Fig. 5. CcrZ activates DnaA-dependent replication initiation.

a, Localization of FtsZ-mTurquoise2 in a thermo-sensitive DnaA strain (dnaA^TS) at permissive (30°C) and non-permissive (40°C) temperatures shows that dnaA inactivation leads to a similar phenotype as ccrZ inactivation. Scale bar, 3 μm. b, TEM of DnaA^TS at non-permissive temperature (40°C) indicates the presence of multiple septa, similarly to a ΔccrZ mutant. Scale bar, 250 nm. c, When replication is driven in a RepN-dependent manner in B. subtilis (oriN), no decrease in ori/ter ratio can be observed in absence of ccrZBs (oriN, ΔccrZss). Mean values are indicated under the boxes. Data are shown as box (25^th to 75^th percentile) and whiskers (1.5x interquartile range) plots ; n=3 independent samples. d, Schematic representation of CcrZ motifs. CcrZ has one putative domain annotated APH (Phosphotransferase enzyme family; PFAM01636). Sequence alignment with several kinases revealed the presence of a conserved P-loop, APH and Brenner’s motifs, found in most phosphotransferases. Locations of mutations made for 3 essential (red) and 2 non-essential (black) conserved residues are shown underneath. e, LicA choline kinase structure complexed with AMP and MES (2-(N-morpholino)ethanesulfonic acid). The five residues indicated in yellow are conserved between CcrZ and LicA (and highly conserved within Firmicutes). f, Mutation of three of these five conserved residues in the putative ATP binding pocket leads to growth defects. g, Localization of CcrZ-H157A-GFP and CcrZ-D177A-GFP is not impaired. Scale bar, 3 μm.