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Philos Trans R Soc Lond B Biol Sci. 2016 Nov 5;371(1707). pii: 20150492. doi: 10.1098/rstb.2015.0492.

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

Author information

1
Biological Physics Research Group, Clarendon Laboratory, Department of Physics, University of Oxford, Oxford OX1 3PU, UK.
2
Department of Biological Chemistry, The Weizmann Institute of Science, 7610001 Rehovot, Israel.
3
Department of Biochemistry, University of Oxford, Oxford OX1 3QU, UK.
4
Biological Physics Research Group, Clarendon Laboratory, Department of Physics, University of Oxford, Oxford OX1 3PU, UK richard.berry@physics.ox.ac.uk.

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'.

KEYWORDS:

bacterial motility; chemotaxis; electroporation; single-molecule fluorescence microscopy

PMID:
27672145
PMCID:
PMC5052738
DOI:
10.1098/rstb.2015.0492
[Indexed for MEDLINE]
Free PMC Article

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