Assembly dynamics of FtsZ rings in Bacillus subtilis and Escherichia coli and effects of FtsZ-regulating proteins

Abstract

FtsZ is the major cytoskeletal component of the bacterial cell division machinery. It forms a ring-shaped structure (the Z ring) that constricts as the bacterium divides. Previous in vivo experiments with green fluorescent protein-labeled FtsZ and fluorescence recovery after photobleaching have shown that the Escherichia coli Z ring is extremely dynamic, continually remodeling itself with a half time of 30 s, similar to microtubules in the mitotic spindle. In the present work, under different experimental conditions, we have found that the half time for fluorescence recovery of E. coli Z rings is even shorter (approximately 9 s). As before, the turnover appears to be coupled to GTP hydrolysis, since the mutant FtsZ84 protein, with reduced GTPase in vitro, showed an approximately 3-fold longer half time. We have also extended the studies to Bacillus subtilis and found that this species exhibits equally rapid dynamics of the Z ring (half time, approximately 8 s). Interestingly, null mutations of the FtsZ-regulating proteins ZapA, EzrA, and MinCD had only modest effects on the assembly dynamics. This suggests that these proteins do not directly regulate FtsZ subunit exchange in and out of polymers. In B. subtilis, only 30 to 35% of the FtsZ protein was in the Z ring, from which we conclude that a Z ring only 2 or 3 protofilaments thick can function for cell division.

FIG. 1.

FRAP of an FtsZ ring in wild-type B. subtilis cells. (A) Time-lapse series of fluorescence images showing the time course of recovery. The arrow shows the half-ring to be bleached. (B) Intensity of the photobleached region over time. The data points have been corrected for background and photobleaching during the observation period. The points represent the fluorescence data, and the solid line is the predicted recovery curve given the first-order rate constant, k, determined by least-squares fitting of the data to the equation given in Materials and Methods. The recovery half time for this series was 7.0 s.

FIG. 2.

FRAP of an FtsZ ring in wild-type E. coli BW27783(pJSB150) cells. (A) Time-lapse series of fluorescence images. (B) Intensity of the photobleached region over time. The half time of recovery for the series shown was 8.9 s.

FIG. 3.

FRAP of FtsZ rings in B. subtilis cells lacking ezrA. (A) Time-lapse series of fluorescence images. (B) Intensity of the photobleached region over time. Recovery half time, 13.0 s.

FIG. 4.

FRAP of FtsZ rings in B. subtilis cells lacking minCD. (A) Time-lapse series of fluorescence images. (B) Intensity of the photobleached region over time. Recovery half time, 10.7 s.

FIG. 5.

Histograms of Z-ring fluorescence recovery times for wild-type (wt) versus mutant cells of B. subtilis (Bs) (A) and E. coli (Ec) (B).