CcrZ is a pneumococcal spatiotemporal cell cycle regulator that interacts with FtsZ and controls DNA replication by modulating the activity of DnaA

Clement Gallay,Renske Van Raaphorst,Gro A Stamsås,Simone Pelliciari,Jan-Willem Veening,Stephan Gruber,Julien Dénéréaz,Alan D Grossman,Morten Kjos,Xue Liu,Stefano Sanselicio,Young Min Soh,Heath Murray,Mary E Anderson

doi:10.1038/s41564-021-00949-1

Abstract

Most bacteria replicate and segregate their DNA concomitantly while growing, before cell division takes place. How bacteria synchronize these different cell cycle events to ensure faithful chromosome inheritance by daughter cells is poorly understood. Here, we identify Cell Cycle Regulator protein interacting with FtsZ (CcrZ) as a conserved and essential protein in pneumococci and related Firmicutes such as Bacillus subtilis and Staphylococcus aureus. CcrZ couples cell division with DNA replication by controlling the activity of the master initiator of DNA replication, DnaA. The absence of CcrZ causes mis-timed and reduced initiation of DNA replication, which subsequently results in aberrant cell division. We show that CcrZ from Streptococcus pneumoniae interacts directly with the cytoskeleton protein FtsZ, which places CcrZ in the middle of the newborn cell where the DnaA-bound origin is positioned. This work uncovers a mechanism for control of the bacterial cell cycle in which CcrZ controls DnaA activity to ensure that the chromosome is replicated at the right time during the cell cycle.

Highlights

E. coli and B. subtilis use different systems for regulating their cell cycles, the way they localize their division site is conserved, as both organisms use a variant of the Min system to prevent polymerization of the tubulin-like protein FtsZ away from the mid-cell[16,17]
Using high-throughput gene silencing with clustered regularly interspaced short palindromic repeats interference (CRISPRi) of all essential genes of S. pneumoniae[26], we examined proteins leading to defects in DNA content on depletion
Our findings show that Cycle Regulator protein interacting with FtsZ (CcrZ) acts as a spatiotemporal link between cell division and DNA replication in S. pneumoniae

Summary

GFP-CcrZ 1 0 –1 –2

CcrZ depletion in S. pneumoniae led to slight morphological defects and modest changes in cell size when analysed by phase-contrast microscopy (Fig. 1d). After 2 h of FtsZ depletion, chromosomal DNA was isolated and oriC/ter ratios were determined This showed that on mis-localization of CcrZ, cells under-replicate (Fig. 4o). To test whether CcrZ controls DNA replication by regulating DnaA activity, we made use of the fact that a B. subtilis ∆ccrZBs mutant underinitiates (Fig. 4h) and a strain was constructed in which DNA replication was driven in a RepN-dependent manner (from a plasmid origin of replication oriN) rather than from DnaA-dependent initiation (from oriC). This showed no significant ori/ter ratio differences when ccrZ was deleted (Fig. 5c), suggesting. In the absence of CcrZ, initiation of DNA replication is mis-timed and occurs too late relative to cellular growth and Z-ring formation, frequently leading to futile division events, mis-segregated chromosomes and anucleate cells (Fig. 6e)

Discussion

Findings

Methods