Nutrient availability is one of the strongest determinants of cell size. When grown in rich media, single-celled organisms such as yeast and bacteria can be up to twice the size of their slow-growing counterparts. The ability to modulate size in a nutrient-dependent manner requires cells to: (1) detect when they have reached the appropriate mass for a given growth rate and (2) transmit this information to the division apparatus. We report the identification of a metabolic sensor that couples nutritional availability to division in Bacillus subtilis. A key component of this sensor is an effector, UgtP, which localizes to the division site in a nutrient-dependent manner and inhibits assembly of the tubulin-like cell division protein FtsZ. This sensor serves to maintain a constant ratio of FtsZ rings to cell length regardless of growth rate and ensures that cells reach the appropriate mass and complete chromosome segregation prior to cytokinesis.

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...

We have investigated the conditions required for polar localization of the CheZ phosphatase by using a CheZ-green fluorescent protein fusion protein that, when expressed from a single gene in the chromosome, restored chemotaxis to a ⌬cheZ strain. Localization was observed in wild-type, ⌬cheZ, ⌬cheYZ, and ⌬cheRB cells but not in cells with cheA, cheW, or all chemoreceptor genes except aer deleted. Cells making only CheA-short (CheA S ) or CheA lacking the P2 domain also retained normal localization, whereas cells producing only CheA-long or CheA missing the P1 and P2 domains did not. We conclude that CheZ localization requires the truncated C-terminal portion of the P1 domain present in CheA S . Missense mutations targeting residues 83 through 120 of CheZ also abolished localization. Two of these mutations do not disrupt chemotaxis, indicating that they specifically prevent interaction with CheA S while leaving other activities of CheZ intact.

SummaryAssembly of the tubulin-like cytoskeletal protein FtsZ into a ring structure establishes the location of the nascent division site in prokaryotes. Factors that modulate FtsZ assembly are essential for ensuring the precise spatial and temporal regulation of cytokinesis. We have identified ClpX, the substrate recognition subunit of the ClpXP protease, as an inhibitor of FtsZ assembly in Bacillus subtilis . Genetic data indicate that ClpX but not ClpP inhibits FtsZ-ring formation in vivo . In vitro , ClpX inhibits FtsZ assembly in a ClpP-independent manner through a mechanism that does not require ATP hydrolysis. Together our data support a model in which ClpX helps maintain the cytoplasmic pool of unassembled FtsZ that is required for the dynamic nature of the cytokinetic ring. ClpX is conserved throughout bacteria and has been shown to interact directly with FtsZ in Escherichia coli . Thus, we speculate that ClpX functions as a general regulator of FtsZ assembly and cell division in a wide variety of bacteria.

ClpX is a well-characterized bacterial chaperone that plays a role in many processes, including protein turnover and the remodeling of macromolecular complexes. All of these activities require ATP hydrolysisdependent, ClpX-mediated protein unfolding. Here we used site-directed mutagenesis in combination with genetics and biochemistry to establish that ClpX inhibits assembly of the conserved division protein FtsZ through a noncanonical mechanism independent of its role as an ATP-dependent chaperone.The highly conserved chaperone ClpX has been implicated in numerous cellular processes, including the turnover of regulatory proteins (6,13,19,20,32), the destruction of unfinished polypeptides (8,30,31), and the remodeling of macromolecular complexes (3-5). While ClpX-mediated proteolysis is dependent on the ClpP protease, ClpX alone is sufficient for remodeling of macromolecular complexes, such as the phage Mu transpososome (3-5). All of these functions share a requirement for ATP hydrolysis-dependent, ClpX-mediated protein unfolding.In Bacillus subtilis, ClpX modulates assembly of the conserved cytoskeletal protein FtsZ to help control the process of cell division (29). FtsZ, a homolog of the eukaryotic cytoskeletal protein tubulin, assembles into a ring structure at the nascent division site in response to an unidentified cell cycle signal. This ring serves as the foundation for assembly of the cell division apparatus (25). In previous work, we determined that ClpX inhibits FtsZ assembly and maintains the pool of subunits available for ring formation (29). Surprisingly, our biochemical data suggested that ClpX inhibits FtsZ assembly through a ClpP-independent mechanism that does not appear to require ATP hydrolysis (29).Here we employed site-directed mutagenesis in combination with genetic and biochemical analyses to establish that ClpX has two functions in vivo: an ATP hydrolysis-dependent chaperone activity, required for the unfolding of proteolytic substrates, and an ATP hydrolysis-independent activity, required for interaction with FtsZ.Generating mutations in ClpX residues required for chaperone activity. To generate mutations that render B. subtilis ClpX defective for ATP-dependent protein unfolding, we took advantage of an alignment with the well-characterized Escherichia coli ClpX protein (Fig. 1A) to engineer single alanine substitutions in B. subtilis ClpX residues that are predicted to be required for ClpX-mediated proteolysis. These substitutions included mutations in the putative Walker A [ClpX(K122A)] and Walker B [ClpX(E182A)] boxes, which in E. coli are required for ATP binding and hydrolysis, respectively, as well as a mutation in the substrate-processing pore loop [ClpX(Y150A)] (12). ATP binding is necessary for formation of the active ClpX hexamer (2, 9, 27), whereas ATP hydrolysis provides the energy required for protein unfolding and translocation (12). Mutations in the E. coli ClpX Walker A and Walker B motifs disrupt ClpX-mediated proteolysis and macromolecular complex remodeling (reviewed in...