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

Abstract

To understand the regulatory mechanism by cyclic diguanylate (c-di-GMP) receptor and transcriptional regulator Clpxoo of expression of endoglucanase gene (engAxoo) in Xanthomonas oryzae pv. oryzae, the bacterial leaf blight pathogen of rice. A plasmid to expressclpxoo gene was constructed and transformed into Escherichia coli for expression by isopropylthio-beta-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 (engAxoo-p) 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. 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. 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.