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PHYSIOLOGY AND BIOTECHNOLOGY

Optimization of Cry3A Yields in Bacillus thuringiensis by Use of Sporulation-Dependent Promoters in Combination with the STAB-SD mRNA Sequence

Hyun-Woo Park, Baoxue Ge, Leah S. Bauer, Brian A. Federici
Hyun-Woo Park
Department of Entomology and
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Baoxue Ge
Interdepartmental Graduate Program in Genetics, University of California, Riverside, California 92521, and
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Leah S. Bauer
USDA Forest Service, North Central Forest Experiment Station, Michigan State University, East Lansing, Michigan 48823
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Brian A. Federici
Department of Entomology and
Interdepartmental Graduate Program in Genetics, University of California, Riverside, California 92521, and
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DOI: 10.1128/AEM.64.10.3932-3938.1998
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  • Fig. 1.
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    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.
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    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.
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    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.
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    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.
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    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

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  • Table 1.

    Comparison of the growth efficiency of B. thuringiensis strains in four media

    StrainNo. of CFU/mla in:
    GYSbPeptonized milkG-TrisNutrient broth
    4Q75.3 × 107 a8.0 × 1083.4 × 1087.0 × 106
    4Q7/pHT31014.3 × 107 b5.9 × 1083.1 × 1082.2 × 106
    DSM 28039.7 × 107 c5.2 × 1085.8 × 1079.7 × 105
    NB1763.8 × 107 d2.2 × 1083.6 × 1074.1 × 106
    4Q7/pPFT3A3.5 × 107 e1.4 × 1081.9 × 1074.1 × 105
    4Q7/pPFT3As2.8 × 107 d1.2 × 1081.1 × 1073.4 × 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

    StrainLength (μm)Width (μm)Thickness (μm)Volb (μm3)
    DSM 28030.701 ± 0.1430.701 ± 0.1430.187 ± 0.0310.100 ± 0.054 a
    NB1760.941 ± 0.1710.941 ± 0.1710.280 ± 0.0600.236 ± 0.138 b
    4Q7/pPFT3A0.817 ± 0.1210.817 ± 0.1210.193 ± 0.0260.132 ± 0.047 a
    4Q7/pPFT3As1.555 ± 0.2731.024 ± 0.0980.434 ± 0.0920.691 ± 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

    StrainMean LC50 (ng/mm2)b (95% fiducial limits)Slope (± SE)Amt of Cry3A (% of solubilized protein)c
    DSM 28033.4 (1.75–5.65)1.88 ± 0.377.7
    4Q7/pPFT3As4.8 (3.25–6.83)1.82 ± 0.2552.9
    • ↵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

    StrainBacterial count (CFU/ml)aCrystal vol/cell (μm3)Total crystal vol/ml (μm3)Ratio
    DSM 28039.71 × 1070.1009.71.0
    4Q7/pPFT3A2.84 × 1070.1323.70.4
    4Q7/pPFT3As3.54 × 1070.69124.62.6
    NB1763.75 × 1070.2368.91.0
    • ↵a Most of the colonies obtained resulted from viable spores.

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Optimization of Cry3A Yields in Bacillus thuringiensis by Use of Sporulation-Dependent Promoters in Combination with the STAB-SD mRNA Sequence
Hyun-Woo Park, Baoxue Ge, Leah S. Bauer, Brian A. Federici
Applied and Environmental Microbiology Oct 1998, 64 (10) 3932-3938; DOI: 10.1128/AEM.64.10.3932-3938.1998

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Optimization of Cry3A Yields in Bacillus thuringiensis by Use of Sporulation-Dependent Promoters in Combination with the STAB-SD mRNA Sequence
Hyun-Woo Park, Baoxue Ge, Leah S. Bauer, Brian A. Federici
Applied and Environmental Microbiology Oct 1998, 64 (10) 3932-3938; DOI: 10.1128/AEM.64.10.3932-3938.1998
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KEYWORDS

Bacillus thuringiensis
Bacterial Proteins
Endotoxins
Promoter Regions, Genetic

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