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Improving the insecticidal activity of Bacillus thuringiensis subsp. aizawai against Spodoptera exigua by chromosomal expression of a chitinase gene

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Abstract

A transcriptionally fused gene comprising the P19 gene from Bacillus thuringiensis subsp. israelensis fused with a chitinase gene (chiBlA) from B. licheniformis was integrated into the B. thuringiensis subsp. aizawai BTA1 genome by homologous recombination. The resulting B. thuringiensis subsp. aizawai strain (INT1) showed growth and sporulation comparable with that of the wild-type strain. INT1 produced four chitinases of different molecular masses (i.e., 66, 55, 39, 36 kDa). Three of these (66, 55, 36 kDa) were derived from the cloned chiBlA gene, whereas the 39-kDa chitinase originated from BTA1. Using surface contamination bioassays, the 50% lethal concentration of lyophilized whole culture broth of INT1 against Spodoptera exigua neonate larvae was 12.2 μg/cm2, compared with 30.8 μg/cm2 for BTA1. Bioassays using filtered culture supernatant of INT1 (110 μg/cm2) together with trypsin-activated purified Cry1C protein of B. thuringiensis (1,280 ng/cm2) showed 75.0% mortality, compared with 56.7% mortality for Cry1C combined with BTA1 at the same concentration. Using scanning electron microscopy, clear perforations were observed in S. exigua fifth instar peritrophic membranes incubated with either crude or purified chitinase, or isolated from fifth instar S. exigua fed purified chitinase since the first instar. These results show that chitinase can increase the activity of B. thuringiensis subsp. aizawai against S. exigua. This is the first documentation of expressing a chimeric chitinase gene on the chromosome of B. thuringiensis; and chromosomal integration might be used as a potential technique for strain improvement.

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Acknowledgements

S.T. is a recipient of the Royal Golden Jubilee Scholarship from the Thailand Research Fund. This work was partially supported by the National Center for Genetic Engineering and Biotechnology (BT-B-06-XG-14-4401) and the Ministry of University Affairs, under the subproject Higher Education for Science and Technology on Agricultural Biotechnology. We thank Mary Cupp and Dunhua Zhang, Department of Entomology and Plant Pathology, Auburn University, for their help and kind support in protein purification. We thank T.W. Flegel, Faculty of Science, Mahidol University, for critical reading of this manuscript.

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Authors and Affiliations

  1. Department of Biotechnology, Faculty of Science, Mahidol University, Phayathai, Bangkok, 10400, Thailand

    S. Thamthiankul & W. Panbangred

  2. Department of Entomology and Plant Pathology, Auburn University, Auburn, Alabama, USA

    W. J. Moar

  3. Department of Biological Sciences, Auburn University, Auburn, Alabama, USA

    M. E. Miller

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  1. S. Thamthiankul
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  2. W. J. Moar
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  3. M. E. Miller
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  4. W. Panbangred
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Correspondence to W. Panbangred.

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Thamthiankul, S., Moar, W.J., Miller, M.E. et al. 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 (2004). https://doi.org/10.1007/s00253-004-1606-6

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  • DOI: https://doi.org/10.1007/s00253-004-1606-6

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