Comparative imaging of a bacterial surface-located GFP fusion protein by epifluorescence and scanning near-field optical microscopy.
IcsA is an autotransporter protein that plays a role in the virulence of Shigella bacteria. We have examined the cellular localization of a fusion of an IcsA fragment to the green fluorescent protein (GFP) expressed in Escherichia coli using a dual epifluorescence and scanning near-field optical microscope. By combining the data obtained from far-field with near-field microscopy of the same sample, discrimination between surface-bound fusion proteins and fusion proteins located in the cellular cytoplasm becomes possible. Furthermore, and for the first time, the inherent advantages in resolution of the near-field images provides highly specific details of the location of a GFP fusion protein on a bacterial cell surface.
Gunning
A P
AP
Institute of Food Research, Norwich Research Park, Colney, Norwich NR4 7UA, U.K. patrick.gunning@bbsrc.ac.uk
Bongaerts
R J M
RJ
Kirby
A R
AR
Hinton
J C D
JC
Morris
V J
VJ
D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D018625:Microscopy, Atomic Force; D008856:Microscopy, Fluorescence / Q000379:methods*; D020527:Microscopy, Scanning Probe / Q000379:methods*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012760:Shigella / Q000235:genetics; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*
D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; C058483:virG protein, Shigella flexneri; D049452:Green Fluorescent Proteins