The Yesso scallop (Patinopecten yessoensis) pathogen, V. splendidus strain JZ6 (Liu et al., 2013), was cultivated on Zobell 2216E agar at 20°C for 24 h. E. coli strains DH5α, BL21 (DE3), SY327 and S17-1 (Table 1) were cultured on LB agar at 37°C for 24 h.

ECPs of V. splendidus JZ6 wild-type and mutant strains were obtained by using the cellophane method (Balebona et al., 1998). Strains were grown in Tryptic Soy Broth (TSB) medium supplemented with 2% w/v sodium chloride (NaCl) at 20°C for 12 h, and 200 μL of the cultures was spread onto Zobell 2216E agar plates, overlayed with sterile cellophane sheets and plates were incubated at 4, 10, 16, 20, 24, and 28°C for 24 h. Bacterial cells were harvested with sterile saline, and the cell suspensions were centrifuged at 12,000g, at 4°C for 10 min. The bacterial cells were collected and stored at −80°C prior to RNA extraction. The supernatants were filtered through 0.22 μm membrane filters and used as the crude ECPs (designated as P₄, P₁₀, P₁₆, P₂₀, P₂₄, and P₂₈). The total protein contents of the ECPs were measured following the bicinchonininc acid (BCA) method (Smith et al., 1985).

The concentration of the six ECPs samples were adjusted at 20 μg/μL, and 10 μL were loaded onto a 12% SDS-polyacrylamide gel for SDS-PAGE. Following electrophoresis, protein bands were visualized with Coomassie bright blue R250. Vsm in ECPs samples was detected by immunoblotting using a monoclonal antibody of JZ6 Vsm (Liu et al., 2016). The ECPs samples were transferred onto a sheet of nitrocellulose transfer membrane (Millipore, USA). The membrane was blocked with 5% skim milk powder solution overnight and incubated with anti-Vsm solution (1:1000, v/v) at room temperature (RT) for 1 h. After being washing with TBST (10 mmol/L Tris-HCl, pH 8.0, 100 mmol/L NaCl and 0.05% (w/v) Tween 20), the membrane was incubated with 1:2000 (v/v) horseradish peroxidase-conjugated anti-mouse IgG (Life Technologies, USA) at RT for 1 h. Finally, the membrane was incubated with SuperSignal®West Pico (Thermo Scientific, USA) and exposed to film. Mouses pre-immune serum was used as negative control.

After SDS-PAGE, protein bands from the ECPs of strain JZ6 were excised and sent to BGI (BGI technology service co., LTD, China) for mass spectrometry (MS) analysis. Proteins were digested with 0.01 μg/μL trypsin, and the resulting peptides were subjected to nanoelectrospray ionization followed by tandem mass spectrometry (MS/MS) in a LTQ Orbitrap Velos (Thermo, USA) coupled online to the HPLC. Raw data files acquired from the Orbitrap were converted into MGF files using Proteome Discoverer 1.2 (Thermo, USA), and protein identification was performed by using Mascot search engine 2.3.02 (Matrix Science, United Kingdom).

Total RNA was extracted with TRIzol reagent (Invitrogen, USA). Based on Promega M-MLV RT Usage information, the first-strand cDNA synthesis was carried out with the DNase I (Promega, USA)-treated total RNA and random primers (TaKaRa, Japan). Synthesis was performed at 42°C for 1 h, then terminated by heating at 95°C for 5 min.

The mRNA levels of the vsm and pepTL at different temperatures were validated by quantitative real-time PCR (qRT-PCR). Specific primers (P1/P2 for vsm and P3/P4 for pepTL) were designed according to the corresponding sequences in the genome of strain JZ6 (Table 2). The comparative C_T method (2^−ΔΔCT method) was used to analyze the expression level (Livak and Schmittgen, 2001). Two 16S rDNA primers for V. splendidus, P5 and P6 (Table 2), were used as internal control to verify successful transcription and to calibrate the cDNA template for corresponding samples. qRT-PCR was performed using Applied Biosystems 7500 (Life technologies, USA), and the collected data were analyzed with 7500 System SDS Software. The assay was conducted in a volume of 20 μL consisting 10 μL of 2 × SYBR® Premix Ex Taq™ II (TaKaRa, Japan), 0.8 μL of each forward and reverse primer (10 μmol/L), 0.4 μL of 50 × ROX reference dye, 2 μL of DNA extract (10 ng/μL) and 6 μL of nuclease-free water. The reaction was performed at 95°C for 30 s, 40 cycles of primer annealing at 95°C for 5 s, primer extension at 60°C for 31 s. Dissociation curve analysis of amplification products was performed at the end of each PCR to confirm that only one PCR product was amplified and detected. All data were given in terms of relative mRNA expressed as mean ± S.E. (N = 4).

Genomic DNA of V. splendidus JZ6 was extracted from 5 mL of an overnight culture using the DNeasy Blood and Tissue Kit according to the manufacturer's protocol (Qiagen, Germany). Specific primers P7, P8, and P9 (Table 2) were designed from the nucleotide sequences encoding the upstream fragment of Vsm's fungalysin/thermolysin propeptide (FTP) domain, protease inhibitory function (PepSY) domain and peptidase M4 domain, respectively. An EcoRI site sequence was added at 5′ end. P10 (Table 2) was designed as a reverse primer with an XhoI site sequence. PCR products were digested with EcoRI and XhoI (NEB, USA), and ligated into the same restriction enzymes sites of expression vector pET28a (+) (Merck, Germany).

The complete open read frame (ORF) encoding PepTL was amplified with primers P11 and P12 (Table 2) containing NcoI and EcoRI recongnition sequences at their 5′ end, respectively. The PCR product was digested with NcoI and EcoRI (NEB, USA), and ligated into the same restriction enzymes sites of expression vector pET22b (+) (Merck, Germany).

The recombinant plasmids were transformed into competent cell E. coli BL21 DE3 (Tiangen, China), and transformants were incubated in LB medium at 37°C with shaking at 220 rpm for 4 h. IPTG was added to the cell cultures to a final concentration of 1 mmol/L once the cultures reach at an OD₆₀₀ of 0.4–0.6, and incubated at 18°C with shaking at 150 rpm for 24 h. The cultures were sonicated (200 W for 30 min) and centrifuged (12,000g, 4°C for 10 min) to obtain the supernatant containing soluble target proteins. Recombinant proteins (rVsmP1, rVsmP2, rVsmP3, and rPepTL) were purified with a Ni Sepharose column (Roche, Switzerland), and dialyzed against Tris buffer (20 mmol/L Tris-HCl, 150 mmol/L NaCl, pH 8.0) for 24 h. The resultant proteins were separated by SDS-PAGE, and visualized with Coomassie Bright Blue R250. The concentrations of purified soluble proteins were quantified by BCA method (Smith et al., 1985).

The protein domains of PepTL and Vsm were predicted by the simple modular architecture research tool (SMART) (http://smart.embl-heidelberg.de/). The presumed structures of both PepTL and Vsm were modeled by using the prediction algorithm I-TASSER (http://zhanglab.ccmb.med.umich.edu/) and displayed by Jmol Viewer (version 14.4.4). The interaction between PepTL and Vsm was in silico performed and displayed by AutoDock Tools version 1.5.6.

The in vitro assay was carried out to assess the proteolytic activity of rPepTL. Three concentrations of rPepTL (5, 10 and 20 μg/μL) were screened in preliminary experiment, and 10 μg/μL of protein was determined to be an optimal concentration. For the experimental group, 50 μL of rPepTL solution containing 10 mmol/L ZnCl₂ was mixed with 50 μL of ECPs P₂₈ (20 μg/μL) at 20°C for 1 h. Fifty microliter of ECPs P₂₈ and ECPs P₁₀ was used as negative and positive control, respectively. After the reaction, the mixtures were separated by SDS-PAGE and analyzed for reaction products as described in Section Analysis of Extracellular Products (ECPs) From V. splendidus JZ6 at Different Temperatures.

The suicide plasmid, pK18mobsacB-Ery (Wang et al., 2015), was used for deletion of the region encoding the active site in PepTL. Two pairs of primers (P13/P14 and P15/P16) were used to amplify the upstream and downstream DNA sequences of the target region from strain JZ6 genomic DNA. The PCR fragments of 170 and 220 bp were purified and fused in an overlap PCR reaction using primers P13 and P16 (Table 2). The fused segment was sequenced and digested with XbaI/SalI (NEB, USA), cloned into the same sites of pK18mobsacB-Ery, and then transformed into E. coli stains SY327 and S17-1. Suicide plasmid pK18Ery-pepTL was mobilized from E. coli S17-1 into V. splendidus JZ6 by intergeneric conjugation.

After mating, cells were spread on 2216E plates containing erythromycin (25 μg/mL) to select for clones in which the suicide vector pK18Ery-pepTL had been integrated into the JZ6 genome via a single crossover event. The mutants were then grown at 20°C with shaking in 2216E medium without any antibiotics for 8 h. To select mutants in which a second recombination event had occurred, the culture was diluted and spread on 2216E medium containing 10% sucrose and incubated at 20°C for 24–36 h. Single colonies were replica-plated onto 2216E and erythromycin containing 2216E plates, and colonies sensitive to erythromycin (25 μg/mL) were collected and confirmed by PCR followed by DNA sequencing.

The significant differences among groups were subjected to one-way analysis of variance (one-way ANOVA) and multiple comparisons by using SPSS 16.0 program. Statistically significant difference was designated at p < 0.05 or p < 0.01.

In our previous study, we demonstrated that Vsm from strain JZ6 was presented in the extracellular protein fraction in two forms differing in their molecular weight (Liu et al., 2016). In the present study, the differences in ECPs from strain JZ6 cultured at different temperatures (4, 10, 16, 20, 24, and 28°C) were monitored by SDS-PAGE. Two bands (JZE1:~35kDa and JZE2: ~45kDa) were observed in these ECPs, and their relative quantities were assessed with Image Lab Software (Bio-Rad, USA). At 4°C, JZE1 was the dominant band observed in ECPs P₄, with an intensity 5.88-fold that of JZE2 (~45kDa) (Figures 1A,C). As the temperature increased, the relative quantity of JZE1 increased at 10°C (1.54-fold of JZE1 in ECPs P₄) and then decreased from 0.89- to 0.11-fold of the level of JZE1 in ECPs P₄ as the temperature increased from 16 to 28°C (Figures 1A,C). The concentration of JZE2 in ECPs increased 0.42-, 0.49-, 0.51-, 0.57-, and 0.67-fold that of JZE1 in ECPs P₄ at 10, 16, 20, 24, and 28°C, respectively (Figures 1A,C). Western-blotting with a monoclonal antibody against JZ6 Vsm also revealed two main bands in ECPs of strain JZ6 (Figure 1B), and their intensity changes were consistent with the tendency observed in the SDS-PAGE assay.

Two bands of JZE1 and JZE2 were analyzed by LC-ESI-MS/MS mass spectrometry. Eight and eleven specific peptide fragments detected in JZE1 and JZE2, respectively, and all of them were identified as Vsm from strain JZ6. The eight specific peptide fragments from JZE1 were located in the M4 domain, and so were eight of the eleven fragments from JZE2. The other three peptides from JZE2 were assigned to the PepSY domain (Table 3). Thus, JZE1 was generated from JZE2 by cleavage.

The relative expression levels of vsm mRNA at 4, 10, 16, 20, 24°C were significantly higher (p < 0.01) than that at 28°C, with the highest level at 4°C (836.82-fold of that at 28°C), and then gradually decreased with the temperature increasing from 10 to 24°C (Figure 2). Similarly, the relative expression level of pepTL was highest at 4°C (191.38-fold, p < 0.01), and gradually decreased from 29.25-fold (p < 0.01) at 10°C to 2.59-fold (p < 0.05) at 24°C, finally reaching its lowest level at 28°C (Figure 2).

PepTL is predicted to perform its proteolytic activity as a homodimer (Figure 3A). A structure similar to that of S. typhimurium peptidase T (PepT, PDB No.1fno), was predicted with 10 α-helix and 15 β-sheet elements, but the conformation of dimerization domain from Ala176 to Phe291 in PepTL was significantly different from that of PepT (Glu206-Asn321) (Figure 3B). The 3D structure of Vsm consisted of the FTP domain, PepSY domain and two M4 domains (Figure 3C). In silico analysis of interaction by AutoDock Tools revealed that the homodimer of PepTL could tightly bind to Vsm, and the binding sites were located in the gap between PepSY and M4 domains of Vsm (Figure 3C).

Three protein segments of Vsm containing different functional domains were expressed to confirm the composition and molecular weight of JZE1 and JZE2. Segment 1 of Vsm (rVsmP1) consisted of 478 amino acids, containing FTP, PepSY and two Peptidase_M4 domains with a predicted molecular weight of ~51.50 kDa (Figure 4A). Segment 2 (rVsmP2) was composed of PepSY and two Peptidase_M4 domains (389 amino acids with a predicted molecular weight of ~44.93 kDa). Segment 3 (rVsmP3) only had two Peptidase_M4 domains (312 amino acids), and its predicted molecular weight was ~34.90 kDa. These recombinant proteins were expressed in E. coli BL21 (DE3) and purified by Ni Sepharose column. After SDS-PAGE analysis, the molecular weights of purified proteins were consistent with their theoretical values (Figure 4B), indicating that rVsmP3 shared a similar protein size with JZE1, and rVsmP2 had the same domain architectures as JZE2.

The full-length ORF of pepTL was of 1107 bp, encoding a polypeptide of 368 amino acids with a predicted molecular weight of ~39.30 kDa. Four different domain architectures of PepTL were analyzed by SMART, and the Peptidase_M20 domain was considered as the PepTL with E-value 3.8 × 10⁻¹³. Recombinant PepTL was expressed in E. coli BL21 (DE3) utilizing pET22b (+). After 24 h IPTG induction, the whole cell lysate was analyzed by SDS-PAGE and a distinct band was revealed with a molecular weight of ~40 kDa (Figure 5, Lane 2) which was in agreement with the predicted molecular mass. The purified and refolded rPepTL was of the same molecular weight (Figure 5, Lane 3). After incubation of ECPs P₂₈ with rPepTL at 20°C for 1 h, the intensity of the band corresponding to JZE2 dramatically decreased and the intensity of the JZE1 band increased (Figure 6). Furthermore, the quantity of rPepTL was less than that in the rPepTL only group (Figure 6). The changes in the intensity of the bands corresponding to JZE1 were also observed in immunoblotting with the monoclonal antibody of Vsm (Figure 6). No autocatalytic activity of ECPs P₁₀ and P₂₈ was observed.

The function of PepTL was also tested by deleting the pepTL gene from strain JZ6. ECPs from both wide type and ΔpepTL mutant were collected and subjected to SDS-PAGE post-culture at 10 and 28°C. Compared with the wild type (Figure 7, Line 1 and 2), the bands for Vsm in ECPs were not significantly different when the ΔpepTL mutant was cultured at 10 and 28°C (Figure 7, Line 3 and 4). The banding patterns of JZE1 and JZE2 were completely consistent with ECPs P₂₈ of wild type, indicating that pepTL gene was crucial for the transformation from JZE2 to JZE1.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.