Cellular Concentrations of the Transporters DctA and DcuB and the Sensor DcuS of Escherichia coli and the Contributions of Free and Complexed DcuS to Transcriptional Regulation by DcuR
Cellular Concentrations of the Transporters DctA and DcuB and the Sensor DcuS of Escherichia coli and the Contributions of Free and Complexed DcuS to Transcriptional Regulation by DcuR
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大肠杆菌转运蛋白 DctA 和 DcuB 以及传感器 DcuS 的细胞浓度以及游离和复合 DcuS 对 DcuR 转录调节的贡献
DOI：
10.1128/jb.00612-17
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发表时间：
2018
影响因子：
3.2
通讯作者：
Unden G
中科院分区：
文献类型：
--
作者：
Wörner S;Surmann K;Ebert-Jung A;Völker U;Hammer E;Unden G
在大肠杆菌中，C4-二羧酸的催化由DcuS-DcuR双组分系统调节。传感器激酶DcuS的功能状态由C4-二羧酸盐（如富马酸盐）控制，并分别与C4-二羧酸转运蛋白DctA和DcuB复合。已知游离DcuS（DcuSF）即使在不存在富马酸盐的情况下也具有恒定活性，而DcuB-DcuS和DctA-DcuS复合物需要富马酸盐来活化。为了阐明转运蛋白对DcuS功能状态和DcuSF和DcuB-DcuS（或DctA-DcuS）浓度的影响，通过质谱法测定需氧或厌氧生长细胞中DcuS、DcuB和DctA的绝对水平。DcuS以恒定的极低水平存在（10 - 20分子DcuS/细胞），而DcuB和DctA的水平较高（最低，200分子/细胞），并在富马酸盐中进一步增加（分别为12.7倍和2.7倍）。与DcuS和DcuB内容与DcuS的功能状态允许估计的DcuS的比例在自由（DcuSF）和复杂的（DcuB-DcuS）状态。出乎意料的是，DcuSF水平总是很低（<总DcuS的2%），排除了早期模型显示DcuSF在非诱导条件下是主要物质。在不存在富马酸盐的情况下，当DcuSF负责基础dcuB表达时，形成高达8%的最大DcuB水平。这些足以用于DcuB-DcuS复合物形成和基础运输活性。在富马酸盐（> 100 μ M）的存在下，DcuB-DcuS复合物驱动大部分dcuB表达，因此负责诱导。重要信息双组分系统（TCS）是细菌传感和适应环境线索的主要装置。TCS的膜结合传感器激酶通常使用功能未知的辅助蛋白。DcuS-DcuR TCS对C4-二羧酸起反应，需要DcuS与C4-二羧酸转运蛋白DctA或DcuB形成复合物。游离DcuS（DcuSF）在自磷酸化中具有组成性活性，并被认为在特定条件下具有主要作用。在此，通过质谱法在活化和非活化条件下测定DcuS、DcuB和DctA的绝对浓度。它们的绝对含量与DcuS的功能状态的关系揭示了它们对DcuS-DcuR在体内的控制的贡献，这是通过其他方法无法获得的，从而导致对先前模型的修订。
In Escherichia coli, the catabolism of C4-dicarboxylates is regulated by the DcuS-DcuR two-component system. The functional state of the sensor kinase DcuS is controlled by C4-dicarboxylates (like fumarate) and complexation with the C4-dicarboxylate transporters DctA and DcuB, respectively. Free DcuS (DcuSF) is known to be constantly active even in the absence of fumarate, whereas the DcuB-DcuS and DctA-DcuS complexes require fumarate for activation. To elucidate the impact of the transporters on the functional state of DcuS and the concentrations of DcuSFand DcuB-DcuS (or DctA-DcuS), the absolute levels of DcuS, DcuB, and DctA were determined in aerobically or anaerobically grown cells by mass spectrometry. DcuS was present at a constant very low level (10 to 20 molecules DcuS/cell), whereas the levels of DcuB and DctA were higher (minimum, 200 molecules/cell) and further increased with fumarate (12.7- and 2.7-fold, respectively). Relating DcuS and DcuB contents with the functional state of DcuS allowed an estimation of the proportions of DcuS in the free (DcuSF) and the complexed (DcuB-DcuS) states. Unexpectedly, DcuSFlevels were always low (<2% of total DcuS), ruling out earlier models that show DcuSFas the major species under noninducing conditions. In the absence of fumarate, when DcuSFis responsible for basaldcuBexpression, up to 8% of the maximal DcuB levels are formed. These suffice for DcuB-DcuS complex formation and basal transport activity. In the presence of fumarate (>100 μM), the DcuB-DcuS complex drives the majority ofdcuBexpression and is thus responsible for induction.IMPORTANCETwo-component systems (TCS) are major devices for sensing by bacteria and adaptation to environmental cues. Membrane-bound sensor kinases of TCS often use accessory proteins of unknown function. The DcuS-DcuR TCS responds to C4-dicarboxylates and requires formation of the complex of DcuS with C4-dicarboxylate transporters DctA or DcuB. Free DcuS (DcuSF) is constitutively active in autophosphorylation and was supposed to have a major role under specific conditions. Here, absolute concentrations of DcuS, DcuB, and DctA were determined under activating and nonactivating conditions by mass spectrometry. The relationship of their absolute contents to the functional state of DcuS revealed their contribution to the control of DcuS-DcuRin vivo, which was not accessible by other approaches, leading to a revision of previous models.
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影响因子：
3.2
作者：
Davies, SJ;Golby, P;Andrews, SC
通讯作者：
Andrews, SC
大肠杆菌柠檬酸循环的分子遗传学方面。
DOI：
--
发表时间：
1987
期刊：
Biochemical Society Symposium
影响因子：
--
作者：
J. S. Miles;J. Guest
通讯作者：
J. Guest
大肠杆菌 DctA/DcuS 传感器复合物中 DctA 和 DcuS 功能的区分：DctA 作为活性开关的功能和 DcuS 作为 C4-二羧酸盐传感器的功能
影响因子：
3.6
作者：
P. Steinmetz;Sebastian Wörner;G. Unden
通讯作者：
G. Unden
大肠杆菌膜和蛋白脂质体中的寡聚传感器激酶 DcuS：化学交联和 FRET 光谱
影响因子：
3.2
作者：
P. Scheu;Yun;J. Bauer;H. Kneuper;T. Basché;G. Unden;W. Erker
通讯作者：
W. Erker
转运蛋白的四级结构和功能。
DOI：
--
发表时间：
2002
期刊：
TIBS -Trends in Biochemical Sciences. Regular ed
影响因子：
--
作者：
L. Veenhoff;E. Heuberger;B. Poolman
通讯作者：
B. Poolman