Hyperproduction of chitinase influences crystal toxin synthesis and sporulation of Bacillus thuringiensis |
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| Authors: | J. Eleazar Barboza-Corona Tomás Ortiz-Rodríguez Norma de la Fuente-Salcido Dennis K. Bideshi Jorge E. Ibarra Rubén Salcedo-Hernández |
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| Affiliation: | 1.Campus Irapuato-Salamanca, División Ciencias de la Vida, Departamento de Ingeniería en Alimentos,Universidad de Guanajuato,Irapuato,Mexico;2.Unidad Académica de Ciencias Químico Biológicas Farmacéuticas,Universidad Autónoma de Nayarit,Tepic,Mexico;3.Escuela de Ciencias Biológicas,Universidad Autónoma de Coahuila,Torreón,Mexico;4.Department of Natural and Mathematical Sciences,California Baptist University,Riverside,USA;5.Department of Entomology,University of California, Riverside,Riverside,USA;6.Departamento de Biotecnología y Bioquímica,Cinvestav-IPN,Irapuato,Mexico |
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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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| Keywords: | Bacillus thuringiensis ChiA74 Cry1Ac Endochitinase Overexpression pcytA/STAB |
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