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Construction of a Promoter-probe Vector for Bacillus thuringiensis: the Identification of cis-acting Elements of the chiA Locus

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Abstract

The expression and application of Bacillus thuringiensis (Bt) chitinase genes have been extensively investigated. However, little information is available regarding the regulation of chitinase gene expression in Bt. In this study, a shuttle promoter-probe vector was constructed incorporating the thermostable β-galactosidase gene bgaB of B. stearothermophilus as the reporter for the study of Bt promoters. Using this plasmid, the activity of the chiA gene promoter in Bt was investigated. Deletion analysis of the putative chiA promoter region revealed that the sequence located ~75 bp DNA from positions −116 to −42, with respect to the translation start site, is the core promoter of chiA gene. Furthermore, a site for chitin induction was identified near position −36. This site for negative regulation was indicated downstream of the RNA polymerase binding sites of the promoter of chiA. The expression of chiA started in cell grown for about 6 h and reached the maximum after 60 h of incubation. Induction of chiA expression by chitin was demonstrated by an increase in β-galactosidase activity of ~2.5-fold.

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Acknowledgments

This study was supported by grants from the National Natural Science Foundation of China (No. 30971957), the Development Program of China (863 program) (No. 2011AA10A203) and the Tianjin Natural Science Foundation (No. 11JCYBJC08300 and 2012-2014). The authors thank Prof. Wei Chen at Jiangnan University of China for the gift of the plasmid pBSK-bgaB.

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Authors and Affiliations

  1. Department of Microbiology, College of Life Sciences, Nankai University, Weijin Road 94, Tianjin, 300071, People’s Republic of China

    Chi-Chu Xie, Yang Luo, Yue-Hua Chen & Jun Cai

  2. Key Laboratory of Molecular Microbiology and Technology, Ministry of Education, Tianjin, 300071, People’s Republic of China

    Yue-Hua Chen & Jun Cai

  3. Tianjin Key Laboratory of Microbial Functional Genomics, Tianjin, 300071, People’s Republic of China

    Yue-Hua Chen & Jun Cai

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  1. Chi-Chu Xie
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  2. Yang Luo
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Correspondence to Yue-Hua Chen.

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Xie, CC., Luo, Y., Chen, YH. et al. Construction of a Promoter-probe Vector for Bacillus thuringiensis: the Identification of cis-acting Elements of the chiA Locus. Curr Microbiol 64, 492–500 (2012). https://doi.org/10.1007/s00284-012-0100-0

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