The ClpP1P2 proteolytic complex is essential in Mycobacterium tuberculosis. Proteolysis by ClpP1P2 requires an associated ATPase, either ClpX or ClpC1. Here, we sought to define the unique contributions of the ClpX ATPase to mycobacterial growth. We formally demonstrated that ClpX is essential for mycobacterial growth, and to understand its essential functions, we identified ClpX-His-interacting proteins by pulldown and tandem mass spectrometry. We found an unexpected association between ClpX and proteins involved in DNA replication, and we confirm a physical association between ClpX and the essential DNA maintenance protein single-stranded-DNA binding protein (SSB). Purified SSB is not degraded by ClpXP1P2; instead, SSB enhances ATP hydrolysis by ClpX and degradation of the model substrate GFP-SsrA by ClpXP1P2. This activation of ClpX is mediated by the C-terminal tail of SSB, which had been implicated in the activation of other ATPases associated with DNA replication. Consistent with the predicted interactions, depletion of clpX transcript perturbs DNA replication. These data reveal that ClpX participates in DNA replication and identify the first activator of ClpX in mycobacteria.

中文翻译：

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ClpX是必不可少的，并由分枝杆菌中的单链DNA结合蛋白激活。

ClpP1P2蛋白水解复合物在结核分枝杆菌中必不可少。ClpP1P2进行蛋白水解需要一个相关的ATPase，即ClpX或ClpC1。在这里，我们试图定义ClpX ATPase对分枝杆菌生长的独特贡献。我们正式证明了ClpX对于分枝杆菌的生长至关重要，并且为了了解其基本功能，我们通过下拉法和串联质谱法鉴定了ClpX-His相互作用蛋白。我们发现了ClpX与参与DNA复制的蛋白质之间存在意想不到的关联，并且我们确认了ClpX与必需的DNA维持蛋白单链DNA结合蛋白（SSB）之间存在物理关联。纯化的SSB不会被ClpXP1P2降解；相反，SSB通过ClpX增强ATP水解，并通过ClpXP1P2增强模型底物GFP-SsrA的降解。ClpX的这种激活是由SSB的C末端尾部介导的，这与DNA复制相关的其他ATP酶的激活有关。与预计的相互作用一致，消耗clpX转录物干扰DNA复制。这些数据表明，ClpX参与DNA复制并鉴定了分枝杆菌中ClpX的第一个激活剂。