Data_Sheet_1_Single-molecule tracking of PprI in D. radiodurans without interference of autoblinking.docx

Autoblinking is a widespread phenomenon and exhibits high level of intensity in some bacteria. In Deinococcus radiodurans (D. radiodurans), strong autoblinking was found to be indistinguishable from PAmCherry and greatly prevented single-molecule tracking of proteins of interest. Here we employed the bright photoswitchable fluorescent protein mMaple3 to label PprI, one essential DNA repair factor, and characterized systematically the fluorescence intensity and bleaching kinetics of both autoblinking and PprI-mMaple3 molecules within cells grown under three different conditions. Under minimal media, we can largely separate autoblinking from mMaple3 molecules and perform reliably single-molecule tracking of PprI in D. radiodurans, by means of applying signal-to-noise ratio and constraining the minimal length for linking the trajectories. We observed three states of PprI molecules, which bear different subcellular localizations and distinct functionalities. Our strategy provides a useful means to study the dynamics and distributions of proteins of interest in bacterial cells with high level of autoblinking.

自闪烁现象在部分细菌中普遍存在，且在某些细菌中表现出极高的强度。在放线菌属的辐射菌（Deinococcus radiodurans，简称D. radiodurans）中，研究发现强烈的自闪烁现象与PAmCherry难以区分，并极大地阻碍了对感兴趣蛋白质的单分子追踪。本研究中，我们采用了明亮的可光控荧光蛋白mMaple3对DNA修复的关键因子PprI进行标记，并在三种不同培养条件下系统地分析了自闪烁及PprI-mMaple3分子在细胞内的荧光强度和漂白动力学。在最小培养基中，我们可以有效地将自闪烁与mMaple3分子分离，并通过应用信噪比以及限制轨迹连接的最小长度，在D. radiodurans中可靠地追踪PprI的单分子。我们观察到PprI分子存在三种状态，这些状态具有不同的亚细胞定位和独特的功能。我们的策略为研究具有高度自闪烁能力的细菌细胞中感兴趣蛋白质的动态和分布提供了有效手段。