Yeo Jongchan, Dippel Andrew B, Wang Xin C, Hammond Ming C
Department of Chemistry, University of California , Berkeley, California 94720, United States.
Department of Molecular & Cell Biology, University of California , Berkeley, California 94720, United States.
Biochemistry. 2018 Jan 9;57(1):108-116. doi: 10.1021/acs.biochem.7b00696. Epub 2017 Nov 2.
Intracellular signaling enzymes drive critical changes in cellular physiology and gene expression, but their endogenous activities in vivo remain highly challenging to study in real time and for individual cells. Here we show that flow cytometry can be performed in complex media to monitor single-cell population distributions and dynamics of cyclic di-GMP signaling, which controls the bacterial colonization program. These in vivo biochemistry experiments are enabled by our second-generation RNA-based fluorescent (RBF) biosensors, which exhibit high fluorescence turn-on in response to cyclic di-GMP. Specifically, we demonstrate that intracellular levels of cyclic di-GMP in Escherichia coli are repressed with excess zinc, but not with other divalent metals. Furthermore, in both flow cytometry and fluorescence microscopy setups, we monitor the dynamic increase in cellular cyclic di-GMP levels upon zinc depletion and show that this response is due to de-repression of the endogenous diguanylate cyclase DgcZ. In the presence of zinc, cells exhibit enhanced cell motility and increased sensitivity to antibiotics due to inhibited biofilm formation. Taken together, these results showcase the application of RBF biosensors in visualizing single-cell dynamic changes in cyclic di-GMP signaling in direct response to environmental cues such as zinc and highlight our ability to assess whether observed phenotypes are related to specific signaling enzymes and pathways.
细胞内信号酶驱动细胞生理和基因表达的关键变化，但其在体内的内源性活性在实时研究单个细胞时仍极具挑战性。在这里，我们表明可以在复杂培养基中进行流式细胞术，以监测单细胞群体分布和环二鸟苷酸信号传导的动态，环二鸟苷酸信号传导控制细菌定植程序。我们的第二代基于RNA的荧光（RBF）生物传感器实现了这些体内生物化学实验，该传感器在响应环二鸟苷酸时表现出高荧光开启。具体而言，我们证明了过量锌会抑制大肠杆菌中环二鸟苷酸的细胞内水平，但其他二价金属则不会。此外，在流式细胞术和荧光显微镜设置中，我们监测了锌耗尽后细胞内环二鸟苷酸水平的动态增加，并表明这种反应是由于内源性二鸟苷酸环化酶DgcZ的去抑制作用。在有锌的情况下，由于生物膜形成受到抑制，细胞表现出增强的细胞运动性和对抗生素的敏感性增加。综上所述，这些结果展示了RBF生物传感器在可视化环二鸟苷酸信号传导中对锌等环境线索的直接响应的单细胞动态变化方面的应用，并突出了我们评估观察到的表型是否与特定信号酶和途径相关的能力。
