Groban Eli S, Clarke Elizabeth J, Salis Howard M, Miller Susan M, Voigt Christopher A
University of California, San Francisco, 94158, USA.
J Mol Biol. 2009 Jul 17;390(3):380-93. doi: 10.1016/j.jmb.2009.05.007. Epub 2009 May 13.
Two-component systems are a class of sensors that enable bacteria to respond to environmental and cell-state signals. The canonical system consists of a membrane-bound sensor histidine kinase that autophosphorylates in response to a signal and transfers the phosphate to an intracellular response regulator. Bacteria typically have dozens of two-component systems. The key questions are whether these systems are linear and, if they are, how cross talk between systems is buffered. In this work, we studied the EnvZ/OmpR and CpxA/CpxR systems from Escherichia coli, which have been shown previously to exhibit slow cross talk in vitro. Using in vitro radiolabeling and a rapid quenched-flow apparatus, we experimentally measured 10 biochemical parameters capturing the cognate and non-cognate phosphotransfer reactions between the systems. These data were used to parameterize a mathematical model that was used to predict how cross talk is affected as different genes are knocked out. It was predicted that significant cross talk between EnvZ and CpxR only occurs for the triple mutant DeltaompR DeltacpxA DeltaactA-pta. All seven combinations of these knockouts were made to test this prediction and only the triple mutant demonstrated significant cross talk, where the cpxP promoter was induced 280-fold upon the activation of EnvZ. Furthermore, the behavior of the other knockouts agrees with the model predictions. These results support a kinetic model of buffering where both the cognate bifunctional phosphatase activity and the competition between regulator proteins for phosphate prevent cross talk in vivo.
双组分系统是一类能使细菌对环境和细胞状态信号作出反应的传感器。典型的双组分系统由一个膜结合传感器组氨酸激酶组成，该激酶会因信号而发生自身磷酸化，并将磷酸基团转移至细胞内反应调节因子。细菌通常有数十种双组分系统。关键问题在于这些系统是否呈线性，以及如果是线性的，系统间的串扰是如何被缓冲的。在这项研究中，我们研究了来自大肠杆菌的EnvZ/OmpR和CpxA/CpxR系统，先前已证明这两个系统在体外表现出缓慢的串扰。我们使用体外放射性标记和快速淬灭流动装置，通过实验测量了10个生化参数，这些参数捕捉了系统间同源和非同源的磷酸转移反应。这些数据被用于参数化一个数学模型，该模型用于预测当不同基因被敲除时串扰是如何受到影响的。据预测，只有在三重突变体DeltaompR DeltacpxA DeltaactA - pta中，EnvZ和CpxR之间才会发生显著的串扰。我们构建了所有这七种基因敲除组合来验证这一预测，结果只有三重突变体表现出显著的串扰，即EnvZ激活后cpxP启动子被诱导了280倍。此外，其他基因敲除的行为与模型预测相符。这些结果支持了一种缓冲动力学模型，即同源双功能磷酸酶活性以及调节蛋白对磷酸基团的竞争在体内都能防止串扰。
