Unique and overlapping roles for ZipA and FtsA in septal ring assembly in Escherichia coli

Abstract

ZipA and FtsA are essential division proteins in Escherichia coli that are recruited to the division site by interaction with FtsZ. Utilizing a newly isolated temperature-sensitive mutation in zipA we have more fully characterized the role of ZipA. We confirmed that ZipA is not required for Z ring formation; however, we found that ZipA, like FtsA, is required for recruitment of FtsK and therefore all downstream division proteins. In the absence of FtsA or ZipA Z rings formed; however, in the absence of both, new Z rings were unable to form and preformed Z rings were destabilized. Consistent with this, we found that an FtsZ mutant unable to interact with both ZipA and FtsA was unable to assemble into Z rings. These results demonstrate that ZipA and FtsA are both required for recruitment of additional division proteins to the Z ring, but either one is capable of supporting formation and stabilization of Z rings.

Fig. 1. The zipA1 (Ts) mutation confers a temperature-sensitive division phenotype. A culture of PS223 [zipA1 (Ts)] growing exponentially at 30°C was shifted to 42°C. Cells were photographed before and 2 h after the shift to 42°C.

Fig. 2. Localization of ZipA, FtsZ and FtsA in zipA1 (Ts) cells. PS223 [zipA1 (Ts)] was grown as in Figure 1. Samples were taken, fixed and processed for fluorescence microscopy as described in Materials and methods. The first, second and third columns are photographs of cells stained with anti-ZipA, anti-FtsZ and anti-FtsA antisera as indicated. Samples were taken at various times after the temperature shift to 42°C as indicated to the left of the figure.

Fig. 3. Localization of FtsK in zipA1 (Ts) cells. PS223 [zipA1 (Ts)] was grown as in Figure 1. Samples were taken at 30°C and after a shift to 42°C, fixed and processed for fluorescence microscopy as described in Materials and methods. The upper panel shows cells before the temperature shift. The middle and lower panels show cells at 5 and 30 min, respectively, after the temperature shift to 42°C.

Fig. 4. Localization of ZipA, FtsZ and FtsA in ftsA12 (Ts) zipA1 (Ts) cells. A culture of PS234 [zipA1 (Ts) ftsA12 (Ts)] growing exponentially at 30°C was shifted to 42°C. Samples were taken before and at the indicated times after the shift to 42°C. The antibodies used for staining are indicated at the top of the figure.

Fig. 5. Localization of FtsZ in ftsA12 (Ts) zipA1 (Ts) cells at 30 and 42°C. Portions of the figures shown in Figures 4 and 7 were magnified ×2. The top two panels show cells of ftsA12 (Ts) zipA1 (Ts) at 30°C (top panel) and 30 min after the temperature shift to 42°C (middle panel). These images illustrate the contrast between the sharp and symmetric appearance of typical Z rings observed at 30°C and the abnormal FtsZ structures observed at 42°C in this strain. The bottom panel shows magnified ftsI23 (Ts) zipA1 (Ts) cells 30 min after a shift to 42°C. These cells display several typical Z rings per cell.

Fig. 6. Distribution of the foci of FtsZ fluorescence in PS234 [zipA1 (Ts) ftsA12 (Ts)] at the non-permissive temperature. Cells from the experiment in Figure 3 were analyzed for the distribution of fluorescent spots. Only the sample taken 30 min after the temperature shift from 30 to 42°C was examined. A total of 156 cells with 334 spots were analyzed. Six cells did not contain any spots. The distance from each spot to the nearest pole was measured and divided by half the total cell length. The graph presented here shows the percentage of spots observed for each fraction of the cell length.

Fig. 7. Localization of FtsZ in ftsI23(Ts) zipA1 (Ts) cells. A culture of PS414 [zipA1 (Ts) ftsI23 (Ts)] was analyzed as described in the legend to Figure 4.

Fig. 8. Localization of FtsZ and mutant FtsZFL. Cultures of PS165 (pSEB135-P_BAD::ftsZ) or PS166 (pSEB135FL-P_BAD::ftsZFL) were placed at 42°C to deplete the endogenous FtsZ. After 90 min 0.02% arabinose was added, and samples were taken 90 min later and stained for FtsZ as described in Materials and methods. Since the level of induction by arabinose is variable from cell to cell in the same culture (Siegele and Hu, 1997), each column shows two cells from each culture, illustrating two different levels of expression of FtsZ (left column) or FtsZFL (right column).

Fig. 9. Dependency pathway for recruitment of cell division proteins to the division site. The first clearly defined step in cell division is the assembly of FtsZ to form the Z ring. FtsA and ZipA colocalize with FtsZ, and as shown in this study the presence of either one is sufficient for Z ring formation. The other proteins are then recruited and both ZipA and FtsA are required.