Kiehler Brittany, Haggett Lindsey, Fujita Masaya
Department of Biology and Biochemistry, University of Houston, Houston, TX, USA.
Microbiologyopen. 2017 Aug;6(4). doi: 10.1002/mbo3.481. Epub 2017 Apr 27.
Sporulation in Bacillus subtilis is induced upon starvation. In a widely accepted model, an N-terminal "sensor" domain of the major sporulation kinase KinA recognizes a hypothetical starvation signal(s) and autophosphorylates a histidine residue to activate the master regulator Spo0A via a multicomponent phosphorelay. However, to date no confirmed signal has been found. Here, we demonstrated that PAS-A, the most N-terminal of the three PAS domains (PAS-ABC), is dispensable for the activity, contrary to a previous report. Our data indicated that the autokinase activity is dependent on the formation of a functional tetramer, which is mediated by, at least, PAS-B and PAS-C. Additionally, we ruled out the previously proposed notion that NAD /NADH ratio controls KinA activity through the PAS-A domain by demonstrating that the cofactors show no effects on the kinase activity in vitro. In support of these data, we found that the cofactors exist in approximately 1000-fold excess of KinA in the cell and the cofactors' ratio does not change significantly during growth and sporulation, suggesting that changes in the cofactor ratio might not play a role in controlling KinA activity. These data may refute the widely-held belief that the activity of KinA is regulated in response to an unknown starvation signal(s).
枯草芽孢杆菌在饥饿时会诱导形成芽孢。在一个被广泛接受的模型中，主要芽孢形成激酶KinA的N端“传感器”结构域识别一个假设的饥饿信号，并使一个组氨酸残基自磷酸化，通过多组分磷酸传递来激活主调控因子Spo0A。然而，迄今为止尚未发现已确认的信号。在此，我们证明，与之前的报道相反，三个PAS结构域（PAS-ABC）中最N端的PAS-A对于该活性是可有可无的。我们的数据表明，自激酶活性依赖于功能性四聚体的形成，这至少由PAS-B和PAS-C介导。此外，我们通过证明这些辅因子在体外对激酶活性没有影响，排除了之前提出的NAD /NADH比值通过PAS-A结构域控制KinA活性的观点。为支持这些数据，我们发现细胞中辅因子的存在量比KinA大约多出1000倍，并且在生长和芽孢形成过程中辅因子的比例没有显著变化，这表明辅因子比例的变化可能在控制KinA活性中不起作用。这些数据可能会反驳一种广泛持有的观点，即KinA的活性是响应未知的饥饿信号而受到调节的。
