Fig. 3. CcrZ directly interacts with FtsZ.

a, Split-luciferase assay using several combinations with CcrZ-LgBit reveals that CcrZ and FtsZ are in very close proximity, as indicated by a high luminescence signal. FtsA, EzrA and ZapA, all three interacting directly with FtsZ, also gave a slight signal. hlpA-LgBit hlpA-SmBit (HlpA-HlpA), here diluted 100 times, is used as positive control. Each dot represents the average of n=15 measurements of a technical replicate, with the size of the dot representing the SEM. b, FtsZ-CcrZ interaction confirmation by bacterial two-hybrid assay. T25 is the empty vector pST25 and T25-FtsZ corresponds to vector pST25-FtsZ used in combination with pUT18-CcrZ (CcrZ-T18) and pUT18-FtsZ (FtsZ-T18). c, Affinity purification of FtsZ-GFP from S. pneumoniae cells (2^nd lane) also pulls down untagged CcrZ (4^th lane). Purification of GFP alone (first lane) did not pull CcrZ down (3^rd lane). d, FtsZ from S. pneumoniae expressed in E. coli co-purifies with CcrZ_Sp-GFP by affinity purification. WC: whole cell extract, S: supernatant, HE: heat eluted products, C: CcrZ-GFP, F: FtsZ. e, Epifluorescence time-lapse microscopy of CcrZ-mKate2 at 37°C in presence (left panel) or absence (right panel) of FtsZ. When FtsZ amounts are reduced, cells increase their size and CcrZ is de-localized from mid-cell. Scale bar, 3 μm.