Understanding the mechanism of the tripartite resistance-nodulation-division (RND) family efflux pumps in Gram-negative bacteria is essential for devising strategies to impair their metal/drug efflux function for antibacterial treatments. Ye it remains poorly defined how the subunits of these multicomponent efflux complexes can/may adjust their assembly forms dynamically to function effectively in a living cell. The long- term goal here is to understand how bacterial membrane efflux pumps can be manipulated for preventative and therapeutic purposes. The overall objective of this application is to perform early-stage research to define the connection between the assembly forms of tripartite RND efflux pumps and the cellular demands for efflux in live cells of the model organism E. coli. On the basis of preliminary studies and literature analysis, a central hypothesis is formulated that tripartite RND efflux pumps exist in a dynamic disassembly-assembly equilibrium in the cell and, in response to cellular demands, shift toward the assembled complexes for more effective efflux. CusCBA and AcrBA-TolC are chosen for study as the representatives of the RND heavy-metal efflux (HME) and hydrophobic-and-amphiphilic efflux (HAE) sub-families, respectively, in E. coli. The research will employ the primary approach of single-molecule tracking via time-lapse stroboscopic imaging, in which the proteins will be tagged with a photoconvertible-fluorescent-protein in live cells, along with the complementary approaches of genetic engineering, ensemble protein interaction studies, protein localization assay, ICP-MS, and protein MassSpec, including collaboration with a biochemist. The rationale for the proposed research is that, once we have tested or even confirmed our central hypothesis, we will be able to better connect the dynamic assembly of CusCBA and AcrBA-TolC with their efflux functions, which may allow us to devise strategies to inhibit their assembly to impair efflux, thus contributing to the prevention and management of bacterial multidrug resistance. The proposed research has three specific aims: 1) Identify the disassembly-assembly mechanism of the metal efflux complex CusCBA of the RND-HME subfamily in live E. coli cells. 2) Identify the metal-responsive elements in the dynamic disassembly-assembly of CusA into CusC3B6A3 in live E. coli cells. 3) Identify the disassembly-assembly mechanism of the drug efflux complex AcrBA-TolC of the RND-HAE subfamily in live E. coli cells. The proposed research is significant because it will fill knowledge gaps in the mechanisms of RND- family efflux pumps, thus advancing the field of efflux pump biology and helping devise strategies to intervene in bacterial drug efflux for antibacterial therapy, and it will advance the understanding of multidrug efflux pumps in general, including related ones in human cells. The proposed research is innovative because it targets the novel mechanism of substrate-responsive dynamic disassembly-assembly for tripartite RND-family pumps and it applies the advanced approach of single-molecule tracking via time-lapse stroboscopic imaging, which is further complemented by other methods.