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

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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.