Bacillus thuringiensis isolates and growth conditions
Local B. thuringiensis kurstaki strains (MnD and BnBt) obtained from the culture collection at Department of Biology, Faculty of Science, Karadeniz Technical University, Turkey, and B. thuringiensis kurstaki HD-1 strain (positive control) obtained from Bacillus Genetic Stock Center were used in this study (Kati et al. 2005, 2007; Sezen et al. 2010). Bt isolates were grown in a nutrient agar (NA; Merck) and Luria Bertani media at 30 °C (for 1 l LB; 10 g tryptone, 5 g NaCl, 5 g yeast extract).
Spodoptera littoralis was grown in the Microbiology Laboratory, Karadeniz Technical University, Trabzon, Turkey, under controlled conditions of 25 ± 2 °C, 60 ± 5% RH, and L16:D8 h. S. littoralis originated from a standardized culture line. Larvae were reared on semi-synthetic diet (266.5 g dried bean, 4 g ascorbic acid, 2.5 g methyl 4 hydroxybenzoate, 1.25 g sorbic acid, 0.5 g B vitamin, 3 g wheat germ, 35 g yeast extract, 14 g agar).
Detection of Vip3 gene by polymerase chain reaction
Total DNA was extracted as explained earlier (Ferrandis et al. 1999). For the screening of the Vip3 gene, degenerate primers were used (Hernández-Rodríguez et al. 2009). Polymerase chain reaction (PCR) was prepared in a final volume of 50 μl. The PCR mixtures included 3 μl of the DNA template, 1.25 U of Taq DNA polymerase (New England BioLabs), 5 μl of 10× reaction buffer, 3 μl 1.5 mM MgCl2, 1 μl 10 mM of each dNTP, and 1 μl 10 mM primers for Vip3 gene screening. The PCR conditions were as follows: 5 min denaturation at 95 °C, followed by 35 cycles of denaturation at 95 °C for 1 min, annealing at 45 °C for 1 min, and extension at 72 °C for 2 min. Final extension was carried out at 72 °C for 10 min. The products were separated by electrophoresis in 1% agarose gel.
Cloning and sequencing of Vip3 gene
The amplified Vip3 DNA fragment was purified, using the NucleoSpin Extract DNA Purification Kit (from Macherey-Nagel), ligated with pGEM-T cloning vector (from Promega) using T4 DNA ligase enzyme, and transformed into Escherichia coli JM101 cells. The clones were selected on LB plates containing ampicillin (50 mg/ml), X-Gal (40 mg/ml), and IPTG (24 mg/ml). The Vip3 gene was analyzed and compared to the updated GenBank data by using BLAST program (http://www.ncbi.nlm.nih.gov/blast).
Purification of Vip3 proteins
Culture of B. thuringiensis kurstaki isolates (BnBt, MnD)
B. thuringiensis isolates were re-inoculated to a 0.1 OD600 nm from an overnight culture on a Teriffic (12% tryptone, 2.4% yeast extract, 0.04% glycerol, 0.17 M KH2PO4, 0.72 M K2HPO4) liquid medium to determine the bacterial growth phase and vegetative phase and were maintained at 30 °C in a shaking incubator. Measurements were taken at OD600 nm with samples taken at specific time intervals for 36 h, and the vegetative phase was determined by plotting the data on the graph.
Isolation of Vip3 proteins from cultured supernatants
Bacterial cultures grown for 24 h were centrifuged at 12.000g for 10 min at 4 °C, and supernatants containing Vip3 proteins were collected (Sattar et al. 2008).
Ammonium sulfate precipitation
Ammonium sulfate [(NH4)2SO4] precipitation method (REF) was followed to precipitate the protein from the culture supernatant. First of all, solid ammonium sulfate was added to the bacterial supernatant at 60% saturation (Sattar et al. 2008). Ammonium sulfate was added to the supernatant slowly at 4 °C by mixing on a magnetic stirrer. Precipitation was followed by centrifugation at 10.000 rpm for 10 min at 4 °C. After centrifugation, the pellets were resolved by using 1 ml of 100 mM pH 7.5 Tris-HCl buffer.
The ammonium sulfate precipitation was subjected to overnight dialysis against 20 mM pH 7.5 Tris-HCl buffer to remove especially the salts and small molecules. Following dialysis, the samples were centrifuged at the highest speed for 10 min to allow the degradation of the proteins.
A column of 50 cm in length and 1.5 cm in diameter was used for ion-exchange chromatography. For this purpose, Q-Sepharose (anion exchanger), an anionic ion exchanger, was used in this process. Tris-HCI buffer (20 mM pH 8) was used as the mobile phase. The gasses of the column material and all buffers used in the experiment were taken in a vacuum pump and then slowly filled into the collar using a pasteurized pipette. After filling, the column was equilibrated with 500 ml of Tris-HCI buffer. The extract was passed through the column to allow the contained proteins to be attached to the column packing material. Subsequently, 50 ml of buffer was passed through the column to remove proteins that were not attached to the column. The salt (NaCl) content of the column was then increased from 0 to 0.6 M. A 200 ml NaCl gradient bridge was used for this. The buffer flow rate was set to 1 ml/min, and the fractions from the column were collected in glass tubes to be 3.5 ml.
The fractions obtained were run on 12% SDS-PAGE. The determined tubes containing protein extract were combined and selected for purity on SDS-PAGE. After completion of the SDS-PAGE run, the gels were stained with fast silver staining technique. The gel image was transferred to the computer using a scanner.
Estimation of the protein
The amount of protein was determined according to the Bradford method (Bradford 1976). Bovine serum albumin (BSA) was used as standard in the calibration curve prepared. The prepared standards and samples were transferred onto 96-well microplates, and measurements were made at 595 nm, using a UV-visible spectroscopy system (Bio-rad).
Trypsin activation of Vip3 proteins
Partially purified Vip3 proteins were activated by commercial trypsin (10%) at 37 °C for 2 h.
Determination of insecticidal activities of Vip3 proteins
The second instar larvae of S. littoralis were used to test the activity of partially purified and trypsin-activated Vip3 proteins of Bt kurstaki isolates (BnBt, MnD). Protein samples were prepared at the concentrations of 10, 25, 50, 100, and 200 ng/μl from both Vip3 proteins. In the experiments, water and NaCl were used as negative controls. Samples containing Vip3 proteins of BnBt and MnD isolates were spread with 10 μl of sample on the 1 cm2 of lettuce leaf for testing on each one larva of S. littoralis. The concentrations of toxin samples were applied per insect. Larvae were starved for 4 h before application. For each concentration of Vip3 protein, 30 larvae were placed in test vessels one by one, and tests were repeated three times. Biotests were carried out at 25 ± 2 °C and 60% RH. Larval mortality was followed up to 10 days and corrected for control mortality, using Abbott’s formula (Abbott 1925).
The data were subjected to ANOVA and subsequently to LSD. The lethal concentrations (LC50 and LC90) were estimated by probit regression analysis (Finney 1971). Statistical analyses were performed using SPSS 20.0 software.