Single-molecule imaging of electroporated dye-labelled CheY in live Escherichia coli.
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'.
Biological Physics Research Group, Clarendon Laboratory, Department of Physics, University of Oxford, Oxford OX1 3PU, UK.
Diana
D
Di Paolo
Department of Biological Chemistry, The Weizmann Institute of Science, 7610001 Rehovot, Israel.
Oshri
O
Afanzar
Department of Biochemistry, University of Oxford, Oxford OX1 3QU, UK.
Judith P
JP
Armitage
Biological Physics Research Group, Clarendon Laboratory, Department of Physics, University of Oxford, Oxford OX1 3PU, UK richard.berry@physics.ox.ac.uk.
Richard M
RM
Berry
D018274:Electroporation / Q000379:methods*; D004926:Escherichia coli / Q000166:cytology*; D029968:Escherichia coli Proteins; D008164:Luminescent Proteins / Q000737:chemistry; D000072236:Methyl-Accepting Chemotaxis Proteins / Q000737:chemistry*; D008856:Microscopy, Fluorescence; D000072760:Single Molecule Imaging / Q000379:methods*
D029968:Escherichia coli Proteins; D008164:Luminescent Proteins; D000072236:Methyl-Accepting Chemotaxis Proteins; C000607606:cheY protein, E coli
bacterial motility; chemotaxis; electroporation; single-molecule fluorescence microscopy
PLoS One. 2013 Sep 16;8(9):e74200
24066121
J Biol Chem. 1989 Oct 5;264(28):16700-12
2506181
Proc Natl Acad Sci U S A. 2004 Aug 31;101(35):12887-92
15306682
Bioconjug Chem. 2003 Jan-Feb;14(1):195-204
12526709
J Bacteriol. 1984 Jun;158(3):1208-10
6373740
J Bacteriol. 2004 Jun;186(12):3687-94
15175281
Nature. 2006 Sep 21;443(7109):355-8
16971952
Chemistry. 2010 Jan 4;16(1):158-66
19950338
Gene. 1996;173(1 Spec No):83-7
8707061
Phys Chem Chem Phys. 2014 Jul 7;16(25):12688-94
24837080
Curr Opin Biotechnol. 2010 Dec;21(6):766-76
21030243
Nat Methods. 2005 Dec;2(12):905-9
16299475
Biophys J. 2013 Dec 3;105(11):2439-50
24314075
Nature. 1982 Apr 29;296(5860):855-7
7040985
Trends Biotechnol. 2006 Apr;24(4):155-62
16488034
MBio. 2014 Jul 01;5(4):e01216-14
24987089
Appl Microbiol Biotechnol. 2006 Sep;72(2):361-7
16397770
Elife. 2015 May 29;4:e07410
26023830
PLoS One. 2014 Jan 15;9(1):e85605
24454899
J Am Chem Soc. 2012 Feb 29;134(8):3720-8
22239252
IEEE Trans Biomed Eng. 1978 May;25(3):303-4
680765
Proc Natl Acad Sci U S A. 2002 Oct 1;99(20):12669-74
12232047
Gene. 1992 Feb 15;111(2):229-33
1347277
Nature. 1998 Jul 9;394(6689):192-5
9671304
Proc Natl Acad Sci U S A. 2010 Jun 22;107(25):11347-51
20498085
Nat Methods. 2009 Jul;6(7):513-4
19503081
Proc Natl Acad Sci U S A. 2008 Apr 29;105(17):6403-8
18427119
PLoS One. 2014 Feb 04;9(2):e87649
24503716
Mol Microbiol. 2000 Aug;37(4):740-51
10972797
Nat Biotechnol. 2003 Jan;21(1):86-9
12469133
J Bacteriol. 2003 Apr;185(7):2354-61
12644507
Biochemistry. 1993 Feb 9;32(5):1212-8
8448132
J Cell Biol. 1984 Apr;98 (4):1556-64
6201494
J Cell Sci. 1992 Jul;102 ( Pt 3):533-41
1506433
Histochem Cell Biol. 2014 Jul;142(1):113-24
24696085
Science. 2012 Jun 22;336(6088):1595-8
22723426
Science. 1986 Oct 10;234(4773):179-86
3018930
FEBS Lett. 2014 Oct 1;588(19):3585-94
24859634
PLoS Biol. 2009 Jun 16;7(6):e1000137
19547746
Nat Methods. 2009 May;6(5):343-5
19363495
Anal Chem. 2012 Feb 7;84(3):1526-32
22243282
Nat Commun. 2012;3:1204
23149748
Science. 2000 Mar 3;287(5458):1652-5
10698740
Nat Methods. 2011 Nov 06;8(12):1027-36
22056676
Science. 1991 Nov 29;254(5036):1374-7
1962196
Proc Natl Acad Sci U S A. 2002 Jun 11;99(12):7877-82
12060735
Chem Soc Rev. 2009 Oct;38(10):2887-921
19771335