Achieving Single-Molecule Tracking of Subcellular Regulation in Bacteria during Real-Time Environmental Perturbations.
Achieving Single-Molecule Tracking of Subcellular Regulation in Bacteria during Real-Time Environmental Perturbations.
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DOI：
10.1021/acs.analchem.2c02899
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发表时间：
2023-01-17
影响因子：
7.4
通讯作者：
Biteen, Julie S.
中科院分区：
文献类型：
--
作者：
Calkins, Anna L.;Demey, Lucas M.;Rosenthal, Brooke M.;DiRita, Victor J.;Biteen, Julie S.
细菌依赖于蛋白质系统来响应外部环境信号进行调节。单分子荧光成像和跟踪已经阐明了这些蛋白质系统在各种细菌中的复杂机制。我们最近调查了霍乱弧菌，革兰氏阴性菌负责人类霍乱疾病，其通过膜定位的转录因子TcpP和ToxR的毒素和毒力因子的生产的调节。这些实验确定了TcpP和ToxR在稳态条件下协同工作，但这些动态相互作用在环境扰动过程中如何变化的测量被限制在琼脂糖垫上的细菌细胞的传统制备所排除。在这里，我们解决了这一技术差距，并通过实施两种替代的样品制备：装置微流体装置和壳聚糖涂层盖玻片，在实时变化过程中获得单分子动力学。我们报告了在微流体装置中活细菌细胞内的单分子跟踪的第一个演示。此外，使用壳聚糖包被的盖玻片，我们表明实时环境变化影响TcpP-PAmCherry动力学，在降至pH 6后约45分钟和诱导ToxR表达后约20分钟激活细菌中的毒力条件。这些新技术的进步使我们有能力进行新的实验，在实时环境扰动期间通过单分子成像和跟踪来研究各种细菌。
Bacteria rely on protein systems for regulation in response to external environmental signals. Single-molecule fluorescence imaging and tracking has elucidated the complex mechanism of these protein systems in a variety of bacteria. We recently investigated Vibrio cholerae, the Gram-negative bacterium responsible for the human cholera disease, and its regulation of the production of toxins and virulence factors through the membrane-localized transcription factors TcpP and ToxR. These experiments determined that TcpP and ToxR work cooperatively under steady-state conditions, but measurements of how these dynamical interactions change over the course of environmental perturbations were precluded by the traditional preparation of bacteria cells confined on agarose pads. Here, we address this gap in technology and access single-molecule dynamics during real-time changes by implementing two alternative sample preparations: device microfluidic device and chitosan-coated coverslips. We report the first demonstration of single-molecule tracking within live bacterial cells in a microfluidic device. Additionally, using the chitosan-coated coverslips, we show that real-time environmental changes impact TcpP-PAmCherry dynamics, activating a virulence condition in the bacteria about 45 minutes after dropping to pH 6 and about 20 minutes after inducing ToxR expression. These new technology advances open our ability for new experiments studying a variety of bacteria with single-molecule imaging and tracking during real-time environmental perturbations.
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DOI：
10.1073/pnas.0409559102
发表时间：
2005-02-22
影响因子：
11.1
作者：
Hung, DT;Mekalanos, JJ
通讯作者：
Mekalanos, JJ
DOI：
10.1007/978-1-4939-7098-8_18
发表时间：
2017-01-01
期刊：
BACTERIAL NUCLEOID: METHODS AND PROTOCOLS
影响因子：
--
作者：
de Leeuw, Roy;Brazda, Peter;Dekker, Nynke H.
通讯作者：
Dekker, Nynke H.
影响因子：
16
作者：
Krukonis, ES;DiRita, VJ
通讯作者：
DiRita, VJ
影响因子：
6.4
作者：
Calkins AL;Demey LM;Karslake JD;Donarski ED;Biteen JS;DiRita VJ
通讯作者：
DiRita VJ
影响因子：
56.9
作者：
Hung, DT;Shakhnovich, EA;Mekalanos, JJ
通讯作者：
Mekalanos, JJ