
Contributor Information
Tadashi Nittami, Email: nittami@ynu.ac.jp.
Jelle Matthijnssens, Katholieke Universiteit Leuven, Leuven, Belgium.
DATA AVAILABILITY
The genome sequences of the three Escherichia phages listed in Table 1 are available in NCBI GenBank under the accession numbers PV018827 and PQ179042. Raw sequence data are available under Sequence Read Archive (SRA) accession numbers SRR33209174 and SRR33223178.
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Associated Data
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Data Availability Statement
The genome sequences of the three Escherichia phages listed in Table 1 are available in NCBI GenBank under the accession numbers PV018827 and PQ179042. Raw sequence data are available under Sequence Read Archive (SRA) accession numbers SRR33209174 and SRR33223178.