Bacterial strains, culture conditions, and plasmids
V. anguillarum strain NB10 (serotype O1) was standard cultured aerobically under conditions of 125 rpm and 25 ℃ in brain heart infusion (BHI) media (Becton Dickinson and Co., Sparks, MD, USA). Two E. coli strains DH5α and BL21 (DE3) for gene cloning and protein expression, respectively, were standard cultured under conditions of 150 rpm and 37°C in Luria-Bertani (LB) broth. All bacterial strains were stocked at -80°C in 25% glycerol and 7% dimethyl sulfoxide (DMSO) until used later. The pET28a(+) vector (Novagen, Madison, WI, USA) containing the T7 promoter and kanamycin resistance gene was used for cloning and expression. The pcDNA™3.1/His/lacZ vector (Invitrogen, Life Technologies, Carlsbad, CA, USA) was used as the template for LacZ gene. The bacterial strains and plasmids used in this study are listed in Table S1.
Differential expression proteome analysis based on environmental stress
Stress condition and of total protein preparation
V. anguillarum NB10 was cultured in BHI broth at 25°C and pH 7 until the OD600 of 0.6 as 5×108 colony forming units per milliliter (CFU mL− 1) was reached. The cells were harvested through centrifugation at 12,000 rpm for 5 min and resuspended in normal (25°C and pH 7) or pH stress-conditioned (25°C and pH 10) media. V. anguillarum NB10 was harvested through centrifugation and vortexed thoroughly for 16 h in a lysis solution containing 7 M urea, 4% 3-(3-cholamidopropyl dimethylammonium) propane sulfonate (CHAPS), 2 M thiourea, 100 mM dithiothreitol (DTT), and 2% Pharmalyte. After centrifugation, the supernatants was recovered and treated using a 2-D Clean-up Kit (GE Healthcare, Piscataway, NJ, USA). The purified total proteins were resuspended in the lysis solution. Protein concentrations were determined using a modified Bradford assay; bovine serum albumin was used as the standard.
Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE)
One-dimensional isoelectric focusing (IEF) and two-dimensional (2D) sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS–PAGE) (2-DE) were performed according to the manufacturer’s protocol (GE Healthcare, Piscataway, NJ, USA). The purified protein samples (200 µg) containing a 0.5% immobilized pH gradient (IPG) buffer (pH 4–7) and 5% bromophenol blue were applied to an immobilized IPG Dry Strip (immobilized pH 4–7, 13 cm; GE Healthcare) and rehydrated for 16 h in lysis solution. The focused IPG strips were equilibrated for 20 min in a solution containing 50 mM Tris–HCl (pH 8.8), 6 M urea, 30% glycerol, 2% SDS, and 1% DTT. Then, the DTT in the solution was replaced with 0.5% iodoacetamide, and the process was continued for an additional 20 min. The equilibrated strips were applied to a 12% polyacrylamide gel (14 × 13 cm, 1.5 mm thick). Silver staining was performed to visualize the proteins, and mass spectrometry was performed according to the manufacturer's protocol (Thermo Fisher Scientific, Cleveland, OH, USA).
Protein identification
For identifying protein, the proteins were identified by after drying the gel, followed by dehydrating in acetonitrile and overnight digestion at 37°C with sequencing-grade trypsin (Promega, Madison, WI, USA). The resulting tryptic peptides were dissolved in 50% acetonitrile containing 0.5% trifluoroacetic acid and desalted using a ZipTip C18 pipette tip (Millipore, Billerica, MA, USA). The peptides were directly eluted onto matrix-assisted laser desorption ionization (MALDI) plates using a-cyano-4-hydroxy-cinnamic acid (CHCA) matrix solution (10 mg mL− 1 CHCA in 0.5% TFA: 50% acetonitrile, 1:1). MALDI-time of flight/mass spectrometry (MALDI-TOF/MS) and MALDI-TOF/MS/MS were performed in reflection mode using a 4700 Proteomics Analyzer (Applied Biosystems, Foster City, CA, USA). The proteins were identified by searching the National Center for Biotechnology Information (NCBI) database using the MASCOT program (http://www.matrixscience.com/; RRID:SCR_014322).
In silico method for identifying protein and promoter region
Genome analysis
The NB10 genome was analyzed using the complete genome sequence deposited at NCBI (accession number: PRJEB5701) to identify FK506 binding protein (FKBP) and its promoter region based on the results of the amino acid sequence confirmed using MALDI-TOF/MS. Genome analysis was performed according to a method reported previously with slight modifications [40]. Briefly, the NB10 genome annotations were performed using Proksee (https://proksee.ca/), Rapid Annotation using Subsystem Technology 2.0 (RAST; https://rast.nmpdr.org/), and Bacterial and Viral Bioinformatics Resource Center (BV-BRC; http://www.bv-brc.org/) services. A comparative annotation analysis of all results was performed to complete the commentary. The circular map was constructed using the BV-BRC circular viewer (https://www.bv-brc.org/view/Genome/).
Three-dimensional homology-modeling and ligand docking
Molecular modeling and docking process were performed according to the procedure described in the reported previously with slight modifications [41]. Briefly, for 3D structural modeling of FKBP, homologous proteins were screened using the AlphaFold v2 protein database (https://alphafold.ebi.ac.uk/) using an AI system that predicts protein 3D structure based on its amino acid sequence (PBD ID; A0A649YL14.1.A, unknown gene, unknown organism). Secondary screening was performed on the protein templates using the NCBI (https://www.ncbi.nlm.nih.gov/structure/) and RCSB (https://www.rcsb.org/) servers. The candidate templates were applied to a protein structure homology modeling server (Swiss-Model; https://swissmodel.expasy.org/) to select templates for the structures that were determined using X-ray crystallography. The final predicted tertiary structures were constructed based on the selected template (PDB ID; 7dek.2.A, Pseudomonas aeruginosa FK506-binding protein PaFkbA). The 3D conformers of tacrolimus (PubChem CID: 445643; C44H69NO12; FK-506) that act as FKBP ligands were visualized using the PubChem Chemical Molecules Database (https://pubchem.ncbi.nlm.nih.gov/). The intermolecular binding sites and affinities of the proteins and ligands were predicted and evaluated using the CB-Dock2 server (https://cadd.labshare.cn/cb-dock2/). Visualization and further analysis of the molecular complexes for parameters such as density maps, trajectories, and structure matching were performed using UCSF ChimeraX (https://www.cgl.ucsf.edu/chimerax/; RRID:SCR_015872) program.
Sequence alignment of the promoter region
The protein corresponding to FKBP of NB10 was traced in other Vibrio species (V. parahaemolyticus ATCC 17802, accession number CP014046.2; V. harveyi ATCC 33843, CP009467.2; V. alginolyticus ATCC 17749, CP006718.1; and V. fluvialis 10M-VF, CP118599.1). The sequences of the FKBP promoter region of each Vibrio were secured through genome comparative analysis. The nucleotide sequence identity matrix and amino acid sequence homology of the fatty acid synthesis-related proteins were calculated using the BioEdit 7.2 program (https://bioedit.software.informer.com/; RRID:SCR_007361).
Constructions of recombinant vector
pVApro (pET-28a(+)/FKBP promoter/lacZ) was constructed by inserting the FKBP promoter and lacZ gene into pET-28a(+) as the backbone vector. The 3.1 Kbp lacZ gene was amplified using PCR (TaKaRa, Kyoto, Japan) with pcDNA™3.1/His/lacZ as the template and complementary primer sets containing BamHI and XhoI restriction enzyme sites for gene cloning (New England Biolabs (NEB), Cambridge, MA, USA). The PCR conditions were 25 amplification cycles of 97°C for 30 s, 58°C for 60 s, and 72°C for 30 s. The amplified lacZ and pET-28a(+) were digested using BamHI and XhoI and inserted into the multiple cloning site (MCS) of pET-28a(+) using T4 DNA ligase (Takara, Kyoto, Japan). Then, pET-28a(+)/lacZ was transformed into E. coli DH5α using heat shock at 42°C for 60 s to confirm the recombinant plasmid. Next, the stepwise deleted FABP promoter regions were amplified using NB10 chromosomal DNA as the template and complementary primer sets containing the BglII and BamHI restriction sites (PCR condition: 25 amplification cycles of 97°C for 30 s, 58°C for 30 s, and 72°C for 30 s). The PCR products and the purified pET-28a(+)/lacZ were digested using BglII and BamHI restriction enzymes, treated with ligase, and transformed again into E. coli DH5α to complete the pVApro/wild – del3. The insertion of each target gene was confirmed via DNA sequencing with the Applied Bio-systems 3730XL using the BigDye(R) Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, CA, USA) [42]. All the primer sets used in this study are listed in Table S2.
Measurements of promoter strength
β-galactosidase assay
The β-galactosidase assay was performed according to the Miller method with minor modifications to quantify the activation level of the promoter in terms of number of Miller units of enzyme activity [43]. Briefly, 3 mL of overnight cultured recombinant E. coli DH5α harboring pVApro/wild (E. coli pVApro/wild) was inoculated into LB-Kanamycin (50 µg/mL) broth (300 mL). The cells were cultured for 6 h at 37°C until the late log phase was 2×109 CFU/mL. The cells were collected through centrifugation at 6,000 rpm for 10 min and immediately suspended in 50 mL of fresh LB medium, which was adjusted to pH 5–10 with 6N HCl and NaOH. The samples for promoter strength measurement were collected after 1, 2, and 4 h of incubation at 25°C. To measure promoter strength in response to temperature stress, the samples were incubated at 37 and 50°C and pH 7, followed by centrifugation at 12,000 rpm for 10 min to remove the supernatant completely. The collected cells were resuspended in 50 mL of 50 mM Tris–HCl (pH 7.0 ± 0.2) buffer and disrupted using a sonicator (Sonics & Materials, Inc., Newtown, CT, USA) at 4°C (3 s pulses at 150 W for 30 min with 2 s gap between pulses). After centrifugation, the supernatant was discarded, and the cell debris and inclusion bodies were used as crude enzyme for determining β-galactosidase activity. Each sample was assayed for β-galactosidase assay with o-nitrophenyl-β-D-galactoside (ONPG) as the substrate as described with minor modifications [44]. The assay mixture (100 µL) containing 5 mM ONPG and crude enzyme solution were incubated for 10 min at 40°C. The reaction was stopped by adding one volume of 1 M Na2CO3. The optical density of the reactants was read using a Microplate Reader (KLAB, Daejeon, Republic of Korea). The Miller formula was used to calculate the Miller units of enzyme per minute per milliliter of the sample. Each sample was processed in triplicate, and the average values were used to calculate the Miller units of the enzyme in each sample.
1 Miller Unit = 1000 * \(\frac{({Abs}_{420}-\left(1.75\text{*} {Abs}_{550}\right))}{\left(t\text{*}v\text{*} {Abs}_{600}\right)}\)
where, Abs420 indicates the absorbance of yellow o-nitrophenol; Abs550 indicates the scatter from cell debris; t indicates the reaction time in minutes; v indicates the volume of culture assayed in milliliters; and Abs600 indicates cell density.
Total RNA extraction and cDNA synthesis
Cultured bacteria were centrifuged at 12,000 rpm for 10 min, and the pellet was re-suspended in 1 ml TRIzol reagent (Invitrogen Lige Technologies, Burlington, Canada) to isolate the total RNA. Subsequent preparation and washing were performed using the Hybrid-RTM RNA Isolation Kit (GeneAll Biotechnology, LTD, Seoul, Korea). DNA was hydrolyzed using RQ DNase I (Promega, Madison, WI, USA). Complementary DNA (cDNA) was synthesized using PrimeScript™ 1st Strand cDNA Synthesis Kit (Takara, Otsu, Japan) under the following conditions: initial reaction with random hexamer at 30°C for 10 min, followed by extension at 42°C for 60 min. The RNA and cDNA were quantified using NanoDrop ND-1000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA).
Gene expression analysis
To validate the in vivo differential expression of genes at the transcriptional level, quantitative real-time PCR (qRT-PCR) was performed using a Thermal Cycler Disc™ Real Time System Lite (model TP700/760, software version V5.0x) (Takara Bio Inc, Otsu, Japan) instrument and SYBR Premix Ex Taq™ (Tli RNaseH Plus, Takara, Kyoto, Japan) [45]. The two-step shuttle PCR protocol was optimized to include by 35 cycles of initial denaturation for 30 s at 95°C, followed denaturation at 95°C for 5 s and annealing and extension at 58°C for 15 s. The PCR mixture (25 µL) contained 12.5 µL of 2x SYBR Premix Ex Taq™, 0.5 µL of each primer (15 µM), 9.5 µl of sterile distilled water, and 2.0 µL of cDNA. V. anguillarum 16S rRNA was used as the housekeeping gene for internal control. The relative quantitative value was expressed in accordance with the 2−△△Ct method [46].
Statistical analysis
All data were subjected to one-way analysis of variance (ANOVA) using Statistical Package for the Social Sciences (SPSS), followed by Duncan's multiple range test. Statistical significance was set at p < 0.05, unless otherwise noted.