The type IV pili retraction motor PilT oscillates from pole to pole during reversal

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and started to decrease only at 120 min. These data imply that the half-life of PilB is much longer than an average reversal period (15 min under our conditions). Thus, PilB that accumulates at the new leading pole just after reversal is not synthesized de novo, but must be transferred from the old leading pole.

Figure 25. PilB levels are unchanged in presence of chloramphenicol for 60 min

A) Immunoblots of PilB accumulation. Total protein from 710⁷ cells per lane was loaded. Top panel depicts PilB accumulation in untreated WT cells; bottom panel PilB accumulation in WT cells treated with chloramphenicol (Cm). B) Quantitative analysis of PilB accumulation. PilB levels in cells grown without Cm are shown in blue bars; in cells treated with Cm in red bars.

In summary, we found that PilB localizes predominantly at the leading cell pole and that PilB localization is likely dynamic with PilB being unipolar at the leading cell pole immediately after a cellular reversal, building up also at the lagging cell pole during a reversal period, and, finally, PilB relocating from the old leading pole to the new leading cell pole.

2.2.6 The type IV pili retraction motor PilT oscillates from pole to pole

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(Figure 26B, marked by grey arrow), suggesting that a fraction of YFP-PilT is cleaved.

Figure 26. YFP-PilT accumulates at WT levels and restores S-motility defect in ΔpilT A) YFP-PilT complements the motility defect in a ΔpilT mutant. Cells were incubated at 32˚ for 24h on 0.5% agar supplemented with 0.5% CTT, and visualized with a stereomicroscope at 50-fold magnification. Scale bar: 5 mm. WT formed colonies with large rafts of cells at the edge typical of T4P-dependent motility whereas DK10409 (ΔpilT) did not form rafts at the edge.

However, SA3045 (ΔpilT/yfp-pilT) displayed a motility phenotype similar to that of the WT.

B) Immunoblot of PilT and YFP-PilT accumulation. Cells from exponentially growing cultures were harvested and samples analyzed as in Figure 14A. Strains used (left to right): DK1622, DK10409, and SA3045. Blot on the left was probed with rabbit anti-PilT antibodies, and blot on the right with monoclonal anti-GFP antibodies, which also recognize YFP. PilT and YFP-PilT proteins are indicated with the arrows. Grey arrows indicate the degradation product of YFP-PilT. Migration of molecular size markers is indicated on the left.

Therefore, we studied PilT localization using YFP-PilT as well as by immunofluorescence microscopy. In cells analyzed directly from suspension both YFP-PilT (Figure 27A and C) and a native YFP-PilT (Figure 27A and B) localized predominantly in a bipolar symmetric pattern. Surprisingly, in cells with bipolar asymmetric or unipolar PilT clusters, a large PilT cluster localized to the lagging, non-piliated cell pole, where it colocalized with the RomR-GFP - marker for the lagging cell pole (Figure 27D).

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Figure 27. PilT localizes in a bipolar symmetric pattern in non-moving cells

A). Localization of YFP-PilT. Cells were transferred from exponentially growing cultures to a thin 0.7% agar pad on a microscope slide, and imaged by fluorescence and phase-contrast microscopy. Top and bottom rows show phase-contrast and fluorescence images, respectively.

Scale bar: 5 μm. B). Localization of PilT by immunofluorescence microscopy. Cells were harvested from the exponentially growing cultures and analyzed as described in Figure 17A using anti-PilT antibodies. Top and bottom rows show phase-contrast and fluorescence images, respectively. C). Histogram of distribution of PilT polar clusters. The data for WT (DK1622) are from immunofluorescence microscopy and for SA3045 from YFP-PilT localization. Data are presented as in Figure 17B. In total N=100 cells were analyzed for each strain.D). Localization of PilT by immunofluorescence microscopy in RomR-GFP containing strain. Large PilT cluster colocalizes with RomR-GFP. Cells were prepared and analyzed as described in (A). N=20 cells were examined. The top row shows phase-contrast image, middle rows fluorescence images, bottom row represents merged image. Scale bar: 5 μm.

Since native PilT and YFP-PilT localized similarly, we determined the localization of PilT in moving cells using YFP-PilT. In moving cells YFP-PilT localized in a unipolar or in a bipolar asymmetric pattern (Figure 28A and B).

Importantly, the large YFP-PilT cluster in moving cells also localized at the lagging cell pole. The YFP-PilT signal at the leading cell pole varied greatly over time in individual cells (Figure 28A: 1:00, 2:00, 2:30 and 4:00-5:00 min) and occasionally disappeared completely. Thus, PilT localization shifts from a predominantly bipolar symmetric pattern in cells analyzed directly from the liquid culture to an asymmetric polar pattern in cells moving on a surface.

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Figure 28. PilT localization is dynamic between reversals

A) Localization of YFP-PilT in moving cells. Cells of SA3045 were transferred from exponentially growing cultures to a thin 0.7% agar pad on a microscope slide, and imaged by fluorescence microscopy at 30 s intervals. Representative cell is shown. The cell moves in a direction indicated by white arrows and does not reverse. Scale bar: 5 μm. B) Quantitative analysis of polar YFP-PilT fluorescence signals. Integrated fluorescence intensities (arbitrary units) of the two background-subtracted polar clusters in the cells in (A) are plotted as a function of time.

To investigate whether PilT localization changes during reversals, YFP-PilT localization was followed in 20 reversing cells. All reversals were accompanied by relocation of the large YFP-PilT cluster from the old lagging pole to the new lagging pole (Figure 29A and B for a representative cell). Quantification of the fluorescence signal of the YFP-PilT clusters during reversals (Figure 29B) revealed that during a reversal the polar signals initially decreased in intensity (from 1:30 to 2:00 min for the cell depicted in Figure 29A) and at the same time the cell stopped moving. As the intensity of the cluster at the old lagging pole continued to decrease, the cell began to move in the opposite direction (at 2:30 for the cell shown in Figure 29A). Importantly, dynamic localization of PilT during reversals was also observed in the presence of 25 μg/mlchloramphenicol. In addition, in SA3045 cells displaying several reversals, large YFP-PilT cluster oscillated from the old lagging pole to the new lagging pole during each reversal (data not shown). Taken together, these observations strongly suggest that the YFP-PilT at the new, post-reversal lagging pole is not synthesized de novo, but originates from the YFP-PilT cluster at the old, pre-reversal lagging pole, i.e. the

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mechanism underlying dynamic YFP-PilT localization involves the transfer of the protein from the old lagging to the new lagging cell pole.

Figure 29. PilT localization is dynamic during reversals in WT

A) Localization of YFP-PilT in moving cells. Cells were prepared and analyzed as in Figure 28A. Representative cell is shown. Cell stops and reverses between 1:30 and 2:30 min. Scale bar: 5 μm. B) Quantitative analyses of polar YFP-PilT fluorescence signals. Integrated fluorescence intensities (arbitrary units) of the two background-subtracted polar clusters in the cells in (A) are plotted as a function of time.

To determine whether the dynamic localization of YFP-PilT depends on the Frz system, we analyzed its localization in moving cells in the hypo-reversing strain SA3029 (frzCD::Tn5lacΩ536, ΔpilT/yfp-pilT). In all SA3029 cells YFP-PilT also localized in unipolar and bipolar asymmetric patterns (N=20), and all cells contained the large YFP-PilT cluster at the lagging pole and displayed bursts of accumulation at the leading pole (Figure 30A and B). Cells of SA3029 did not reverse, and YFP-PilT did not relocate between poles. Therefore, Frz is dispensable for the unipolar and bipolar

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asymmetric PilT localization but required for relocating PilT clusters during reversals.

Figure 30. PilT dynamic localization during reversal depends on Frz system

A) Localization of YFP-PilT in hypo-reversing moving cells (SA3029). Cells were prepared and analyzed as in Figure 28A. Representative cell is shown. Scale bar: 5 μm. B) Quantitative analysis of polar YFP-PilT fluorescence signals in cells of SA3029. Integrated fluorescence intensities (arbitrary units) of the two background-subtracted polar clusters in the cells in (A) are plotted as a function of time.