The AdhR regulatory protein is an activator of ⁵⁴-dependent transcription of adhA1 and adhA2 genes, which are required for alcohol synthesis in Clostridium beijerinckii. Here, we identified the signal perceived by AdhR and determined the regulatory mechanism of AdhR activity. By assaying the activity of AdhR in N-terminally truncated forms, a negative control mechanism of AdhR activity was identified in which the central AAA⁺ domain is subject to repression by the N-terminal GAF and PAS domains. Binding of Fe²⁺ to the GAF domain was found to relieve intramolecular repression and stimulate the ATPase activity of AdhR, allowing the AdhR to activate transcription. This control mechanism enables AdhR to regulate transcription of adhA1 and adhA2 in response to cellular redox status. The mutants deficient in AdhR or ⁵⁴ showed large shifts in intracellular redox state indicated by the NADH/NAD⁺ ratio under conditions of increased electron availability or oxidative stress. We demonstrated that the Fe²⁺-activated transcriptional regulator AdhR and ⁵⁴ control alcohol synthesis to maintain redox homeostasis in clostridial cells. Expression of N-terminally truncated forms of AdhR resulted in improved solvent production by C. beijerinckii.

IMPORTANCE Solventogenic clostridia are anaerobic bacteria that can produce butanol, ethanol, and acetone, which can be used as biofuels or building block chemicals. Here, we show that AdhR, a ⁵⁴-dependent transcriptional activator, senses the intracellular redox status and controls alcohol synthesis in Clostridium beijerinckii. AdhR provides a new example of a GAF domain coordinating a mononuclear non-heme iron to sense and transduce the redox signal. Our study reveals a previously unrecognized functional role of ⁵⁴ in control of cellular redox balance and provides new insights into redox signaling and regulation in clostridia. Our results reveal AdhR as a novel engineering target for improving solvent production by C. beijerinckii and other solventogenic clostridia.

AdhR调节蛋白是adhA1和adhA2基因⁵⁴依赖性转录的激活剂，这是拜氏梭菌中酒精合成所必需的。在这里，我们确定了AdhR感知到的信号，并确定了AdhR活性的调节机制。通过测定N-末端截短形式的AdhR的活性，鉴定了AdhR活性的阴性对照机制，其中中央AAA ⁺结构域受到N-末端GAF和PAS结构域的抑制。Fe ^2+的结合发现对GAF结构域的“抑制”减轻分子内阻遏并刺激AdhR的ATP酶活性，从而允许AdhR激活转录。这种控制机制使AdhR能够响应细胞氧化还原状态来调节adhA1和adhA2的转录。缺乏AdhR或⁵⁴的突变体在电子可利用性增加或氧化应激增加的条件下，NADH / NAD ⁺比值表明细胞内氧化还原状态发生了较大变化。我们证明了Fe ²⁺激活的转录调节因子AdhR和⁵⁴控制酒精的合成，以维持梭菌细胞中的氧化还原稳态。NhT截短形式的AdhR的表达导致拜氏梭菌提高了溶剂产量。

重要说明产溶剂梭状芽胞杆菌是厌氧细菌，可产生丁醇，乙醇和丙酮，可用作生物燃料或化学原料。在这里，我们显示AdhR，⁵⁴依赖性转录激活剂，感知细胞内的氧化还原状态，并控制拜氏梭菌中的醇合成。AdhR提供了一个GAF域的新示例，该域可协调单核非血红素铁以检测和转导氧化还原信号。我们的研究揭示了⁵⁴在控制细胞氧化还原平衡中以前未被认识的功能作用，并为梭菌中的氧化还原信号传导和调节提供了新的见解。我们的结果表明，AdhR是改善拜氏梭菌溶剂生产的新工程目标。 和其他产溶剂梭状芽胞杆菌。