Functional dichotomy and distinct nanoscale assemblies of a cell cycle-controlled bipolar zinc-finger regulator
- Institute of Genetics and Genomics in Geneva (iGE3), Faculty of Medicine, University of Geneva, Switzerland
- École Polytechnique Fédérale de Lausanne, Switzerland
- Institute for Cell and Molecular Biosciences, Newcastle University, United Kingdom
Figures
Figure 1
Cell cycle profile and phylogeny of ZitP and CpaM.
(A) Scheme depicting the polarized factors PopZ, ZitP and CpaM during the cell cycle of the dimorphic bacterium C. crescentus. (B) Pilus assembly pathways and global dependencies of the two master …
Figure 2 with 2 supplements
Functional dichotomy in ZitP and effects on polar morphogenesis.
(A) Bacteriophage infection assays of WT, ΔzitP, ΔzitP;fliGD306G and ΔcpaM mutant cells. Cells harbour empty pMT335 or a complementing plasmid (pMT335 backbone) and were grown in the absence of …
Figure 2—figure supplement 1
Master regulator-dependent promoters in ΔzitP.
Relative β-galactosidase activity (in percentage) of various lacZ-fused promoters in WT and ΔzitP cells.
Figure 2—figure supplement 2
CtrA- and (p)ppGpp-independent influence of the ΔzitP motility defect.
(A) Relative β-galactosidase activity of lacZ-based promoter-probe reporters to the promoters of pilA and CC_1982 in WT, ΔzitP and cpaM cells. (B) Relative β-galactosidase activity of lacZ-based …
Figure 3 with 8 supplements
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 Pxyl promoter on a plasmid integrated at the chromosomal xylX locus in ΔzitP …
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 …
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 Pxyl promoter on a plasmid integrated at the …
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 Pvan-zitP-TAP plasmid. After electrophoresis of TAP extracts, the gel was …
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 …
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 …
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 …
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. ZitPWT, ZitPCS or ZitPGAP were …
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 Pxyl at the xylX locus. …
Figure 4
Cell cycle regulation of ZitP and CpaM localization.
(A) Immunoblots showing the levels of ZitP, CpaM and master cell cycle regulators along the C. crescentus cell cycle in a synchronized WT population. The upper scheme depicts C. crescentus cell …
Additional files
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Supplementary file 1
Bacterial strains, plasmids and oligonucleotides list
- https://doi.org/10.7554/eLife.18647.017
