INTRODUCTION

Top Abstract Introduction Materials & Methods Results Discussion References

Cell division in bacteria occurs by the coordinated invagination of the cell envelope layers that make up the cell wall. Work in the past few years has revealed that this process is mediated by a membrane-associated, cytoskeleton-like organelle which assembles at the prospective division site before the onset of septal invagination and which remains associated with the ingrowing cell wall until septal closure (24, 32). In Escherichia coli, septum formation requires at least nine gene products, FtsA, -I, -K, -L, -N, -Q, -W, -Z, and ZipA, which are specifically dedicated to this process. All these essential division proteins have recently been shown to be part of the septal ring structure in E. coli (2-4, 17, 25, 35, 39, 40) with the exception of FtsL and -Q. A distant homolog of FtsQ (DivIB), however, was recently localized to the septal ring of Bacillus subtilis (19), and it may be expected that FtsQ, as well as FtsL, is associated with the organelle during some stage of the division cycle in E. coli.

The phylogenetically highly conserved FtsZ protein is a major component of the septal ring and plays a key role in the division process. The protein is a tubulin-like GTPase which can form large protofilament-like polymers in vitro (9, 15, 23, 26-28, 31, 38, 41). In vivo, FtsZ moves from the cytoplasm to accumulate in a ring-like structure at the prospective division site early in the division cycle (4). Based on the in vitro properties of FtsZ, this ring is thought to be formed by a self-assembly reaction and to consist of a homopolymeric form of the protein. How formation of the FtsZ ring is initiated and by what mechanism it is normally restricted to certain sites on the cell envelope remains unclear. It is conceivable, however, that it involves the interaction of FtsZ with a hypothetical membrane factor (factor X) which marks potential division sites and stimulates polymerization of the protein at these sites.

We recently used an in vitro assay to search for factors in E. coli which interact directly with the FtsZ protein. This approach led to the discovery of a previously unknown division protein, which we called ZipA (17). ZipA is essential for cell division, and our results indicate not only that ZipA and FtsZ associate with each other in vitro but that this interaction also occurs in vivo and is required for cell constriction. Evidence suggests that ZipA is a bitopic integral membrane protein of type Ib of which the N terminus traverses the inner membrane once and of which the rest of the protein resides in the cytoplasm. Furthermore, localization studies indicate that ZipA and FtsZ interact primarily within the septal ring structure. Thus, like FtsZ, a ZipA-Gfp fusion protein was found to accumulate very early in the division cycle in a ring structure at the prospective division site and to remain associated with the invaginating septum during the cell constriction process (17).

In addition to its interaction with ZipA, recent two-hybrid experiments (14, 37) as well as localization studies (3, 25) have indicated that FtsZ also directly interacts with the FtsA protein, supporting previous genetic experiments suggesting such an interaction (8, 13). FtsA is a phosphoprotein which is peripherally associated with the membrane. It appears to belong to a family of ATPases which also includes actin, sugar kinases, and Hsp70 proteins (5, 33), but its role in the division process is presently not understood.

The observations that ZipA is an early component of the septal ring and that it binds to both FtsZ and the cytoplasmic membrane raise the possibility that the protein is directly involved in the assembly of FtsZ and/or other components, such as FtsA, into the septal ring organelle. In assessing the possible roles of ZipA in the division process, we have used both indirect immunofluorescence and Gfp tagging techniques to study the interdependence of ZipA, FtsZ, and FtsA localization. To this end, we constructed a set of strains allowing us to determine the cellular location of each of these three proteins in cells from which one of the other two had been specifically depleted.

Our results show that ZipA fails to accumulate in a ring shape in the absence of FtsZ. Conversely, depletion of ZipA does not abolish formation of FtsZ rings but leads to a significant reduction in the number of rings per unit of cell mass. In addition, ZipA does not appear to require FtsA for assembly into the septal ring and vice versa. It is suggested that septal ring formation starts by assembly of the FtsZ ring, after which ZipA and FtsA join this structure in a mutually independent fashion through direct interactions with the FtsZ protein.

	MATERIALS AND METHODS

Top Abstract Introduction Materials & Methods Results Discussion References

Strains. Unless stated otherwise, cells were grown at 37°C in Luria-Bertani (LB) medium supplemented, where appropriate, with antibiotics at concentrations of 50 µg/ml (ampicillin, kanamycin, and spectinomycin), 25 µg/ml (chloramphenicol), and 12.5 µg/ml (tetracycline).

lacZ36M15

dadR trpE trpA tna minCDE recA

Plasmids and phages. Relevant features of the plasmids and phages used in this study are presented in Fig. 1 and Table 1. Plasmids pCX41, pDB280, pDB346, pDB355, pDB361, and pCH32 are all derivatives of pSC101. Plasmids pCX41 (36) and pCH32 (17) were described previously. Plasmid pDB280 was obtained by inserting a fragment containing the complete ftsA and partial ftsQ and ftsZ genes into pMAK705 (18). To create the ftsA⁰ allele, pDB280 was cut with BglII, and the ends were filled in using Klenow fragment. Religation yielded pDB283 and resulted in the replacement of the unique BglII site in ftsA with a ClaI site and a frameshift mutation in the ftsA open reading frame.

View larger version (20K): [in this window] [in a new window]   FIG. 1.   Chromosomal inserts of plasmids and phages. The physical maps of ftsA and ftsZ (a) and of zipA (b) are shown at the top of the panel. The positions of AflII (Af), BamHI (B), BclI (Bc), BglII (Bg), Bsu36I (Bs), ClaI (C), EcoRI (E), HindIII (H), and NsiI (N) restriction sites are indicated. Inserts of plasmids and phages are presented below each map. As indicated, the transcription of inserts in some of the constructs was placed under control of the lac promoter (Plac), or PR. Note that in addition to the zipA fragment shown, pCH32 carries the ftsZ gene immediately downstream of this fragment (17).

R

R

R

Antisera. Polyclonal anti-FtsZ antiserum was raised against native FtsZ protein, which had been purified as described previously (9). Anti-FtsA antiserum was raised against a 35.6-kDa FtsA-His fusion consisting of the N-terminal 319 amino acids (aa) of FtsA fused to the peptide AAALE(H)₆. Synthesis of this protein was induced by growth of strain BL21(DE3)/plysS/pDB273 in the presence of IPTG. Virtually all of the FtsA-His protein was present in inclusion bodies. Cells were broken in a French pressure cell, and inclusion bodies were collected by low-speed centrifugation. The pellet fraction was treated with 4.0 M guanidine-HCl, and solubilized material was fractionated by nickel affinity chromatography. Fractions highly enriched for FtsA-His were pooled and further purified on a MonoQ column by fast protein liquid chromatography in the presence of 4.0 M urea. Anti-ZipA antiserum was raised against a soluble 35.9-kDa His-ZipA fusion protein in which the first 38 aa of ZipA, including its transmembrane domain, were replaced by the peptide MG(H)₁₀SSGHIEGRHMASMTGGQQMGRI. Synthesis of this fusion protein was induced by growth of strain BL21(DE3)/plysS/pCH15 in the presence of IPTG. The protein was purified in the absence of chaotropic agents by nickel affinity chromatography followed by anion exchange chromatography on a MonoQ column. Polyclonal antisera against FtsZ, FtsA, and ZipA were obtained by immunizing rabbits (12). For immunofluorescence staining, the FtsA and ZipA antisera were further affinity purified as previously described (3). Anti-FtsZ monoclonal antibodies have been described (34).

HID of FtsZ, FtsA, and ZipA. Cultures of heat-induced depletion (HID) strains PB143/pCX41, CH2/pDB280, and CH5/pCH32 and the wild-type control strain CH3/pMAK700 were grown overnight at 30°C in LB medium with appropriate antibiotics. Cultures were diluted 1:200 in fresh LB medium without antibiotics, incubated at 30°C for 1.5 h, and then shifted to the nonpermissive temperature (42°C) for plasmid replication for 3 to 4 h. Optical densities at 600 nm (OD₆₀₀) were between 0.4 and 0.8.

CID of FtsZ, FtsA, and ZipA. For depletion of FtsZ, the cold-induced depletion (CID) strain PB143/pDB346 was grown overnight at 42°C. Cultures were diluted to an OD₆₀₀ of 0.02 in LB medium and grown at 42°C for 1 h. Cultures were shifted to 30°C and further incubated for approximately 8 h until an OD₆₀₀ of 0.7 to 0.9 was reached. For depletion of FtsA, strain CH2/pDB355 was grown overnight at 37°C and diluted to a starting OD₆₀₀ of 0.04. Cultures were incubated immediately at 30°C for approximately 8 h to an OD₆₀₀ of 0.7 to 0.9. For depletion of ZipA, strain CH5/pDB361 was grown overnight at 42°C and diluted to an OD₆₀₀ of 0.01. Cultures were incubated immediately at 30°C for approximately 10 h to an OD₆₀₀ of 0.8 to 1.0. For quantitative immunoblotting assays (see below), the wild-type control strains CH3/pDB346, CH3/pDB355, and CH3/pDB361 were treated identically to PB143/pDB346, CH2/pDB355, and CH5/pDB361, respectively.

Immunoblotting. Strains were grown as described above. Cells were harvested by centrifugation, resuspended in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) buffer (12) to the equivalent of 20.0 OD₆₀₀ units per ml, and incubated at 100°C for 5 min. Total protein concentration in each lysate was determined by the method of Bradford using bovine immunoglobulin G (IgG) as a standard. Each SDS-PAGE gel contained 70 µg of a depleted extract in one lane and different amounts (70, 35, 18, 9, 4, and 2 µg) of the appropriate control extract in adjacent lanes. Western blotting and antigen detection with rabbit polyclonal anti-FtsZ, anti-FtsA, and anti-ZipA antiserum were performed essentially as described (12) except that goat anti-rabbit IgG conjugated to alkaline phosphatase was used as secondary antibody. Blots were digitized by optical scanning, and the integrated density of each band of interest was measured using NIH-Image 1.60 software. Values corresponding to the control lanes were used to prepare a standard curve, on the basis of which the value corresponding to the depleted extract was calculated.

Immunofluorescence microscopy. The wild-type strain PB103 was grown in LB medium at 37°C to an OD₆₀₀ of 0.6. The HID strains PB143/pCX41, CH2/pDB280, and CH5/pCH32 were grown at nonpermissive temperature as described above. Cells were prepared for immunofluorescent staining as described previously (3). The samples were incubated overnight at 4°C with either a 1:2,000 dilution of an anti-FtsZ mouse monoclonal antibody cocktail consisting of equimolar amounts of monoclonal antibodies 4, 11, and 12 (34), or a 1:25 or 1:100 dilution of affinity-purified rabbit polyclonal antibody to FtsA or ZipA, respectively. Cy3-conjugated secondary antibody (Sigma) was used at a 1:4,000 (anti-mouse IgG) or 1:2,000 (anti-rabbit IgG) dilution. Cells were viewed and digitally imaged as previously described (17), with the exception that 535- to 585-nm excitation and 595- to 670-nm barrier filters were used.

Gfp localization. Lysogenic derivatives of CID strains PB143/pDB346, CH2/pDB355, and CH5/pDB361 were grown at nonpermissive temperature as described above for their nonlysogenic parents, except that IPTG was present during incubation at the nonpermissive temperature. Lysogenic derivatives of wild-type strains PB103 and CH3 were also grown in the presence of IPTG at 30°C to an OD₆₀₀ of 0.8 to 1.0. IPTG was used at 25 µM (CH50 and CH75) or 50 µM (DR120). Cells were chemically fixed, viewed, and imaged as previously described (17).

Quantitation of cell lengths and fluorescent rings. From each field of cells we obtained two digital images using fluorescence and differential interference contrast optics, respectively (17). The first image was used to count the number of ring structures per cell and the second was used to determine cell length by using Segmented Ruler (Tim Rand, Yale University) software. In case only a portion of a long filament was captured within the field of view, cell segments larger than 30 µm were counted as one cell. In each case, such cell segments made up less than 30% of the number of cells scored, but we note that the average length values of the populations containing long filaments in Tables 2 and 3 should be considered minima.

	RESULTS

Top Abstract Introduction Materials & Methods Results Discussion References

Localization of ZipA, FtsZ, and FtsA. Assembly of ZipA, FtsZ, and FtsA into the septal ring was monitored by the application of both indirect immunofluorescence and Gfp tagging techniques.

View larger version (48K): [in this window] [in a new window]   FIG. 2.   Localization of ZipA, FtsA, and FtsZ in wild-type cells. Fluorescence (a through f) and corresponding differential interference contrast (a' through f') micrographs showing the location in normally dividing cells of both native (a to c) and Gfp tagged (d to f) ZipA, FtsA, and FtsZ proteins, respectively. Cells shown are from strains PB103 (wild type [wt]) (a to c), CH3(CH50)/pDB355 [wt(Plac::zipA-gfp)/cI857 PR::ftsA+] (d), PB103(CH75) [wt(Plac::gfp-ftsA)] (e), and CH3(DR120)/pDB361 [wt(Plac::gfp-ftsZ)/cI857 PR::zipA+] (f). Cells in panels a to c were grown at 37°C prior to immunofluorescence staining with the appropriate antibodies. Cells in panels d to f were grown at 30°C in the presence of 25 µM (d and e) or 50 µM (f) IPTG, and were observed immediately after chemical fixation. Results with a number of other normally dividing lysogens [e.g., PB103(CH50), CH3(CH75), and PB103(DR120)] were similar to those shown in panels d to f. Bar in panel a represents 2.0 µm.

Strains for HID or CID of division proteins. Throughout this study, we used two types of strains which allowed for the specific, temperature-dependent depletion of ZipA, FtsZ, or FtsA (Table 1). For convenience, we use the acronyms HID (for heat-induced depletion) to indicate one type and CID (for cold-induced depletion) to indicate the other type.

View larger version (77K): [in this window] [in a new window]   FIG. 3.   Depletion of division proteins. Immunoblots showing cellular levels of FtsZ (a and d), FtsA (b and e), or ZipA (c and f) after growth of the corresponding HID, CID, and wild-type control strains at either 42°C (a to c) or 30°C (d to f). Lanes 1 contained 70 µg of total protein of depletion strains PB143/pCX41 (a), CH2/pDB280 (b), CH5/pCH32 (c), PB143/pDB346 (d), CH2/pDB355 (e), and CH5/pDB361 (f). Lanes 2 through 7 contained 70, 35, 18, 9, 4, and 2 µg of protein, respectively, of control strains CH3/pMAK700 (a to c), CH3/pDB346 (d), CH3/pDB355 (e), and CH3/pDB361 (f). Proteins were detected with polyclonal antisera. The anti-FtsA serum showed some reactivity to an abundant 40-kDa species (most likely EF-Tu) which migrated slightly faster than FtsA (b and e). Measurements of band intensities indicated that, in each case, the cellular concentration of the depleted division protein was less than 15% of its normal concentration.

R

R

R

R

Localization of ZipA in FtsZ and FtsA filaments. In a previous report, we showed that a ZipA-Gfp protein localized to the septal ring structure in wild-type dividing cells (17). As demonstrated above by immunofluorescence microscopy with affinity-purified anti-ZipA antibodies, the same is true for native ZipA. To study whether this localization pattern is dependent on the FtsZ protein, we first observed the distribution of ZipA-Gfp in cells from which FtsZ had been specifically depleted. For this experiment, the FtsZ^CID strain PB143/pDB346 (ftsZ⁰/cI857 P_R::ftsZ) was lysogenized with phage CH50 (P_lac::zipA-gfp), containing zipA-gfp downstream of the IPTG-inducible lac promoter (17). Cells were grown at 30°C in the presence of 25 µM IPTG and observed by fluorescence microscopy. As shown in Fig. 4a, cells formed long nonseptate filaments due to depletion of the FtsZ protein. Moreover, ZipA-Gfp failed to accumulate in ring structures but appeared to be evenly distributed along the periphery of the filaments. Of 133 filaments, representing a total cell length of 2,564 µm, only a single ring structure was seen in each of six relatively short filaments (Table 3). These results suggested that ZipA-Gfp was still associated with the cytoplasmic membrane, but required FtsZ in order to localize to the septal ring.

View larger version (86K): [in this window] [in a new window]   FIG. 4.   Localization of ZipA in FtsZ and FtsA filaments. Fluorescence (a to d) and differential interference contrast (c' to d') micrographs showing the location of ZipA-Gfp (a and c) or native ZipA (b and d) in filaments depleted for FtsZ (a and b) or FtsA (c and d). Cells shown are from the FtsZCID strain PB143(CH50)/pDB346 [ftsZ0(Plac::zipA-gfp)/cI857 PR::ftsZ+] (a), the FtsZHID strain PB143/pCX41 [ftsZ0/repA(Ts) ftsZ+] (b), the FtsACID strain CH2(CH50)/pDB355 [ftsA0(Plac::zipA-gfp)/cI857 PR::ftsA+] (c), and the FtsAHID strain CH2/pDB280 [ftsA0/repA(Ts) ftsA+] (d). Cells in panels b and d were grown at 42°C prior to staining with affinity-purified anti-ZipA antiserum. Cells in panels a and c were grown at 30°C in the presence of 25 µM IPTG and were observed immediately after chemical fixation. Bar represents 5.0 (a and b) or 11.7 (c and d) µm.

R

Localization of FtsZ in ZipA and FtsA filaments. The finding that ZipA failed to localize to the septal ring after depletion of FtsZ suggested either that the incorporation of both proteins into the structure depended on the protein partner or that formation of the FtsZ ring can occur independently of ZipA. To assess which of these possibilities is most likely correct, we studied the localization of FtsZ in cells from which ZipA had been depleted. To this end, the ZipA^CID strain CH5/pDB361 (zipA::aph/cI857 P_R::zipA) was lysogenized with phage DR120 (P_lac::gfp-ftsZ), and cells of a resulting lysogen were shifted from 42 to 30°C in the presence of 50 µM IPTG. As illustrated in Fig. 5a, many of the resulting ZipA-depleted filaments showed multiple fluorescent ring structures, suggesting that FtsZ rings were still being formed. Not all filaments showed rings, however, and of 64 filaments examined, rings were absent in 12 (19%) cells. In addition, the majority of filaments contained fewer ring structures than one might expect. Thus, whereas in the ring-containing portion of a wild-type population the ratio of cell length to FtsZ ring was approximately 2.7 µm, this ratio was significantly higher (8.6 µm) in the portion of ZipA^CID filaments that contained ring structures. In addition, there was a large variation in the value of this ratio among individual filaments, ranging from 4.9 to 40.2 µm (Table 3).

View larger version (114K): [in this window] [in a new window]   FIG. 5.   Localization of FtsZ in ZipA and FtsA filaments. Fluorescence (a through d) and differential interference contrast (a' to d') micrographs showing the location of Gfp-FtsZ (a and c) or native FtsZ (b and d) in filaments depleted for ZipA (a and b) or FtsA (c and d). Cells shown are from the ZipACID strain CH5(DR120)/pDB361 [zipA::aph(Plac::gfp-ftsZ)/cI857 PR::zipA+] (a), the ZipAHID strain CH5/pCH32 (zipA::aph/repAts ftsZ+ zipA+) (b), the FtsACID strain CH2(DR120)/pDB355 [ftsA0(Plac::gfp-ftsZ)/cI857 PR::ftsA+] (c), and the FtsAHID strain CH2/pDB280 [ftsA0/repA(Ts) ftsA+] (d). Cells in panels b and d were grown at 42°C prior to staining with FtsZ-specific monoclonal antibodies. Cells in panels a and c were grown at 30°C in the presence of 50 µM IPTG and were observed immediately after chemical fixation. Bar in panel a represents 5.0 µm.

R

Localization of FtsA in FtsZ and ZipA filaments. In a previous study, it was shown that formation of the FtsZ ring is a prerequisite for the localization of FtsA to the septal ring structure (3). Consistent with the results of this study, we found that FtsA failed to localize in filaments from which FtsZ had been depleted. Thus, no fluorescent rings were observed in filaments of the FtsZ^CID strain PB143/pDB346 (ftsZ⁰/cI857 P_R::ftsZ), lysogenic for CH75 (P_lac::gfp-ftsA) and grown at 30°C in the presence of 25 µM IPTG (Fig. 6a; Table 3). Similarly, FtsA-specific antibodies failed to stain septal ring structures in the vast majority (96%) of FtsZ^HID filaments of strain PB143/pCX41 [ftsZ⁰/repA(Ts) ftsZ⁺] (Table 2).

View larger version (116K): [in this window] [in a new window]   FIG. 6.   Localization of FtsA in FtsZ and ZipA filaments. Fluorescence (a to c) and differential interference contrast (a' to c') micrographs showing the location of Gfp-FtsA (a and b) or native FtsA (c) in filaments depleted of FtsZ (a) or ZipA (b and c). Cells shown are from the FtsZCID strain PB143(CH75)/pDB346 [ftsZ0(Plac::gfp-ftsA)/cI857 PR::ftsZ+] (a), the ZipACID strain CH5(CH75)/pDB361 [zipA::aph(Plac::gfp-ftsA)/cI857 PR::zipA+] (b), and the ZipAHID strain CH5/pCH32 [zipA::aph/repA(Ts) ftsZ+ zipA+] (c). Cells in panel c were grown at 42°C prior to staining with affinity-purified anti-FtsA antiserum. Cells in panels a and b were grown at 30°C in the presence of 25 µM IPTG and were observed immediately after chemical fixation. Bar represents 5.0 (a and b) or 11.0 (c) µm.

R

	DISCUSSION

Top Abstract Introduction Materials & Methods Results Discussion References

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, 3, 6, 22, 29, 35, 39, 40). 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 (reference 1 and this study), FtsI (1, 29, 35, 39), FtsN (2), FtsK (40), FtsQ (1), and FtsW (6, 22) filaments.

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. 4 to 6). 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. 3]) 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, 2, 6, 22, 29, 39, 40).

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 ZipA^HID and 8.9 µm in ZipA^CID) and FtsA-depleted (6.2 µm in FtsA^HID and 6.3 µm in FtsA^CID) 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 2). 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 3). 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, 2, 6, 22, 29, 39, 40), 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.