Growth Rate-Dependent Regulation of Medial FtsZ Ring Formation

Abstract

FtsZ is an essential cell division protein conserved throughout the bacteria and archaea. In response to an unknown cell cycle signal, FtsZ polymerizes into a ring that establishes the future division site. We conducted a series of experiments examining the link between growth rate, medial FtsZ ring formation, and the intracellular concentration of FtsZ in the gram-positive bacterium Bacillus subtilis. We found that, although the frequency of cells with FtsZ rings varies as much as threefold in a growth rate-dependent manner, the average intracellular concentration of FtsZ remains constant irrespective of doubling time. Additionally, expressing ftsZ solely from a constitutive promoter, thereby eliminating normal transcriptional control, did not alter the growth rate regulation of medial FtsZ ring formation. Finally, our data indicate that overexpressing FtsZ does not dramatically increase the frequency of cells with medial FtsZ rings, suggesting that the mechanisms governing ring formation are refractile to increases in FtsZ concentration. These results support a model in which the timing of FtsZ assembly is governed primarily through cell cycle-dependent changes in FtsZ polymerization kinetics and not simply via oscillations in the intracellular concentration of FtsZ. Importantly, this model can be extended to the gram-negative bacterium Escherichia coli. Our data show that, like those in B. subtilis, average FtsZ levels in E. coli are constant irrespective of doubling time.Temporally, cell division must be tightly coupled to chromosome replication, chromosome segregation, and cell growth to ensure that both daughter cells inherit complete genomes and are of the appropriate size and shape. In eukaryotes, the precise orchestration of cyclin-dependent kinases in conjunction with ubiquitin-mediated proteolysis guarantees that each stage of the cell cycle is firmly integrated with the next (22, 28). In bacteria, in which DNA synthesis, chromosome segregation, and cell division can overlap, the factors that govern cell cycle transitions are not clearly defined.The earliest known event in bacterial cell division is the assembly of the tubulin-like protein FtsZ into a ring structure at the nascent division site in response to an unidentified cell cycle signal (35,45). FtsZ ring formation has been shown to be required for the recruitment of other division proteins, including components of the cell wall, to the septal site in both Escherichia coli and Bacillus subtilis (10,35,45,55). Fluorescence microscopy of wild-type and mutant E. coli cells indicates that FtsZ first localizes to midcell as a small focus of protein that then extends bidirectionally around the circumference of the cell (2). This observation suggests the presence of a nucleation site that lowers the critical concentration of FtsZ required to initiate polymerization. In response to a second, also unidentified, cell cycle signal the FtsZ ring constricts like a drawstring at the leading edge of the invaginating septum to help mediate cytokinesis (35).Alth...

FtsZ and the division of prokaryotic cells and organelles

“…The factors that are involved in this cell-cycle timing of septation are not known, although DNA replication seems to be important for the proper expression of several genes, including ftsZ 69 . In E. coli and B. subtilis, FtsZ levels do not change significantly during the cell cycle 70 , which indicates that the activity of FtsZ is cellcycle controlled. By contrast, FtsZ levels in C. crescentus vary dramatically because of proteolysis and resynthesis 71 ; nevertheless, the artificial increase of FtsZ levels at the wrong time in the cell cycle does not trigger Z-ring assembly or change the timing of Z-ring constriction 72 .…”

In Vivo Imaging of a Bacterial Cell Division Protein Using a Protease‐Assisted Small‐Molecule Labeling Approach

“…M9 media has been extensively used to study FtsZ gene expression. [10,11] Growth in M9 minimal media resulted in an increase in doubling time from ~20 min (in LB) media to ~ 80 min. However, this change in growth rate does not alter the levels of FtsZ as indicated in a previous report by Levin and coworkers.…”

“…In E. coli, the formation of the cytokinetic ring is governed by changes in the polymerization kinetics FtsZ during the course of the cell cycle rather than by oscillations in intracellular concentrations . [11] Next, we optimized the FtsZ expression levels such that the Z-ring formation by the FtsZ protein can be observed under our labeling conditions. Since overproduction of FtsZ at levels only 2-7 fold higher than wild type from multicopy plasmids have been known to result in minicell formation, [12] we used a P BAD vector system for FtsZ expression studies.…”

ClpXP and ClpAP control the Escherichia coli division protein ZapC by proteolysis

“…Although the intracellular concentration of FtsZ remains essentially unchanged at varying growth rates, the frequency of Z-ring formation is altered in a growth-rate-dependent manner, suggesting that FtsZ polymerization dynamics control growth rate regulation of FtsZ-ring formation in E. coli (Weart & Levin, 2003). Given that both ClpXP and ZapC regulate Z-ring assembly dynamics, we reasoned that changes in growth rate might differentially influence ClpXP-mediated ZapC stability.…”

“…Hydrolysis of the bound nucleotide leads to depolymerization of the FtsZ protofilaments (Erickson et al, 2010;Mukherjee & Lutkenhaus, 1998). As FtsZ levels are essentially constant throughout the cell cycle, spatiotemporal control of division is exercised largely through the assembly/ disassembly of the Z-ring (Rueda et al, 2003;Weart & Levin, 2003). A number of proteins that interact with FtsZ contribute to its function by modulating the dynamics of FtsZassembly (Adams & Errington, 2009;Huang et al, 2013;Lutkenhaus et al, 2012;Ortiz et al, 2016).…”