Binding of transcription regulator Clpxoo to promoter of endoglucanase gene engAxoo was inhibited by c-di-GMP in Xanthomonas oryzae pv． oryzae．

Abstract:

Abstract: ［Objective］ To understand the regulatory mechanism by cyclic diguanylate (c-di-GMP) receptor and transcriptional regulator Clpxoo ofexpression of endoglucanase gene (engAxoo) in Xanthomonas oryzae pv．oryzae，the bacterial leaf blight pathogen of rice．［Methods］A plasmid to expressclpxoo gene was constructed and transformed into Escherichia coli for expression by isopropylthio-β-D-galactoside (IPTG) induction． The recombinant protein was purified by Ni-NTA resin． The binding affinity between purified Clpxoo protein and the promoter of endoglucanase gene (engAxoop) was determined by electrophoretic mobility shift assay using fluoresce in (FAM) -labeled probes．The role of c-di-GMP on the binding was also examined．［Results］Under the optimized conditions，Clpxoo was expressed and purified successfully． Mobility shift of engAxoo-p in the presence of Clpxoo was observed，indicating that specific binding occurred between them．Moreover，addition of c-di-GMP molecules in the above reaction system abolished such binding．［Conclusion］ Once interacting with the signal molecule c-di-GMP，Clpxoo conformational structure may change substantially，which results in inhibition of binding to engAxoo-p; The optimized methods for Clpxoo protein purification and electrophoretic mobility shift assay (EMSA) can be used for subsequent identification of Clp regulon in a larger scale．