The septal ring in E. coli
is a complex structure consisting of at least seven different protein products. Important challenges concerning this organelle are to understand its precise molecular architecture, its mode of assembly, and the mechanism whereby it mediates the coordinated invagination of the cell envelope layers during septum formation. The order in which the different components assemble to form a mature septal ring has been studied in several laboratories by determining the localization of FtsA, -I, -N, -K, and -Z in filaments in which one of the essential division proteins has either lost function due to a conditional mutation or drug treatment or is largely lacking, due to a specific block in gene expression (2
). The picture emerging from these studies is that, first, FtsZ assembles at the prospective division site to form the FtsZ ring. FtsA then joins the FtsZ ring, followed by FtsI, FtsK and FtsN. The best evidence supporting early assembly of the FtsZ ring has come from the observations that FtsA (reference 3
and this study), FtsI (35
), FtsK (40
) and FtsN (2
) completely failed to localize properly in FtsZ−
filaments whereas, on the other hand, FtsZ rings were still present in FtsA−
and this study), FtsI−
), and FtsW−
We recently identified ZipA as a novel division factor and showed that it is an integral inner membrane protein which interacts directly with FtsZ. A ZipA-Gfp fusion protein, furthermore, localized to the septal ring at an early stage of the division cycle (17
). Here, we confirmed this localization pattern for native ZipA by immunofluorescence microscopy.
The combined properties of ZipA raised the possibility that the protein plays a role in assembly of the FtsZ ring by attracting FtsZ to the prospective division site. The principal conclusion of this study is that this possibility is most likely incorrect. In FtsZ-depleted filaments, the bulk of both native ZipA or ZipA-Gfp clearly failed to accumulate at potential division sites but, rather, appeared to be evenly distributed over the cell membrane. Conversely, both native FtsZ and a Gfp-FtsZ fusion protein still assembled into ring structures in the majority of ZipA-depleted filaments. It is most likely, therefore, that the localization of ZipA to the septal ring is secondary to that of FtsZ.
In support of the conclusion that FtsA localization is also dependent on FtsZ (3
), we found that FtsA and Gfp-FtsA failed to localize after depletion of FtsZ, but that FtsZ rings could still be formed in FtsA-depleted filaments. Interestingly, however, ZipA rings were still present in FtsA-depleted filaments, and FtsA rings were still present in ZipA-depleted filaments, indicating that the incorporation of ZipA into the septal ring does not require the prior localization of FtsA and vice versa. Combined with the knowledge that both ZipA and FtsA localize early in the division cycle and that both can bind FtsZ directly, we propose that both proteins become associated with the FtsZ ring either during or very soon after formation of the latter. In this regard, it is interesting that whereas the vast majority of FtsZ- or ZipA-depleted filaments appeared completely smooth, more than half of the FtsA-depleted filaments (HID or CID) showed one or more marked indentations of the cell wall (Fig. to ). This suggests that despite the early localization of FtsA, the early stages of septation are less sensitive to depletion of the protein than are later stages.
Although the scenario proposed above represents the most straightforward interpretation of our results, it is difficult to completely rule out an essential role for ZipA in the assembly of the FtsZ ring. Although the ZipA-depleted filaments used in this study clearly contained an insufficient level of ZipA to support cell division, it cannot be excluded that this level (~10% of normal [Fig. ]) may have been sufficient to actively stimulate formation of FtsZ rings. This same argument also applies to FtsA, -I, -N, -K, -Q, and -W in the studies in which FtsZ localization was observed after inactivation or depletion of one of these division proteins (1
In these same studies, the ratio of cell length to the number of FtsZ rings in most types of Fts− filaments was much higher than expected, and a large variability in this ratio between individual filaments was observed. Similarly, we observed that the average length-to-FtsZ ring ratios in the ring-containing populations of both ZipA-depleted (17.2 μm in ZipAHID and 8.9 μm in ZipACID) and FtsA-depleted (6.2 μm in FtsAHID and 6.3 μm in FtsACID) filaments was significantly higher than that in wild-type cell (~2 to 3 μm). In addition, this ratio varied widely between individual filaments. For instance, after heat-induced depletion of ZipA, 72% of the filaments (ranging in size from 10.1 to 83.1 μm) showed from one to five FtsZ rings per cell with a length-to-ring ratio ranging from 3.3 to 89.0 μm, and the remaining 28% (ranging in size from 10.3 to 76.9 μm) showed no ring at all (Table ). After CID of ZipA, 81% of the population (ranging in size from 5.7 to 85.5 μm) contained from one to nine Gfp-FtsZ rings per cell with a ratio ranging from 4.9 to 40.2 μm, and the remaining 19% (ranging in size from 16.8 to 63.4 μm) were devoid of rings (Table ). The length-to-ZipA ring ratio in FtsA− filaments and length-to-FtsA ring ratio in ZipA− filaments were also relatively high and variable which, given the high and variable length to FtsZ ring ratio in FtsA− and ZipA− filaments, is consistent with the interpretation that the incorporation of FtsA and ZipA into the septal ring is dependent on formation of the FtsZ ring component.
Why the numbers of FtsZ rings are so low and variable in ZipA-depleted cells (and other types of filaments) relative to wild-type cells is an intriguing question. The possibility that depletion of ZipA simply leads to a reduction in the cellular concentration of FtsZ was ruled out by quantitative immunoblot analyses showing that depletion of 90% of ZipA affected the FtsZ concentration by less than 5% (not shown). Inefficient fixation and/or staining of cells could lead to an underestimation of the number of ring structures by immunofluorescence, but we consider it unlikely that this was a determining factor in our experiments, especially since we obtained largely comparable results using Gfp tagged proteins as septal ring markers. One reasonable hypothesis is that binding of ZipA to the FtsZ ring stabilizes the structure, for instance, by providing an anchor to the cell membrane. If correct, and when combined with the results of previous studies (1
), this would mean that most of the septal ring components contribute substantially to the stability of the structure. As pointed out by Pogliano et al. (29
), an interesting alternative possibility is that the state of maturation or activity of a septal ring at one potential division site somehow affects the formation or stability of rings at additional sites in the cell. Clearly, more work is needed to test these possibilities.
The results of this study indicate that ZipA is not required for the initiation of FtsZ ring formation or for the recruitment of FtsA to the ring but suggest that it contributes significantly to the stability of the organelle. Many additional roles for the protein are possible. Because ZipA is very likely among the first factors to become associated with the FtsZ ring, it is conceivable that the protein helps recruit other components to the structure. It will, therefore, be interesting to determine the localization pattern of other Fts proteins in ZipA-depleted cells. Filaments lacking ZipA are smooth, which is consistent with a role for the protein throughout the cell wall invagination process. It is possible that ZipA simply ensures that the inner membrane remains anchored to the shrinking FtsZ ring. In addition, the protein may well play a more active role(s), such as stimulating constriction of the FtsZ ring or coordinating the activity of the FtsZ ring in the cytoplasm with the peptidoglycan synthesizing machinery in the periplasm. Additional experimentation is needed to define the role(s) of ZipA more precisely.
To date, assembly of the FtsZ ring at the prospective division site is the first recognizable step in the formation of the septal ring organelle. Although we cannot be absolutely certain, this and other studies have rendered it unlikely that any of the other known division proteins, including ZipA, perform an essential function prior to this step. To determine what defines a potential division site and how FtsZ assembly is initiated at this site remain important unanswered questions.