Pseudomonas syringae pv. actinidiae was previously isolated by our research group from the stems of a kiwifruit plant affected by kiwifruit canker and was used as the indicator strain for this experiment. The PSA strain was preserved in the Luria-Bertani (LB) liquid medium containing 20% glycerol at −80°C. Pichia pastoris X33 was acquired from Sangon Biotech (Shanghai).

Indicator strains included the following: Proteusbacillus vulgaris ATCC-6896, Escherichia coli ATCC-35150, Burkholderia cepacia CGMCC-27262, Staphylococcus aureus CMCC-26003, Streptococcus agalactiae ATCC-12403, Bacillus subtilis ATCC-6896, Bacillus safensis CGMCC-27263, Aspergillus niger ATCC-16404, and Candida albicans CMCC-98001. All strains were purchased from Baiou Bowei Biotechnology Co., Ltd. (Beijing).

Rhizosphere soil samples from healthy kiwifruit were collected from Songbai town, Yongshun County, Xiangxi Tujia and Miao Autonomous Prefecture, Hunan Province, at coordinates N28°54′, E110°4′.

Rhizobacteria were isolated using the plate count method in the Luria-Bertani (LB) medium (Zhou et al., 2012; Huang et al., 2013). We crushed the soil samples into granules, mixed them evenly, and weighed 1 g of the soil samples into conical flasks containing sterile water for dilutions of 10⁻³ times, 10⁻⁴ times, and 10⁻⁵ times. The samples were shaken uniformly at 180 r/min for 3 min at room temperature, and then, 50 μL of the mixtures was spread onto the LB medium. After cultivating for 7 days at 28°C, different morphological characteristics of the bacterial strains were randomly selected and purified through repeated streaking onto fresh LB plates. The purified colonies were preserved in the LB liquid medium containing 20% glycerol at −80°C.

Preparation of PSA-containing plates (Chen et al., 2022): PSA was cultured overnight in the LB medium at 22°C. Then, 100 mL of fresh PSA inoculum was added to 500 mL of the LB medium cooled to 55°C, mixed quickly, and poured onto the plates to create PSA-containing plates. The isolated rhizobacteria were placed onto the PSA-containing plates. The medium was incubated at 22°C until clear zones of inhibited PSA growth became visible.

The following describes the preparation of cell-free fermentation broth (Teixeira et al., 2009): Bacteria against PSA were cultured in 5 mL of LB liquid medium at 28°C, 180 r/min. After adding 100 μL of the inoculum to 50 mL of LB liquid medium, the culture was fermented at 28°C and 180 r/min. Subsequently, the fermentation broth was centrifuged at 10000 r/min for 10 min at 4°C. Finally, the fermentation broth was filtered through a microporous membrane of 0.22 μm to remove the remaining bacterial cells.

The antagonistic activity of the bacterial fermentation supernatants against PSA was evaluated using the Oxford cup method (Li et al., 2021). After placing Oxford cups on the PSA-containing plates, 200 μL of the fermentation supernatant was pipetted into each Oxford cup. The plates were cultured at 22°C, and bacteriostasis activity was observed after 48 h.

The cellular morphology of the rhizobacteria was observed using Gram staining. Meanwhile, the rhizobacteria were cultured in the LB medium at 28°C for 72 h to observe their colony morphologies.

Genomic DNA from the antagonistic rhizobacteria was extracted using the Rapid Bacterial Genomic DNA Isolation Kit, following the manufacturer’s protocol (Huang et al., 2013). The 16S rRNA gene sequence of the antagonistic strains was amplified by the polymerase chain reaction (PCR) using PA (5′-AGAGTTTGATCCTGGCTCAG-3′) and PB (5′-TTAAGGTGATCCAGCC GCA-3′) primers (Zhao et al., 2021). The reaction mixture was as follows: 25 μL of Taq PCR Mix (2×), 0.4 μM of F/R primers, 0.1 μM of bacterial DNA, and sterile water to a final volume of 50 μL. The PCR procedure included pre-denaturation at 95°C for 2.5 min, followed by denaturation at 95°C for 15 s, annealing at 55°C for 30 s, and extension at 72°C for 1 min for a total of 35 cycles. A final extension was performed at 72°C for 10 min. The amplicons were sequenced, and the sequence was obtained and identified using the NCBI identification service.¹ The 16S rRNA gene sequences of the Wq-1 strain and type strains of closely related strain species were aligned using MEGA 7.0 software. After removing positions containing gaps and missing nucleotides at both ends of the aligned sequences, the final aligned sequences were constructed into a phylogenetic tree using the neighbor-joining (NJ) method (Waghmode et al., 2011).

Ammonium sulfate precipitation: After the fermentation broth was refrigerated at 4°C for 30 min, it was filtered through a microporous membrane of 0.22 μm, and the cooled cell-free fermentation broth was placed on a magnetic stirrer. Ammonium sulfate was then slowly added to 5% of the volume of the fermentation broth each time until the precipitation of the proteins was observed.

Ultrafiltration (Forestier et al., 2022): The ultrafiltration centrifuge tube was pretreated following the instructions. A 500 μL protein sample was added to the centrifuge filter and centrifugated at 4°C and 5,000 r/min for 40 min. First, the sample was filtered using a 30 kDa filter, followed by a 10 kDa filter. The original volume was restored with double-distilled water, and the sample was collected. The inhibitory activity of these isolated samples was determined using the Oxford cup method for PSA.

The gel was prepared using a 15% SDS-PAGE gel preparation kit, following the manufacturer’s protocol (Babar et al., 2022). The electrode buffer was prepared using the following components: 15.1 g of Tris, 94 g of Glycine, 5 g of SDS, and 5,000 mL of H₂O. Dye solution preparation included the following: 1.5 g of coomassie brilliant blue R250, 300 mL of carbinol, 60 mL of glacial acetic acid, and 140 mL of H₂O. The preparation of decolorizing solution included the following: 300 mL of carbinol, 300 mL of glacial acetic acid, and 400 mL of H₂O. The electrophoretic conditions were set at 120 V for 100 min.

After cutting the target strip, it was rinsed in a large amount of sterile water, placed into a 1 mL centrifuge tube, and stored in sterile water at 4°C. The target strip was then stored in Drikold and sent to the APTBIO company. The samples were analyzed using LC–MS/MS.

The identified amino acid sequence was analyzed using BLAST searches in the NCBI, UniProt, and ADP3 databases (Wang, 2023), and all similar sequences were downloaded. The sequence similarity of the protein with homologous proteins and bacteriocins was assessed using the Clustal W program.

B. laterosporus Wq-1 was cultured in the LB liquid medium overnight at 28°C, 180 r/min for 24 h. The fermentation broth was centrifuged at 8,000 r/min for 5 min. The collected cells were sent to Sangon Biotech (Shanghai) for genome sequencing using the PacBio RS II platform. GeneMarkS (Besemer et al., 2001) software was used to analyze the whole genome sequence of Wq-1 and annotate the function elements of the gene and protein (Coudert et al., 2023).

To facilitate expression, the signal peptide of protein 01021 was excised, and the protein 01021 codon was optimized. The enzyme cutting sites EcoR I and Acc I were selected, and 6 × his tags were added to the C-end of the protein to facilitate purification. The synthesized gene was linked to pPICZa, and the recombinant plasmid was named PA-01021. The plasmid PA-01021 was then transfected into E. coli DH5a to obtain E. coli DH5a-PA-01021.

The E. coli DH5a-PA-01021 strain was cultured in LB liquid medium (containing 100ug/mL zeocin) overnight at 28°C, 180 r/min for 24 h. The fermentation broth was centrifuged at 8,000 r/min for 5 min, and the cells were collected. The PA-01021 plasmid was extracted from E. coli DH5a-PA-01021 using a plasmid extraction kit. The PA-01021 plasmid was linearized by selecting Pme I. P. pastoris X33 was cultured in 5 mL YPD liquid medium at 28°C, 280 r/min for 24 h. The P. pastoris X33 cells were cleaned with sterile water and 1 mol/L of sorbitol solution. Then, 200 μL of the P. pastoris cells was mixed with 10 μL of the linearized PA-01021 plasmid, and the mixed solution was then transferred into a 2 mm electroporation cuvette for electroporation transformation.

The electro-transformed P. pastoris X33-PA-01021 was spread on the YPD medium containing 100 ug/ml zeocin and was cultured at 28°C for 48 h. A single colony was selected and cultured at 28°C for two generations. Then, the total DNA of P. pastoris X33-PA-01021 was extracted using the Rapid Yeast Genomic DNA Isolation Kit, and subsequently we identified it by PCR. The PCR procedure was similar to 2.5. The 01021 gene was amplified by PCR using primers AOX5 (5′-GACTGGTTCCAATTGACAAGC-3′) and AOX3 (5′-GGCAAATGGCATTCTGA CAT-3′), and the PCR products were then sent to Sangon Biotech (Shanghai) for sequencing.

The P. pastoris X33 PA-01021 transformant was cultured in 50 mL BMGY medium overnight at 28°C, 280 r/min for 16 h. The fermentation broths were centrifuged for 5 min at 4°C 6000 r/min, and the cells were collected. The cells were resuspended in 50 mL of BMMY medium. The expression was induced by adding methanol with a final concentration of 0.5%, and then every 24 h, methanol with a final concentration of 0.5% was added. P. pastoris X33 transferred to blank pPICZa was used as the control group. After 72 h, the fermentation broths were centrifuged for 5 min at 4°C and 10,000 r/min, and the supernatant was collected. SDS-PAGE was used to detect its expression condition, and the inhibitory activity of the supernatant was detected for PSA using the Oxford cup method.

The P. pastoris X33 PA-01021 fermentation supernatant was collected. The extracellular protein was collected through ammonium sulfate precipitation. The protein was then re-dissolved in 0.1 mol of phosphate buffered saline (PBS) solution and desalted for future use. Equilibrium buffer, wash buffer, and elution buffer were configured according to the instructions for Ni-NTA Agarose. The pH of all buffers and the protein sample was adjusted to 8.0. The Ni-NTA Agarose was transferred to 20 mL of a gravity column. The column was flushed with 5-column volumes of ultra-pure water to remove ethanol, and the equilibrium buffer was added to balance the Ni-NTA Agarose. The protein samples were then added and incubated at room temperature for 2 h, and the gravity column valve was opened to allow the solution to flow out. Then, the wash buffer with 5-column volumes was used to eluate the impurity protein. Finally, the elution buffer was used to eluate the target protein, and the eluent and outflow liquid were collected. The purification effect was identified using SDS-PAGE. The purified sample strips were sent to Sangon Biotech (Shanghai) for LC–MS/MS identification.

The purified sample strips were rinsed and decolorized. The sample strips were rinsed with NH₄HCO₃ and C₂H₃N to dehydrate them. DTT was added to the samples for reduction, and an IAA solution was added for alkylation. Then, the strips were washed. Finally, the sample strips were dehydrated. Trypsin was added to the sample, followed by the addition of NH₄HCO₃ (containing 10% ACN). The mixture was then digested overnight. Once enzymolysis was complete, extraction liquid (67% C₂H₃N, 2% CH₂O₂) was added, followed by ultrasonic treatment, centrifugation, concentration, and drying. Sample processing was then completed.

The samples were redissolved in Nano-LC mobile phase A (0.1% CH₂O₂) for online LC–MS analysis. The samples were loaded onto a nanoViper C18 pre-column (3 μM, 100 Å) and then rinsed for desalting. The liquid phase was provided by Easy-nLC 1,200 nL Liquid Phase System (ThermoFisher, United States). The samples were then separated using an analytical column (C18 reverse-phase chromatographic column, 75 μM × 25 cm, C18-2 μM, 100 Å) after desalting on a pre-column. The gradient used in the experiment involved increasing mobile phase B (80% C₂H₃N, 0.1% CH₂O₂) from 5 to 38% within 30 min. Mass spectrometry was performed using the Thermo Fisher Q Exactive system, combined with the nanometric spray Nano Flex ion source (ThermoFisher, United States). The spray voltage was 1.9 kV, and the heating temperature of the ion transport tube was 275°C. The scanning mode of the mass spectrometry was data-dependent analysis, with a primary MS resolution of 70,000, a scanning range of 350-2000 m/z, and a maximum injection time of 100 ms. Up to 20 secondary spectra with charges ranging from 2+ to 5+ were acquired during each DDA cycle, with a maximum injection time of 50 ms for secondary mass spectrometry ions. The collision chamber energy (high-energy crash-induced dissociation, HCD) was set to 28 eV for all precursor ions, and the dynamic exclusion was set to 25 s.

The WIFF file of the MS was converted into an MGF format file using ProteoWizard (3.0.10577 64-bit), and the MGF file was imported into Mascot (V2.3.02) for protein identification. The search parameters were as follows: the database was UniProt, the enzyme was trypsin, and the maximum allowable missing cutting site was 1. The fixed modification included carbamidomethylation (C); the variable modifications included acetylation (Protein N-term), deamidation (NQ), Gln- > pyro-Glu (N-term Q), Glu- > pyro-Glu (N-term E), and oxidation (M). The MS tolerance was 20 ppm, and the MS/MS tolerance was 0.05 Da. Identification was considered successful when the protein score C.I. % was greater than 95%.

To determine the stability of the bacteriostatic proteins exposed to ultraviolet light, the samples were treated using a UV lamp for 1 h, 3 h, 5 h, 8 h, and 11 h. The remaining antibacterial activity of the samples was determined using the Oxford cup method. The indicator was PSA, while the control was a sample without UV treatment.

The bacteriostatic proteins were kept at 100°C for 1 h, 121°C for 30 min, and 25°C for 15 days, respectively. Among them, the proteins that were kept at 100°C were treated in a water bath, and the proteins that were kept at 121°C were treated in a sterilizer (Naimah et al., 2018). After the treatment, the samples were cooled to room temperature, and the residual antibacterial activity was assessed using the Oxford cup method. The used indicator was PSA. An unheated protein sample was used as the control.

To determine the pH stability of the bacteriostatic proteins, the pH buffer of the purified samples was adjusted to a range from 2 to 11 (in increments of 2 pH) using 4 M NaOH or 4 M HCl (Qi et al., 2021). The samples were then incubated at 25°C for 4 h, after which the pH of each sample was readjusted to 7.0. The remaining antibacterial activity of the samples was determined using the Oxford cup method. The used indicator was PSA, and the control was a sample without pH treatment.

To determine the antimicrobial spectrum of the bacteriostatic proteins, the antibacterial activity of the purified samples was determined using the Oxford cup method. The control was double-distilled water.

The soil samples were gradually diluted and then evenly spread onto LB medium. After 7 days, a substantial number of microbial colonies appeared. Among the different dilution gradients of the soil samples, the 10⁻⁴ gradient exhibited the most effective separation, with evenly distributed microbial colonies and optimal gaps between them. A total of 93 strains of diverse microorganisms were isolated from the rhizosphere soil samples of kiwifruit, including 30 strains of fungi, seven strains of actinomycetes, and 56 strains of bacteria. After 63 bacteria were co-cultured separately on PSA-containing medium at 22°C for 3 days, it was found that four bacteria demonstrated significant inhibitory activity against PSA. The diameter of their inhibition zones against PSA ranged from 9 to 19 mm. The growth cycle on the LB medium varied from 3 to 7 days. No evident inhibitory activity was observed in the remaining strains of the isolates.

The Oxford Cup method was used to assess the PSA-inhibiting activity of the cell-free fermentation broths derived from the four bacteria. The results revealed that the cell-free fermentation broths of three bacteria exhibited significant inhibitory effects on PSA. The inhibition zone diameter of these three bacteria against PSA ranged from 9 to 15 mm. Notably, the bacterial cell and cell-free fermentation broth of one white bacterium displayed the highest antagonistic activity, with a 15 mm inhibition zone diameter (Figure 1A). Consequently, the white bacterium was selected for further investigation of its bacterial characteristics and antagonistic activity.

The white bacterium was cultivated in the LB medium at 28°C for 72 h. Its colony morphology was characterized by a milky-white appearance with concentric circles, as well as surface drying and folding (Figure 1B). A fresh colony was taken for Gram staining, and under a microscope, it was observed that the cells were short and rod-shaped, and the Gram stain was positive. The PCR amplification product size of the Wq-1 strain was 1,360 bp (NCBI accession: OR618324.1), and 16S rDNA sequences analysis showed a maximum identity of 99.46% with Brevibacillus laterosporus NECC11213 (PP779784.1). In addition, in the phylogenetic tree (Figure 2), strain Wq-1 clustered with type strain B. laterosporus 5LHD-1. Strain Wq-1 was conclusively identified as B. laterosporus based on both molecular and morphological characteristics. Therefore, the Wq-1 strain was designated as B. laterosporus Wq-1.

Ammonium sulfate was slowly introduced into the cell-free fermentation broth of Wq-1. Protein precipitation was initiated when the ammonium sulfate concentration reached 20%. As the concentration of ammonium sulfate in the cell-free fermentation broth increased, more protein precipitates became evident. Upon reaching 70% ammonium sulfate concentration in the cell-free fermentation broth, the amount of protein precipitates stabilized in the fermentation solution.

The experiment showed that when the ammonium sulfate concentration reached 35%, the protein precipitates harvested through centrifugation exhibited antibacterial activity. However, after collecting the remaining protein precipitates between 35 and 70% ammonium sulfate concentration, the isolated protein no longer retained antibacterial activity. Therefore, the bacteriostatic proteins in the fermentation broth of B. laterosporus Wq-1 were all precipitated when the ammonium sulfate concentration reached 35%.

The protein precipitates collected through centrifugation were subsequently re-dissolved in a 0.05 mol/mL PBS solution (pH 7.0). Notably, only approximately 0.02 g of precipitate dissolved per 1 g of precipitate. Then, the soluble portion was separated from the insoluble portion. To prevent interference from salt in the sample during subsequent experiments, the protein sample was desalted using a 3 kDa ultrafiltration centrifuge tube.

The soluble and insoluble fractions of the protein were tested for their bacteriostasis activity against PSA using the Oxford Cup method. The experiment revealed that the inhibitory activity of the insoluble fraction was approximately 20% compared to the dissolved fraction. Therefore, the majority of the bacteriostatic protein precipitates in the cell-free fermentation broth of Wq-1 were re-dissolved in a 0.05 mol/mL PBS solution, with only a small amount of bacteriostatic proteins remaining in the insoluble precipitation. Thus, the soluble precipitated fractions were selected for subsequent purification.

The protein samples were initially placed in a 30 kDa ultrafiltration centrifuge tube for first-stage purification to obtain <30 kDa and > 30 kDa fractions. Bacteriostatic experiments indicated that the inhibitory activity against PSA was concentrated in the <30 kDa fraction. Subsequently, the <30 kDa fraction underwent second-stage separation using a 10 kDa ultrafiltration centrifuge tube to obtain <10 kDa and > 10 kDa fractions. The bacteriostatic experiments revealed that the bacteriostatic activity was concentrated in the >10 kDa fraction (see Supplementary material). In conclusion, the molecular weight of the antibacterial protein in the cell-free fermentation broth of Wq-1 ranged from 10 to 30 kDa.

To precisely determine the molecular weight of the antibacterial protein of strain Wq-1, the ultrafiltered activity fraction was analyzed using SDS-PAGE. The results showed the presence of a single band at 12 kDa in the ultrafiltered activity fraction (Figure 3). In summary, the antimicrobial substances produced by B. laterosporus Wq-1 were inhibitory proteins with a molecular weight of 12 kDa.

The 12 kDa protein band is cut into a centrifuge tube, this protein is then enzymatically cleaved and detected by LC–MS/MS. The results showed that two peptide segments had high scores and IBAQ values in the 12 kDa active protein sample. Meanwhile, the whole genome sequence (NCBI accession: CP136163.1) of B. laterosporus Wq-1 was analyzed using GeneMarkS software, and a total of 5,025 proteins were predicted. The two peptide segments (AFGITVTPLLPV and YGTLNYIR) identified by LC–MS/MS were compared with the GeneMarkS software output result of the Wq-1 genome sequence (Figure 4), and one protein was obtained, protein ID: PROKKA-01021. Therefore, we concluded that the active substance against PSA in the cell-free fermentation broth of B. laterosporus Wq-1 was protein 01021.

The sequence of protein 01021 was analyzed using BLAST searches in the NCBI, UniProt, and ADP 3 databases. The homologous sequence was compared with the amino acid sequence of protein 01021. The analysis revealed that protein 01021 is most closely related to an uncharacterized protein (NCBI accession number WP168420414.1), with 100% similarity. The second closest match was BLB8, with 99.14% similarity (Figure 5A). The amino acid sequence of protein 01021 was compared with those of other bacteriocins. The comparison showed that that protein 01021 has very low similarity to Laterosporulin (Similarity 11.8%) and the other seven bacteriocins produced by B. laterosporus. The most similar sequence identified was Garvicin Q, with only 19.4% similarity (Figure 5B).

The primitive length of the coding gene for protein 01021 was 351 bp. After removing the signal peptide and adding the enzyme cutting sites EcoR I and Acc I and 6 × his tags, the gene size was 297 bp. The PA-01021 recombinant plasmid was then constructed. After transferring the plasmid PA-01021 into P. pastoris X33, seven transformants were selected. The PCR results showed that all seven transformants successfully incorporated the gene 01021, and they were named Q1, Q2, Q3, Q4, Q5, Q6, and Q7, respectively.

The bacteriostatic experiments showed that the recombinant protein 01021 expressed by P. pastoris-Q1, Q3, and Q5 effectively inhibited PSA, while the control group exhibited no bacteriostatic activity. In summary, protein 01021 was successfully expressed in P. pastoris X33 and demonstrated high activity in inhibiting PSA.

The recombinant bacteriocin carried a 6 × his tag, so it was purified using the Ni-NTA resin. SDS-PAGE analysis revealed that, compared to the control group, a clear band appeared at 12 kDa, which was consistent with the theoretical molecular weight of the bacteriocin. The purified recombinant bacteriocin also displayed a clear band at 12 kDa.

LC–MS/MS analysis of the purified protein sample revealed that there were peptide segments in the sample (Table 1). All peptides were matched with JUQZ-1, and the sequence coverage rate was 61.6%. The scores of four peptide segments (TSNETWNIGSHIR, EVIKVEYDSSTQFNK, TAVQPGSASIYVYK, and YGTLNYIR) were greater than 26 (Table 1). The identification result was highly reliable.

The physical properties of bacteriostatic proteins are key criteria for evaluating their practical value. Some experiments were conducted to characterize the ultraviolet, pH, and temperature tolerance of the purified bacteriostatic proteins.

Remarkably, when exposed to UV irradiation for 1 h, 3 h, 5 h, 8 h, and 11 h, these purified bacteriostatic proteins demonstrated sustained antibacterial activity, with no significant decrease in antagonistic activity (Figure 6A). Furthermore, the bacteriostatic activity of the isolated proteins showed no significant decline after 1 h of treatment at 100°C. After a 30 min treatment at 121°C, the bacteriostatic activity remained at 40%, and it maintained 100% activity after 15 days at room temperature (25°C; Figure 6B). The bacteriostatic activity of the antimicrobial protein was preserved at pH 2.0 and pH 11.0 after a 30-min treatment at 25°C. Throughout the pH range from 2.0 to 11.0, the antimicrobial protein showed minimal loss of bacteriostatic activity (Figure 6C).

The antimicrobial spectrum of the protein JUQZ-1 against nine strains is shown in Table 2. JUQZ-1 was found to have broad-spectrum antimicrobial activity against both Gram-positive and Gram-negative bacteria. However, the sensitivity of the Gram-positive bacteria to JUQZ-1 was lower than that of the Gram-negative bacteria to JUQZ-1. Meanwhile, Aspergillus niger and Candida albicans were completely resistant to JUQZ-1. Overall, JUQZ-1 is a broad-spectrum antimicrobial protein.