Unusual mechanisms of metal regulation in bacteria: from single molecules to single cells to cell communities

确定细胞如何调节锌等过渡金属的吸收和外流是 阐明金属动态平衡的细胞机制。细菌模型系统为 了解调控机制。在大肠杆菌中，对锌离子有反应的金属调节剂锌锌受体感觉到锌的过剩 并激活锌外流系统(例如，锌锌离子A)，而Zur感觉锌充足并抑制锌吸收系统 (例如，ZnuABC)，使这种必需金属在细胞内保持在适当的生理水平。过去的研究表明 对蛋白质分子的结构、功能和调控机制提供了重要的见解 细胞金属浓度，包括金属调节剂和金属摄取/外排转运体等。 机械路径仍然知之甚少，特别是关于空间和时间上的协调 蛋白质和/或DNA之间的相互作用，这些蛋白质和/或DNA可能位于细胞的不同位置。这里的长期目标是 是为了了解细胞中的金属调节如何被操纵以达到预防和治疗的目的。 为了实现这一目标，国际和平研究所建立了一个国际公认的独特的研究计划，该计划适用于 并开发了先进的单分子/单细胞成像方法来询问和了解 在体外和活细胞中细菌金属调节的机制，这一机制被散装进一步增强 生物化学/生物物理和蛋白质/基因工程方法以及与 生物学家和工程师。这项研究导致了第一个金属机制的发现- 反应转录调控和金属外流。这次更新的目的是推进研究和 从单分子和单细胞到细胞群落对细菌金属调控的理解， 包括三个目标，集中在大肠杆菌中锌的调节：(1)确定锌摄取的“通过DNA”机制-- VS-外流调节；(2)确定ZnuABC在细胞内摄取锌的机制；(3)解剖细胞-细胞 细菌群落内锌稳态的相互作用。这项研究具有重要意义，因为它将提供 新的机制洞察：金属调节剂如何在DNA上相互作用，超越现在 “设定点”机制范式；锌多组分转运体的时空配位 摄取；以及自下而上细胞群落内锌稳态中的细胞-细胞相互作用；因为这些 洞察力将加深我们对金属细胞生物学的总体理解，包括人类的相关过程 细胞，从而为确定疾病的原因或制定预防疾病提供基础知识 涉及类似的调节过程，或用于设计策略以破坏细菌的锌稳态以用于抗菌剂 治疗。这项研究具有创新性，因为它在金属监管中产生了新的机械概念， 在微生物群落中的摄取/排出和涌现行为，因为它应用了新的单一的 分子/细胞成像方法以及微流控和光遗传操作。

Defining how cells regulate the uptake and efflux of transition metals such as Zn is a key component in elucidating cellular mechanisms of metal homeostasis. Bacterial model systems provide paradigms for understanding regulation mechanisms. In E. coli, the Zn2+-responsive metalloregulator ZntR senses Zn excess and activates Zn efflux systems (e.g., ZntA), while Zur senses Zn sufficiency and represses Zn uptake systems (e.g., ZnuABC), to keep this essential metal at appropriate physiological levels in the cell. Past research has provided significant insights into the structure, function, and mechanism of the protein players in regulating cellular metal concentrations, including metalloregulators, and metal uptake/efflux transporters, etc. Yet, many mechanistic pathways are still poorly understood, especially regarding spatially and temporally coordinated interactions among proteins and/or DNA that can reside at different locations in the cell. The long-term goal here is to understand how metal regulation in the cell can be manipulated for preventive and therapeutic purposes. Toward this goal, the PI has established an internationally recognized and unique research program that applies and develops advanced single-molecule/single-cell imaging approaches to interrogate and understand the mechanisms of bacterial metal regulation both in vitro and in live cells, which are further enhanced by bulk biochemical/biophysical and protein/genetic engineering approaches and by established collaborations with biologists and engineers. The research has led to the discoveries of first-of-their-kind mechanisms of metal- responsive transcriptional regulation and metal efflux. The objective of this renewal is to advance the study and understanding of bacterial metal regulation from single molecules and single cells toward cell communities, comprising three aims that focus on Zn regulation in E. coli: (1) define a “through-DNA” mechanism for Zn uptake- vs-efflux regulation; (2) define the mechanism of ZnuABC for Zn uptake in the cell; and (3) dissect cell-cell interactions in Zn homeostasis within bacterial communities. The research is significant because it will provide novel mechanistic insights into: how metalloregulators can act on each other on DNA, beyond the present paradigm of “set-point” mechanism; the spatiotemporal coordination of multicomponent Zn transporters for Zn uptake; and the cell-cell interactions in Zn homeostasis within a bottom-up cell community; and because these insights will deepen our understanding of cell biology of metals in general, including related processes in human cells, thus providing fundamental knowledge for identifying causes or developing preventions of diseases that involve similar regulation processes or for devising strategies to impair bacterial Zn homeostasis for antimicrobial treatments. The research is innovative because it generates novel mechanistic concepts in metal regulation, uptake/efflux, and emergent behaviors in microbial communities and because it applies novel single- molecule/cell imaging methods as well as microfluidic and optogenetic manipulations.