Protein diffusion in Escherichia coli

Conrad Mullineaux
Professor of Microbiology
School of Biological and Chemical Sciences
Queen Mary, University of London
Mile End Road,
London E1 4NS
UK

We use Fluorescence Recovery after Photobleaching to measure the diffusion of fluorescently-tagged proteins in cells of Escherichia coli. For example, we have expressed Green Fluorescent Protein in the cytoplasm, or attached to membrane proteins, or with a TAT leader sequence that drives its export to the periplasm. (Collaboration with Colin Robinson, University of Warwick. ref: Mullineaux et al (2006)). Measurement of GFP diffusion gives us information on the physical nature of these three environments in the cell. The information will be important for modelling dynamic processes in E. coli: how well can we understand bacterial cells as physical systems?

The video clips below show FRAP measurements of GFP diffusion in E. coli cells that have been elongated by treatment with the cell division inhibitor cephalexin. In each case, a line is bleached across the centre of the cell. The spread and recovery of the line give quantitative information on the diffusion of GFP. Where diffusion is rapid, the bleached line is already broad when the first post-bleach image is recorded.

A. GFP in the cytoplasm. Shown at real speed. Frames are 30 microns across. GFP diffusion coefficient about 8 microns2/s

B. GFP in the periplasm. Shown at real speed. Frames are 30 microns across. GFP diffusion coefficient about 3 microns2/s

C. GFP fused to a protein in the cytoplasmic membrane (TatA). Shown speeded up 4 x. Frames are 22 microns across. GFP diffusion coefficient about 0.13 microns2/s.

Note the very rapid movement of GFP in the cytoplasm. Proteins of this size (GFP is 27 kDa) can diffuse very quickly in bacterial cells. Note also that GFP diffusion is only a little slower in the periplasm than in the cytoplasm. Contrary to some previous ideas, the periplasm is a relatively fluid environment. Dr Anja Nenninger was funded by BBSRC to continue this project. We want to know how diffusion coefficients are influenced by protein size, and by other factors that influence the environments inside the cell. We have now published a systematic study of the size-dependence of protein mobility in the E. coli cytoplasm which quantifies for the first time the extent to which the addition of a GFP tag slows down the diffusion of the protein it is attached to. Reassuringly, we find that the effect is small, although it is quantifiable - the GFP tag slows the protein by about 13% at most (Nenninger et al., 2010).

Some key publications on this topic:

Ray, N., Nenninger, A., Mullineaux, C.W. and Robinson, C. (2005) Location and mobility of twin-arginine translocase subunits in the Escherichia coli plasma membrane. J. Biol. Chem. 280, 17961-17968.

Mullineaux, C.W., Nenninger, A., Ray, N. and Robinson, C. (2006) Diffusion of Green Fluorescent Protein in three cell environments in Escherichia coli. J. Bacteriol. 188, 3442-3448.

Nenninger, A., Mastroianni, G. and Mullineaux, C.W. (2010) Size dependence of protein diffusion in the cytoplasm of Escherichia coli. J. Bacteriol. 192, 4535-4540.