General and Molecular Microbiology, Institute for Chemistry and Biology of the Marine Environment (ICBM), Carl von Ossietzky University of Oldenburg, Oldenburg, Germany.
General and Molecular Microbiology, Institute for Chemistry and Biology of the Marine Environment (ICBM), Carl von Ossietzky University of Oldenburg, Oldenburg, Germany
Appl Environ Microbiol. 2018 Aug 31;84(18). doi: 10.1128/AEM.01018-18. Print 2018 Sep 15.
The denitrifying betaproteobacterium "" EbN1 regulates the capacity to anaerobically degrade -ethylphenol (via -hydroxyacetophenone) with high substrate specificity. This process is mediated by the σ-dependent transcriptional regulator EtpR, which apparently recognizes both aromatic compounds, yielding congruent expression profiles. The responsiveness of this regulatory system was studied with -hydroxyacetophenone, which is more easily administered to cultures and traced analytically. Cultures of EbN1 were initially cultivated under nitrate-reducing conditions with a growth-limiting supply of benzoate, upon the complete depletion of which -hydroxyacetophenone was added at various concentrations (from 500 μM down to 0.1 nM). Depletion profiles of this aromatic substrate and presumptive effector were determined by highly sensitive micro-high-performance liquid chromatography (microHPLC). Irrespective of the added concentration of -hydroxyacetophenone, depletion commenced after less than 5 min and suggested a response threshold of below 10 nM. This approximation was corroborated by time-resolved transcript profiles (quantitative reverse transcription-PCR) of selected degradation and efflux relevant genes (e.g., , encoding a subunit of predicted -ethylphenol methylenehydroxylase) and narrowed down to a range of 10 to 1 nM. The most pronounced transcriptional response was observed, as expected, for genes located at the beginning of the two operon-like structures, related to catabolism (i.e., ) and potential efflux (i.e., ). Aromatic compounds are widespread microbial growth substrates with natural as well as anthropogenic sources, albeit with their concentrations and their bioavailabilities varying over several orders of magnitude. Even though degradation pathways and underlying regulatory systems have long been studied with aerobic and, to a lesser extent, with anaerobic bacteria, comparatively little is known about the effector concentration-dependent responsiveness. EbN1 is a model organism for the anaerobic degradation of aromatic compounds with the architecture of the catabolic network and its substrate-specific regulation having been intensively studied by means of differential proteogenomics. The present study aims at unraveling the minimal concentration of an aromatic growth substrate (-hydroxyacetophenone here) required to initiate gene expression for its degradation pathway and to learn in principle about the lower limit of catabolic responsiveness of an anaerobic degradation specialist.
反硝化β-变形菌 EbN1 具有高度的底物特异性，可调节厌氧降解 -乙基苯酚（通过 -羟基苯乙酮）的能力。该过程由 σ 依赖性转录调节因子 EtpR 介导，该因子显然可以识别这两种芳香族化合物，从而产生一致的表达谱。通过 -羟基苯乙酮研究了该调控系统的响应性，因为 -羟基苯乙酮更容易添加到培养物中并进行分析追踪。最初，将 EbN1 培养物在硝酸盐还原条件下培养，并用苯甲酸进行生长限制供应，当苯甲酸完全耗尽时，以各种浓度（从 500 μM 降至 0.1 nM）添加 -羟基苯乙酮。通过高灵敏度微高效液相色谱（microHPLC）测定这种芳香族底物和推定效应物的消耗曲线。无论添加的 -羟基苯乙酮浓度如何，消耗都在不到 5 分钟后开始，表明响应阈值低于 10 nM。这种近似值通过选定的降解和外排相关基因（例如，编码预测的 -乙基苯酚亚甲基羟化酶的一个亚基）的时程转录谱（定量反转录-PCR）得到证实，并缩小到 10 到 1 nM 的范围。正如预期的那样，观察到最明显的转录反应是与两个类似操纵子结构相关的基因，这些基因与分解代谢（即）和潜在的外排（即）有关。芳香族化合物是广泛存在的微生物生长底物，具有天然和人为来源，尽管它们的浓度和生物可利用性在几个数量级上有所不同。尽管已经对好氧菌和在较小程度上对厌氧菌的降解途径和潜在的调控系统进行了长期研究，但对于效应物浓度依赖性响应性的了解相对较少。EbN1 是一种用于研究芳香族化合物厌氧降解的模式生物，其代谢网络的结构及其底物特异性调控已通过差异蛋白基因组学进行了深入研究。本研究旨在揭示启动其降解途径基因表达所需的芳香族生长底物（此处为 -羟基苯乙酮）的最小浓度，并从原则上了解厌氧降解专家的代谢响应的下限。
