Escherichia coli SD405 and 100 additional clinical isolates of E. coli were obtained from the Phage Research Center Laboratory, Liaocheng University. Each strain was streaked onto MacConkey agar plates for activation after retrieval from the strain bank and incubated inverted at 37°C overnight. A single colony from each plate was inoculated into 5 mL of LB broth in centrifuge tubes and incubated at 37°C and 200 rpm for 8–12 h to ensure active growth.

The isolation and purification of the phage was tested using a modified version of the method described by Abdelrahman et al. (2022). A 200 μL sample of sewage-derived phage was added to a centrifuge tube containing 5 mL of LB broth and 200 μL of the corresponding E. coli host culture. The mixture was incubated at 37°C and 220 rpm for 4 h. After incubation, the culture was centrifuged at 5000 rpm for 5 min, and the supernatant was filtered through a 0.22 μm membrane to obtain the fresh phage lysate. For purification, a single plaque is picked using a sterile pipette tip, transferred into SM buffer, and vortexed thoroughly. The mixture is then filtered through a 0.22 μm filter. The filtrate is combined with host bacteria to prepare double-layer plates again. The process of picking a single plaque is repeated for 4–6 times to obtain purified phage.

A high-titer phage lysate was prepared using the double-layer agar amplification method. A logarithmic-phase bacterial culture was mixed with the phage at the optimal multiplicity of infection (MOI) and incubated at 37°C in a water bath for 20 min. Luria-Bertani (LB) semi-solid agar (approximately 50°C) was added, mixed, and poured onto LB solid agar plates. After solidification, the plates were inverted and incubated at 37°C for 8–10 h. Once phage plaques fully developed, 3–5 mL of SM buffer was added to the plate, followed by shaking at 100 rpm for 4 h. The eluate was collected and clarified by low-speed centrifugation (8,000 × g, 10 min, 4°C) to remove bacterial debris. The buffer was then filtered through a 0.22 μm membrane.

For titer determination, 10-fold serial dilutions (e.g., 10^–1 to 10⁻⁸) of the lysate were prepared in LB broth. A 100 μL aliquot of each dilution was mixed with 100 μL of a fresh logarithmic-phase host culture, to make a final mixture of 200 μL. This mixture was then blended with LB semi-solid agar (55°C), and immediately overlaid onto LB plates. After overnight incubation at 37°C, plaques were counted. The phage titer (PFU/mL) was calculated using the formula: Titer = (number of plaques) × (dilution factor) × 10.

For morphological observation by transmission electron microscopy (TEM), phage particles were concentrated and purified. The filtered lysate was treated with polyethylene glycol 8000 (PEG 8000) at a final concentration of 10% (w/v) and incubated at 4 °C for 1 h to overnight. The mixture was then centrifuged at 12,000 × g for 30 min at 4 °C. The resulting pellet was resuspended in a minimal volume of SM buffer. When higher purity was required, an additional purification step using CsCl density gradient centrifugation or ultrafiltration concentrators was performed. The concentrated phage preparation was applied to carbon-coated copper grids, negatively stained with 2% (w/v) phosphotungstic acid (pH 7.0), and examined using transmission electron microscopy (TEM) (Li et al., 2022).

To determine the phage Lysis Profile, we utilized the method described by Kizheva et al. (2025). The host range of the phage was determined using the double-layer agar drop method. A 5 mL aliquot of LB semi-solid agar (approximately 55°C) was poured into each of multiple 10 mL centrifuge tubes containing 100 mL of different E. coli cultures, mixed thoroughly, and poured onto a pre-prepared LB agar plate, followed by gentle shaking for even distribution. Then, 5 μL of phage lysate was dropped onto the solidified surface and allowed to stand for 10 min before incubation at 37°C for 8–10 h. The formation of clear plaques indicated that the phage could lyse the bacterial strain, whereas no plaque formation indicated the absence of lytic activity. Some strain information was consistent with previously published reports from our laboratory (Huang et al., 2025).

For bacterial strains that tested positive in the spot assay, productive infection was further assessed by determining the efficiency of plating (EOP) (Campos et al., 2025). Overnight cultures of each test strain and the primary host strain, E. coli SD405, were freshly grown in LB broth at 37 °C for 12 h. Phage lysates were serially diluted (10^–3 to 10⁻⁷), mixed with different host bacteria, and titers were determined using a standard double-layer agar plaque assay, followed by overnight incubation at 37 °C. All assays were performed in triplicate.

The EOP was calculated as the mean plaque-forming units (PFU) on a test strain divided by the mean PFU on the primary host, E. coli SD405. An EOP value ≥ 0.5 was considered to indicate high production efficiency, reflecting strong replication in the test host. Values between 0.1 and 0.5 were categorized as medium efficiency, corresponding to a moderate level of productive infection.

To determine the optimal multiplicity of infection (MOI) of the bacteriophages, we utilized the method described by Li et al. (2019), The MOI, defined as the ratio of phage particles to host bacterial cells, was determined as follows. The host bacterial suspension was adjusted to 10⁸ CFU/mL, and phage suspensions were adjusted to 10⁸, 10⁷, 10⁶, and 10⁵ PFU/mL. Equal volumes of host and phage suspensions were mixed to achieve MOI values of 10, 1, 0.1, 0.01, and 0.001, respectively. The mixtures were incubated at 37°C in a shaking incubator for 3 h, followed by centrifugation at 12,000 rpm for 2 min and filtration through a 0.22 μm membrane. The resulting phage lysates were serially diluted (10-fold), and the phage titer was determined using the double-layer agar method. The MOI yielding the highest titer was recorded as the optimal MOI for that phage.

The stability of PH was determined using a modification of previously described methods (Wu et al., 2025). The pH stability of the phage was assessed by adjusting LB broth to pH 1–14 using hydrochloric acid or sodium hydroxide. A phage lysate with a titer of 4.74 × 10⁸ PFU/mL was used for the assay. Specifically, 100 μL of this phage lysate was mixed with 900 μL of LB broth at the designated pH and incubated at 37°C for 1, 2, and 3 h, respectively. The mixture was then centrifuged at 12,000 rpm for 5 min, filtered through a 0.22 μm membrane, and serially diluted (10-fold) for titer determination using the double-layer agar method. Each experiment was performed in triplicate to ensure reproducibility.

The chloroform stability of the phage was tested using a modified version of the method described by Gao et al. (2023). The phage suspension used had a titer of 3.1 × 10⁸ PFU/mL. Phage suspensions (1.0, 0.9, 0.8, 0.7, 0.6, and 0.5 mL) were added to 1.5 mL microcentrifuge tubes, and chloroform solutions (0, 10, 20, 30, 40, and 50%) were added to reach a final volume of 1 mL. The samples were mixed thoroughly and incubated at 37°C for 30 min, 1 h, and 2 h, respectively. The mixtures were then centrifuged at 12,000 rpm for 10 min. Aliquots (100 μL) from each tube were serially diluted (10-fold), and the phage titer was determined using the double-layer agar method. Each test was performed in triplicate.

The stability of temperature was determined using a modification of previously described methods (Cao et al., 2023). A phage lysate with a titer of 5.2 × 10⁸ PFU/mL was used for the assay. To evaluate thermal stability, 500 μL of phage lysate was incubated in a water bath at 4, 25, 40, 50, 60, 70, and 80°C. Samples were collected at 20, 40, and 60 min for each temperature. The lysates were serially diluted (10-fold), and phage titers were determined using the double-layer agar method. Each experiment was conducted in triplicate to ensure reproducibility.

To determine the one-step growth curve of the bacteriophages, we utilized the method described by Wan et al. (2025), A phage lysate with a titer of 1.5 × 10⁸ PFU/mL was used. The one-step growth curve was determined by mixing the host bacterial culture and phage lysate at the optimal MOI and incubating at 37°C for 5 min. The mixture was centrifuged at 12,000 rpm for 5 min, and the supernatant was discarded. The pellet was washed twice with preheated LB broth (37°C), resuspended in 1 mL of preheated LB broth, and transferred to 100 mL of preheated LB medium. Incubation was carried out at 37°C for 120 min in a constant-temperature shaker. Beginning at 0 min, samples were collected every 10 min, filtered through a 0.22 μm membrane, and used to obtain phage lysates. After 10-fold serial dilution, titers were determined using the double-layer agar method. The burst size (PFU/cell) was calculated using the following equation: Burst size = (Final phage titer – Initial phage titer)/Initial number of infected host bacteria. All experiments were performed in triplicate.

Phage DNA was extracted using the E.Z.N.A.^® DNA Kit (OMEGA, United States). Sequencing libraries were prepared using the TruSeq™ Nano DNA Sample Prep Kit (Illumina, United States), and whole-genome sequencing was conducted on the Illumina NovaSeq 6000 platform by Shanghai Lingen Biotechnology Co., Ltd. (Shanghai, China). Raw data quality control was performed using Trimmomatic v0.36 (Bolger et al., 2014), and ABySS v2.2.0 was used for genome assembly. Protein-coding genes were predicted using GeneMarkS v4.17 (Besemer and Borodovsky, 2005), a tool optimized for viral genomes. The predicted gene products were functionally annotated through BLASTp searches against the NCBI non-redundant protein database (NR) to assign putative functions. Functional classification information was further retrieved from the COG, GO, and KEGG databases based on the BLASTp results. Protein domain analysis (e.g., Pfam) and identification of specific enzyme families (e.g., CAZy) were similarly performed using information derived from these BLASTp alignments. Sequence similarity was assessed via BLASTp searches against the NCBI database¹ (Altschul et al., 1997). tRNA genes were predicted using tRNAscan-SE v2.0² (Lowe and Chan, 2016). Virulence and antibiotic resistance genes were identified using the Virulence Factor Database³ and the Comprehensive Antibiotic Resistance Database (CARD)⁴ (Cao et al., 2023). The lifestyle of phage vB_EcoM_SD286 was computationally predicted using the bioinformatics tool PhaBOX (Shang et al., 2022). The complete genome sequence of the phage was submitted to GenBank, and BLASTn was used to compare phage vB_EcoM_SD286 with sequences available in the database. Phage tBLASTx analysis was conducted via the Virus Proteome Tree (Viptree) server⁵ (Nishimura et al., 2017) to construct a phage proteome tree based on whole-genome similarity (SG). Phylogenetic analysis was performed using the VICTOR web service⁶ (Meier-Kolthoff and Göker, 2017), a method commonly used for phage genome-based phylogeny and classification. Based on the NCBI BLASTn results, 15 homologous sequences with the highest similarity were selected. Pairwise genomic distances and similarities were calculated using the VIRIDIC platform⁷ (Moraru et al., 2020).

Based on the amino acid sequence of the gp38 protein, its basic physicochemical characteristics were analyzed using the ProtParam platform⁸ (Gasteiger et al., 2003). The hydrophilicity and hydrophobicity of gp38 were evaluated using ProtScale.⁹ Transmembrane domain prediction was performed using the TMHMM Server v2.0¹⁰ (Santos Junior et al., 2020), and signal peptide analysis was conducted using NovoPro SignalP¹¹ (Almagro Armenteros et al., 2019). The secondary and tertiary structures of gp38 were predicted using PSIPRED¹² (Buchan et al., 2024) and the SWISS-MODEL homology modeling server¹³ (Waterhouse et al., 2018), respectively. Furthermore, the three-dimensional structures of the three proteins relevant to the subsequent experiments were predicted using AlphaFold3¹⁴ (Jumper et al., 2021).

Construction of the recombinant plasmid was performed using a modified version of the method described by Azizoglu et al. (2016). Primers targeting the gp38 gene of phage vB_EcoM_SD286 were designed using SnapGene software, based on the previously sequenced genome. Restriction enzyme sites BamHI and XhoI were incorporated into the primers. The forward primer 5’-CGGGATCCATGGCACAAGCGAAAGCTGACC-3’, and reverse primer 5’-CCCTCGAGTTTACTCGGCTTCTTCCGTAGCT-3’ were synthesized by Sangon Biotech (Shanghai, China). Using the designed gp38-F/R primers and phage DNA as the template, Polymerase Chain Reaction (PCR) amplification was performed to obtain the target fragment. The PCR amplification was performed under the following conditions: Initial denaturation at 95°C for 5 min, followed by 30 cycles at 94°C for 1 min, 67.9°C for 1 min, 72°C for 0.5 min, and a final extension step at 72°C for 10 min. Both the pET-28a(+) vector and the gp38 insert were digested with BamHI and XhoI (Takara Bio Inc.), followed by ligation at 16°C overnight using T4 DNA ligase (Takara Bio Inc.). The recombinant constructs were verified by colony PCR and double-enzyme digestion. Positive colonies were confirmed by Sanger sequencing, and successfully constructed recombinant plasmids pET-28a(+)-gp38 were stored at −80°C for future use.

Following an identical experimental procedure, recombinant plasmids for the control proteins were constructed. The primers used were as follows:

For gp66 (GenBank: YAO58176.1): Forward 5’-CGGGATC CATGTATATGGCTGATGTAC-3’, Reverse 5’-CCAAGCTTG TTACGATACGTTGCTTACG-3’; For lysozyme (lys) (GenBank: YP_009845677.1): Forward 5’-ATGAAAGTGCAAT ACAAAGATATTGAT-3’, Reverse 5’-CTATCGGGTACGTTCT AAAACG-3’.

Appropriate annealing temperatures and extension times were optimized for each protein respectively. The resulting recombinant plasmids were stored at −80°C.

The recombinant plasmid pET-28a(+)-gp38 was transformed into E. coli BL21(DE3) competent cells (Sangon Biotech) to generate the recombinant strain BL21-pET-28a(+)-gp38. The same transformation procedure was followed for the expression of the gp66 and lys proteins. An overnight culture of the recombinant strain was inoculated at a 1:100 ratio into LB broth supplemented with kanamycin (Solarbio) and incubated until the logarithmic phase (OD₆₀₀ = 0.4–0.6). Isopropyl β-D-Thiogalactopyranoside (IPTG) was added to a final concentration of 0.5 mM, and induction was carried out for 5 h. The culture was centrifuged at 4°C and 12,000 rpm for 10 min, and the cell pellet was collected. The cells were washed twice with phosphate-buffered saline (PBS), sonicated at 4°C, and centrifuged again at 4°C and 12,000 rpm for 15 min. The supernatant and pellet were collected separately, and protein expression in both fractions was analyzed using sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE).

The SDS-PAGE gels were prepared using a 12.5% ExpressCast PAGE precast gel kit. After cleaning and assembling the glass plates, the separating gel was prepared according to the instructions, poured to an appropriate height, and overlaid with water for 20 min. Following the removal of the water layer and drying, the stacking gel was added, and a comb was inserted until complete polymerization. The polymerized gel was installed in the electrophoresis tank and immersed in running buffer. The boiled protein samples were cooled to room temperature prior to loading, with 10 μL of sample loaded per well alongside 5 μL of protein marker as a reference. Electrophoresis was initially performed at 80 V until the dye front entered the separating gel(typically about 20 min), after which the voltage was increased to 120 V and maintained until the dye front reached the bottom of the gel. Upon completion, the gel was removed and stained with Coomassie Brilliant Blue for 2–4 h (or overnight), followed by destaining until clear protein bands were visible. Finally, the gel was photographed and analyzed.

For purification, nickel affinity chromatography was performed to isolate inducibly expressed recombinant proteins. The inclusion bodies were dissolved in column equilibration buffer (100 mM NaH₂PO₄, 10 mM Tris-Cl, 8 M urea, pH 8.0) and incubated at room temperature for 30–60 min. The mixture was then centrifuged at 12,000 rpm for 30 min to remove insoluble material. A nickel–agarose column was pre-equilibrated by rinsing with five column volumes of deionized water, followed by five column volumes of equilibration buffer. The supernatant was loaded onto the column, and unbound material was washed off with five column volumes of washing buffer (100 mM NaH₂PO₄, 10 mM Tris-Cl, 10 mM imidazole, 8 M urea, pH 8.0). The target protein was then eluted using five column volumes of elution buffer (100 mM NaH₂PO₄, 10 mM Tris-Cl, 250 mM imidazole, 8 M urea, pH 8.0). Refolding of inclusion body proteins was performed following previously reported methods (Ni et al., 2016; Ye et al., 2019). Protein purity across the elution fractions was assessed by SDS–PAGE, and the molecular size was reconfirmed. The protein concentration was determined using the Bradford Protein Assay Kit (Beyotime Biotechnology). The purified recombinant gp38 protein was stored at −80°C until further use.

The effect of the gp38 protein on phage adsorption was verified through competitive adsorption assays using E. coli SD405 (10⁸ CFU/mL) and phage vB_EcoM_SD286 (10⁸ PFU/mL). The optimal MOI for vB_EcoM_SD286 was 0.01, and the incubation period was 20 min. The procedure was adapted from previously reported methods (Ge et al., 2023), as described below. Group 1: E. coli SD405 (1 mL) was mixed with 10 μL of vB_EcoM_SD286 and incubated at 37°C in a shaker for 15 min. Group 2: E. coli SD405 (1 mL) was mixed with 140 μL of gp38 protein (1.6 mg/mL) and incubated at 37°C for 20 min, followed by the addition of 10 μL of vB_EcoM_SD286 and further incubation for 15 min under the same conditions. Group 3: SM buffer (1 mL) was mixed with 10 μL of vB_EcoM_SD286 and incubated at 37°C for 15 min. Group 4: E. coli SD405 (1 mL) was mixed with 70 μL of gp38 protein (0.8 mg/mL) and incubated at 37°C for 20 min. Then, 10 μL of vB_EcoM_SD286 was added, mixed thoroughly, and incubated at 37°C for 15 min. LB broth was added to equalize the final volume with that of Group 2. After incubation, all mixtures were centrifuged at 4°C and 12,000 rpm for 10 min. The supernatants were collected, and the titers of free vB_EcoM_SD286 were determined using the double-layer agar method. A negative control was included using an unrelated protein that neither affected phage adsorption nor bound to the host bacterium, and a positive control was established using gp66, which shares homology with the tail filament protein gp41 previously reported in vB_EcoM_SD350 (Huang et al., 2025).

To assess potential bacteriolytic activity of the proteins, bacterial viability was evaluated. E. coli SD405 (1 mL at 10⁸ CFU/mL) was incubated with each test protein—gp38, gp66, or Lys—at a final concentration of 1.6 mg/mL, along with a control treated with an equal volume of LB broth. All samples were incubated at 37 °C for 20 min, with consistent final reaction volumes across groups. Following incubation, the mixtures were serially diluted 10-fold in sterile LB broth, and 100 μL of each dilution was plated onto LB agar. Plates were incubated overnight at 37 °C, and bacterial concentration was determined by colony counting (CFU/mL). Each test was performed in triplicate.

This experiment was conducted with modifications based on established methods (Ding et al., 2024). Host bacteria (100 μL) were inoculated into 5 mL of LB broth and incubated at 37°C and 220 rpm until reaching an OD₆₀₀ of 0.3 (approximately 10⁸ CFU/mL). The culture (5 mL) was transferred to a 10 mL centrifuge tube, centrifuged at 8,000 rpm for 10 min at room temperature, and the supernatant was discarded. The bacterial pellet was washed three times with 1 mL of 1 × PBS and resuspended in 1 × PBS containing gp38 protein to yield a final concentration of 10⁸ CFU/mL. The bacteria–protein mixture was incubated for 0.2, 0.5, 2, 4, 8, 12, 16, and 20 min, followed by rapid centrifugation at 4°C and 12,000 × g for 30 s. The supernatant was collected, and protein concentration was determined using a BCA protein assay kit. A negative control consisted of protein solution not incubated with bacteria, and a positive control consisted of bovine serum albumin (BSA, 1.5 mg/mL) incubated with host bacteria.

All data were analyzed using GraphPad Prism v8.4.3. Statistical significance was evaluated by multiple t-tests, and all experiments were performed in triplicate. Results are presented as mean ± standard deviation (SD) (Wen et al., 2024).

Phage vB_EcoM_SD286 was isolated from sewage at a farm in Shandong Province and underwent six rounds of purification. Using the double-layer agar plate method, the titer of vB_EcoM_SD286 was 1.5 × 10⁸ PFU/mL, producing uniformly sized phage plaques (Figure 1A). TEM revealed that vB_EcoM_SD286 belongs to the tailed phage order, muscle-tailed phage family, with a head shaped like a regular icosahedron and a tail exhibiting helical symmetry (Figure 1B). E. coli strains isolated from different regions between 2018 and 2024 by our laboratory were used to determine the host range of vB_EcoM_SD286 through the double-layer plate spot method. The phage was found to infect 39 E. coli strains, showing a coverage rate of 39% (Table 1).

The bacteriophage vB_EcoM_SD286 showed productive infection in only a subset of the tested E. coli strains, while the majority of isolates proved resistant (Table 1). High replication efficiency, defined as an EOP value ≥ 0.5, was observed on several strains, including E. coli SD288, SD350, SD324, DZC1-1, and DZC10-1. An additional strain, E. coli SD339, supported plaque formation with an EOP above 0.1, albeit with visibly fewer plaques.

Overall, vB_EcoM_SD286 displays a narrow host range, with productive infection confined to a limited number of susceptible hosts. Among the 39 strains tested, only six—besides the primary host strain—were effectively infected. The vast majority of strains exhibited complete resistance, yielding EOP values of zero. These results indicate that vB_EcoM_SD286 is a highly specific phage whose infectivity is restricted to a narrow set of host strains.

At an MOI of 0.01, the titer of vB_EcoM_SD286 was highest (approximately 5.36 × 10⁹ PFU/mL), confirming this as the optimal MOI (Figure 2A). The phage retained high titers across a broad pH range (5–11) for up to 180 min, though its activity declined in a time-dependent manner at pH 4 and pH 12. Exposure to pH 3 or pH 13 reduced the titer to undetectable levels within 1 h (Figure 2B). Treatment with chloroform at volume ratios ranging from 0 to 50% for 30, 60, or 120 min did not significantly affect phage viability, further confirming the absence of lipid components in its structure (Figure 2C). In temperature stability assays, vB_EcoM_SD286 remained stable after incubation at 4 °C, 25 °C, and 40–50 °C for 20, 40, or 60 min. A slight titer reduction was observed after 20 min at 60 °C. At 70 °C, the titer decreased sharply in a time- and temperature-dependent manner, and the phage was completely inactivated within 20 min at 80 °C (Figure 2D). A one-step growth curve revealed a latency period of approximately 25 min, with the titer increasing rapidly between 25 and 40 min. From 40 to 60 min, the titer increased gradually until reaching a plateau after 60 min, with a burst size of approximately 33 PFU/cell (Figure 2E).

The circular diagram of the phage vB_EcoM_SD286 genome is shown in Figure 3. Whole-genome sequencing revealed that phage vB_EcoM_SD286 is a linear double-stranded DNA molecule with a total length of 52,891 bp and a GC content of 46.06%. No tRNA genes, virulence factors, or antibiotic resistance genes were detected (Figure 3). We utilized PhaBOX (PhaTYP), a state-of-the-art deep learning tool, to analyze the genome. The analysis classified vB_EcoM_SD286 as “virulent phage” with high confidence. Genome annotation identified 74 open reading frames (ORFs), including 35 ORFs on the forward strand and 39 ORFs on the reverse strand. Among them, 31 ORFs encoded proteins with known functions, while 43 ORFs were predicted as hypothetical proteins (Table 2).

Based on the 5633 phage genomes recorded in the Viptree database, vB_EcoM_SD286 was analyzed and an evolutionary tree was constructed using whole-genome similarity scores (SG) (Figure 4A). Thirty-nine phage sequences showing the highest similarity to vB_EcoM_SD286 were used to construct a local phylogenetic tree, excluding sequences with low or no similarity (Figure 4B). The analysis revealed that vB_EcoM_SD286 (PX226730.1) is most closely related to Salmonella phage UPF_BP2 (NC_048649), followed by Salmonella phage BP63 (NC_031250). Both UPF_BP2 and BP63 belong to the Caudoviricetes class and the Rosemountvirus genus. To further clarify the relationship between Escherichia phage vB_EcoM_SD286 and related phages, a Virus Classification and Tree Building (VICTOR) analysis was conducted based on whole-genome sequences (Figure 4C). The phylogenetic tree showed that vB_EcoM_SD286 clustered in the same branch as Escherichia phages vB_EcoM_IntR, vB_EcoM_swi3, and vB_EcoM_SD350. According to the International Committee on Taxonomy of Viruses (ICTV) classification system and phylogenetic analysis, vB_EcoM_SD286 was assigned to the Caudoviricetes class and Rosemountvirus genus.

Fifteen highly homologous phage genomes were extracted from NCBI and analyzed using VIRIDIC. As shown in Figure 4D, Escherichia phage vB_EcoM_SD286 exhibited high genomic similarity with all other strains (most exceeding 94%), especially with Escherichia phage vB_EcoM_SD350 (OR911937.1), showing 99.4% similarity, indicating an extremely close genetic relationship.

Further genomic structural analysis revealed that vB_EcoM_SD286 exhibited an aligned genome fraction close to 1.0 compared with the reference strain, along with highly consistent genome length ratios. These findings indicate that vB_EcoM_SD286 possesses a complete genomic structure, showing conservatism and high colinearity within this phage lineage.

Using the ProtParam server to predict and analyze the physicochemical properties of gp38, it was determined that the protein contains 123 amino acids, with a molecular mass of 13.75306 kDa, a theoretical isoelectric point (pI) of 9.65, and a half-life of > 10 h in E. coli. The instability index was 33.98, indicating that gp38 is a stable protein. The ProtScale server was used to predict the hydrophilicity and hydrophobicity of gp38 (Figure 5A). Most amino acid residues were found in hydrophilic regions, indicating that gp38 is a stable hydrophilic protein. Using the TMHMM Server v2.0 and Novopro Server, the transmembrane domains and signal peptides of gp38 were predicted. TMHMM analysis showed no transmembrane helices, with a 49.6% probability that the N-terminus is located within the membrane and no significant membrane localization tendency (Figure 5B). The entire sequence was predicted to be located outside the membrane. The probability of a signal peptide was 0.379%, far below the 5% threshold, clearly indicating the absence of a signal peptide (Figure 5C). Secondary structure prediction using PSIPRED showed that α-helices, β-sheets, and random coils accounted for 44.7, 28.5, and 26.8%, respectively (Figure 5D). Tertiary structure prediction using SWISS-MODEL indicated that gp38 exhibits an irregular overall shape (Figure 5E). Furthermore, the three-dimensional structures of the three relevant proteins—gp38, gp66, and lysozyme(lys)—were predicted using AlphaFold3 and are presented in Figure 5F. Based on analysis of the three-dimensional structure, the gp38 protein predominantly exists as a monomer. In the structural model of gp38, the location of the DUF domain (residues 6–119) is explicitly indicated.

The gp38 fragment was amplified by PCR from the vB_EcoM_SD286 genome using specific primers. The gp38 gene fragment was inserted into the BamHI and XhoI sites to construct the recombinant plasmid pET-28a(+)-gp38, which was then expressed in the E. coli BL21(DE3) strain, yielding BL21-pET-28a(+)-gp38. The expression strain was induced with IPTG, and the cells were sonicated to release the target protein. SDS–PAGE analysis revealed that gp38 was clearly expressed in the precipitate fractions at different IPTG concentrations (Figure 6A). Therefore, 37°C and 0.5 mmol/L IPTG were selected as the optimal induction conditions for subsequent experiments.

After large-scale induction, inclusion bodies were collected and subjected to Ni–NTA affinity column purification. The target protein fraction eluted with imidazole was analyzed by SDS–PAGE, confirming high purification efficiency. The purified protein was then refolded through multi-step urea gradient dialysis renaturation. SDS–PAGE analysis of samples before and after denaturation and renaturation confirmed successful inclusion body protein refolding (Figure 6B). The concentration of the refolded gp38 protein was measured using the Bradford protein assay kit, yielding a final concentration of 1.15 mg/mL.

The control proteins gp66 and lys were expressed, purified, and refolded using an identical experimental workflow. All proteins were subsequently prepared for functional assays.

To validate the effect of recombinant gp38 protein on the adsorption of phage vB_EcoM_SD286 to host bacteria, competitive adsorption experiments were conducted to evaluate its inhibitory effect by measuring free phage titers. The results showed that recombinant gp38 protein significantly inhibited the adsorption of phage vB_EcoM_SD286. As shown in Figure 7A, at a gp38 concentration of 1.6 mg/mL, the free phage titer was significantly higher than that of the negative control group (without added competitor), indicating that gp38 competitively occupied receptor sites on the surface of E. coli SD405, thereby blocking normal phage adsorption. In contrast, no competitive inhibition was observed in the lysozyme (Lys) control group (Figure 7c), ruling out non-specific interference and confirming the specific competitive adsorption activity of gp38.

The inhibitory effects of two gp38 protein concentrations (1.6 and 0.8 mg/mL) were also compared. Results showed that 1.6 mg/mL gp38 produced a more pronounced competitive inhibition (Figure 7a), suggesting a concentration-dependent mechanism. Additionally, the gp66 protein, used as another control (Figure 7b), did not exhibit significant competitive inhibition, further supporting the specificity of gp38 function.

To rule out the possibility that the observed inhibition of phage adsorption resulted from bacteriolytic activity, the effect of gp38 on bacterial viability was assessed. As shown in Figure 7B, treatment with gp38 did not reduce the CFU/mL of E. coli SD405 compared to the untreated control, confirming that gp38 lacks bacteriolytic properties under the experimental conditions. These results collectively demonstrate that gp38 specifically inhibits phage adsorption through a receptor-blocking mechanism, independent of host cell lysis.

To further validate this effect, the binding capacity of the gp38 protein to host bacteria was evaluated. Quantitative analysis, calibrated against a negative control (PBS + gp38), confirmed a highly specific, rapid, and efficient binding interaction between gp38 and the host bacteria. As shown in Figure 7C, the binding amount between gp38 and the bacteria stabilized within 2–4 min, likely reaching saturation, and remained stable beyond 16 min without significant variation. After correction by the negative control, the BSA group effectively excluded non-specific binding, confirming that the data accurately reflected true protein–bacterium interactions. These findings indicate that the gp38 protein functions as a critical bacterial receptor-binding component, with a rapid and specific binding process completing within minutes. Collectively, the results suggest that gp38 exhibits RBP-like functionality and is likely the receptor-binding protein of phage vB_EcoM_SD286.