title: In vivo analysis of DNA binding and ligand interaction of BlcR, an IclR-type repressor from Agrobacterium tumefaciens.
abstract: Agrobacterium tumefaciens BlcR represses transcription of the blcABC operon, which is involved in metabolism of γ-butyrolactone, and this repression is alleviated by succinate semialdehyde (SSA). BlcR exists as a homodimer, and the blcABC promoter DNA contains two BlcR-binding sites (IR1 and IR2) that correspond to two BlcR dimers. In this study, we established an in vivo system to examine the SSA-responsive control of BlcR transcriptional regulation. The endogenous blcR, encoded in the pAtC58 plasmid of A. tumefaciens C58, was not optimal for investigating the effect of SSA on BlcR repression, probably due to the SSA degradation mediated by the pAt-encoded blcABC. We therefore introduced blcR (and the blcABC promoter DNA, separately) exogenously into a strain of C58 cured of pAtC58 (and pTiC58). We applied this system to interrogate BlcR-DNA interactions and to test predictions from our prior structural and biochemical studies. This in vivo analysis confirmed the previously mapped SSA-binding site and supported a model by which DNA coordinates formation of a BlcR tetramer. In addition, we identified a specific lysine residue (K59) as an important determinant for DNA binding. Moreover, based on isothermal titration calorimetry analysis, we found IR1 to play the dominant role in binding to BlcR, relative to IR2. Together, these in vivo results expand the biochemical findings and provide new mechanistic insights into BlcR-DNA interactions.
authors: Department of Molecular and Cellular Biochemistry, 212 S. Hawthorne Dr., Simon Hall, Indiana University, Bloomington, IN 47405, USA.
authors: Yi
authors: Y
authors: Pan
authors: Department of Biology, 212 S. Hawthorne Dr., Simon Hall, Indiana University, Bloomington, IN 47405, USA.
authors: Yi
authors: Y
authors: Wang
authors: Department of Biology, 212 S. Hawthorne Dr., Simon Hall, Indiana University, Bloomington, IN 47405, USA.
authors: Clay
authors: C
authors: Fuqua
authors: Department of Molecular and Cellular Biochemistry, 212 S. Hawthorne Dr., Simon Hall, Indiana University, Bloomington, IN 47405, USA.
authors: Lingling
authors: L
authors: Chen
mesh_terms: D015107:4-Butyrolactone / Q000378:metabolism; D016960:Agrobacterium tumefaciens / Q000235:genetics / Q000378:metabolism*; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D004269:DNA, Bacterial / Q000235:genetics / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D008024:Ligands; D009876:Operon; D012097:Repressor Proteins / Q000235:genetics / Q000378:metabolism*; D019802:Succinic Acid / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; D008024:Ligands; D012097:Repressor Proteins; D019802:Succinic Acid; D015107:4-Butyrolactone