Single-molecule imaging of electroporated dye-labelled CheY in live E. coli

Journal article

For the last two decades, the use of genetically fused Fluorescent Proteins has greatly contributed to the study of chemotactic signalling 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; they may interfere with the native interactions of the protein, and the chromophores of FPs have low brightness and photostability and fast photobleaching rates. A recently developed technique for the electroporation of fluorescently labelled proteins in live bacteria has enabled us to bypass these limitations and study the in vivobehaviour of CheY at the single molecule level. Here we show that purified CheY proteins labelled with organic dyes can be internalized into E. colicells in controllable concentrations and imaged with video fluorescence microscopy. The use of this approach is illustrated by showing single CheY molecules diffusing within cells and interacting with the sensory clusters and the flagellar motors in real time.

Authors: Berry, R; Di Paolo, D; Afanzar, O; Armitage, J

• Publication status: Published
• Peer review status: Peer reviewed
• Publisher: Royal Society
• Journal: Philosophical Transactions B
• Volume: 371
• Issue: 1707
• Publication date: 2016-09-26
• Acceptance date: 2016-07-02
• DOI: 10.1098/rstb.2015.0492
• Keywords: electroporation; chemotaxis; bacterial mobility; single-molecule fluorescence microscopy