title: [Construction of screening system for mutation of negative regulatory genes in Streptomyces].
abstract: OBJECTIVE
  We aimed to create a novel report system for screening the mutation of the negative regulatory genes, especially for those repressing the expression of cryptic antibiotics clusters.
  
  
  METHODS
  We used marker-free gene disruption strategy, which combines with the "REDIRECT (Rapid Efficient Directed Recombination Time Saving)" technology and in vivo site-specific recombination by Streptomyces phage phiBT1 integrase, to construct a scbR2/inoA double mutant strain of S. coelicolor M145. This strain was used as the host of the report system. For the construction of the reporter plasmid, the ScbR2 repressed promoter of cpkO from CPK (cryptic polyketide) cluster was used to drive the expression of a promoterless conserved gene inoA of S. coelicolor. Then the reporter plasmid was introduced into the host strain described above to test the availability of inoA as a reporter gene in this system.
  
  
  RESULTS
  The scbR2/inoA double mutant strain gave rise to a bald pheno type on MM medium in the absence of inositol, and produced yellow pigmented secondary metabolite by the disruption of scbR2 to release the repression of cpkO, a pathway specific activator gene situated in CPK cluster. After introducing the reporter plasmid into this test stain, the resulting strain recovered the phenotype as wild-type strain, indicating that the promoter of cpkO can drive the expression of inoA in scbR2 mutant and consequently restore the biosynthesis of inositol.
  
  
  CONCLUSIONS
  Our results indicated that inoA can be used as a novel reporter gene for Streptomyces, especially for detecting the activation of the "silent" promoter. This report system might be available for screening the mutation of the negative regulatory genes for the cryptic secondary metabolic gene clusters.
authors.affiliation: Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China. zhuyu_126@126.com
authors.forename: Yu
authors.initials: Y
authors.lastname: Zhu
authors.affiliation: Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China.
authors.forename: Chi
authors.initials: C
authors.lastname: Feng
authors.affiliation: Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China.
authors.forename: Huarong
authors.initials: H
authors.lastname: Tan
authors.affiliation: Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China.
authors.forename: Yuqing
authors.initials: Y
authors.lastname: Tian
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D015536:Down-Regulation; D015964:Gene Expression Regulation, Bacterial; D020868:Gene Silencing; D005809:Genes, Regulator; D017930:Genes, Reporter; D009154:Mutation; D010957:Plasmids / Q000235:genetics; D011995:Recombination, Genetic; D048372:Streptomyces coelicolor / Q000235:genetics* / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins