Figure 3
The chimeric Intimin-FAST protein expressed by E. coli cells becomes fluorescent upon binding to the membrane permeant fluorogen HBR-3,5DM. (a) FAST is fused to a protein of interest (POI). The fluorogen HBR-3,5DM supplied in the culture medium becomes fluorescent when it binds to FAST and reveals the POI. (b) Total internal reflection fluorescence microscopy (TIRF) generates an evanescent wave at the glass-gel interface in the sample. The short penetration depth of the evanescent wave (typically 200 nm) allows to remove the unwanted background but only reveals a section of bacteria close to the coverslip. IM and OM are the inner and outer membrane respectively. (c) Brightfield (right) and TIRF (left) images of E. coli MG1655 wild-type (WT) and Intimin-FAST cells. Exponentially growing cells (OD600 = 0.7) WT or containing pNeae2-FAST were deposited on a 1% agarose LB pad containing 20 µM of HBR-3,5DM with no (first two rows) or with 0.5 mM IPTG (bottom row). The background of the fluorescence images has been subtracted. All fluorescent images are displayed with the same contrast. Scale bar is 5 µm. Representative images of one experiment are presented. (d) Quantification of the fluorescent signal for WT and intimin-FAST cells in presence or absence of IPTG. Error bars represent standard errors of three biological replicates each containing at least 100 bacteria. Statistical analysis was performed using unpaired t tests (****, p value < 0.0001).
