Yuehua Chen, Jin Shi, Na Zhang, Jun Cai and Mingchun Li
Department of Microbiology, College of Life Sciences, Nankai University, Tianjin, China
At present, the chitinase gene (chi) expression have been mainly studied in Streptomyces, and relatively less reported in Bacillus. A cis-acting element has been founded in the upstream of chiA and chiB in Bacillus thuringenesis subsp. israelensis strain 75(Bti 75) by this lab. Which was similar to “dre” (DasR responsive element) in B. subtilis. Substitution or deletion one base of this dre sequence would convert inducible expression of chi expression into constitutive expression. As was reported, the regulation of YvoA was similar to DasR, which was a key regulator of metabolism of chitin and its hydrolysis products in Bt.
Based on the homology modeling（http://www.swissmodel.expasy.org/） and molecular docking (Discovery Studio Client 2.5) results, the yvoA gene was mutated by site-specific mutagenesis, then transform into E. coli XL21. The mutated YvoA was expressed and purified. The interactions of the mutation YvoA with DNA or effector was detected by use of Electrophoretic mobility shift assay (EMSA).
This study carried out totally 11 mutative sites of amino acid. The results showed that the structural basis of HTH domains in N terminal was essential for YvoA binding to DNA. When the N terminal Glu17 was mutated into Gly17, YvoA lost its ability to bind to dre site of DNA.
The C terminal Arg135 was mutated into Gly135, YvoA lost its ability to bind to the effector GlcN-6-P. The study on the effector-binding domain in C terminal of YvoA demonstrated the amino acid carried the positively charged played a key role in “anchoring” the molecules on GlcN-6-P.
Keywords: B. thuringiensis; chitinase; YvoA; site-directed-mutagenesis; acive site.