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

细菌中金属调节的不寻常机制：从单分子到单细胞再到细胞群落

• 批准号: 10713045
• 负责人: Peng Chen
• 金额: $ 33.13万
• 依托单位: CORNELL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-07-01 至 2027-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10713045
• 关键词: Abbreviations; Bacteria; Bacterial Model; Behavior; Biochemical; Biological; Biological Models; Biophysics; Cell Communication; Cells; Cellular biology; Chemicals; Collaborations; Communities; Complex; Cytosol; DNA; Development; Disease; Engineering; Environment; Escherichia coli; Foundations; Genetic Engineering; Goals; Gram-Negative Bacteria; Green Fluorescent Proteins; Growth; Homeostasis; Human; Impairment; In Vitro; International; Invaded; Knowledge; Left; Life; Location; Mechanics; Membrane; Metals; Microfluidics; Micronutrients; Mission; Molecular Chaperones; National Institute of General Medical Sciences; Outcome; Pathway interactions; Physiological; Prevention; Preventive; Process; Progress Reports; Proteins; Public Health; Reaction; Regulation; Repression; Research; Scheme; Structure; System; Therapeutic; Toxic effect; Transcriptional Regulation; Transition Elements; Zinc deficiency; antimicrobial; bacterial community; cell community; cellular imaging; disorder prevention; efflux pump; human disease; human pathogen; imaging approach; imaging modality; innovation; insight; microbial community; model organism; novel; optogenetics; pathogen; periplasm; polydimethylsiloxane; programs; single molecule; single-molecule FRET; spatiotemporal; uptake

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.

定义细胞如何调节过渡金属（例如锌）的吸收和流出是 阐明金属稳态的细胞机制。细菌模型系统提供了范例 了解监管机制。在大肠杆菌中，Zn2+ 响应型金属调节剂 ZntR 可以感知 Zn 过量 并激活锌流出系统（例如 ZntA），而 Zur 感知锌充足并抑制锌吸收系统 （例如，ZnuABC），以将这种必需金属在细胞中保持在适当的生理水平。过去的研究有 对蛋白质调节作用的结构、功能和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/流出转运蛋白等。然而，许多 机制途径仍然知之甚少，特别是在空间和时间协调方面 位于细胞不同位置的蛋白质和/或 DNA 之间的相互作用。长期目标在这里 目的是了解如何操纵细胞中的金属调节以达到预防和治疗目的。 为了实现这一目标，PI 建立了一项国际公认的独特研究计划，该计划适用于 并开发先进的单分子/单细胞成像方法来询问和了解 体外和活细胞中的细菌金属调节机制，通过批量进一步增强 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。该研究首次发现了金属- 响应性转录调控和金属流出。此次更新的目的是推进研究和 了解从单分子和单细胞到细胞群落的细菌金属调节， 包括三个重点关注大肠杆菌中锌调节的目标：(1) 定义锌吸收的“通过 DNA”机制 vs-流出调节； (2)明确ZnuABC在细胞内摄取Zn的机制； (3) 解剖细胞间 细菌群落内锌稳态的相互作用。这项研究意义重大，因为它将提供 新颖的机制见解：超越目前，金属调节剂如何在 DNA 上相互作用 “设定点”机制的范例；多组分锌转运蛋白的时空协调 吸收；以及自下而上的细胞群落中锌稳态的细胞间相互作用；并且因为这些 见解将加深我们对金属细胞生物学的理解，包括人类的相关过程 细胞，从而为确定病因或制定疾病预防措施提供基础知识 涉及类似的调节过程或制定策略来损害细菌锌稳态以实现抗菌 治疗。这项研究具有创新性，因为它产生了金属调节方面的新颖机械概念， 摄取/流出以及微生物群落中的紧急行为，并且因为它应用了新颖的单- 分子/细胞成像方法以及微流体和光遗传学操作。

项目成果

Concentration- and chromosome-organization-dependent regulator unbinding from DNA for transcription regulation in living cells.

• DOI: 10.1038/ncomms8445
• 发表时间: 2015-07-06
• 期刊: Nature communications
• 影响因子: 16.6
• 作者: Chen TY;Santiago AG;Jung W;Krzemiński Ł;Yang F;Martell DJ;Helmann JD;Chen P
• 通讯作者: Chen P

A 'through-DNA' mechanism for metal uptake-vs.-efflux regulation.

金属摄取与流出调节的“通过 DNA”机制。

• DOI: 10.1101/2023.12.05.570191
• 发表时间: 2023
• 期刊: bioRxiv : the preprint server for biology
• 作者: Chakraborty,UditKumar;Park,Youngchan;Sengupta,Kushal;Jung,Won;Joshi,ChandraP;Francis,DanielleH;Chen,Peng
• 通讯作者: Chen,Peng

Facilitated Unbinding via Multivalency-Enabled Ternary Complexes: New Paradigm for Protein-DNA Interactions.

• DOI: 10.1021/acs.accounts.7b00541
• 发表时间: 2018-04-17
• 期刊: Accounts of chemical research
• 影响因子: 18.3
• 作者: Chen TY;Cheng YS;Huang PS;Chen P
• 通讯作者: Chen P

Mechanical stress compromises multicomponent efflux complexes in bacteria

• DOI: 10.1073/pnas.1909562116
• 发表时间: 2019-12-17
• 期刊: PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
• 影响因子: 11.1
• 作者: Genova, Lauren A.;Roberts, Melanie F.;Hernandez, Christopher J.
• 通讯作者: Hernandez, Christopher J.

Quantifying Multistate Cytoplasmic Molecular Diffusion in Bacterial Cells via Inverse Transform of Confined Displacement Distribution.

• DOI: 10.1021/acs.jpcb.5b08654
• 发表时间: 2015-11-12
• 期刊: The journal of physical chemistry. B
• 作者: Chen TY;Jung W;Santiago AG;Yang F;Krzemiński Ł;Chen P
• 通讯作者: Chen P