Polar accumulation of pyoverdin and exit from stationary phase

Abstract

Pyoverdin is a water-soluble metal-chelator synthesized by members of the genus Pseudomonas and used for the acquisition of insoluble ferric iron. Although freely diffusible in aqueous environments, preferential dissemination of pyoverdin among adjacent cells, fine-tuning of intracellular siderophore concentrations, and fitness advantages to pyoverdin-producing versus nonproducing cells, indicate control of location and release. Here, using time-lapse fluorescence microscopy to track single cells in growing microcolonies of Pseudomonas fluorescens SBW25, we show accumulation of pyoverdin at cell poles. Accumulation occurs on cessation of cell growth, is achieved by cross-feeding in pyoverdin-nonproducing mutants and is reversible. Moreover, accumulation coincides with localization of a fluorescent periplasmic reporter, suggesting that pyoverdin accumulation at cell poles is part of the general cellular response to starvation. Compatible with this conclusion is absence of non-accumulating phenotypes in a range of pyoverdin mutants. Analysis of the performance of pyoverdin-producing and nonproducing cells under conditions promoting polar accumulation shows an advantage to accumulation on resumption of growth after stress. Examination of pyoverdin polar accumulation in a multispecies community and in a range of laboratory and natural species of Pseudomonas, including P. aeruginosa PAO1 and P. putida KT2440, confirms that the phenotype is characteristic of Pseudomonas.

Keywords: Pseudomonas; accumulation; imaging; iron; polarization; pyoverdin.

Figure 1.

Accumulation of pyoverdin in P. fluorescens SBW25 at the cell pole. (A) Snapshots of experiments described in (Zhang and Rainey 2013) where fitness assays of pyoverdin producing SBW25 and a nonproducing pvdS defective mutant yield contrasting results depending on the culture medium. In both cases the environment is unstructured and ancestral SBW25 producer cells are rare, inoculated at  1%. A fitness advantage to nonproducing cells in KB was previously reported, but the reverse in CAA (Zhang and Rainey 2013). In KB (top) pyoverdin nonproducing cells rarely showed evidence of accumulation of pyoverdin, whereas (bottom) this was common in CAA cultured cells where accumulation is visible at the pole. Images were obtained from 3 µl samples of these experiments imaged under fluorescence light to visualize the distribution of pyoverdin. All scale bars correspond to 10 µm. (B) Fluorescence time-lapse images of a growing microcolony of SBW25 in a SMM agarose pad. Images represent selected time points including, respectively: the initial inoculum, exponential growth, end of exponential growth (i.e., the final number of cells in the colony) and end of time-lapse acquisition (18 h total). (C) Mean fluorescence intensity along the long axis of cells in a growing microcolony, when the last generation of cells is born (left) and at the end of acquisition (t = 18 h, right). Black dotted line represents the fluorescence profile of individual cells, the red line represents a smoothed mean of all the cells. Between 0 h and 18 h accumulation of pyoverdin is evident, especially at the pole.

Figure 2.

Polar accumulation is a reversible phenotype associated with arrest of cell division. (A) Polarization in different growth stages of a microcolony. Mean age of the cells in a growing microcolony of SBW25 (black line, top) and the corresponding frequency of polarized cells for each time point (black line, bottom). Colored panels represent the growth stages of the microcolony, from left to right: lag phase (generation F0), exponential phase (generation F1), and stationary phase (generation F2). N = 35, 159, 187, respectively. In all cases data has been filtered to exclude cells with segmentation errors or other artifacts that preclude proper analysis. Note that as microcolonies begin to form in exponential phase and cells are no longer isolated, overlap between adjacent cells creates regions of high fluorescence that could lead to classification errors. Nevertheless, visual inspection reveals that cells remain in a homogeneous state during exponential growth, with polarization onset being clearly associated to stationary phase. To maintain a consistent physiological response, in this study cells are manipulated from a starting point corresponding to early stationary phase (Fig. S4). Elongation rate and accumulation of fluorescence at the cell pole of individual cells in a growing microcolony. Data extracted from (B), markers represent individual cells in different growth phases of the colony (F1, exponential, triangle markers; F2, stationary, circle markers) and the old (white markers) and new (black markers) cell pole. Elongation rate is represented by the average over the lifetime of a cell. Accumulation of fluorescence at the pole is represented by the maximum ratio over the lifetime of a cell of the sum of the pixels in the pole region and central region of a cell. These regions are defined by segmenting the cell and dividing it in 3 portions over the long axis, where the external 1/3 represent each pole and the remaining 1/3 represents the center. (C) Polarization in response to chemical stresses related and unrelated to iron metabolism. Bars represent the frequency of polarized cells at the start of treatment and after 8h of treatment with either 100 µg/ml 2,2′-dipyridil (DP) (light bars) or 5 µg/ml tetracycline (dark bars). No cell division was observed during treatment. N = 99 (t = 0 h), 94 (t = 8 h) and N = 94 (t = 0 h), 79 (t = 8 h) for DP and tetracyline treatments respectively. (D) Depolarization and subsequent growth of cells pre-treated with an iron chelator. Plot represents colony growth and polarization as in (A). Cells were treated with 100 µg/ml DP during 4h, washed and inoculated on a fresh SMM agarose pad. Data corresponds to five technical replicates i.e., five positions on the agarose pad. Red arrows indicate the time point where the colony overall starts growing (top) and where the majority of the cells are depolarized (bottom). Total initial number of cells N = 87.

Figure 3.

Untangling the mechanism of polarization. (A) A recent study (Shi et al. 2021) showed that upon starvation the cytoplasm of E. coli cells shrinks creating extra space in the periplasm at one of the cell poles. To check if a similar phenomenon happens in P. fluorescens SBW25 we expressed a red fluorescent mScarlet protein fused to the localization signal peptide of dsbA, a periplasmic protein. The image obtained at t = 18 h shows co-localization of mScarlet (bottom, red) and pyoverdin fluorescence (top, blue, scale bar represents 10 µm). (B) Quantitative analysis of time-resolved images at 30 min and 18 h. Cells were segmented and intensity of fluorescence was measured along the long axis. Fluorescence at the poles was compared with the signal produced at the center of each cell; this was done for both pyoverdin (x-axis) and mScarlet (y-axis). Dots represent the intensity ratio for both fluorescent molecules. For measurements at 18 h the values for both the new pole (green dots) and old pole (yellow dots) are represented. Polar information is not available for freshly inoculated cells. The gray dashed line highlights the values where the center of the cell and the poles are not substantially different, i.e., their fluorescence ratio is close to 1. Deviation from this value indicates polar accumulation of the molecules. C) Cartoon depicting a simplified version of the pyoverdin pathway in which candidate genes for polarization are shown. 1. Pyoverdin (Pvd) synthesis starts in the periplasm in response to iron scarcity mediated by the transcription factor PvdS. Pyoverdin is then secreted to the bacterial periplasm, where it matures and becomes fluorescent. 2. Periplasmic pyoverdin is exported into the external medium by a complex that includes the transporter OpmQ. There, it chelates insoluble iron (Fe³⁺). 3. Ferripyoverdin complexes (no longer fluorescent) are then imported back into the periplasm after binding to the receptor FpvA. This receptor is known to also bind free pyoverdin (Ringel and Brüser 2018). In the periplasm, iron is extracted and pyoverdin is again recycled into the external medium by OpmQ. Polarization might also be determined by genes unrelated to the pyoverdin pathway: 4. SBW25 is known to secrete polymers such as cellulose that might trap pyoverdin (Spiers et al. 2002). 5. Pyoverdin could accumulate at the cell poles due to the rod shape of SBW25. (D) Mutants associated to the main processes depicted in (A) and their phenotype with regards to pyoverdin polarization. Mutants were grown on an agarose pad as described and fluorescence images displaying pyoverdin were taken at the time points indicated in the photo. The pyoverdin nonproducing mutant pvdSG229A(D77N) was co-inoculated with SBW25 to enable access of the mutant to pyoverdin. Mutants were tagged with a red fluorescent protein, one mutant colony is displayed in the image.

Figure 4.

Pyoverdin accumulation facilitates recovery of growth after stress. (A) Time until first division (lag time) of SBW25 (green) and pyoverdin defective mutant pvdS229(D77N), termed Pvd^- (gray) under different treatments and conditions. Prior to inoculation cells were either grown in the usual culture medium SMM (no stress on left panel) or treated with DP for 4h (stress on right panel). Cells were then inoculated on a fresh agarose pad, supplemented with 0.45 mM Fe₂[SO₄]₃: these are labeled “+ Fe” (yellow background); unsupplemented SMM treatments are labeled “SMM” (white background). Dots represent individual cell values, box plots represent the associated distribution (median, 25th and 75h percentiles) N = 145, 261, 199, 193, 111, 168, 156, 156, respectively, from left to right. (B) Time until first division of SBW25 (green) and Pvd^- mutants co-inoculated in a fresh agarose pad after separate stress treatment (4h in DP). Pvd^- mutants were labeled with a red fluorescent protein to allow identification of individual cells. Dots and box plots as in (A). N = 175 total cells.

Figure 5.

Accumulation visible by polarization is evident in SBW25 in a multispecies community and is a common phenotype in related species of the genus Pseudomonas. (A) Polarized SBW25 cells in a multispecies community with interdependencies. SBW25 and a cellulose-degrading Bacillus strain isolated from a compost heap in Paris, France (Quistad et al. 2020) were co-cultured in glass vials with minimal medium and cellulose paper as the only carbon source. The community was periodically sampled to assess the polarization state of SBW25. A representative image obtained after 28 days of growth is displayed, where both strains are visible (left, phase contrast image) and in a magnified region where pyoverdin distribution in SBW25 is visualized (right, fluorescence image). (B) Qualitative accumulation assessment in species of Pseudomonas other than SBW25. A collection of laboratory (left) and natural (right) strains were tested for accumulation as evident by polarization. Accumulation by polarization in commonly used laboratory strains: P. aeruginosa PAO1 tested in SMM agarose pad with 1000 µg/ml DP for 7:30 h; P. putida KT2440 tested in SMM agarose pad for 24h. Polarization test in natural isolates, left to right and top to bottom: U106 (liquid KB medium for 24 h); U177 (SMM agarose pad for 24 h); U149 (liquid SMM with DP 100 µg/ml for 24 h); U180 (liquid SMM with DP 100 µg/ml for 24 h); U181 (SMM agarose pad for 16 h); T24 V1a (SMM agarose pad for 24 h); T24 V9b (SMM agarose pad for 24 h); T24 H1b (SMM agarose pad for 24 h); T24 H9b (liquid SMM for 24 h). Natural isolates were collected in Oxford, UK (Zhang et al. 2020) (first and second row) and in Paris, France (Quistad et al. 2020) (Bottom row)(locations are roughly marked with a pink circle on the map). Color represents the identified Pseudomonas species. Insets highlight cells with clearly accumulated pyoverdin.