Article Figures & Data
Figures
- Fig. 1.
Major steps in the construction of vectors for expressing the cry3A gene with and without the STAB-SD sequence under the control of cyt1A promoters. (A) AHindIII fragment from B. thuringiensis subsp. morrisoni (strain tenebrionis) containing the cry3A gene was cloned into pUC13, generating pUC13-Btt. (B) Copies of cry3A with and without the STAB-SD sequence were generated by PCR and cloned into theSmaI site downstream from the dual cytA promoters of the E. coli-B. thuringiensis shuttle vector pHT3101. This yielded the expression vectors pPFT3As and pPFT3A for expressing, respectively, cry3A with and without the STAB-SD sequence under the control of the dual sporulation-dependentcytA promoters.
- Fig. 2.
Comparison of Cry3A yields by SDS-PAGE for two test media. Lanes: 1 and 2, B. thuringiensis 4Q7 transformed with pPFT3As grown, respectively, in GYS and peptonized milk; 3 and 4, NB176, the mutant strain of B. thuringiensis subsp. morrisoni (strain tenebrionis) with a higher cry3A copy number, grown, respectively in GYS and peptonized milk; 5, molecular mass markers. Pelleted patriculates from equal volumes of culture media (GYS or peptonized milk) in which test strains were grown were loaded in each lane.
- Fig. 3.
Analysis of Cry3A production of wild-type, mutant, and engineered strains of B. thuringiensis by SDS-PAGE. Sedimented crystals, spores, and cellular debris obtained from equal volumes of culture medium at the end of sporulation were loaded into each lane. Lanes: 1, molecular mass markers; 2,B. thuringiensis 4Q7 transformed with pPFT3A (cry3A without the STAB-SD sequence under the control of cytA promoters); 3, 4Q7 transformed with pPFT3As (cry3A with the STAB-SD sequence under the control ofcytA promoters); 4, wild-type B. thuringiensis subsp. morrisoni (strain tenebrionis) DSM 2803; 5, 4Q7 transformed with pHT3101; 6, NB176, the mutant strain of B. thuringiensis subsp.morrisoni (strain tenebrionis) with a highercry3A copy number. The ratios at the bottom of the lanes were determined by densitometry scanning of the gel; they indicate the ratio of Cry3A per unit of GYS in comparison to that produced by the DSM 2803 strain.
- Fig. 4.
Electron micrographs of sporulated wild-type and engineered strains of B. thuringiensis, illustrating crystals typical of these strains. (A) Wild-typeB. thuringiensis subsp.morrisoni (strain tenebrionis) DSM 2803. (B) Acrystalliferous strain (4Q7) of B. thuringiensis subsp. israelensistransformed with pPFT3A (cry3A without the STAB-SD sequence under the control of cytA promoters). (C and D) Cross section (C) and sagittal section (D) through 4Q7 cells transformed with pPFT3As (cry3A with the STAB-SD sequence under the control of cytA promoters). All micrographs are at the same magnification. Bar, 300 nm.
- Fig. 5.
Electron micrographs comparing the thickness of typical Cry3A crystals produced by NB176 and the acrystalliferous 4Q7 strain ofB. thuringiensis subsp.israelensis transformed with pPFT3As. (A) NB176. (B) 4Q7/pPFT3As. Both micrographs are the same magnification. Bar, 300 nm.
Tables
- Table 1.
Comparison of the growth efficiency of B. thuringiensis strains in four media
Strain No. of CFU/mla in: GYSb Peptonized milk G-Tris Nutrient broth 4Q7 5.3 × 107 a 8.0 × 108 3.4 × 108 7.0 × 106 4Q7/pHT3101 4.3 × 107 b 5.9 × 108 3.1 × 108 2.2 × 106 DSM 2803 9.7 × 107 c 5.2 × 108 5.8 × 107 9.7 × 105 NB176 3.8 × 107 d 2.2 × 108 3.6 × 107 4.1 × 106 4Q7/pPFT3A 3.5 × 107 e 1.4 × 108 1.9 × 107 4.1 × 105 4Q7/pPFT3As 2.8 × 107 d 1.2 × 108 1.1 × 107 3.4 × 105 ↵a Standard deviations (±) were as follows: GYS (4Q7, 2.5 × 106; 4Q7/pHT3101, 2 × 106; DSM 2803, 1.1 × 106; NB176, 2.9 × 106; 4Q7/pPFT3A, 4.6 × 106; 4Q7/pPFT3As, 2.0 × 106), peptonized milk (4Q7, 2.5 × 108; 4Q7/pHT3101, 1.8 × 107; DSM 2803, 1.9 × 107; NB176, 1.7 × 107; 4Q7/pPFT3A, 8.9 × 105; 4Q7/pPFT3As, 1.1 × 106), G-Tris (4Q7, 4.4 × 106; 4Q7/pHT3101, 1.9 × 106; DSM 2803, 4.5 × 106; NB176, 9.2 × 105; 4Q7/pPFT3A, 8.9 × 105; 4Q7/pPFT3As, 1.1 × 106), and nutrient broth (4Q7, 3 × 105; 4Q7/pHT3101, 1.3 × 105; DSM 2803, 1.4 × 104; NB176, 2.9 × 105; 4Q7/pPFT3A, 4.3 × 104; 4Q7/pPFT3As, 4.3 × 104).
↵b Values followed by different letters were significantly different at P = 0.05.
- Table 2.
Dimensions and volumes of Cry3A crystals produced by different strainsa
Strain Length (μm) Width (μm) Thickness (μm) Volb (μm3) DSM 2803 0.701 ± 0.143 0.701 ± 0.143 0.187 ± 0.031 0.100 ± 0.054 a NB176 0.941 ± 0.171 0.941 ± 0.171 0.280 ± 0.060 0.236 ± 0.138 b 4Q7/pPFT3A 0.817 ± 0.121 0.817 ± 0.121 0.193 ± 0.026 0.132 ± 0.047 a 4Q7/pPFT3As 1.555 ± 0.273 1.024 ± 0.098 0.434 ± 0.092 0.691 ± 0.242 c ↵a Means for each crystal dimension were calculated separately, whereas the volumes for those of DSM 2803 (n = 17), NB176 (n = 20), and 4Q7/pPFT3A (n = 19), were calculated for individual crystals by assuming that the length and width of each crystal were equal. Therefore, multiplying the mean dimensions for these three strains will yield values slightly different from those presented in the Volume column. The length and width of crystals produced by 4Q7/pPFT3As, however, were not equal, and thus the volume value is the product of the mean dimensions.
↵b Values followed by different letters were significantly different at P = 0.05.
- Table 3.
Toxicity of Cry3A produced by DSM 2803 and 4Q7/pPFT3As to C. scriptaa
Strain Mean LC50 (ng/mm2)b (95% fiducial limits) Slope (± SE) Amt of Cry3A (% of solubilized protein)c DSM 2803 3.4 (1.75–5.65) 1.88 ± 0.37 7.7 4Q7/pPFT3As 4.8 (3.25–6.83) 1.82 ± 0.25 52.9 ↵a Cry3A was solubilized from lyophilized powders of each strain grown on GYS medium and assayed against second instars feeding on leaf discs.
↵b These values are not significantly different.
↵c This value represents the proportion that Cry3A constituted of the total protein solubilized from the lyophilized powders of these strains.
- Table 4.
Calculated crystal yields per unit of culture medium for different Cry3A strains
Strain Bacterial count (CFU/ml)a Crystal vol/cell (μm3) Total crystal vol/ml (μm3) Ratio DSM 2803 9.71 × 107 0.100 9.7 1.0 4Q7/pPFT3A 2.84 × 107 0.132 3.7 0.4 4Q7/pPFT3As 3.54 × 107 0.691 24.6 2.6 NB176 3.75 × 107 0.236 8.9 1.0 ↵a Most of the colonies obtained resulted from viable spores.
