Histidine kinases (HK) switch between conformational states that promote kinase and phosphatase activities to regulate diverse cellular processes. Past studies have shown that these functional states can display heterogeneity between cells in microbial communities and can vary at the subcellular level. Methods to track and correlate the kinase conformational state with the phenotypic response of living bacteria cells will offer new opportunities to interrogate bacterial signaling mechanisms. As a proof of principle, we incorporated both mClover3 (donor) and mRuby3 (acceptor) fluorescent proteins into the Caulobacter crescentus cell-cycle HK CckA as an in vivo fluorescence resonance energy transfer (FRET) sensor to detect these structural changes. Our engineered FRET sensor was responsive to CckA-specific input signals and detected subcellular changes in CckA signal integration that occurs as cells develop. We demonstrated the potential of using the CckA FRET sensor as an in vivo screening tool for HK inhibitors. In summary, we have developed a new HK FRET sensor design strategy that can be adopted to monitor in vivo changes for interrogation of a broad range of signaling mechanisms in living bacteria.

中文翻译：

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组氨酸激酶FRET传感器的设计，用于检测活细菌内的复杂信号整合。

组氨酸激酶（HK）在构象状态之间切换，从而促进激酶和磷酸酶活性来调节多种细胞过程。过去的研究表明，这些功能状态可以显示微生物群落中细胞之间的异质性，并且可以在亚细胞水平上变化。跟踪激酶构象状态并使之与活细菌细胞表型反应相关的方法将为研究细菌信号传导机制提供新的机会。作为原理上的证明，我们将mClover3（供体）和mRuby3（受体）荧光蛋白都掺入了新月形杆菌细胞周期HK CckA中荧光共振能量转移（FRET）传感器可检测这些结构变化。我们设计的FRET传感器对CckA特定的输入信号有反应，并检测到随着细胞发育而发生的CckA信号整合中的亚细胞变化。我们证明了将CckA FRET传感器用作HK抑制剂的体内筛选工具的潜力。总而言之，我们已经开发出一种新的HK FRET传感器设计策略，该策略可用于监视体内变化以询问活细菌中多种信号传导机制。