Two-component signal transduction systems are the main mechanism by which bacteria sense and respond to their environment, and their membrane-located histidine protein kinases generally constitute the sensory components of these systems. Relatively little is known about their fundamental mechanisms and precise nature of the molecular signals sensed, because of the technical challenges of producing sufficient quantities of these hydrophobic membrane proteins. This study evaluated the heterologous production, purification and activities of the 16 intact membrane sensor kinases of Enterococcus faecalis. Following the cloning of the genes into expression plasmid pTTQ18His, all but one kinase was expressed successfully in Escherichia coli inner membranes. Purification of the hexa-histidine 'tagged' recombinant proteins was achieved for 13, and all but one were verified as intact. Thirteen intact kinases possessed autophosphorylation activity with no added signal when assayed in membrane vesicles or as purified proteins. Signal testing of two functionally-characterized kinases, FsrC and VicK, was successful examplifying the potential use of in vitro activity assays of intact proteins for systematic signal identification. Intact FsrC exhibited an approximately 10-fold increase in activity in response to a two-fold molar excess of synthetic GBAP pheromone, whilst glutathione, and possibly redox potential, were identified for the first time as direct modulators of VicK activity in vitro. The impact of DTT on VicK phosphorylation resulted in increased levels of phosphorylated VicR, the downstream response regulator, thereby confirming the potential of this in vitro approach for investigations of modulator effects on the entire signal transduction process of two-component systems.

中文翻译：

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来自粪肠球菌的完整膜传感器激酶家族的表达、纯化和活性。

双组分信号转导系统是细菌感知和响应环境的主要机制，它们位于膜上的组氨酸蛋白激酶通常构成这些系统的感觉组分。由于产生足够数量的这些疏水性膜蛋白的技术挑战，对其基本机制和感测到的分子信号的精确性质知之甚少。本研究评估了粪肠球菌 16 种完整膜传感器激酶的异源生产、纯化和活性。将基因克隆到表达质粒 pTTQ18His 后，除了一种激酶外，所有激酶都在大肠杆菌内膜中成功表达。六组氨酸“标记”重组蛋白的纯化达到了 13，除了一个之外，其他所有的都被证实完好无损。当在膜囊泡中或作为纯化的蛋白质进行检测时，13 种完整激酶具有自磷酸化活性，没有附加信号。两种功能表征激酶 FsrC 和 VicK 的信号测试成功地证明了完整蛋白质的体外活性测定在系统信号识别中的潜在用途。完整的 FsrC 表现出大约 10 倍的活性增加，以响应两倍摩尔过量的合成 GBAP 信息素，而谷胱甘肽和可能的氧化还原电位首次被确定为体外 VicK 活性的直接调节剂。DTT 对 VicK 磷酸化的影响导致磷酸化 VicR（下游反应调节器）水平升高，