Bacterial Strains and Plasmids
Escherichia coli TOP10 (Invitrogen, Carlsbad, CA, USA) was used for constructing recombinant plasmids. The genomic DNA of P. syringae pv. syringae (ICMP3023) provided full length of inaV gene. The attenuated V. anguillarum strain MVAV6203 developed in our previous work was used as the carrier host for surface display. Plasmid mTn5gusA-pgfp21 carrying the gfpuv gene (Xi et al. 1999) provided the gene source for construction of GFP derivatives. Plasmid pUC18 (TaKaRa, Japan) was used as a parent plasmid for construction of truncated inaV hybrids. The strains and plasmids used in this study are shown in Table 1.
Table 1 Strains and plasmids used in this study
Plasmids Construction
Recombinant plasmids harboring various inaV hybrids were constructed as follows using the primers in Table 2. The genomic DNA of P. syringae pv. syringae (ICMP 3023) containing full length of inaV gene was used as a template to amplify the N- and C-terminal domain by using primer nos. 1–7. The amplified fragments were digested with corresponding enzymes and inserted into the same sites of plasmid vector pUC18 to generate three different display plasmids, named as pUC-Nhis, pUC-NChis, and pUC-NhisC. The 720 bp of gfpuv fragments was amplified from plasmid mTn5gusA-pgfp12 using primer nos. 8–12 and digested with corresponding enzymes and inserted into appropriate positions of the display plasmids to generate three reporter plasmids named: pUC-NGFP, pUC-NCGFP, and pUC-NGFPC (Fig. 1). The cytosolic GFP expression plasmid was also constructed by fusion the gfp gene into the MCS of pUC18 by EcoRI and PstI digestion, which resulted in the pUC-GFP as a control plasmid for either expression or localization assays.
Table 2 Primers used in the construction
Culture Condition and Fusion Protein Expression
The E. coli TOP10 strain harboring plasmid was grown in Luria–Bertani (LB) medium (Tryptone 1%, yeast extract 0.5%, NaCl 0.5%) supplemented with ampicillin to a final concentration of 100 μg/ml. Cells were grown in 250-ml flasks with a 50-ml working volume in a shaker at 200 rpm and 37°C. The V. anguillarum strain MVAV6203 harboring plasmid was grown in LB2 medium (Tryptone 1%, yeast extract 0.5%, NaCl 2%) supplemented with ampicillin to a final concentration of 200 μg/ml. Cells were grown in 250-ml flasks with a 50-ml working volume in a shaker at 200 rpm and 30°C. For expression, overnight cultures were subcultured at 1:100 and cultured at 30°C for 1 day.
Measurement of Whole Cell Fluorescence
Cell density (OD600) was measured at 600 nm on an ultraviolet–visible (UV/Vis) spectrophotometer. The whole cell GFP fluorescence was determined by a fluorescence microplate reader (GENios Pro, Tecan, Mannedorf, Zurich, Switzerland) at excitation of 485 nm and emission at 535 nm. All the samples were diluted to OD600 of 1.0 before fluorescence was measured using the phosphate-buffered saline (PBS). The fluorescence signal of the untransformed cells of attenuated V. anguillarum strain diluted under the same condition (OD600 = 1.0) was set as background and subtracted from the overall fluorescence to give the GFP fluorescence. For comparison, all the GFP fluorescence data were normalized by the average fluorescence signal.
Cell Fractionation
Bacterial cell fractionation was performed by the sodium lauroyl sarcosine (SLS) method with minor modifications (Lattemann et al. 2000). Cells harboring display plasmid were cultured for 24 h. Cells were harvested by centrifugation at 10,000×g for 2 min, washed by PBS three times, and resuspended in Tris-HCl buffer (50 mM, pH 8.0, containing 0.3% NaCl). The cell suspension was, then, treated with an ultrasound sonication for 5 min on ice. To remove unbroken cells and debris, the whole cell lysate was centrifuged at 5,000×g for 5 min. To obtain total membrane fraction, the supernatant was pelleted by centrifugation at 20,000×g and 4°C for 1 h using an ultracentrifuge (Sigma, Osterode, Germany). The supernatant was regarded as soluble cytoplasmic fraction. For further outer membrane fractionation, the pellet (total membrane fraction) was resuspended with 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid buffer (10 mM, pH 7.4) containing 1% SLS for solubilizing inner membrane and incubated at room temperature for 30 min, and then the outer membrane fraction was repelleted by ultracentrifugation for 1 h at 20,000×g and 4°C. The supernatant was regarded as the inner membrane fraction, and after a repeat for outer membrane fractionation, the pellet was regarded as the outer membrane fraction. After cell fractionation, equal volumes of the cytoplasmic fraction and outer membrane fraction were saved for further analyses.
Enzyme-Linked ImmunoSorbent Assay
The cytoplasmic fraction and outer membrane fraction were all diluted to the same OD (OD 280 nm = 1.0) on a NanoDrop ND-1000 UV-Vis spectrophotometer (Nano-Drop Technologies, USA). Microtitre plate wells were coated with 50 μl of each fraction solution by overnight incubation at 4°C. Excess protein was discarded and wells were blocked with 200 μl of PBS containing 3% bovine serum albumin (BSA) for 1 h at 37°C. After removing the blocking solution and washing three times with PBS-T (PBS buffer, pH 7.2, containing 0.05% Tween-20), the wells were incubated for 1.5 h at 37°C with rabbit-anti-GFP (Proteintech, USA) antibody at a dilution of 1:5,000 (v/v). The complex were washed three times with PBS-T and then incubated for 1.5 h at 37°C with horseradish peroxidase-conjugated goat-anti-rabbit antibody (Jackson ImmunoResearch Laboratories, USA) at a dilution of 1:10,000 (v/v). Finally, the wells were washed three times again, and (3,3′,5,5′-Tetramethylbenzidine) TMB solution (TIANGEN Biotech, Beijing, China) was added as a color-developing substrate. With the addition of 2 M H2SO4, the reaction was terminated. The absorbance of each well was measured at 450 nm by a microplate reader (Bio-Rad model 550, Hercules, CA, USA).
Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis and Western Blot
The cell fraction (cytoplasmic and outer membrane) in equal volume was mixed with sample loading buffer, boiled for 5 min, and resolved by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. For western blot analysis, gel was electroblotted onto polyvinylidene-fluoride membrane using a Mini Protean 3 Cell (Bio-Rad, Hercules, CA, USA) at 100 V for 3 h. The membrane was then blocked with PBS-T-BSA buffer (PBS buffer, pH 7.2, containing 0.05% Tween-20 and 2% (w/v) BSA) at 37°C for 1 h. For immunodetection, the membrane was incubated for 1.5 h at 37°C in antibody solution (PBS-T-BSA buffer) containing polyclonal rabbit-anti-GFP antibody (1:3,000 (v/v); Proteintech) and probed with secondary goat-anti-rabbit immunoglobulin G conjugated with horseradish peroxidase (1:5,000 (v/v); Jackson ImmunoResearch Laboratories). After successive washing with PBS-T, the TMB solution (TIANGEN Biotech) was added and the reaction was quenched with 2 M H2SO4.