Abstract
For the past two decades, the use of genetically fused fluorescent proteins (FPs) has greatly contributed to the study of chemotactic signalling in Escherichia 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 vivo behaviour of CheY at the single-molecule level. Here we show that purified CheY proteins labelled with organic dyes can be internalized into E. coli cells 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.This article is part of the themed issue ‘The new bacteriology’.
Highlights
Many species of motile bacteria use rotating extracellular filaments to propel themselves through liquid media
In Escherichia coli, the motor is powered by a transmembrane flux of Hþ and the electrochemical energy is converted into work through a ring of stator units pushing on a central rotor
Electrocompetent cells were incubated with the fluorescent dye-labelled CheY for 5 – 10 min in a 0.1 cm cuvette; a 12 kV cm21 electric field was applied to allow for internalization of some fluorescent molecules in solution; following electroporation, cells were recovered in super optimal broth with catabolite repression (SOC) at 308C, 450 r.p.m. in a Thermomixer (Eppendorf ) or on 1% EZ rich defined medium agarose pads for an amount of time varying according to the experiment being carried out
Summary
Many species of motile bacteria use rotating extracellular filaments to propel themselves through liquid media. Single-molecule photobleaching steps in the fluorescence timetraces of E. coli cells electroporated with a 15 nM concentration of CheY(Cys)-Cy3B and Atto647-(Cys)CheY were taken as a measure of fluorophore brightness in vivo. Electrocompetent cells were incubated with the fluorescent dye-labelled CheY for 5 – 10 min in a 0.1 cm cuvette; a 12 kV cm electric field was applied to allow for internalization of some fluorescent molecules in solution; following electroporation, cells were recovered in super optimal broth with catabolite repression (SOC) at 308C, 450 r.p.m. in a Thermomixer (Eppendorf ) or on 1% EZ rich defined medium agarose pads for an amount of time varying according to the experiment being carried out (minutes to hours). A mercury lamp with appropriate excitation and emission filters for the fluorescent dyes being imaged was used
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