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.

FIG. 1.

Alternative models for the regulation of FtsZ ring formation. (A) FtsZ production is regulated in a cell cycle-dependent fashion. As the cell approaches the appropriate time for ring formation (asterisk), intracellular FtsZ levels rise above the critical concentration (horizontal dotted line) for polymerization and allow for the assembly of the FtsZ ring. As the division cycle is completed (vertical lines), FtsZ levels rapidly drop to predivision levels, thereby preventing inappropriate ring formation. (B and C) Alternatively, FtsZ levels remain constant throughout the division cycle. The initiation of ring formation is, instead, controlled by the modulation of FtsZ polymerization dynamics. At the appropriate time, the cell stimulates the assembly of the FtsZ ring from a previously existing pool of monomers by either initiating a positive stimulus of FtsZ polymerization (B, arrows) or by relieving the repression of FtsZ polymerization present during the predivisional period (C, striped boxes).

FIG. 2.

The frequency of FtsZ ring formation varies with growth rate in B. subtilis. Wild-type cells were grown in either LB medium (A to C) or minimal succinate (D to F). (A and D) Cell walls stained with wheat germ agglutinin conjugated to fluorescein isothiocyanate. (B and E) FtsZ immunolocalized by using affinity-purified primary antibody and secondary antibody conjugated to Cy-3. (C and F) Drawings of merged images. Scale bar, 2 μm.

FIG. 3.

Plot of the frequencies of cells with medial FtsZ rings versus doubling times. Wild-type B. subtilis cells (JH642; filled circles) were grown in LB medium, minimal glucose, minimal sorbitol, and minimal succinate to produce doubling times of 26, 41, 74, and 94 min, respectively. The FtsZ ring frequencies for these cultures were 85% (1,366 of 1,601) for LB medium, 75% (828 of 1,104) for minimal glucose, 53% (698 of 1,305) for minimal sorbitol, and 34% (449 of 1,326) for minimal succinate. A B. subtilis strain expressing ftsZ solely from a xylose-inducible promoter (BW121; unfilled squares) was grown in LB medium, minimal glycerol, minimal sorbitol, and minimal succinate to produce doubling times of 23, 48, 75, and 99 min, respectively. The FtsZ ring frequencies for these cultures were 86% (421 of 492) for LB medium, 68% (349 of 513) for minimal glycerol, 52% (263 of 509) for minimal sorbitol, and 30% (159 of 531) for minimal succinate.

FIG. 4.

FtsZ levels remain constant regardless of growth rate. (A) Representative growth curves for wild-type B. subtilis cells grown in LB medium, minimal glucose (Gluc), minimal sorbitol (Sorb), or minimal succinate (Succ) (from left to right, respectively). Samples were harvested at an OD₆₀₀ of ∼0.600, which is marked in the figure by an asterisk. Arrows, when present, indicate where a culture has exited from exponential-phase growth. OD600 is shown on the y axis. (B) Representative quantitative immunoblot of FtsZ from wild-type B. subtilis cells grown in LB medium, minimal glucose, minimal sorbitol, and minimal succinate. Samples were harvested during exponential growth and normalized to total protein at gel loading. Relative concentrations of FtsZ, normalized to those for wild-type cells grown in LB medium, are shown below. Margins of error were calculated by using a t test with a 95% confidence level and are based on data from three independent experiments (i.e., three separate sets of cultures).

FIG. 5.

Overexpression of FtsZ following induction of P_spachy-ftsZ. (A) Quantitative immunoblot of FtsZ from wild-type (WT) cells (left) and PL950 cells bearing P_spachy-ftsZ in the absence (middle) and presence (right) of 10 M IPTG. Samples were harvested during mid-exponential growth, and lysates were normalized to total protein concentration at gel loading. Relative concentrations of FtsZ, normalized to those for the wild type, are shown below. (B) FtsZ localization in cells overexpressing FtsZ. (Left) DNA. (Middle) FtsZ. (Right) Cartoon depicting the position of the FtsZ rings relative to the nucleoids. Note the doublet of FtsZ in the lower cell. Scale bar, 0.5 μm.

FIG. 6.

FtsZ levels in E. coli remain constant regardless of growth rate. (A) Representative growth curves for wild-type E. coli cells grown in LB medium, minimal glucose, minimal glycerol, and minimal succinate (from left to right, respectively). Samples were harvested at an OD₆₀₀ of ∼0.250, which is marked by an asterisk. Arrows, when present, indicate where a culture has exited from exponential-phase growth. OD600 is shown on the y axis. (B) Representative quantitative immunoblot of FtsZ from wild-type E. coli cells grown in LB medium, minimal glucose, minimal glycerol, and minimal succinate. Samples were harvested during exponential growth and normalized to an OD₆₀₀ of ∼0.250 at gel loading. Relative concentrations of FtsZ, normalized to those for wild-type cells grown in LB medium, are shown below. Margins of error were calculated by using a t test with a 95% confidence level and are based on data from three independent experiments (i.e., three separate sets of cultures).