Microbiology and Wine Research, Institute for Molecular Physiology, Johannes Gutenberg University Mainz, Mainz, Germany.
Department of Functional Genomics, Interfaculty Institute of Genetics and Functional Genomics, University Medicine Greifswald, Greifswald, Germany.
J Bacteriol. 2018 Jan 24;200(4). doi: 10.1128/JB.00612-17. Print 2018 Feb 15.
In , the catabolism of C-dicarboxylates is regulated by the DcuS-DcuR two-component system. The functional state of the sensor kinase DcuS is controlled by C-dicarboxylates (like fumarate) and complexation with the C-dicarboxylate transporters DctA and DcuB, respectively. Free DcuS (DcuS) 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 DcuS and 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 (DcuS) and the complexed (DcuB-DcuS) states. Unexpectedly, DcuS levels were always low (<2% of total DcuS), ruling out earlier models that show DcuS as the major species under noninducing conditions. In the absence of fumarate, when DcuS is responsible for basal expression, 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 of expression and is thus responsible for induction. Two-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 C-dicarboxylates and requires formation of the complex of DcuS with C-dicarboxylate transporters DctA or DcuB. Free DcuS (DcuS) 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-DcuR , which was not accessible by other approaches, leading to a revision of previous models.
在 中，C-二羧酸的分解代谢受 DcuS-DcuR 双组分系统调控。传感器激酶 DcuS 的功能状态受 C-二羧酸（如富马酸）和与 C-二羧酸转运蛋白 DctA 和 DcuB 的复合物分别控制。游离的 DcuS（DcuS）即使在没有富马酸的情况下也被认为是持续活跃的，而 DcuB-DcuS 和 DctA-DcuS 复合物则需要富马酸才能激活。为了阐明转运蛋白对 DcuS 功能状态以及 DcuS 和 DcuB-DcuS（或 DctA-DcuS）浓度的影响，通过质谱法测定了有氧或无氧生长细胞中 DcuS、DcuB 和 DctA 的绝对水平。DcuS 以非常低的恒定水平存在（每个细胞 10 到 20 个 DcuS 分子），而 DcuB 和 DctA 的水平较高（最低 200 个分子/细胞），并随富马酸进一步增加（分别增加 12.7 倍和 2.7 倍）。将 DcuS 和 DcuB 的含量与 DcuS 的功能状态相关联，可估算游离（DcuS）和复合物（DcuB-DcuS）状态下 DcuS 的比例。出乎意料的是，DcuS 水平一直很低（<2%的总 DcuS），排除了早期模型，该模型表明在非诱导条件下 DcuS 是主要物质。在没有富马酸的情况下，当 DcuS 负责基础 表达时，形成多达 8%的最大 DcuB 水平。这些足以形成 DcuB-DcuS 复合物并发挥基础转运活性。在富马酸存在（>100 μM）时，DcuB-DcuS 复合物驱动大多数 表达，因此负责诱导。双组分系统（TCS）是细菌感应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 C-二羧酸做出响应，需要形成 DcuS 与 C-二羧酸转运蛋白 DctA 或 DcuB 的复合物。游离的 DcuS（DcuS）在自动磷酸化中持续活跃，并且在特定条件下被认为具有主要作用。在这里，通过质谱法在激活和非激活条件下确定了 DcuS、DcuB 和 DctA 的绝对浓度。它们的绝对含量与 DcuS 功能状态的关系揭示了它们对 DcuS-DcuR 调控的贡献，这是其他方法无法获得的，从而导致对以前模型的修正。
