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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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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DOI: https://doi.org/10.1007/s10482-009-9332-9