Department of Molecular and Cellular Biochemistry, 212 S. Hawthorne Dr., Simon Hall, Indiana University, Bloomington, IN 47405, USA.
Department of Biology, 212 S. Hawthorne Dr., Simon Hall, Indiana University, Bloomington, IN 47405, USA.
Microbiology (Reading). 2013 Apr;159(Pt 4):814-822. doi: 10.1099/mic.0.065680-0. Epub 2013 Feb 28.
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.
根癌农杆菌 BlcR 抑制参与 γ-丁内酯代谢的 blcABC 操纵子的转录，琥珀酸半醛 (SSA) 缓解这种抑制。BlcR 作为同源二聚体存在，blcABC 启动子 DNA 包含两个 BlcR 结合位点 (IR1 和 IR2)，它们对应于两个 BlcR 二聚体。在这项研究中，我们建立了一个体内系统来研究 SSA 对 BlcR 转录调控的响应控制。根癌农杆菌 C58 的 pAtC58 质粒编码的内源性 blcR 不适合研究 SSA 对 BlcR 抑制的影响，可能是由于 pAt 编码的 blcABC 介导的 SSA 降解。因此，我们将 blcR（和 blcABC 启动子 DNA，分别）外源性引入 C58 菌株中，该菌株已消除了 pAtC58（和 pTiC58）。我们应用该系统研究 BlcR-DNA 相互作用，并验证我们之前的结构和生化研究的预测。这种体内分析证实了先前映射的 SSA 结合位点，并支持了一种模型，即 DNA 协调 BlcR 四聚体的形成。此外，我们确定了一个特定的赖氨酸残基 (K59) 作为 DNA 结合的重要决定因素。此外，基于等温滴定量热法分析，我们发现与 IR2 相比，IR1 在与 BlcR 结合中起主导作用。总之，这些体内结果扩展了生化发现，并为 BlcR-DNA 相互作用提供了新的机制见解。
