1. IntroductionPhages are viruses that infect bacterial cells, and 1 × 1031 phages are estimated to be found on earth. These viruses are extremely abundant and ubiquitous in nature and can be found in aquatic environments, soil, and human/animal ecosystems [1,2,3,4]. Phages can be sorted into obligately lytic and temperate phages according to their life cycles. Similar to other viruses, phages can highjack host cells to replicate. Obligately lytic phages release their assembled phage progeny into the environment via host cell lysis [5]. Temperate phages can integrate their DNA into host chromosomes or exist as plasmids and, in most cases, have no lytic effect on their hosts [6]. The increasingly prevalent antibiotic resistance of bacteria has resulted in phages receiving increased attention as a substitute for traditional antibiotics. Phages have the advantages of strong specificity, fewer adverse effects, and relatively low drug development costs [7,8,9]. Obligately lytic phages have been widely used to inactivate and control pathogens in the food, agriculture, veterinary, clinical, and industrial sectors, among other fields. Temperate phages, such as lambda phages, have both obligately lytic and lysogenic cycles [10]. Most temperate phages encode transposases, integrases, virulence factors, and other potential safety hazards, resulting in limited applications [11,12,13]. Under most conditions, phage integration in lysogenic cells is stable enough to maintain its existence even after multiple cell divisions. Only a small number of phages can be induced spontaneously, and the induction of most prophages requires certain conditions, such as ultraviolet radiation, high osmotic pressure, H2O2, and mitomycin C [14,15,16,17]. Studies have shown that the integration of prophages increases growth, antibiotic resistance, biofilm formation, adhesion, motility, environmental stress response, and virulence of their hosts [18,19,20,21]. However, some phage genes may have opposite effects on the growth of host cells since the expression of phage proteins affects the availability of host cell resources [22].Phages are the main components (>97%) of human enteroviruses. It is estimated that there are approximately 108–109 phage particles per gram of human feces and 1015 particles in the gastrointestinal tract [23,24]. There is considerable diversity among phages, and phages in the human intestine are mainly non-enveloped dsDNA Caudovirales, ssDNA Microviridae and ssDNA filamentous phages [25,26]. Metagenomic studies have shown that most intestinal bacteria carry prophage genes [25,27]. By infecting specific populations of bacteria, phages can alter the structures of microbiota or their phenotypes, leading to intestinal homeostasis or dysbiosis and even chronic infectious and autoimmune diseases [28,29]. Temperate phages can strongly promote the virulence and adaptability of bacterial hosts by encoding virulence genes and then play a pro-inflammatory role in the intestines [4,30]. Temperate phages can increase the adaptability of symbiotic bacteria by eliminating repeated infections or lysogenic transformations to help maintain a healthy intestinal environment [31]. The induction of temperate phages leads to the death of bacterial hosts and changes population dynamics and intestinal ecological balance [32]. Temperate phages play an important role in research because of their fundamental role in intestinal health.

Research on intestinal phages has focused chiefly on metagenomics, with few studies reporting on a single individual. In this study, a novel temperate Escherichia coli phage from the gut was isolated. The biological and genomic characteristics, life cycles, and host virulence of the isolated phages were analyzed to gain valuable insights into intestinal temperate phages.

2. Materials and Methods 2.1. Bacteria and Culture ConditionsE. coli YO1 was isolated from fecal samples at Yangzhou University in 2021 and cultured in Luria-Bertani (LB) broth at 37 °C and 220 rpm. The 16S rRNA genes were amplified using the primers 27F and 1492R for identification. dinB, icdA, pabB, polB, putP, trpA, trpB, and uidA were used for multi-locus sequence typing (MLST) [33]. O antigen was determined by Gao Song’s team from Veterinary College of Yangzhou University using agglutination tests [34]. H antigen was determined by PCR and sequencing of fliC using primer fliCF/fliCR [35]. K1 antigen was determined by PCR and sequencing of kpsM using primer K1F/K1R [36]. The important virulence genes aatA, aggR, eae, bfpA, ltA, stA, ipaH, stx1, and stx2 of enteropathogenic E. coli (EPEC), enteroaggregative E. coli (EAEC), Shiga toxin-producing E. coli (STEC), enterotoxigenic E. coli (ETEC), and enteroinvasive E. coli (EIEC) were identified by PCR [37]. Antibiotic resistance was determined using standard Kirby-Bauer disk diffusion. Data analyses were performed according to the guidelines of the standardized protocol of the National Committee for Clinical Laboratory Standards and designated as R (resistant), I (intermediate sensitive), and S (sensitive). The primers used in this study are listed in Table S1. 2.2. Phage Isolation and Purification

Phage vB_EcoP_ZX5 was isolated from the fecal samples at Yangzhou University in 2021. Briefly, 1 g of fecal sample was completely mixed with 1 mL of PBS and centrifuged at 8000× g for 5 min. The resulting supernatants were passed through a 0.22 μm filter. Then, the supernatant filtrates were added to 5 mL of logarithmic phase (OD600 = 0.6) E. coli YO1 and cultured at 37 °C for 2–4 h. The lysate was centrifuged at 12,000× g for 2 min, and the phages in the supernatants were purified using the double plate method. Briefly, 300 μL of E. coli YO1 and 100 μL of a ten-fold dilution series of phages were added to 5 mL of top LB soft agar and then poured onto an LB agar plate and cultured at 37 °C overnight. The next day, a single plaque was picked from the plate, inoculated into 5 mL of logarithmic phase host, and cultured at 37 °C for 2–4 h. The cultures were centrifuged at 12,000× g for 2 min, and the lysates were passed through a 0.22-μm filter. These steps were repeated at least three times until a single phage was obtained.

PEG/NaCl was used to enrich phage particles. The phages were added to 100 mL of logarithmic phase host and cultured at 37 °C and 220 rpm for 2–4 h. The cultures were centrifuged at 8000× g for 10 min. Then, DNase I and RNase A (1 µg/mL) were added to the lysate and incubated at 37 °C for 30 min. Next, 1 M NaCl was added to the supernatant, which was then placed in an ice bath for 1 h. After centrifugation at 8000× g for 10 min, 10% (w/v) polyethylene glycol 8000 (PEG 8000) was added to the lysate and precipitated overnight at 4 °C. After centrifugation at 8000× g for 20 min, the precipitate was resuspended in PBS. The phage suspension was stored at 4 °C.

2.3. DNA Isolation and Sequencing

Phage vB_EcoP_ZX5 DNA was extracted using the Virus Genomic DNA/RNA Extraction Kit (Tiangen, Beijing, China). Whole-genome sequencing of the phage was performed by Shanghai Bioengineering Co. Ltd. Phage DNA fragments with a length of approximately 500 bp were randomly interrupted by a Covaris ultrasonic crusher (Covaris, Woburn, MA, USA) and then purified using Hieff NGS™ DNA selection beads (Yeasen, Shanghai, China). The sequencing library was constructed using the NEB Next® Ultra™ DNA Library Prep Kit for Illumina® (NEB, Ipswich, MA, USA), including terminal repair, adaptor ligation, DNA purification, and library amplification. After passing the quality test, the DNA library was sequenced using an Illumina hiseqpe150 sequencing platform. The original sequencing data were filtered at first and then were assembled using New Blew 3.0.

2.4. Genome AnalysisThe tRNAs in the genome were predicted using tRNAscan-SE 2.0 (http://lowelab.ucsc.edu/tRNAscan-SE/, accessed on 6 April 2022) [38]. The coding domain sequences (CDSs) were predicted using the RAST annotation server web (https://rast.nmpdr.org/, accessed on 10 March 2022) and aligned using BLASTX in NCBI (https://blast.ncbi.nlm.nih.gov, accessed on 6 April 2022) [39]. Virulence and drug resistance genes were compared using VFPB (http://www.mgc.ac.cn/VFs/main.htm, accessed on 6 April 2022) and CARD (https://card.mcmaster.ca, accessed on 6 April 2022) [40,41]. The genome was visualized using Easyfig 2.2.5 [42]. Genomic blast analysis was performed using the BLAST Ring Image Generator (BRIG) [43]. Multiple sequence alignment of the genomes was performed using Easyfig 2.2.5. Dot plots of the whole-genome analysis were generated using Gepard [44]. Phylogenetic tree analysis of the phage terminase large subunit was performed by MEGA 5.0 using the neighbor-joining method with 1000 bootstraps. The promoters of phage were predicted by Promoter Hunter (http://www.phisite.org/main/index.php?nav=tools&nav_sel=hunter, accessed on 10 May 2022). 2.5. Transmission Electron Microscopy (TEM)

The purified phage vB_EcoP_ZX5 was incubated on carbon grids (200 mesh) for 10 min, stained with 2% phosphotungstic acid for 1 min, and dried for 30 min. The morphology of the phage was examined using transmission electron microscopy (TEM) (HT7800; Hitachi, Tokyo, Japan).

2.6. Phage Adsorption TestThe phage vB_EcoP_ZX5 was added to a logarithmic phase E. coli YO1 (1 × 108 CFU/mL) at an multiplicity of infection (MOI) of 0.1 and cultured at 37 °C [45]. The phage titer in the culture supernatant was measured every 5 min. The experiment was repeated in triplicate. The adsorption time and efficiency of the phages were then calculated. 2.7. One-Step Growth Curve

The phage vB_EcoP_ZX5 was added to a logarithmic phase E. coli YO1 (1 × 108 CFU/mL) at an MOI of 0.1. The mixture was incubated at 37 °C for 10 min and then centrifuged at 12,000× g for 1 min to remove free phages. The precipitate was resuspended in LB broth (time zero) and cultured at 37 °C and 220 rpm. Three parallel samples were collected every 10 min and centrifuged at 12,000× g for 1 min. Phage titer in the supernatant was determined using the double agar plate method. Burst size was calculated as follows: burst size = number of released phages/number of infected bacteria. The experiments were repeated in triplicate.

2.8. Phage Stability Studies 2.8.1. Thermal Stability Test

To test for thermal stability, 300 µL of phage suspension (108 PFU/mL) was incubated at 4 °C, 40 °C, 50 °C, 60 °C, and 70 °C for 20, 40, and 60 min. The resulting phage titers were determined using the double-layer plate method. The experiments were repeated in triplicate.

2.8.2. pH Stability Test

To test for pH stability, 300 µL of phage suspension (108 PFU/mL) was incubated at different pH buffers (pH 1–13) and then incubated at 37 °C for 20, 40, and 60 min. The resulting phage titers were determined using the double-layer plate method. The experiments were repeated in triplicate.

2.9. Screening and Subculture of Phage Lysogenic Bacteria

Phage vB_EcoP_ZX5 was added to the logarithmic phase E. coli YO1 with an MOI of 0.1 and co-cultured in LB broth at 37 °C for 12 h. The cultures were then scribed to obtain monoclonal bacteria. The monoclonal strains were randomly selected and cultured in LB broth at 37 °C for 12 h. Next, the lysogenized bacteria were verified by PCR amplification of phage integrase (1F/1R) and terminase large subunit (2F/2R). The lysogenized bacterial cultures were subcultured again. These steps were repeated to obtain lysogenic E. coli YO1+ after ten passages. Primer 3F and 3R are from the 3′-end of guaA in E. coli YO1, and primer 3F2 and 3R2 are from the 5′-end of integrase in phage vB_EcoP_ZX5. Primer 3F/3R was used to amplify the bacterial attachment site (attB) in E. coli YO1, resulting in a 286bp DNA1 fragment. Primers 3F/3R2 and 3F2/3R were used to amplify the phage integration site (attP) attL and attR in E. coli YO1+, respectively, resulting in a 214bp DNA2 fragment and a 281bp DNA3 fragment. The sequences of DNA1, DNA2 and DNA3 were verified by Sanger sequencing. The titer of free phages in the lysogenized bacterial culture supernatant was determined using a double-layer plate method.

2.10. Phage Infection Characteristics

Different doses of phages (MOI = 0.1, 1 and 10) were added to the logarithmic phase host (108 CFU/mL) and cultured in LB broth at 37 °C and 220 rpm. The number of phages was determined every 30 min. The titer of total phages and prophages were determined by qPCR using primers 4F/4R and 3F2/3R. The titer of free phages in the supernatant was determined using a double-layer plate method. Primer 4F/4R was used to amplify the sequence of terminase large subunit in phage vB_EcoP_ZX5, which can amplify 230 bp fragments in all phage genomes including lytic and lysogenic cycle. Because primers 3F2 and 3R are derived from phage and bacteria, respectively, 3F2/3R can only amplify 281 bp fragments in lysogenic strains integrated with phage vB_EcoP_ZX5 in lysogenic cycle. The experiment was repeated three times. qPCR reactions were performed using a Roche Light Cycler 480 II PCR instrument (Roche, Basel, Switzerland) and ChamQ Universal SYBR qPCR Master Mix (Vazyme, Nanjing, China), according to the manufacturer’s instructions. The cycling conditions were a step at 95 °C for 30 s, followed by 40 cycles of 10 s at 95 °C and 30 s at 60 °C, and finally 95 °C for 15 s, 60 °C for 60 s, 95 °C for 15 s, and 37 °C for 2 min.

2.11. Construction of Overexpression Plasmids

The candidate genes that affected the phage vB_EcoP_ZX5 lytic–lysogenic cycles were verified by gene overexpression. Plasmid pGEX-6P-1 (GenBank: U78872.1) carrying GST tag was used to express phage genes. Gp4 anti-repressor, gp12 integrase, and gp21 repressor with his tag were amplified using primers 5F/5R to 7F/7R and cloned into the plasmid pGEX-6P-1 using the ClonExpress II One Step Cloning Kit (Vazyme). The products were transformed into DH5α competent cells, and the monoclonal strains were cultured for sequencing identification (8F/8R). The positive plasmid pGEX-6P-x was extracted using Plasmid Mini Kit (Vazyme) and stored at −20 °C. E. coli YO1 was cultured in the logarithmic phase, and competent cells were prepared. The plasmids were electro-transformed into E. coli YO1 using an electroporator (Eppendorf, Hamburg, Germany). E. coli YO1 transformed with plasmid pGEX-6P-x was identified by PCR (8F/8R) and was cultured to logarithmic phase in LB broth at 37 °C. E. coli YO1 carrying the empty plasmid pGEX-6P-1 containing GST tag (GST) was used as a negative control. Isopropyl ß-D-1-thiogalactopyranoside (IPTG) at a final concentration of 1 nM was added to the bacterial culture and then grown for 6 h. The protein expression of plasmid pGEX-6P-x was characterized by western blotting using anti-His-Tag mouse monoclonal antibody (Cwbio, Taizhou, China) and goat anti-mouse IgG conjugated to horse-radish peroxidase (Cwbio).

2.12. Effect of Gene Overexpression on Phage Cycles

E. coli YO1 with plasmid pGEX-6P-x was cultured to logarithmic phase in LB broth at 37 °C. IPTG at a final concentration of 1 nM and phage vB_EcoP_ZX5 at a final concentration of 107 PFU/mL were added to the bacterial culture and then grown for 6 h. After adding phages, 3 mL of cultures were collected every hour. Next, 1 mL of culture was rapidly frozen and stored at –80 °C to determine the titer of total phages and prophages using qPCR. Then, 1 mL of culture was centrifuged at 12,000× g for 2 min, and the titer of free phages in the supernatants was determined using the double-layer plate method. Lastly, 1 mL of culture was centrifuged at 12,000× g for 2 min, and the bacterial precipitate was stored at –80 °C until the transcription level of phage genes was analyzed by qPCR. The experiment was repeated three times.

The total RNA of the bacteria was extracted by TRNzol (Tiangen, Beijing, China) as follows: the bacterial precipitation was resuspended with 200 μL of TRNzol, 200 μL of chloroform was added and centrifuged at 12,000× g for 15 min, the supernatant was transferred to a new centrifuge tube with 200 μL of isopropanol, the tube was placed in an ice bath for 1 h and then centrifuged at 12,000× g for 15 min, the resulting supernatant was discarded, and the RNA was dissolved in RNase-free water. RNA samples were reverse-transcribed using HiScript® III RT SuperMix for qPCR (Vazyme). The transcription levels of phage integrase (9F/9R), repressor (10F/10R), anti-repressor (11F/11R), lysin (12F/12R) and holin (13F/13R) were measured by qPCR. The experiment was repeated three times. The cycling conditions were a step at 95 °C for 30 s, followed by 35 cycles of 10 s at 95 °C and 30 s at 60 °C, and finally 95 °C for 15 s, 60 °C for 60 s, 95 °C for 15 s, and 37 °C for 2 min.

2.13. Bacterial Growth Curve and Resistance to Environmental Stress

Overnight cultured E. coli YO1 and E. coli YO1+ were inoculated into a fresh medium (107 CFU/mL), and the OD600 of bacteria was measured using a Nanophotometer Ultramicro Spectrophotometer (Implen, Munich, Germany) every 30 min. The experiment was repeated three times.

The responses of the E. coli YO1 and E. coli YO1+ to external stress were evaluated by measuring the change in the bacterial count under different stresses. The logarithmic phase bacteria (5 × 107 CFU/mL) were treated with acid buffer (pH 3), alkaline buffer (pH 10), and 30 mM H2O2 at 37 °C for 10 min. The logarithmic phase bacteria were treated at 50 °C for 10 min to test for heat stress. After treatment, cell viability was measured using the plate counting method. The experiment was repeated three times.

2.14. Biofilm Assay

The overnight cultured E. coli YO1 (group YO1) and E. coli YO1+ (group YO1+) were transferred to fresh medium in 96-well polystyrene plates (Corning Costar, Corning, NY, USA) at a ratio of 1:100, setting PBS as blank control. The bacteria of each group were cultured at 37 °C, in which the culture medium was replaced with fresh LB every 24 h. After three days of culture, biofilm formation was measured using crystal violet staining. Each well was washed thrice with ddH2O and dried. Crystal violet (200 μL) was added to each well and incubated for 10 min. This was followed by washing each well thrice with ddH2O three times and drying. Glacial acetic acid (200 μL) was added to each well and incubated at for 10 min. The OD600 of each well was measured using a smart microplate reader (Infinite M200 Pro; Tecan, Männedorf, Switzerland). The experiment was repeated three times.

2.15. Adhesion and Invasion AssaysHeLa cells were used to analyze the adhesion and invasion abilities of E. coli YO1 or E. coli YO1+ [46]. In short, HeLa cells were inoculated on a 24-well plate and were cultured in a 5% (v/v) CO2 incubator at 37 °C to form monolayer cells (105 cells per well). The logarithmic phase E. coli YO1 or E. coli YO1+ cells were resuspended in the FBS free DEME medium (107 CFU/mL), and then co-incubated with HeLa cells at 37 °C for 2h. For adhesion test, the cells were washed three times with PBS and then lysed with 0.5% (v/v) Triton X-100. The number of bacteria was calculated on LB plates. For the invasion test, after the cells were co-incubated with bacteria, ampicillin antibiotics (100 μg/mL) were added and incubated at 37 °C for 2 h to kill the bacteria on the surfaces of cell. Cells were cleaned by PBS for three times and then lysed with 0.5% (v/v) Triton X-100. 2.16. Analyses of Serum Sensitivity and Virulence of E. coli YO1

Female BABL/C mice, aged 6–8 weeks, were purchased from the Experimental Animal Center of Yangzhou University. All animal experiments were performed in strict accordance with the Regulations for the Administration of Affairs Concerning Experimental Animals, approved by the Animal Welfare and Research Ethics Committee of Yangzhou University.

The blood of healthy mice was collected and immediately stored at 37 °C for 2 h and then centrifuged at 2000× g for 5 min to collect serum. A total of 100 μL of E. coli YO1 and E. coli YO1+ (104 CFU/mL) were mixed with 100 μL of mouse serum. After incubation at 37 °C for 1 h, the cell viability was determined. The experiment was repeated three times.

E. coli YO1 and E. coli YO1+ were cultured overnight and resuspended in PBS after centrifugation. The mice were divided into three groups, with six mice per group. Mice were intraperitoneally injected with E. coli YO1 (109 CFU), E. coli YO1+ (109 CFU), or PBS at a total volume of 100 μL. The survival of the mice was recorded every day. Three mice were euthanized two days after infection. The main organs were collected and homogenized by dissection, and the bacterial load was measured using the plate counting method.

4. Conclusions

In this study, the temperate phage vB_EcoP_ZX5 and its host E. coli YO1 (O87:H52:K1) were isolated from human fecal samples. The biological characteristics, genomic characteristics, and lytic–lysogenic cycles of the phage were evaluated. The phage vB_EcoP_ZX5 was found to have a short tail and belong to the genus Uetakevirus and the family Podoviridae. The phage vB_EcoP_ZX5 integrated into the 3′-end of guaA of E. coli YO1 to form lysogenized bacteria that existed stably. The integration of vB_EcoP_ZX5 had no significant impact on the growth rate, biofilm, stress environment response, serum sensitivity, antibiotic sensitivity adherence to HeLa cells, or virulence of E. coli YO1. In order to identify the key proteins affecting phage lytic–lysogenic cycles, the changes in phage gene (repressor, endolysin, and holin) transcription level and phage titer (total phages, free phages, and prophages) caused by overexpression of ORF12 integrase, ORF21 repressor protein, or ORF4 anti-repressor protein were analyzed. The results show that the high level of ORF21 repressor completely prevented the phage from entering the lytic cycle, whereas the ORF4 anti-repressor protein could enhance the phage from entering the lytic cycle. Our findings contribute to the enrichment of the temperate phage information library, and the mystery of how repressor and anti-repressor proteins take effect on phages’ life cycles remains to be unraveled.