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Hyperproduction of chitinase influences crystal toxin synthesis and sporulation of Bacillus thuringiensis

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Abstract

Bacillus thuringiensis HD-73 was transformed with the endochitinase gene chiA74 under the control of a strong promoter (pcytA) and a 5′ mRNA stabilizing (STAB-SD) sequence (HD-73-pEBchiA74). Expression levels were compared with those observed from the wild type strain (HD-73) and the recombinant HD-73 strain expressing chiA74 under the control of its native promoter (HD-73-pEHchiA74). The chitinolytic activity of HD-73-pEBchiA74 was markedly elevated, being ~58- and 362-fold higher than, respectively, HD-73-pEHchiA74 and parental HD-73, representing the highest levels of chitinase expression in recombinant B. thuringiensis reported to date. Parasporal crystals measured under transmission electron microscopy showed that HD-73 produced crystals of 1.235 (±0.214) and 1.356 (±0.247) μm in length when the bacterium was grown in respectively, NBS and NBS with glucose. Otherwise, HD-73-pEBchiA74 synthesized crystals of 1.250 (±0.222) and 1.139 (±0.202) μm in length when cultivated in NBS and NBS with glucose, respectively, values that showed a diminution of ~10 and 20% compared with crystals produced by HD-73-pEHchiA74 grown under the same conditions. Comparison of viable spore counts per ml showed that HD-73-pEBchiA74 produced fewest viable spores (1.5 × 109, 1.3 × 109), compared to HD-73-pEHchiA74 (4.9 × 109, 5.3 × 109) and HD-73 (6.8 × 109, 8.8 × 109) when grown in NBS and NBS supplemented with glucose, respectively. No change in cellular protease activity was observed despite the overproduction of the chitinase.

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Acknowledgments

This research was supported by grant (SEP-CONACYT-2003-C02-44990, México) to J.E.B.-C. The authors are grateful for the essential material provided by Brian A. Federici (University of California, Riverside) and Hyun-Woo Park (Florida A&M University) for the pPF-CH vector and whose group developed the pcyt1A/STAB-SD expression system. In addition, we thank Gabriela Casique-Arroyo, Laura Cortez, Regina Basurto and Aurora Verver for their excellent technical support. Tomas Ortiz-Rodríguez and Norma de la Fuente-Salcido are, respectively, undergraduate and graduate students supported by CONCYTEG and CONACYT, México, fellowships.

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Authors and Affiliations

  1. Campus Irapuato-Salamanca, División Ciencias de la Vida, Departamento de Ingeniería en Alimentos, Universidad de Guanajuato, Apartado postal 311, 36500, Irapuato, Guanajuato, Mexico

    J. Eleazar Barboza-Corona, Tomás Ortiz-Rodríguez, Norma de la Fuente-Salcido & Rubén Salcedo-Hernández

  2. Unidad Académica de Ciencias Químico Biológicas Farmacéuticas, Universidad Autónoma de Nayarit, Tepic, Nayarit, Mexico

    Tomás Ortiz-Rodríguez

  3. Escuela de Ciencias Biológicas, Universidad Autónoma de Coahuila, 27440, Torreón, Coahuila, Mexico

    Norma de la Fuente-Salcido

  4. Department of Natural and Mathematical Sciences, California Baptist University, 8432 Magnolia Avenue, Riverside, CA, 92504, USA

    Dennis K. Bideshi

  5. Department of Entomology, University of California, Riverside, Riverside, CA, 92521, USA

    Dennis K. Bideshi

  6. Departamento de Biotecnología y Bioquímica, Cinvestav-IPN, Campus Guanajuato, 36500, Irapuato, Guanajuato, Mexico

    Jorge E. Ibarra

Authors
  1. J. Eleazar Barboza-Corona
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  2. Tomás Ortiz-Rodríguez
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  3. Norma de la Fuente-Salcido
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  4. Dennis K. Bideshi
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  5. Jorge E. Ibarra
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  6. Rubén Salcedo-Hernández
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Correspondence to J. Eleazar Barboza-Corona.

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Barboza-Corona, J.E., Ortiz-Rodríguez, T., de la Fuente-Salcido, N. et al. Hyperproduction of chitinase influences crystal toxin synthesis and sporulation of Bacillus thuringiensis . Antonie van Leeuwenhoek 96, 31–42 (2009). https://doi.org/10.1007/s10482-009-9332-9

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