title: [Binding of transcription regulator Clpxoo to promoter of endoglucanase gene engAxoo was inhibited by c-di-GMP in Xanthomonas oryzae pv. oryzae].
abstract: OBJECTIVE
  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.
  
  
  METHODS
  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.
  
  
  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.
  
  
  CONCLUSIONS
  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.
authors.forename: Bo
authors.initials: B
authors.lastname: Li
authors.forename: Fang
authors.initials: F
authors.lastname: Tian
authors.forename: Huamin
authors.initials: H
authors.lastname: Chen
authors.forename: Maosen
authors.initials: M
authors.lastname: Wu
authors.forename: Chenyang
authors.initials: C
authors.lastname: He
mesh_terms: D002480:Cellulase / Q000235:genetics*; D003001:Cloning, Molecular; D002373:Cyclic AMP Receptor Protein / Q000235:genetics / Q000378:metabolism*; D006152:Cyclic GMP / Q000031:analogs & derivatives* / Q000494:pharmacology; D024202:Electrophoretic Mobility Shift Assay; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D014974:Xanthomonas / Q000235:genetics*
chemical_list: D002373:Cyclic AMP Receptor Protein; D011994:Recombinant Proteins; C062025:bis(3',5')-cyclic diguanylic acid; D002480:Cellulase; D006152:Cyclic GMP