Imaging Efflux Machineries for Metal Defense in Live Bacteria
Abstract
Metal efflux pumps, such as the tripartite efflux pump CusCBA, provide bacteria protection from environmental toxic metals such as copper and silver. The CusCBA multisubunit complex spans the periplasmic space in gram negative bacteria. On the basis of preliminary studies, it is hypothesized that CusCBA exists in a dynamic disassembly-assembly equilibrium with a shift toward the assembled form in response to increasing cellular Cu levels. The objective of the proposed research is to define the connection between the assembly forms of CusCBA and the cellular demands for Cu efflux in live E. coli cells, as well as the dynamic interconversions among the different assembly forms under various Cu efflux demands, with the long-term goal of understanding how bacterial membrane efflux pumps can be manipulated for antibacterial treatments. Following fluorescent photoconvertible tagging of the CusA protein, single molecule tracking will be performed via time-lapse stroboscopic imaging to define the resolvable number of diffusion states (i.e., assembly states) in live E. coli as a function of Cu exposure. This will provide a measure of the % CusCBA complexes that are freely diffusing in the periplasm with those that are membrane bound and fully (or incompletely) complexed. In addition, the tagged CusA copy number in individual cells will be determined (using fluorescence counting) to deconvolute effects of copy number heterogeneity on the distribution of the Cus complexes in different diffusion states. Complementary approaches to confirm the observations will include the use of membrane fractionation /Western blot, an analysis of Cu loading to CusA using antibody pulldown and inductively coupled plasma mass spectrometry (ICP-MS) and the examination of mutants of cueO (Cu oxidase) which renders bacterial cells more sensitive to Cu stress. In addition, the Cu-responsive elements at within CusA and CusB will be identified via mutational analysis at specific Met residues of these two proteins. These mutants will be assayed for the loss of the ÒslowÓ diffusion state. Finally, the single-molecule tracking data analysis and results will be validated using diffusion simulations in a continuous curved 2-D surface.
Document Details
- Document Type
- DoD Grant Award
- Publication Date
- Jan 12, 2017
- Source ID
- W911NF1510268
Entities
People
- Peng Chen
Organizations
- Army Contracting Command
- Cornell University
- United States Army