Imaging of Single Dye-Labeled Chemotaxis Proteins in Live Bacteria Using Electroporation

Abstract

For the last 2 decades, the use of genetically fused fluorescent proteins (FPs) has greatly contributed to the study of chemotactic signaling in E. coli, including the activation of the response regulator protein CheY and its interaction with the flagellar motor. However, this approach suffers from a number of limitations, both biological and biophysical. For example, not all fusions are fully functional when fused to a bulky FP, which can have a similar molecular weight to its fused counterpart. FPs may interfere with the native interactions of the protein, and their chromophores have low brightness and photostability, and fast photobleaching rates. Electroporation allows for internalization of purified CheY proteins labeled with organic dyes into E. coli cells in controllable concentrations. Using fluorescence video microscopy, it is possible to observe single CheY molecules diffusing within cells and interacting with the sensory clusters and the flagellar motors in real time.

New Twists and Turns in Bacterial Locomotion and Signal Transduction

“…The very brief exposure times required to image fluorescently labeled fast-moving proteins preclude the use of the otherwise standard approach of constructing a GFP fusion protein; moreover, the motility of small proteins like CheY (129 amino acids [aa]) is likely to be altered by fusion with GFP (238 aa). Helen Miller and Jia Khoo (Judith Armitage and Richard Berry labs, University of Oxford) presented a new method to monitor cytoplasmic proteins inside cells (48). The protein of interest, in this case CheY, was purified and labeled with the small fluorescent dye iFluor 633 using cysteine-maleimide chemistry and then introduced back into the bacteria by an electroporation method similar to that used for transfection of DNA into eukaryotic cells.…”