IBG-1: Biotechnology, Institute of Bio- and Geosciences, Forschungszentrum Jülich, Jülich, Germany.
Department of Biological Chemistry, Faculty of Agriculture, Yamaguchi University, Yamaguchi, Japan.
Appl Environ Microbiol. 2021 May 11;87(11). doi: 10.1128/AEM.00195-21.
Gene expression in the obligately aerobic acetic acid bacterium responds to oxygen limitation, but the regulators involved are unknown. In this study, we analyzed a transcriptional regulator named GoxR (GOX0974), which is the only member of the fumarate-nitrate reduction regulator (FNR) family in this species. Evidence that GoxR contains an iron-sulfur cluster was obtained, suggesting that GoxR functions as an oxygen sensor similar to FNR. The direct target genes of GoxR were determined by combining several approaches, including a transcriptome comparison of a Δ mutant with the wild-type strain and detection of GoxR binding sites by chromatin affinity purification and sequencing (ChAP-Seq). Prominent targets were the genes encoding a cytochrome oxidase with low O affinity, which were repressed by GoxR, and the operon, which was activated by GoxR. The operon encodes a transhydrogenase (), an NADH-dependent oxidoreductase (GOX0313), and another oxidoreductase (GOX0314). Evidence was obtained for GoxR being active despite a high dissolved oxygen concentration in the medium. We suggest a model in which the very high respiration rates of due to periplasmic oxidations cause an oxygen-limited cytoplasm and insufficient reoxidation of NAD(P)H in the respiratory chain, leading to inhibited cytoplasmic carbohydrate degradation. GoxR-triggered induction of the operon enhances fast interconversion of NADPH and NADH by the transhydrogenase and NADH reoxidation by the GOX0313 oxidoreductase via reduction of acetaldehyde formed by pyruvate decarboxylase to ethanol. In fact, small amounts of ethanol were formed by under oxygen-restricted conditions in a GoxR-dependent manner. serves as a cell factory for oxidative biotransformations based on membrane-bound dehydrogenases and as a model organism for elucidating the metabolism of acetic acid bacteria. Surprisingly, to our knowledge none of the more than 100 transcriptional regulators encoded in the genome of has been studied experimentally until now. In this work, we analyzed the function of a regulator named GoxR, which belongs to the FNR family. Members of this family serve as oxygen sensors by means of an oxygen-sensitive [4Fe-4S] cluster and typically regulate genes important for growth under anoxic conditions by anaerobic respiration or fermentation. Because has an obligatory aerobic respiratory mode of energy metabolism, it was tempting to elucidate the target genes regulated by GoxR. Our results show that GoxR affects the expression of genes that support the interconversion of NADPH and NADH and the NADH reoxidation by reduction of acetaldehyde to ethanol.
基因表达在专性需氧乙酸菌 响应氧气限制，但涉及的调节剂是未知的。在这项研究中，我们分析了一个转录调节剂命名为 GoxR（GOX0974），这是唯一的成员在该物种的延胡索酸 - 硝酸盐还原调节剂（FNR）家族。获得了 GoxR 含有铁 - 硫簇的证据，表明 GoxR 作为类似于 FNR 的氧气传感器发挥作用。通过结合几种方法，包括与野生型菌株相比 Δ突变体的转录组比较和染色质亲和纯化和测序（ChAP-Seq）检测 GoxR 结合位点，确定了 GoxR 的直接靶基因。突出的靶标是编码低 O 亲和力细胞色素氧化酶的 基因，这些基因被 GoxR 抑制， 和操纵子被 GoxR 激活。 操纵子编码一种转氢酶（），一种 NADH 依赖性氧化还原酶（GOX0313）和另一种氧化还原酶（GOX0314）。尽管培养基中溶解氧浓度很高，但仍获得了 GoxR 活性的证据。我们提出了一个模型，即由于周质氧化作用， 非常高的呼吸速率导致细胞质缺氧和呼吸链中 NAD（P）H的再氧化不足，导致细胞质碳水化合物降解受到抑制。GoxR 触发的 操纵子诱导通过转氢酶增强 NADPH 和 NADH 的快速转换，并且通过丙酮酸脱羧酶形成的乙醛还原为乙醇来增强 GOX0313 氧化还原酶的 NADH 再氧化。事实上，在 GoxR 依赖性方式下，在氧气限制条件下， 形成少量乙醇。作为基于膜结合脱氢酶的氧化生物转化的细胞工厂，并作为阐明乙酸细菌代谢的模式生物。令人惊讶的是，据我们所知，直到现在，在 基因组中编码的 100 多个转录调节剂中，没有一个被实验研究过。在这项工作中，我们分析了一种名为 GoxR 的调节剂的功能，它属于 FNR 家族。该家族的成员通过一个氧气敏感的[4Fe-4S]簇作为氧气传感器，通常通过无氧呼吸或发酵来调节缺氧条件下生长所必需的基因。因为 具有专性需氧呼吸的能量代谢模式，因此很有必要阐明 GoxR 调节的靶基因。我们的结果表明，GoxR 影响支持 NADPH 和 NADH 相互转换以及通过还原乙醛为乙醇来再氧化 NADH 的基因的表达。
