Deciphering Protein Dynamics of the Siderophore Pyoverdine Pathway in <i>Pseudomonas aeruginosa</i>

<div><p><i>Pseudomonas aeruginosa</i> produces the siderophore, pyoverdine (PVD), to obtain iron. Siderophore pathways involve complex mechanisms, and the machineries responsible for biosynthesis, secretion and uptake of the ferri-siderophore span both membranes of Gram-negative bacteria. Most proteins involved in the PVD pathway have been identified and characterized but the way the system functions as a whole remains unknown. By generating strains expressing fluorescent fusion proteins, we show that most of the proteins are homogeneously distributed throughout the bacterial cell. We also studied the dynamics of these proteins using fluorescence recovery after photobleaching (FRAP). This led to the first diffusion coefficients ever determined in <i>P. aeruginosa</i>. Cytoplasmic and periplamic diffusion appeared to be slower than in <i>Escherichia coli</i> but membrane proteins seemed to behave similarly in the two species. The diffusion of cytoplasmic and periplasmic tagged proteins involved in the PVD pathway was dependent on the interaction network to which they belong. Importantly, the TonB protein, motor of the PVD-Fe uptake process, was mostly immobile but its mobility increased substantially in the presence of PVD-Fe.</p></div

A. Scheme depicting the PVD pathway involving biosynthesis, iron uptake and gene expression.

<p>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>B.</b> <b>Fluorescence microscopy analysis of fluorescent fusions with, from left to right and up to down, OpmQ, FpvF, PvdQ, TonB, PvdT, PvdA and PvdS.</b> 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 <i>pvdS-yfp</i>, <i>mcherry-pvdT</i> and <i>mcherry-opmQ</i> were recorded using a high sensitivity camera. Images of <i>fpvF-mcherry</i> in both epifluorescence (left panel) and TIRF (right panel) are shown. Epifluorescence images of <i>pvdA-yfp</i>, <i>mcherry-pvdQ</i> and <i>pvdQ-mcherry</i> are presented. For <i>tonB-mcherry,</i> 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 <i>pvdA</i> promoter (PAO1(pMMB-<i>mcherry</i>)) see in Supplemental Materials (Figure 5-SM).</p

Strains used in this study.

<p>Strains used in this study.</p

Summary of FRAP experiment analysis.

<p>Summary of FRAP experiment analysis.</p

Illustration of FRAP data treatment for PvdQ-mCHERRY and comparison of the fast diffusing of PvdS-YFP with the slow diffusing TonB-mCHERRY.

<p>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 <i>pvdQ-mcherry</i>. 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).</p

An ABC Transporter with Two Periplasmic Binding Proteins Involved in Iron Acquisition in <i>Pseudomonas aeruginosa</i>

Pyoverdine I is the main siderophore secreted by <i>Pseudomonas aeruginosa</i> PAO1 to obtain access to iron. After extracellular iron chelation, pyoverdine-Fe uptake into the bacteria involves a specific outer-membrane transporter, FpvA. Iron is then released in the periplasm by a mechanism involving no siderophore modification but probably iron reduction. The proteins involved in this dissociation step are currently unknown. The pyoverdine locus contains the <i>fpvCDEF</i> operon, which contains four genes. These genes encode an ABC transporter of unknown function with the distinguishing characteristic of encompassing two periplasmic binding proteins, FpvC and FpvF, associated with the ATPase, FpvE, and the permease, FpvD. Deletion of these four genes partially inhibited cytoplasmic uptake of ⁵⁵Fe in the presence of pyoverdine and markedly slowed down the <i>in vivo</i> kinetics of iron release from the siderophore. This transporter is therefore involved in iron acquisition by pyoverdine in <i>P. aeruginosa</i>. Sequence alignments clearly showed that FpvC and FpvF belong to two different subgroups of periplasmic binding proteins. FpvC appears to be a metal-binding protein, whereas FpvF has homology with ferrisiderophore binding proteins. <i>In vivo</i> cross-linking assays and incubation of purified FpvC and FpvF proteins showed formation of complexes between both proteins. These complexes were able to bind <i>in vitro</i> PVDI-Fe, PVDI-Ga, or apo PVDI. This is the first example of an ABC transporter involved in iron acquisition <i>via</i> siderophores, with two periplasmic binding proteins interacting with the ferrisiderophore. The possible roles of FpvCDEF in iron uptake by the PVDI pathway are discussed