Shiga-toxin-producing Escherichia coli (STEC) is an important zoonotic pathogen that causes several diseases in human, such as watery or bloody diarrhea, hemorrhagic colitis, and even fatal hemolytic uremic syndrome (HUS)1. Shiga toxin (Stx) is the major virulence factor of STEC, two Stx types and several subtypes which vary in toxicity have been identified2,3. Stx2k is a newly reported subtype of clinical relevance, and it was exclusively reported in China so far4. Stx is encoded by stx gene, which was located in the late region of lysogenized lambdoid prophages, thus Stx phages play an important role in STEC pathogenesis5. Although numerous Stx prophage sequences have been predicted as part of STEC genomes6, little information is available about the genomic characterization of Stx phages induced from STEC strains7.
In this study, we isolated and characterized a Stx2k phage named phiSTEC1575-Stx2k, the phage was induced from a patient-derived strain STEC1575, using E. coli K-12 derivative strain MC1061 as host strain8. Freshly prepared logarithmic-phase bacterial cultures were treated with mitomycin C (0.5 μg/mL) for 18 hours at 37 °C9. Lysates were filtered through a 0.22- μm nitrocellulose filter. The phiSTEC1575-Stx2k was purified five times by picking single plaques using the double-layer agar plaque method10. The phages were concentrated by adding PEG8000 (20%) and NaCl (2.5 M) to the lysate and incubated overnight at 4 °C11. After staining with 2% phosphotungstic acid, the morphology of phiSTEC1575-Stx2k was observed using a transmission electron microscope (Tecnai12, FEI, USA). As shown in Fig. 1, phiSTEC1575-Stx2k has an isometric polyhedral head (approximately 54 nm in diameter) and a noncontractile tail (approximately 149 nm long), indicating that it belongs to the family Siphoviridae within the order Caudovirales.
To investigate the genomic characteristics of phiSTEC1575-Stx2k, its genomic DNA was extracted by the phenol-chloroform12. Whole-genome sequencing was performed using a MGISEQ-2000 sequencing platform (MGI Tech Co., Ltd., Shenzhen, China) with 150 bp paired-end reads at Shenzhen BGI Co., Ltd. Low-quality (Q-value ≤ 20) reads and adapter sequences were filtered and the sequence was assembled using SPAdes v3.9.013. The phage genome was annotated using RAST14. BLASTp was used for further verification of the predicted proteins (https://blast.ncbi.nlm.nih.gov/Blast.cgi). SnapGene Viewer 5.1.7 (GSL Biotech; available at snapgene.com) was used to generate a map of the phage genome. The complete genome sequence of Escherichia coli strain STEC1575 was determined as previous described4. PHASTER was used to identify prophages in bacterial genome sequences15.
The phiSTEC1575-Stx2k has a circular dsDNA genome of 46,647 bp and a G+C content of 51%. The genome sequence of phiSTEC1575-Stx2k was deposited in the GenBank database under the accession number OQ243220. At the nucleotide level, BLASTn searching showed that the genome sequence was closest to the prophage from E. coli strain RHB24-C21 (CP057485.1), with 69% coverage and 99.27% identity.
The annotation showed that phiSTEC1575-Stx2k possessed 80 open reading frames (ORFs) and three tRNA genes (Supplementary Table S1). Forty-four functional domains were predicted, which could be classified into six modules according to their function, including phage integration, DNA packaging and replication, regulation, toxicity, bacterial lysis, and morphogenesis (Fig. 2A). The phage phiSTEC1575-Stx2k resides as a prophage in the E. coli STEC1575 chromosome (BioSample number SAMN32736966). The complete genome sequences of the phage and prophage were compared using Easyfig v.2.0 (Fig. 2B) and found they were identical. The phage phiSTEC1575-Stx2k integrates into the tRNA-dihydrouridine synthase (GenBank: ABV08454.1) coding gene dusA (Fig. 2C). The 21-bp prophage attachment sites 5’-TTG CAC CAA TGC TCG ATA GGA-3’ on the left (attL) and 5’-TTG CTC CTA TGC TCG ACT GGA-3’ on the right (attR) were found at the chromosome of STEC1575; a 7-bp segment (5’-ATGCTCG-3’) was completely conserved among the prophage attachment site attL, attR and the phage attachment site attP, representing the ‘core’ recombination site for integration or excision as previously reported 16,17. The attR (chromosome) or attP (phage) attachment site is located 184 bp upstream of the phage integrase (ORF80).
In conclusion, a temperate phage named phiSTEC1575-Stx2k was induced from a patient-derived STEC strain. To our knowledge, this is the first report of genomic sequence and characterization of temperate phage from a Stx2k-producing E. coli.