Abstract
Bacillus thuringiensis subsp. kurstaki HD-73 was transformed with the homologous endochitinase gene chiA74 of B. thuringiensis subsp. kenyae LBIT-82 under the regulation of its own promoter and Shine–Dalgarno sequence. The plasmid, pEHchiA74, which harbors chiA74, was detected by southern blot analysis and showed high segregational stability when the recombinant strain was grown in a medium without antibiotic. The recombinant bacterium transformed with pEHchiA74 showed an improvement in chitinolytic activity three times that of the wild-type strain. Expression of ChiA74 did not have any deleterious effect on the crystal morphology and size, but sporulation and Cry1Ac production in rich medium (nutrient broth with glucose) was reduced by approximately 30%. No significant increase in the toxicity of the transformant bacterium toward Plutella xylostella was detected using the same amount of total protein. However, it is possible that ChiA74 synthesis compensated for the decrease in net Cry1Ac synthesis and toxicity observed with the recombinant strain.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Arantes O, Lereclus D. (1991) Construction of cloning vectors for Bacillus thuringiensis. Gene 108:115–119
Arora N, Ahmad T, Rajagopal R, Bhatnagar RK (2003) A constitutively expressed 36 kDa exochitinase from Bacillus thuringiensis HD-1. Biochem Biophys Res Commun 307:620–625
Barboza-Corona JE, Nieto-Mazzocco E, Velázquez-Robledo R, Salcedo-Hernández R, Bautista M, Jiménez B, Ibarra JE (2003) Cloning, sequencing, and expresión of the chitinase gene chiA74 from Bacillus thuringiensis. Appl Environ Microbiol 69:1023–1029
Crickmore N (2005) Using worms to better understand how Bacillus thuringiensis kills insects. Trend Microbiol 13:347–350
Crickmore N (2006) Beyond the spore - past and future developments of Bacillus thuringiensis as a biopesticide. J Appl Microbiol 101:616–619
Driss F, Kallassy-Awad M, Zouari N, Jaoua S (2005) Molecular characterization of a novel chitinase from Bacillus thuringiensis subsp kurstaki. J Appl Microbiol 99:945–953
Federici BA (2005) Insecticidal bacteria An overwhelming success for invertebrate pathology. J Invertebr Pathol 89:30–38
Felse PA, Panda T (1999) Regulation and cloning of microbial chitinase genes. Appl Microbiol Biotechnol 51:141–151
Granados RR, Fu Y, Corsaro B, Hughes PR (2001) Enhancement of Bacillus thuringiensis toxicity to lepidopterous species with the enhancing from Trichoplusia ni granulovirus. J Invertebr Pathol 20:153–159
Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685
Lereclus D, Arantes O, Chaufaux J, Lecadet MM (1989) Transformation and expression of a cloned δ-endotoxin gene in Bacillus thuringiensis FEMS Microbiol Lett 60:211–218
Lertcanawanichakul M, Wiwat C, Bhumiratana A, Dean DH (2004) Expression of chitinase-encoding genes in Bacillus thuringiensis and toxicity of engineered B. thuringiensis subsp aizawai toward Lymantria dispar larvae. Current Microbiol 48:175–181
Lin Y, Xiong G (2004) Molecular cloning and sequence analysis of the chitinase gene form Bacillus thuringiensis serovar alesti. Biotechnol Lett 26:635–639
Park HW, Delécluse A, Federici BA (2001) Construction and characterization of a recombinant Bacillus thuringiensis subsp israeliensis strain that produces Cry11B. J Invertebr Pathol 78:37–44
Ramírez-Reyes A, Escudero-Abarca BI, Aguilar-Uscanga G, Hayward-Jones PM, Barboza-Corona JE (2004) Antifungal activity of Bacillus thuringiensis chitinases and its potential for the biocontrol of phytophatogenic fungi in soybean seeds. J Food Science 69: M131–M134
Regev A, Keller M, Strizhov N, Sneh B, Prudovsky E, Chet I, Ginzberg I, Koncz-Kalman Z, Koncz Z, Schell J, Zilberstein A (1996) Synesrgistic activity of a Bacillus thuringiensis δ-endotoxin and a bacterial endochitinase against Spodoptera littoralis. Appl Environ Microbiol 62:3581–3586
Ruiz-Sanchez A, Cruz-Camarillo R, Salcedo-Hernández R, Ibarra J, Barboza-Corona JE (2005) Molecular cloning and purification of an endochitinase from Serratia marcescens (Nima). Mol Biotechnol 31:103–111
Sirichotpakorn N., Rongnoparut P., Choosang K. and Panbangred. 2001. Coexpression of chitinase and the cry11Aa1 toxin genes in Bacillus thuringiensis serovar israelensis. J Inverterbr Pathol 78:160–169
Smirnoff WA (1971) Effect of chitinase on the action of Bacillus thuringiensis. Can Entomol 103:1829–1831
Tantimavanich S, Pantuwatana S, Bhumiratana A, Panbangred W (1997) Cloning of a chitinase gene into Bacillus thuringiensis subsp aizawai for enhanced insecticidal activity. J Gen Appl Microbiol 43:341–347
Thamthiankul S, Suan-Ngay S, Tantimavanich S, Panbangred W (2001). Chitinase from Bacillus thuringiensis subsp. pakistani. Appl Microbiol Biotechnol 56:395–401
Thamthiankul S, Moar WJ, Miller ME, Panbangred W (2004) Improving the insecticidal activity of Bacillus thuringiensis subsp aizawai against Spodoptera exigua by chromosomal expression of a chitinase gene. Appl Microbiol Biotechnol 65:183–192
Wiwat C, Lertcanawanichakul M, Siwayapram P, Pantuwatana S, Bhumiratana A (1996) Expression of chitinase-encoding genes from Aeromonas hydrophila and Pseudomonas maltophilia in Bacillus thuringiensis subsp israeliensis. Gene 179:119–126
Zhong W.F., Jiang L.H., Yan W.Z., Cai P.Z, Zhang Z.X. and Pei. Y. 2003. Cloning and sequence of chitinase gene from Bacillus thuringiensis subsp. israeliensis. Yi Chuan Xue Bao 30:364–369 (In Chinese)
Acknowledgements
This research was supported by grant SEP-2003-C02-44990. Also, Gabriela Casique is an undergraduate student supported by CONACyT, México, fellowship. The authors are grateful for the essential material provided by Eduardo Salazar and Jorge Ibarra and the technical assistance of Olga Gutierrez-Acosta and Laura Cortez-Preciado.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Casique-Arroyo, G., Bideshi, D., Salcedo-Hernández, R. et al. Development of a recombinant strain of Bacillus thuringiensis subsp. kurstaki HD-73 that produces the endochitinase ChiA74. Antonie van Leeuwenhoek 92, 1–9 (2007). https://doi.org/10.1007/s10482-006-9127-1
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s10482-006-9127-1