Figure 1.
A. Scheme depicting the PVD pathway involving biosynthesis, iron uptake and gene expression.
For details and explanations, refer to the “Introduction” section. The results obtained in this work on protein dynamics are indicated as follows: the stars in red, purple and blue indicate the proteins with rapid, moderate and slow dynamics, respectively. B. Fluorescence microscopy analysis of fluorescent fusions with, from left to right and up to down, OpmQ, FpvF, PvdQ, TonB, PvdT, PvdA and PvdS. Cells were grown twice in minimal medium, washed in minimal medium and spotted onto slides coated with agarose made up in minimal medium. Brightfield images, when available, are presented on the left. Due to low fluorescent signals, epifluorescence images of pvdS-yfp, mcherry-pvdT and mcherry-opmQ were recorded using a high sensitivity camera. Images of fpvF-mcherry in both epifluorescence (left panel) and TIRF (right panel) are shown. Epifluorescence images of pvdA-yfp, mcherry-pvdQ and pvdQ-mcherry are presented. For tonB-mcherry, from left to right, brightfield, epifluorescence and TIRF mode images are presented (scale bar 2 µm). For the fluorescence miscrocopy pictures of PAO1 strain harboring a plasmid encoding a cytoplasmic mCHERRY fluorescent protein expressed under the control of the pvdA promoter (PAO1(pMMB-mcherry)) see in Supplemental Materials (Figure 5-SM).
Table 1.
Strains used in this study.
Figure 2.
Illustration of FRAP data treatment for PvdQ-mCHERRY and comparison of the fast diffusing of PvdS-YFP with the slow diffusing TonB-mCHERRY.
A. One-dimensional profiles along the long cell axis after photobleaching (t = 0 s), during recovery (t = 1 s) and after full recovery (t = 2 s) of strain pvdQ-mcherry. Inset: Fluorescence images extracted from the acquired FRAP stream. From left to right: before photobleaching, after photobleaching and after recovery. B. Experimental Fourier amplitudes for mode n = 1 as a function of time of PvdQ-mCHERRY and the fitted exponential decay (solid line) used to determine the diffusion coefficient. C. Experimental Fourier amplitudes for mode n = 1 as a function of time for PvdS-YFP (gray circles) and TonB-mCHERRY (black circles) and the corresponding fitted exponential decay (hashed and solid lines, respectively).
Table 2.
Summary of FRAP experiment analysis.
Figure 3.
Recovery curves for TonB-mCHERRY in the absence (•) or presence (▪) of PVD-Fe.
The fluorescence intensity from FRAP stream experiments were normalized using the FRAP Analysis plugin in Image-JNIH software [66]. The averaged recovery profiles for TonB-mCHERRY (▪, n = 87) and TonB-mCHERRY after addition of 10 µM PVD-Fe (•, n = 4) are presented.