Figure 3. Distinct ZitP nanoscale assemblies and localization determinants.

(A) Photo-activated light microscopy (PALM) imaging of Dendra2-ZitP or Dendra2-CpaM expressed from the xylose-inducible P_xyl promoter on a plasmid integrated at the chromosomal xylX locus in ΔzitP or ΔcpaM cells exposed to xylose 3 hours before imaging. Scale bar: 1 µm. (B) PALM imaging of Dendra2-ZitP in WT or ΔpopZ::Ω cells. We induced expression of Dendra2-ZitP from the xylose-inducible P_xyl promoter on a plasmid integrated at the chromosomal xylX locus by the addition of xylose 3 hours before imaging. Scale bar: 1 µm. Scale bar of zoomed images: 0.5 µm. (C) Co-immunoprecipitation (co-IP) of ZitP or CpaM with polyclonal antibodies to CpaM or ZitP, respectively. Immunoprecipitates and cell lysates from WT, ΔzitP or ΔcpaM cells were probed for the presence of ZitP or CpaM. (D) Projected area of the Dendra2-ZitP polar complex as determined by PALM from Dendra2-ZitP expressed in WT and ΔpopZ::Ω cells. Black lines indicate medians. Statistical significance from Mood’s median test: n.s, p>0.05; ***p<0.001. (E) ZitP polar binding times in WT and ΔpopZ::Ω cells, measured via single particle tracking PALM. Error bars indicate 95% confidence interval of the fit to the data (Figure 3—figure supplement 6D). Statistical significance from a 2 sample t-test: ***p=p<0.001. (F) Epifluorescence (Dendra2) and Nomarski (DIC) images depicting the localization of Dendra2-ZitP or Dendra2-CpaM in ΔpopZ::Ω, ΔdivJ, divKcs, ΔpleC, ΔcpaE or ΔpodJ cells. Expression of Dendra2-ZitP or Dendra2-CpaM was induced from the chromosomal xylX locus with xylose 4 hours before imaging. Scale bars: 1 µm. (G) (H) Epifluorescence (Dendra2) and Nomarski (DIC) images depicting the localization of the motility-deficient and pilus-proficient Dendra2-ZitP^CS variant (G) or the motility-proficient and pilus-deficient Dendra2-ZitP^1-133 variant (H) in ΔzitP cells. Arrow heads pinpoint stalked poles. We induced expression of Dendra2-fusions from the xylose-inducible P_xyl promoter on a plasmid integrated at the chromosomal xylX locus by the addition of xylose 4 hours before imaging. Scale bars: 1 µm.

DOI: http://dx.doi.org/10.7554/eLife.18647.007

Figure 3—figure supplement 1. Extrinsic determinant for the localization of ZitP and CpaM.

(A) Epifluorescence (Dendra2) and Nomarski (DIC) images depicting the localization of Dendra2-ZitP and Dendra2-CpaM variants in asynchronious ΔzitP or ΔcpaM cells, respectively. We induced expression of Dendra2 fusions expressed from the xylose-inducible P_xyl promoter on a plasmid integrated at the chromosomal xylX locus. Scale bars: 1 µm. (B) Subcellular localisation of Dendra2-ZitP in the ΔcpaM mutant. Cells were imaged in epifluorescence (GFP channel) and bright field mode (DIC). We induced expression of Dendra2-ZitP from P_xyl on plasmids integrated at the chromosomal xylX locus by the addition of xylose 4 hours before imaging. Scale bar: 1 µm. (C) (D) Quantification of Dendra2-ZitP (C) or Dendra2-CpaM (D) localization states (diffuse, monopolar or bipolar) in WT or polarity mutants cells. Dendra2 fusions were expressed from the xylose-inducible P_xyl promoter on a plasmid integrated at the chromosomal xylX locus. The total cell count (n) for each strain is shown above related stacked bars. Values are expressed in percentage of whole cell population.

DOI: http://dx.doi.org/10.7554/eLife.18647.008

Figure 3—figure supplement 2. ZitP and CpaM polar localization by PALM.

(A) PALM images of ZitP or CpaM localization in ΔzitP and ΔcpaM cells, respectively. We induced expression of Dendra2-fusions from the xylose-inducible P_xyl promoter on a plasmid integrated at the chromosomal xylX locus by the addition of xylose 3 hours before imaging. Scale bars: 0.5 µm. (B) PALM images of CpaM localization in WT and ΔzitP cells. We induced expression of Dendra2-fusions from the xylose-inducible P_xyl promoter on a plasmid integrated at the chromosomal xylX locus by the addition of xylose 3 hours before imaging. Scale bars: 1 µm. (C) ZitP localization, with zoomed images of poles in WT and ΔpopZ::Ω cells. Scale bar 0.5 µm.

DOI: http://dx.doi.org/10.7554/eLife.18647.009

Figure 3—figure supplement 3. Tandem affinity purification of ZitP.

Tandem affinity purification (TAP) performed on WT cell extracts bearing an empty pCWR512 plasmid (control) or the P_van-zitP-TAP plasmid. After electrophoresis of TAP extracts, the gel was silver-stained as guided by the manufacturer (SilverQuest, Invitrogen). Arrows indicate bands that were extracted and sent for mass spectrometry analyses. They indicate as well the main identified proteins.

DOI: http://dx.doi.org/10.7554/eLife.18647.010

Figure 3—figure supplement 4. CpaE localization in ΔzitP and ΔcpaM mutant cells.

(A) Epifluorescence (YFP-CpaE) and Nomarski (DIC) images depicting the localization of the pilus component CpaE N-terminally fused to YFP expressed form the native cpaE locus (yfp-cpaE) in WT, ΔzitP and ΔcpaM cells. The scale bars represent 1 µm. (B) Quantification of YFP-CpaE localization profile (diffuse, monopolar or bipolar) in the aforementioned strains. The total cell count (n) for each strain is shown above related stacked bars. Values are expressed in percentage of whole cell population.

DOI: http://dx.doi.org/10.7554/eLife.18647.011

Figure 3—figure supplement 5. Quantitative analysis of ZitP cluster shape and area.

(A) Measurement of polar cluster area. Automated clustering of Dendra2-ZitP localization using DBSCAN. Red dots, identified clusters; black crosses, low-density localizations outside of polar clusters. (B) Image processing operations for area measurement. Identified clusters (i) were converted to a binary image (ii) which was then processed via morphological closing (iii) to make the cluster area measurement less sensitive to noise and molecule sampling rate. (C) Measured circularity, solidity and eccentricity of WT stalked pole, WT other (swarmer) pole and polar foci in ΔpopZ::Ω cells. (D) Measured area of WT stalked pole, WT other (swarmer) pole and polar foci in ΔpopZ::Ω cells compared to the observed area of simulated zero-area clusters. Observed zero-area cluster size is non-zero due to experimental noise. Stars indicate statistical significance: n.s, p>0.05; *p<0.05; **p<0.01; ***p<0.001.

DOI: http://dx.doi.org/10.7554/eLife.18647.012

Figure 3—figure supplement 6. Binding time estimation by stroboscopic single particle tracking of ZitP.

(A) Exemplar histograms of effective on-time (ie. combination of actual binding lifetime with photobleaching lifetime) in different time-lapse conditions for Dendra2-ZitP in WT cells. Lines are fits by a single exponential model. (B) Observed effective on-time of polar Dendra2-ZitP as a function of time-lapse duration in WT and ΔpopZ cells. Fitting of the data with Eq 4 (Gebhardt model) shows large systematic errors. By accounting for finite camera integration time (Eq 5) we obtained good fits to the data. Error bars indicate 1 s.d. (C) Simulation showing the effect of finite camera integration time on observed on-time. Observed on-time (obs. on-time) shows a strong plateau at a minimum observable on-time, compared to the true on-time (sim. on-time), confirming that the plateau observed experimentally most likely arises from finite camera integration time. By accounting for this effect (Eq 5) we obtained good fits to the data. Error bars indicate 1 s.d. (D Comparison of observed effective on-time of polar Dendra2-ZitP as a function of time-lapse duration in WT and ΔpopZ cells, showing fitting with the revised binding time model (Eq 5). Error bars indicate 1 s.d.

DOI: http://dx.doi.org/10.7554/eLife.18647.013

Figure 3—figure supplement 7. Intrinsic determinants for ZitP localization and function.

(A) Epifluorescence (Dendra2) and Nomarski (DIC) images depicting the localization profile of Dendra2-CpaM in ΔzitP cells complemented with ZitP-expression plasmids. ZitP^WT, ZitP^CS or ZitP^GAP were expressed from pMT335 (without vanillate). We used empty pMT335 (-) as a control. Scale bars: 1 µm. (B) Quantification of Dendra2-CpaM localization states (diffuse, monopolar or bipolar) in ΔzitP cells expressing ZitP^WT, ZitP^CS or ZitP^GAP from pMT335 (without vanillate). We used empty pMT335 (-) as a control. Dendra2 fusions were expressed from the xylose-inducible P_xyl promoter on a plasmid integrated at the chromosomal xylX locus. The total cell count (n) for each strains are shown above related stacked bars. Values are expressed in percentage of whole cell population. (C) Reciprocal co-immunoprecipitation (IP) of ZitP variants or CpaM protein with polyclonal antibodies to CpaM or ZitP, respectively. Immunoprecipitates and cell lysates were probed for the presence of ZitP or CpaM by immunoblotting (IB). Extracts were made from ΔzitP and ΔcpaM cells expressing ZitP^WT, ZitP^CS, ZitP^GAP or CpaM from pMT335 grown without vanillate. (D) Quantification of Dendra2-ZitP^CS and Dendra2-ZitP^GAP localization states (diffuse, monopolar or bipolar) in the ΔzitP cells from the xylose-inducible P_xyl promoter on a plasmid integrated at the chromosomal xylX locus. The total cell count (n) is shown above the stacked bar. Value is expressed in percentage of whole cell population.

DOI: http://dx.doi.org/10.7554/eLife.18647.014

Figure 3—figure supplement 8. Effect of DUF3426 on ZitP function.

(A) Relative β-galactosidase activity of lacZ-based promoter-probe reporters to the pilA and CC_1982 promoter in WT and zitP cells expressing Dendra2-ZitP variants from P_xyl at the xylX locus. Xylose was added to the medium. (B) Epifluorescence (Dendra2) and phase images depicting the localization profile of Dendra2-CpaM in ΔzitP cells complemented with pMT463-derived plasmids expressing either WT ZitP (ZitP^WT) or ZitP^1-133. We used empty pMT463 (-) as a control. Scale bars: 1 µm. (C) Epifluorescence (Dendra2) and phase contrast overlays depicting the localization of Dendra2- ZitP^GAP from the xylX locus in ΔzitP cells. Scale bars: 1 µm

DOI: http://dx.doi.org/10.7554/eLife.18647.015