INTRODUCTION

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The first recognized event in bacterial cell division is assembly of the key division protein FtsZ into a ring (the Z-ring) which spans the circumference of the cell and resides just underneath the cytoplasmic membrane. Other division proteins are then recruited to the Z-ring, forming the septal ring organelle, which mediates cell wall invagination (for recent reviews, see references 19 and 32). The biochemical properties and tertiary structure (15, 25, 26) of FtsZ reveal that the protein shares many features with eukaryotic tubulins. Like tubulin, FtsZ is a GTPase (5, 21, 31, 37) which forms dynamic polymer filaments upon binding GTP (9, 16-18, 22-24, 39). Furthermore, hydrolysis of the nucleotide substrate is required for depolymerization and thus contributes to the dynamic nature of the filaments (22, 23). Unlike tubulin, however, FtsZ filaments are homopolymers of a single peptide species, and FtsZ readily binds nucleotide (5, 21, 31) as well as polymerizes (17, 22) in the absence of divalent metal.

We initially identified the ZipA protein of Escherichia coli based on its ability to directly bind FtsZ in vitro and showed it to be an essential cytoplasmic membrane protein which associates with the Z-ring in vivo (10). Depletion of ZipA leads to the formation of nonseptate filaments. FtsZ rings can still assemble in these filaments, although the number of rings per cell mass is significantly smaller than expected (11, 13). These observations suggest that ZipA is required for constriction of the septal ring and that the protein may also play a role in stabilizing the ring structure. Support for a stabilizing role of ZipA has also come from the finding that moderate overexpression of the protein suppresses the instability of the Z-ring in strains with the temperature-sensitive ftsZ84 allele (30).

To better understand the interaction between FtsZ and ZipA, we have tested the ability of deletion derivatives of these proteins to interact both in vitro and in vivo. We show that a small domain at the extreme C terminus of FtsZ and a significantly larger C-terminal domain of ZipA are both required and sufficient for the specific interaction between the two proteins. In agreement with a recent report (30), we also found that addition of purified ZipA protein to FtsZ polymers causes the arrangement of polymers into extensive networks of thick bundles in which individual polymers are present in a staggered side-by-side fashion. Using deletion derivatives of ZipA, we show that the interaction between the minimal FtsZ binding domain of ZipA and FtsZ polymers is also sufficient to induce bundling of the polymers.

The existence of some homology between a highly charged domain in ZipA and a domain in MAP-Tau proteins involved in binding microtubules has led to the assertion that ZipA may be considered a MAP-Tau homolog (30). Our results, however, show that this domain of ZipA is dispensable for both binding to FtsZ and mediating the bundling of FtsZ polymers.

We also tested the ability of various deletion derivatives of ZipA to substitute for the native protein in supporting cell division in vivo. Interestingly, ZipA derivatives which lacked the small N-terminal membrane anchor or in which the membrane anchor was replaced with that of another type Ib protein of E. coli (DjlA), were incapable of substituting for the native protein. Thus, whereas the C-terminal portion of ZipA is sufficient for binding and bundling of FtsZ polymers, it is clearly not sufficient to support cell division. In particular, our results suggest that the membrane anchor domain of ZipA is not merely required to anchor the protein to the membrane but may have more specific properties which are essential to proper functioning of ZipA in the division process.

	MATERIALS AND METHODS

Top Abstract Introduction Materials and Methods Results Discussion References

Strains. Strains PB103 (dadR trpE trpA tna), PB143/pDB346 [PB103 ftsZ⁰ recA::Tn10/cI857(Ts) P_R::ftsZ⁺], CH5/pCH32 [PB103 zipA::aph recA::Tn10/repA(Ts) ftsZ⁺ zipA⁺], and CH5/pDB361 (zipA::aph recA::Tn10/cI857 P_R::zipA⁺) have been described previously (11). Strains BL21(DE3)/plysS [ompT r_B m_B(P_lacUV5::T7gene1)/T7lysS⁺)] and HMS174(DE3) [r m⁺ recA Rif^r (P_lacUV5::T7gene1)] were purchased from Novagen. Unless stated otherwise, cells were grown at 37°C in Luria-Bertani (LB) medium supplemented, where appropriate, with antibiotics at 50 µg/ml (ampicillin, kanamycin, and spectinomycin), 25 µg/ml (chloramphenicol), and 12.5 µg/ml (tetracycline).

Plasmid construction. Plasmids pMLB1113 (6), pDB319, pDB324, pDB326, and pDR10 (10), pDR112 (28), pDR107a, pDR107b, and pDR107c (29), pAR(RI)59/60 (1), pGFPS65T (12), and pPSG961-31 (3) have been described previously. Plasmids pET16b, pET21a, pET21b, pET21c, and plysS were purchased from Novagen, and pUC4-KIXX was purchased from Pharmacia.

(i) FtsZ plasmids. To construct plasmid pDB312, we performed PCR using primers 5'-GGAGGATCCCATATGTTTGAACCAATGGAAC-3' and 5'-TTCCGGTCGACTCTTAATCAGCTTGCTTACG-3' introducing BamHI, NdeI, and SalI sites (underlined) flanking the ftsZ open reading frame (ORF). Digestion with NdeI and SalI resulted in an 1,156-bp fragment, which was ligated to similarly treated pET21a, yielding pDB312.

(ii) ZipA plasmids. For pDB322, zipA was amplified by PCR with primers 5'-ACAGAGATCCATATGATGCAGGATTTGCGTCTG-3' and 5'-TTAACCAAGCTTAAGTGTATCAGGCGTTGG-3', designed to introduce a NdeI site (underlined) at the translation start codon of zipA. The PCR product was treated with NdeI and HindIII, and the 994-bp fragment was ligated to NdeI- and HindIII-digested pET21a, resulting in pDB317. The 676-bp AgeI-HindIII fragment of pDB317 was then replaced with the 1,015-bp AgeI-HindIII fragment of pDB315, yielding pDB318. To obtain pDB319, pDB318 was treated successively with AflII plus HindIII, Klenow enzyme, and ligase, thereby removing all lig sequences and retaining a HindIII site. Plasmid pDB319 encodes the complete ZipA protein under the control of the T7 promoter. To place zipA expression under the control of the lac promoter, the 1,089-bp BglII-HindIII fragment of pDB319 was ligated to BamHI- and HindIII-digested pMLB1113, yielding pDB322.

Protein purification. BL21(DE3)/plysS cells carrying the appropriate pET-derived plasmid were grown overnight in LB medium with 50 µg of ampicillin per ml, 25 µg of chloramphenicol per ml, and 0.1% glucose. Cultures were diluted 1:200 in 500 ml of LB with 50 µg of ampicillin per ml and 0.04% glucose and grown at 30 or 37°C to an optical density at 600 nm (OD₆₀₀) of 0.4 to 0.5. Isopropyl--D-thiogalactoside (IPTG) was added to 0.84 mM, and growth continued for another 2 h. Cells were harvested by centrifugation, washed once in 0.9% saline, and resuspended in cell breakage buffer (specified below) to a final volume of 5 ml. Cell suspensions were subjected to three rapid freeze-thaw cycles using a dry ice-acetone bath and a 37°C water bath. Cell lysis was monitored by phase-contract microscopy. The cell lysates were briefly sonicated to reduce viscosity and fractionated into pellet (P200) and supernatant (S200) fractions by centrifugation at 200,000 × g for 3 h at 5°C. After purification, the proteins were rapidly frozen in a dry ice-acetone bath and stored at 80°C.

FtsZ polymerization. FtsZ was added to polymerization buffer (50 mM morpholineethanesulfonic acid [MES], 50 mM KCl, 10 mM MgCl₂, 1 mM GTP [pH 5.8]) and incubated at 30°C for 5 min. A ZipA derivative (or ZipA storage buffer for ZipA controls) was added, and incubation was continued for another 10 min. The final reaction volume was 50 µl, and the final concentration of each protein was 5 µM. A 10-µl volume of the reaction mixture was spotted on a glow-discharged, carbon-coated copper grid (300 mesh). After 20 s, the grid was wicked dry with filter paper, stained with 1% uranyl acetate for 45 s, and wicked dry again. For some reactions, GDP was used instead of GTP, and/or MgCl₂ was omitted and EDTA was added to 2 mM. Polymerization of FtsZ(1-314)-H was carried out in the same way, except that the pH of the buffer was 6.0. The grids were viewed and photographed on a JEOL 100CX or JEOL 1200CX transmission electron microscope at 80 kV. Negatives were scanned using a Dimage Scan Multi (Minolta), and images were manipulated using Adobe Photoshop.

Affinity blotting. Purified HFKT-FtsZ and ZipA-FKH were phosphorylated as described previously (10) to specific activities of 1.0 × 10⁷ and 4.3 × 10⁶ cpm/µg, respectively. Subsequent procedures were performed as described previously (10).

Protease accessibility. The preparation of spheroplasts and determination of protease accessibility were done essentially as described previously (2). Briefly, an overnight culture of strain PB103 was diluted 100-fold in 20.0 ml of LB broth and grown 37°C to an OD₆₀₀ of 0.5. The cells were harvested by centrifugation and resuspended in 0.15 ml of 30 mM Tris-Cl (pH 8.0) containing 25% sucrose. The resuspended cells were mixed with an equal volume of a solution containing 10 mM EDTA and 0.4 mg of lysozyme per ml in water, and the mixture was incubated at room temperature (RT) for 3.0 min. As judged by phase-contrast microscopy, virtually all the cells had been converted to spheroplasts at this point. Aliquots of 50 µl were either left untreated or treated with protease and/or detergent by addition of proteinase K to 150 µg/ml (1.6 µl of a 5-mg/ml stock) and/or Triton-X100 to 0.1% (1.0 µl of a 5% stock), respectively. After 5 min at RT, 5.0 µl of a 50 mM solution of phenylmethylsulfonyl fluoride in ethanol was added to each sample, and after an additional 5 min at RT, each sample was mixed with 55 µl of 2× SDS-PAGE sample buffer. Samples were incubated at 100°C for 10 min, aliquots (20 µl per lane) were used to prepare three identical SDS-PAGE gels, and proteins were blotted to nitrocellulose filters. One filter was incubated with monoclonal antibody 4H4 to detect TonB (27). FtsZ was detected with a polyclonal antiserum (11), and ZipA was detected by incubation of the remaining filter with radiolabeled HFKT-FtsZ (10).

Quantitative immunoblotting. To determine the cellular levels of plasmid-encoded ZipA and derivatives (see Table 2), cells were harvested by centrifugation, resuspended in SDS-PAGE electrophoresis sample-lysis buffer (7) to the equivalent of 13.3 OD₆₀₀ units per ml, and incubated at 100°C for 5 min. The resulting lysates were further diluted in sample buffer as needed. Samples were separated by SDS-PAGE and transferred to nitrocellulose filters. The filters were incubated with affinity-purified ZipA antibodies and subsequently with enhanced chemiluminescence reagents using an Amersham-Pharmacia ECL kit as recommended by the manufacturer. Chemiluminescent signals were visualized and quantified using a Fluor-S MAX Multiimager system and Quantity One software (Bio-Rad). Each filter contained a reference lane loaded with 10.0 × 10³ OD₆₀₀ equivalent (corresponding to 8.0 µg of total protein) of a PB103/pMLB1113H extract to establish the level of chromosomally encoded native ZipA in wild-type cells. Adjacent lanes were loaded with 2.0 × 10³ and 1.0 × 10³ OD₆₀₀ equivalents (corresponding to 1.6 and 0.8 µg of total protein, respectively) of each relevant extract to determine the level of plasmid-encoded antigen. Signals showed optimal linearity with the amount of antigen under these conditions, as established in pilot experiments. Since the ZipA derivatives migrated close to the position of native ZipA in the gels, the combined signal was measured. Based on the signal in the reference lane, and accounting for dilution factors, the contribution of chromosomally encoded native ZipA was calculated and subtracted from the total signal to obtain the contribution of plasmid encoded ZipA.

	RESULTS

Top Abstract Introduction Materials and Methods Results Discussion References

A small C-terminal domain of FtsZ is required and sufficient for interaction with ZipA. We previously detected the ZipA protein by an affinity-blotting procedure in which a radiolabeled derivative of the FtsZ protein was incubated with Western blots containing ZipA on nitrocellulose filters (10). To delineate the portion of FtsZ involved in the interaction with ZipA, we used a converse approach in which radiolabeled ZipA was incubated with immobilized FtsZ. As probe we used a derivative of ZipA (ZipA-FKH) consisting of the complete ZipA peptide carrying a C-terminal tag peptide (FKH) which includes a substrate site for heart muscle kinase (K) and a stretch of six histidine residues (H). Purified ZipA-FKH was radiolabeled with ³²P and incubated with filters containing native FtsZ as well as a variety of derivatives in which portions of FtsZ were fused to Gfp and/or polyhistidine tags (Fig. 1).

View larger version (25K): [in this window] [in a new window]   FIG. 1.   FtsZ plasmids used to define the ZipA-binding domain. (a) The physical map of ftsZ and portions of flanking genes in E. coli are shown at the top of the panel. The positions of BclI (Bc), EcoRI (E), EcoRV (Rv), and HindIII (H) restriction sites are indicated. The positions in the E. coli FtsZ polypeptide of two domains (N and C) and a connecting core helix (residues ca. 177 to 201) were inferred from the crystal structure of FtsZ from Methanococcus jannaschii (14, 15). Inserts of plasmids are presented below the map, and the FtsZ residues they encode are given at the right of each insert. All plasmids were derivatives of pET21, such that transcription of inserts was under control of the T7lac promoter (Novagen). Plasmid pDB312 encodes native FtsZ. All others encode either full-length or portions of FtsZ fused to various tags, as indicated. H6, stretch of six histidine residues; HFKT, combination tag peptide which includes a stretch of 10 histidine residues and a substrate site for heart muscle kinase (10); T, T7.tag peptide. (b) The ability of these FtsZ derivatives to bind radiolabeled ZipA-FKH was determined by affinity blotting. +, protein binds ZipA; , protein does not bind ZipA.

View larger version (117K): [in this window] [in a new window]   FIG. 2.   Binding of radiolabeled ZipA to a C-terminal domain of FtsZ. Purified proteins (lanes 1 to 4) and whole-cell extracts (lanes 5 to 8) were separated on two identical SDS-PAGE gels. One gel was stained with Coomassie brilliant blue to visualize protein bands (top), and proteins in the other gel were blotted to a nitrocellulose filter which was subsequently incubated with radiolabeled ZipA-FKH (bottom). Lanes 1 to 4 contain 50 pmol of native FtsZ(1-383) (lane 1), FtsZ(1-383)-H (lane 2), FtsZ(1-372)-H (lane 3), or FtsZ(1-314)-H (lane 4). Lanes 5 to 8 contain 10 µl of extract of cells overexpressing Gfp-T-FtsZ(1-383) (lane 5), Gfp-T-FtsZ(289-383) (lane 6), Gfp-T-FtsZ(364-383) (lane 7), or Gfp-T-FtsZ(374-383) (lane 8). Extracts were prepared from cells of strain BL21(DE3)/plysS containing the appropriate plasmid (Fig. 1) after growth in the presence of IPTG and by resuspension of cells in SDS-PAGE lysis buffer to the equivalent of 20.0 OD600 units. Bands corresponding to the overexpressed proteins are indicated by an asterisk in the upper panel. The positions of molecular mass standards (66, 45, 36, 29, and 24 kDa [top to bottom]) are indicated on the left of the panels.

FtsZ interacts with the cytoplasmic C-terminal domain of ZipA in vitro. Membrane fractionation studies, in combination with the deduced primary sequence of ZipA, indicated that the protein is a bitopic integral inner membrane species of type lb, with the N terminus anchored in the membrane and the rest of the protein in the cytoplasm (10). To further validate this assignment, spheroplasts derived from wild-type strain PB103 were incubated with proteinase K in the presence or absence of the detergent Triton X-100, and the fate of ZipA was monitored by affinity blotting with radiolabeled HFKT-FtsZ. As controls, we performed immunoblot analyses on the same samples to determine the fates of FtsZ, a cytoplasmic protein, and of TonB, a type II transmembrane protein of which the bulk is present in the periplasm (27). Addition of proteinase K to detergent-permeabilized spheroplasts led to the complete degradation of all three proteins (Fig. 3, lanes 3), confirming that all three are good substrates for the protease. In contrast, treatment of intact spheroplasts with protease led to the specific degradation of TonB whereas the bulk of both FtsZ and ZipA remained unaffected (lanes 1). These results support a largely cytoplasmic localization of ZipA and indicate that if any part of ZipA is exposed to the periplasm (such as the extreme N-terminal portion), it must be very small.

View larger version (23K): [in this window] [in a new window]   FIG. 3.   Protease inaccessibility of ZipA in intact spheroplasts. Spheroplasts of PB103 cells were either left untreated (lanes 2) or treated with protease (lanes 1 and 3) and/or detergent (lanes 3 and 4). Samples were used to prepare three identical Western blots. TonB (top) and FtsZ (bottom) were detected with specific antibodies; ZipA (middle) was detected by incubation of the blot with radiolabeled HFKT-FtsZ.

View larger version (27K): [in this window] [in a new window]   FIG. 4.   ZipA plasmids used to define the FtsZ-binding domain. (a) The physical map of zipA and portions of flanking genes in E. coli are shown at the top of the panel. The positions of AflII (Af), BamHI (B), HindIII (H), KpnI (K), and PvuII (Pv) restriction sites are indicated. The four domains of the ZipA polypeptide we previously proposed are denoted N (N-terminal membrane anchor), +/ (highly charged domain), P/Q (proline- and glutamine-rich domain), and C (C-terminal domain). The +/ domain includes the MAP-Tau repeat-like sequence proposed by RayChaudhuri to mediate binding to and bundling of FtsZ polymers (30). Inserts of plasmids are presented below the map, and the ZipA residues they encode are given at the right of each insert. All plasmids were derivatives of pET21, such that transcription of inserts was under the control of the T7lac promoter (Novagen). As indicated, inserts encode either full-length or portions of ZipA fused to various tags. H10, stretch of 10 histidine residues; FKH, combination tag which includes a stretch of six histidine residues and a substrate site for heart muscle kinase. See also the legend to Fig. 1. (b) The ability of these ZipA derivatives to bind radiolabeled HFKT-FtsZ was determined by affinity blotting. +, protein binds FtsZ; , protein does not bind FtsZ. The ability of proteins to bundle FtsZ polymers was assessed by electron microscopy in all cases. The results obtained with the three Gfp-tagged derivatives were confirmed by fluorescence microscopy. +, numerous bundles and bundle networks observed. , no bundles observed.

View larger version (93K): [in this window] [in a new window]   FIG. 5.   Binding of radiolabeled FtsZ to a C-terminal domain of ZipA. Purified proteins (50 pmol/lane) were separated on two identical SDS-PAGE gels. One gel was stained with Coomassie brilliant blue to visualize protein bands (top), and proteins in the other gel were blotted to a nitrocellulose filter which was subsequently incubated with radiolabeled HFKT-FtsZ (bottom). Lanes contained ZipA(1-328)-H (lane 1), ZipA(1-302)-H (lane 2), ZipA(23-328) (lane 3), ZipA(23-279) (lane 4), T-ZipA(70-328) (lane 5), T-ZipA(186-328)-H (lane 6), Gfp-T-ZipA(186-328)-H (lane 7), or Gfp-T-ZipA(212-328)-H (lane 8). The positions of molecular mass standards (66, 45, 36, 29, 24, 20, and 14 kDa [top to bottom]) are indicated on the left of the panels.

The FZB domain directs ZipA to the septal ring in vivo. We previously showed that ZipA localizes to the septal ring organelle in a FtsZ-dependent manner (10, 11). As shown above, the FZB domain of ZipA is required and sufficient for binding to FtsZ in vitro. To test whether this domain is also sufficient to direct the ZipA protein to the FtsZ ring in vivo, we examined the cellular localization of derivatives containing various portions of ZipA fused to Gfp (Fig. 6 and 7). As observed previously (10, 11), virtually all fluorescence was seen associated with the septal ring structure in the vast majority of cells of strain PB103/pCH50 [wt/P_lac::zipA(1-328)-gfp], which express a fusion of the full-length ZipA peptide with Gfp (Fig. 7A). In contrast, in PB103/pCH148 [wt/P_lac::zipA(1-302)-gfp] cells, which express a derivative lacking the C-terminal 26 aa of ZipA, fluorescence failed to accumulate at the septal ring. Rather, this fusion appeared to be evenly distributed along the entire periphery of the cell (Fig. 7B). Combined with the observations that ZipA(1-302)-H fractionates with the insoluble fraction of cells and that the protein fails to bind FtsZ in vitro, this distribution indicates that whereas the fusion is still anchored to the cytoplasmic membrane by the ZipA N-terminal membrane domain, it no longer recognizes FtsZ that has accumulated within the septal ring.

View larger version (27K): [in this window] [in a new window]   FIG. 6.   Plasmids used to sublocalize ZipA derivatives. (a) The C-domain of ZipA is renamed FZB (FtsZ-binding domain) to indicate that this domain coincides with the portion of ZipA found here to be required and sufficient for binding FtsZ (see the text). Inserts were cloned into the vector pMLB1113 such that expression of the fusion proteins is under control of the lac promoter and lacIq. D, transmembrane domain corresponding to residues 1 to 32 of DjlA. See also the legend to Fig. 1. (b) Cellular location of fusion proteins in strain PB103. R, virtually all fluorescence associated with the septal ring; M, fluorescence evenly distributed along the entire cell membrane; C, fluorescence evenly distributed throughout the cytoplasm; C/R, a significant portion of total fluorescence throughout the cytoplasm and the rest associated with the septal ring.

View larger version (46K): [in this window] [in a new window]   FIG. 7.   Localization of Gfp-tagged ZipA derivatives in wild-type cells. Cells were chemically fixed and observed under fluorescence (A to F) and differential interference contrast (A' to F') optics. Images faithfully reflected the distributions of fluorescence seen prior to fixation. Panels show cells of strain PB103 (wild type) expressing ZipA derivatives from plasmids pCH50 [Plac::zipA(1-328)- gfp] (A), pCH148 [Plac::zipA(1-302)-gfp] (B), pCH80 [Plac::zipA(23-328)-gfp] (C), pCH138 [Plac::gfp-t-zipA(186-328)] (D), pCH93 [Plac::gfp-t-zipA(212-328)] (E), or pCH174 [Plac::djlA(1-32)-zipA(23-328)-gfp] (F). Cells were grown in the presence of 5 µM (A), 10 µM (B), 25 µM (C), or 100 µM (D to F) IPTG. None of the plasmids interfered noticeably with the normal division phenotype under these conditions. Bar, 3 µm.

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View larger version (99K): [in this window] [in a new window]   FIG. 8.   Localization of Gfp-tagged ZipA derivatives in FtsZ cells. Filaments of the FtsZ CID strain PB143/pDB346 (ftsZ0/cI857 PR::ftsZ) carrying pCH50 [Plac::zipA(1-328)-gfp] (A), pCH80 [Plac::zipA(23-328)-gfp] (B), or pCH174 [Plac::djlA(1-32)-zipA(23-328)-gfp] (C) are shown. Cells were grown at 30°C (leading to depletion of FtsZ) in the presence of 5 µM (A and B) or 100 µM (C) IPTG. The resulting filaments were chemically fixed and observed under fluorescence optics. Images faithfully reflected the distributions of fluorescence seen prior to fixation. Bar, 3 µm.

ZipA-induced bundling of FtsZ protein filaments in vitro. Using an optimized purification protocol for FtsZ, Mukherjee and Lutkenhaus (23) recently showed that the protein can readily form homopolymeric protein filaments in a strictly GTP-dependent fashion and in the absence of additional promoting agents such as DEAE-dextran, cationic phospholipids, or high concentrations of Ca²⁺, which had been used in earlier studies (9, 24, 35, 39). In addition, it was shown that whereas FtsZ can bind nucleotide and form filaments in the absence of divalent metals (5, 21, 23, 31), Mg²⁺ is required for hydrolysis of GTP (5, 21, 31), which, in turn, is correlated with the ability of filaments to depolymerize (22).

View larger version (188K): [in this window] [in a new window]   FIG. 9.   ZipA-induced bundling of FtsZ polymer filaments. Purified native FtsZ (A to E) and FtsZ(1-314)-H (F and G) was incubated in the presence of 1.0 mM GTP and in the presence of either 10 mM Mg2+ (A, B, and D to G) or 2 mM EDTA (C). After 5 min, buffer (A and F), ZipA(23-328) (B, C, and G), Gfp-T-ZipA(186-328)-H (D), or Gfp-T-ZipA(212-328)-H (E) was added. After an additional 10 min, samples were applied to a microscope grid, stained with uranyl acetate, and examined under an electron microscope. Each protein in the reactions was present at 5 µM. The inset in panel B represents a portion (arrow) of the bundle network in more detail, emphasizing the side-by-side arrangement of polymers within the bundles. Polymers or polymer bundles were completely absent in control reactions in which GTP or FtsZ were omitted (data not shown). Bar, 76 nm (C), 100 nm (A, E to G, and inset in B), 125 nm (D), or 600 nm (B).

Domains required for bundling. RayChaudhuri reported on the bundling activity of ZipA during the course of this study and proposed that ZipA is a MAP-Tau homolog, based on some homology between residues 45 to 70 of ZipA and a MAP-Tau repeat motif involved in the binding of microtubule-associated proteins (MAPs) to microtubules (30). To simultaneously test this proposal and delineate the protein domains required for FtsZ polymer bundling, we next assayed the ability of various other purified ZipA derivatives to induce bundling (Fig. 4B and 9).

Detection of FtsZ polymer bundles by fluorescence microscopy. Since the size of the bundle networks we observed by EM was significant, we reasoned that they should also be visible by light microscopy. We first tested this by using the Gfp-tagged ZipA derivative HT-ZipA(39-328)-Gfp. Consistent with the results above, this protein readily bound FtsZ on affinity blots (Fig. 4b) and partially accumulated at the septal ring in cells of strain PB103/pCH77 [wt/Plac::t-zipA(39-328)-gfp] (Fig. 6b). As shown in Fig. 10A, addition of purified HT-ZipA(39-328)-Gfp to preformed FtsZ polymers led to the formation of large protein structures which could indeed be easily visualized by fluorescence microscopy. Fluorescence was concentrated in structures of variable shapes ranging from single thick rods to very extensive networks of thinner filaments. When using FtsZ and HT-ZipA(39-328)-Gfp at equimolar concentration (6.0 µM), we observed very large and highly fluorescent networks which were difficult to image in a single plane of focus. When we reduced the concentration of the fluorescent protein to 0.6 µM, as was done for the experiments in Fig. 10, numerous structures were still observed, but they tended to be smaller and were subsequently easier to image in some detail. Analyses of these structures by EM showed thick bundles of FtsZ polymers, identical in appearance to the ones described above (Fig. 10B). Formation of these structures, furthermore, was clearly dependent on the presence of FtsZ polymers. In control experiments in which FtsZ and/or GTP was omitted or in which GDP was substituted for GTP, the fluorescence signal was largely homogenous. In addition, some fluorescence was concentrated in what appeared as small vesicular structures (Fig. 10C and D). Analyses by EM confirmed the absence of FtsZ polymers and polymer bundles in these samples (data not shown).

View larger version (121K): [in this window] [in a new window]   FIG. 10.   Detection of polymer bundles by fluorescence microscopy. Purified native FtsZ (6.0 µM) was incubated in the presence of GTP (1 mM) and Mg2+ (10 mM) at 30°C. After 5 min, purified H-T-ZipA(39-328)-Gfp was added to 0.6 µM. After an additional 10 min, one sample was applied to a microscope slide and observed immediately by fluorescence microscopy (A) and another was used for observation by EM (B). The fluorescent samples shown in panels C to F were prepared identically, except that GTP was replaced with GDP (C), FtsZ was replaced with buffer (D), or H-T-ZipA(39-328)-Gfp was replaced with either Gfp-T-ZipA(186-328)-H (E) or Gfp-T-ZipA(212-328)-H (F). EM grids prepared from the reactions shown in panels C to F showed extensive bundle networks as in panel A (E), no bundles but many individual FtsZ protofilaments (F), or no individual protofilaments or polymer bundles (C and D) (data not shown). Bar, represents 0.1 µm (B) or 3.4 µm (A and C to F).

The membrane anchor of ZipA is required for function. The results above showed that the FZB domain of ZipA is sufficient and required to bind and bundle FtsZ polymers in vitro and to associate with the FtsZ ring in vivo. To test whether the FZB domain is also sufficient to support cell division, we assessed the ability of various deletion derivatives of ZipA to complement the division defect of cells carrying a chromosomal zipA null allele. To this end, we used the ZipA HID (heat-induced depletion) strain CH5/pCH32 [zipA::aph recA::Tn10/repA(Ts) ftsZ⁺ zipA⁺] in which the chromosomal copy of zipA is destroyed and ZipA is expressed from plasmid pCH32. This plasmid is temperature sensitive for replication, such that growth of these cells at 42°C results in depletion of ZipA and a subsequent block in septation (11). CH5/pCH32 was transformed with plasmids expressing various portions of ZipA under the control of the lac promoter (Fig. 11), and the ability of transformants to form colonies at 42°C in the presence of IPTG (at a wide range of concentrations) was determined (Table 1).

View larger version (28K): [in this window] [in a new window]   FIG. 11.   Plasmids used to test correction of ZipA by ZipA derivatives. (a) Inserts were cloned into the vector pMLB1113 such that expression of the proteins is under control of the lac promoter and lacIq. See also the legends to Fig. 1 and 6. (b) The ability of the plasmids to correct a ZipA phenotype was determined as described in the text. +, correction; , no correction. (c) Shown are the N-terminal domains of ZipA, DjlA, and DjlA(1-32)-ZipA(23-328). Transmembrane segments as predicted by the Dense Alignment Surface method (4) (http://www.biokemi.su.se/~server/DAS/) are boxed. Residue numbers are indicated in parentheses. The arrow marks the junction between DjlA and ZipA residues in the DjlA-ZipA fusion.

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	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion References

In this study we have used a variety of techniques to identify protein domains that are both required and sufficient for the specific interaction between ZipA and FtsZ in vitro as well as in vivo. The results allow us to narrow down the domains involved to the 20 C-terminal residues (aa 364 to 383) of FtsZ and the 143 C-terminal residues (aa 186 to 328) of ZipA. Our findings support and extend the recent reports of Liu et al., who detected an interaction between ZipA(176-328) and FtsZ(1-383) but not FtsZ(1-320) in two-hybrid assays (13), and of Ma and Margolin, who found that a ZipA-Gfp fusion failed to colocalize with FtsZ(1-371) in filaments that had been depleted of native FtsZ (20).

Structure determinations indicate that both tubulin and FtsZ roughly consist of two structural domains which are separated by a long helix corresponding to residues 177 to 201 in the E. coli peptide. The N-terminal two-thirds of both tubulin and FtsZ include all residues which contact the nucleotide and also show the highest degree of primary sequence conservation among peptides from different organisms (14, 15, 26). Alignments of E. coli FtsZ with peptides from various organisms show that the region with a high degree of conservation extends from the N terminus up to approximately residue 314. It was shown previously that FtsZ(1-320) (36) and FtsZ(1-316) (20) contain all elements required for nucleotide-dependent polymer formation, and we found here that the same is true for FtsZ(1-314). This region is followed by a region which is variable in length and sequence in diverse organisms and which extends approximately to residue 369 in E. coli. This variable region is followed, in turn, by a 10-residue peptide which, once again, shows a high degree of conservation and which was named the C-terminal core domain by Ma and Margolin (20). Although FtsZ derivatives lacking this domain can polymerize, they fail to support cell division but instead display a prominent trans-dominant negative phenotype (references 8, 20, and 36 and data not shown), demonstrating the domain is essential for FtsZ function. In E. coli this small domain (DYLDIPAFLR) corresponds to residues 370 to 379. Deletion of the C-terminal 10 residues of FtsZ removes most of this core domain and, as shown here, also abolishes the interaction with ZipA, implicating residues within the core domain in providing a binding interface for ZipA.

Interestingly, the core domain peptide has also been implicated in the interaction between FtsZ and FtsA in Caulobacter crescentus, Bacillus subtilis, and E. coli. Thus, mutation or deletion of the C-terminal ends of FtsZ from each of these species was found to abolish an interaction with FtsA, as detected by two-hybrid analyses (8, 20, 36) and colocalization studies (20). We show here that the C-terminal 20 residues of FtsZ include all elements that are not only required but also sufficient for binding to ZipA. This may not be the case for binding of FtsZ to FtsA, however, since deletion of N-terminal portions of FtsZ also abolished interaction with FtsA in two-hybrid assays (36), suggesting that FtsA may require multiple and/or larger binding surfaces on FtsZ for stable engagement. ZipA and FtsA were shown to be recruited to the FtsZ ring in a mutually independent fashion (11, 13). The finding that the C-core is required for recruitment of both proteins raises the issue of whether ZipA and FtsA bind in a mutually exclusive fashion or whether both can be engaged at the same time. The latter seems unlikely, given the small size of the C-terminal peptide, but formation of a tripartite complex cannot yet be excluded. Assuming that ZipA and FtsA do potentially compete for binding FtsZ, it is presently unclear whether such competition would be physiologically relevant, given the large number of FtsZ molecules estimated to make up the FtsZ ring (18).

RayChaudhuri first reported that ZipA induces extensive bundling of FtsZ polymers in vitro (30). We have confirmed this by both EM and fluorescence microscopy and have found that bundling also occurs in the absence of free Mg²⁺. Furthermore, our results show that the FZB domain of ZipA is both required and sufficient for this bundling activity. This domain does not include the MAP-Tau repeat motif, which is embedded in the highly charged domain near the N terminus of ZipA and which was proposed to mediate FtsZ binding and bundling (30). Although it is possible that this motif plays some role in ZipA function, it is clearly not required or sufficient for binding and bundling FtsZ polymers. Therefore, although ZipA and MAPs have some interesting functional analogies (30), they should not be considered homologs.

It is not clear how binding of the FZB domain of ZipA stimulates lateral associations between FtsZ protofilaments. The assembly of FtsZ protofilaments into bundles or sheets also occurs in the presence of DEAE-dextran (9, 24), at millimolar (>7 mM) concentrations of Ca²⁺ (16, 22, 39), or on a surface of cationic lipids (9). Since these treatments all involve cationic substances and since FtsZ is an acidic protein, it is likely that bundling of FtsZ polymers in these cases is stimulated by a reduction of electrostatic repulsion between individual protofilaments due to charge compensation by the cationic counterions. These counterions are thought to exist in a condensed state, where they are free to diffuse along the polymer axis but are inhibited from diffusing away (22, 33, 34, 38). Because the ZipA protein (as well as its FZB domain) is actually slightly acidic and because the protein binds only to a specific site on the polymer subunit, such nonspecific charge compensation cannot easily explain ZipA mediated bundling. It is possible, however, that ZipA shields repulsive charges on some specific residues and/or that binding of ZipA provokes conformational changes within polymer subunits, causing repulsive groups to become buried or attractive groups to become exposed. Alternatively, bundling might involve noncovalent cross-linking of adjacent polymers through FZB-FZB interactions. Further work is needed to explore these possibilities.

Previous evidence suggests that one role of ZipA is to provide stability to the FtsZ ring (11, 13, 30). The in vitro bundling activity of ZipA provides an attractive mechanism of how the protein might contribute to Z-ring stability in vivo. A strengthening of the lateral association between FtsZ protofilaments would increase the structural integrity and stress-bearing capacity of the whole septal ring structure, which is likely to be important both before and during septal invagination. However, our results suggest that binding and bundling of FtsZ is not the only relevant property of ZipA. In particular, our findings that removal of the membrane anchor domain of ZipA or its replacement with the equivalent domain of DjlA is not tolerated indicate that the N-terminal domain of ZipA may be involved in an essential activity additional to its role as a membrane tether. One possibility is that this domain mediates an essential interaction with another membrane component. Other transmembrane septal ring proteins would be logical candidates for such a component. If so, however, such an interaction must be either weak or dependent on the binding of the FZB domain with the FtsZ ring, since the ZipA(1-302)-Gfp fusion, which lacks just a small portion of the FZB domain, showed no obvious preference for the septal ring (Fig. 6b and 7B).

The FZB domain and the membrane anchor are the most highly conserved regions among ZipA peptides of different species (10), which is consistent with the notion that both domains play essential roles. The charged and PQ-rich domains are not as well conserved, but whether or not these domains are essential for function still needs to be addressed.