title: Positive and negative regulation of GlnR in validamycin A biosynthesis by binding to different loci in promoter region.
abstract: Validamycin A (VAL-A) is a C7N aminocyclitol antibiotic produced by Streptomyces hygroscopicus var. jinggangensis 5008, which has been widely used as antifungal agent against rice sheath blight disease. VAL-A biosynthesis has been proven to be affected by γ-butyrolactone and temperature. Herein, we showed that GlnR, a global regulator in nitrogen metabolism, is specifically associated with valK-valA intergenic promoter region by DNA-affinity chromatography and MS-based protein identification. Subsequent EMSA and DNase I footprinting assays revealed two GlnR binding sites in this promoter region. Targeted disruption of glnR in S. hygroscopicus 5008 led to a significant increase in the transcription of VAL-A structural genes, albeit the VAL-A production was reduced by 80 % and the sporulation of the mutant was impaired. Compared with the wild-type 5008, site-specific mutagenesis of GlnR binding site I enhanced VAL-A production by 2.5-fold, whereas the mutation of GlnR binding site II resulted in a 50 % reduction of VAL-A yield. Moreover, tandem mutation of site I in the site II mutant led to a 66 % increase of VAL-A production. The result suggested that GlnR not only serves as an inhibitor by binding site I but also as an activator by binding site II for VAL-A biosynthesis. Furthermore, overexpression of glnR in the site I mutant JG45 improved VAL-A production for 41 % compared with the control strain containing the vector. Therefore, the obtained data illustrate a novel regulatory feature of the global regulator GlnR. GlnR is firstly proved to act simultaneously as an activator and a repressor in validamycin biosynthesis by binding to different loci within a promoter region of the gene cluster.
authors: State Key Laboratory of Microbial Metabolism and School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai, 200240, China.
authors: Shuang
authors: S
authors: Qu
authors: Qianjin
authors: Q
authors: Kang
authors: Hang
authors: H
authors: Wu
authors: Lei
authors: L
authors: Wang
authors: Linquan
authors: L
authors: Bai
mesh_terms: D001665:Binding Sites; D018983:DNA Footprinting; D004252:DNA Mutational Analysis; D004269:DNA, Bacterial / Q000378:metabolism; D024202:Electrophoretic Mobility Shift Assay; D015964:Gene Expression Regulation, Bacterial; D055786:Gene Knockout Techniques; D007294:Inositol / Q000031:analogs & derivatives* / Q000096:biosynthesis; D016297:Mutagenesis, Site-Directed; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D012097:Repressor Proteins / Q000235:genetics / Q000378:metabolism*; D013302:Streptomyces / Q000235:genetics* / Q000378:metabolism*; D015534:Trans-Activators / Q000235:genetics / Q000378:metabolism*
chemical_list: D004269:DNA, Bacterial; D012097:Repressor Proteins; D015534:Trans-Activators; C030790:validamycin A; D007294:Inositol