The hydrogen-oxidizing "Knallgas" bacterium Ralstonia eutropha can thrive in aerobic and anaerobic environments and readily switches between heterotrophic and autotrophic metabolism, making it an attractive host for biotechnological applications including the sustainable H2-driven production of hydrocarbons. The soluble hydrogenase (SH), one out of four different [NiFe]-hydrogenases in R. eutropha, mediates H2 oxidation even in the presence of O2, thus providing an ideal model system for biological hydrogen production and utilization. The SH reversibly couples H2 oxidation with the reduction of NAD+ to NADH, thereby enabling the sustainable regeneration of this biotechnologically important nicotinamide cofactor. Thus, understanding the interaction of the SH with the cellular NADH/NAD+ pool is of high interest. Here, we applied the fluorescent biosensor Frex to measure changes in cytoplasmic [NADH] in R. eutropha cells under different gas supply conditions. The results show that Frex is well-suited to distinguish SH-mediated changes in the cytoplasmic redox status from effects of general anaerobiosis of the respiratory chain. Upon H2 supply, the Frex reporter reveals a robust fluorescence response and allows for monitoring rapid changes in cellular [NADH]. Compared to the Peredox fluorescence reporter, Frex displays a diminished NADH affinity, which prevents the saturation of the sensor under typical bacterial [NADH] levels. Thus, Frex is a valuable reporter for on-line monitoring of the [NADH]/[NAD+] redox state in living cells of R. eutropha and other proteobacteria. Based on these results, strategies for a rational optimization of fluorescent NADH sensors are discussed.

中文翻译：

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使用Frex荧光传感器区分富氧罗汉果细胞中由于缺氧和H2供给而引起的细胞内NADH / NAD +水平变化。

氢氧化的“纳氏气”细菌富营养的Ralstonia eutropha可以在需氧和厌氧的环境中生长，并且可以在异养和自养代谢之间轻松切换，使其成为生物技术应用（包括可持续的H2驱动的碳氢化合物生产）的有吸引力的宿主。富营养红景天中四种不同的[NiFe]氢化酶之一中的可溶性氢化酶（SH）即使在存在O2的情况下也能介导H2氧化，因此为生物制氢和利用生物提供了理想的模型系统。SH可逆地将H2氧化与NAD +还原为NADH结合，从而使这种具有生物技术重要性的烟酰胺辅因子得以可持续再生。因此，非常了解SH与细胞NADH / NAD +库的相互作用。这里，我们应用了荧光生物传感器Frex来测量在不同供气条件下富营养红景天细胞胞质[NADH]的变化。结果表明，Frex非常适合区分SH介导的细胞质氧化还原状态的变化与呼吸链一般厌氧菌的影响。供应H2后，Frex报告基因显示出强大的荧光反应，并可以监测细胞[NADH]的快速变化。与Peredox荧光报告基因相比，Frex的NADH亲和力降低，这可以防止传感器在典型细菌[NADH]水平下饱和。因此，Frex是宝贵的报告者，可用于在线监测富营养芽孢杆菌和其他蛋白细菌活细胞中的[NADH] / [NAD +]氧化还原状态。根据这些结果，