Complete genome of a novel virulent phage ST0 lysing Escherichia coli H8

Phage ST0 lysing Escherichia coli H8 was isolated from wastewater and sequenced using an Illumina HiSeq system. Genomic analyses revealed that it was virulent phages and contained a circular double-stranded DNA genome, consisting of 170,496 nucleotides with an average G + C content of 37.67%. This study may provide possible alternative materials for phage therapy. Electronic supplementary material The online version of this article (doi: 10.1186/s40793-017-0304-9) contains supplementary material, which is available to authorized users.


Introduction
A large number of antibiotics were produced and widely used in medical and agricultural areas. These substances in the environment didn't tend to be biodegradable, and were easily stored and accumulated in water and soil environment and even in the atmospheric environment [1][2][3]. Recently, antibiotics had been recognized as the emerging environmental pollutants, because of their potential undesirable effects on the ecosystem and human health [4][5][6], such as antibiotic resistance. The resistance of bacteria to current antibiotics increased the difficulty in medical treatment, which accounted for 23,000 deaths annually in the USA. The spread of antibiotic resistant bacteria and antibiotic resistance genes in the environment was a major public health issue. Obviously, strict control of the use of antibiotics and the development of a possible alternative medicine seemed extremely urgent.
Compared with antibiotics, phage therapy had the advantages of high specificity, few side effects and capacity for low-dosage use and so on [7]. In particular, it was alternative and effective to adopt phage therapy to treat diseases caused by antibioticsresistant bacteria strains [8]. Currently phage therapy mainly had single phage treatment, multiple phage treatment and combined therapy of phage and antibiotics. More recently, bacteriophages had been intensively studied and potential application for the control of Escherichia coli in livestock, aquaculture and food products [9][10][11].
In this work, phage ST0 against Escherichia coli H8 was isolated from industrial wastewater in China. Its morphology, complete genome sequence and bioinformatics analysis were explored. This could provide a better understanding to the development of a possible alternative medicines and biocontrol agents.

Classification and features
Escherichia coli H8 (ST100), the host to isolate virulent phages, carrying shiga toxin genes (stx1, stx2) was obtained from the Chinese Center for Disease Control and Prevention. Phage ST0 was isolated from a sewage treatment plant of wastewater in Beijing. The isolation, propagation and titration of phage was done according to the methods described previously [12]. Phage ST0 generated clear plaques on doublelayer plate (Fig. 1a), indicating that it was a virulent phage. The diameter of plaques was 1-2 mm. The transmission electron microscopy image (Fig. 1b) showed that phage ST0 had an icosahedral head approximately 120 nm in long diameter and 80 nm in short diameter. Its long tail was about 120 nm in length and 20 nm in diameter. Phylogenetic analysis based on complete genome sequences revealed that phage ST0 was closely related to Enterobacteria phage HX01 (Accession JX536493.1), whereas the score was relatively low (Fig. 2). A summary of the isolation and general phylogenetic features of phage ST0 are shown in Table 1.

Genome sequencing information
Genome project history Phage ST0 infecting Escherichia coli was isolated and sequenced because of its potential for use in phage therapy. The genome sequence and annotation are available in GenBank (MF044457). These data were summarized in Table 2.

Growth conditions and genomic DNA preparation
Phage ST0 was isolated from a wastewater sample that was filtered through a 0.22-μm polycarbonate membrane filter (Millipore, Bedford, MA, USA). The host strain Escherichia coli H8 was cultured at 37°C using LB medium [13]. Phage DNA was extracted as described by Sambrook and Russell [14]. The phage lysates were concentrated in polyethylene glycol 8000 and bacterial nucleic acids were removed from phage lysates by DNase I (Sigma-Aldrich, Oakville, Canada) and RNaseA (Sigma-Aldrich). Then the phage particles were amplified and stored in SM buffer (100 mM NaCl, 8 mM MgSO4, 50 mM Tris-HCl [pH 7.5]) at 4°C.

Genome sequencing and assembly
DNA was sequenced using the Illumina HiSeq 2500 platform in Beijing Fixgene Tech Co., Ltd. More than 5000-fold coverage of the phage genome is generated by sequencing the cloned fragments. The paired-end reads were assembled using the abyss v. 1.3.6. Possible tRNAs in the genome were determined using tRNAscan-SE. These data were summarized in Table 3.

Genome annotation
The potential ORFs were predicted using PHASTER [15]. Putative protein function of ORFs was annotated by BLASTp against NCBI database and HMMER search against the COG database [16] (These data were summarized in Table 4). The map of a circular representation of phage ST0 genome was generated using CGView Server. Neighbor joining tree was drawn by MEGA 5.05 [17].

Genome properties
The complete genome sequence of phage ST0 had been deposited in GenBank with the accession number MF044457. The complete genome sequence of phage ST0 consisted of 170,496 bp and was circular double-stranded DNA with an average GC content of 37.67%. There were ten tRNAs detected in this genome indicating that phage ST0 could be reliant on its tRNAs after entering into the hosts.
A total of 269 ORFs were predicted in this complete genome, compared with those from the NCBI database ( Fig. 3; Additional file 1: Table S1). These ORFs showed more than 94% identity with 18 different phage strains. Of those, 41 ORFs were predicted in the minus strand and others were in the plus strand. Eighty nine putative ORFs were predicted to have unkown functions.

Insights from the genome sequence
Phage ST0 possessed replication-related genes encoding DNA polymerase (ORF64), DNA primase/helicase   Evidence codes -IDA inferred from direct assay, TAS traceable author statement (i.e., a direct report exists in the literature). These evidence codes are from the Gene Ontology project [20] ORF231 and ORF259). Terminase was observed in the phage ST0 genome, and plays the essential role in the double-stranded DNA packaging process. Terminase generally composed of two subunits identifies the pre-capsid protein and the specific packaging sites, providing energy to packaging process through hydrolysis of ATP [17,18]. This showed that phage ST0 possibly depended on its own terminase to obtain these kinds of function, while many phages lacked this enzyme.
Phage ST0 may be dependent on its own gene transcription and translation, because it possessed RNArelated enzymes such as tRNA synthetase modifier (ORF2), putative thioredoxin (ORF28), RNA polymerase sigma factor (ORF49), RNA ligase (ORF152 and ORF200) and other related proteins (ORF59, ORF63,  The total is based on the total number of protein coding genes in the genome ORF123, ORF140, ORF165, ORF181) to regulate gene expression. Forty one genes that were related to the structure of phage ST0 were identified in its genome, including phage virion protein and tail tube. The ORFs related to structure were mainly concentrated between 106,926 bp and 153,860 bp, which facilitated the rapid assembly of phages and also reflected the lowest energy principle in nature.
The presence of lysozyme (ORF257) and holing (ORF132) indicated that phage ST0 was a lytic phage. Moreover, other several active factors played a quite important role in host cell lysis and inhibition of host cell growth, such as exonuclease (ORF102), inhibitor of host transcription (ORF153), nudix hydrolase (ORF256) and rIIA protein (ORF113, ORF114 and ORF169).

Conclusions
The morphology, complete genome sequence and bioinformatics analysis showed phage ST0 was a novel virulent phage infecting and lysing Escherichia coli H8, which may provide a better understanding to the development of a possible alternative medicines and biocontrol agents.