ZapA uses a two-pronged mechanism to facilitate Z ring formation in Escherichia coli

Abstract

The tubulin-like protein FtsZ assembles into the Z ring that leads to the assembly and activation of the division machinery in most bacteria. ZapA, a widely conserved protein that interacts with FtsZ, plays a pivotal role in organizing FtsZ filaments into a coherent Z ring. Previous studies revealed that ZapA forms a dumbbell-like tetramer that binds cooperatively to FtsZ filaments and aligns them in parallel, leading to the straightening and organization of FtsZ filament bundles. However, how ZapA interacts with FtsZ remains obscure. Here, we reveal that ZapA uses a two-pronged mechanism to interact with FtsZ to facilitate Z ring formation in Escherichia coli. We find that mutations affecting surface-exposed residues at the junction between adjacent FtsZ subunits in a filament as well as in an N-terminal motif of FtsZ weaken its interaction with ZapA in vivo and in vitro, indicating that ZapA binds to these regions of FtsZ. Consistent with this, ZapA prefers FtsZ polymers over monomeric FtsZ molecules and site-specific crosslinking confirmed that the dimer head domain of ZapA is in contact with the junction of FtsZ subunits. As a result, disruption of the putative interaction interfaces between FtsZ and ZapA abolishes the midcell localization of ZapA. Taken together, our results suggest that ZapA tetramers grab the N-terminal tails of FtsZ and bind to the junctions between FtsZ subunits in the filament to straighten and crosslink FtsZ filaments into the Z ring.

Keywords: FtsZ; Z ring; Z ring organization; ZapA; bacterial cell division.

Figure 1

Overexpression of ZapA disrupts Z ring organization and inhibits cell division. (A) Overexpression of ZapA blocks colony formation. Plasmid pEXT22 or pSD319 (P _tac ::zapA) was transformed into strain W3110, and ZapA toxicity was assessed using a spot test. A 2 μl aliquot from each 10‐fold serial dilution was spotted onto LB plates with glucose (Glu) or IPTG and incubated at 37°C overnight before imaging. (B) Representative images of FtsZ‐mNG localization in the absence or presence of ZapA overexpression. Strain W3110 was transformed with a plasmid harboring ftsZ‐mNG under the control of an anhydrotetracycline‐inducible promoter and an empty vector or a plasmid carrying zapA under the control of an IPTG‐inducible promoter. FtsZ‐mNG was expressed at the basal level (without ATc), and ZapA was induced with 500 μM IPTG. Samples were taken and immobilized on an LB agarose pad for imaging. ATc, anhydrotetracycline. (C) Z ring width in the presence or absence of ZapA overexpression. Samples in (B) were analyzed as described in the Materials and Methods. Number of Z rings analyzed (WT: n = 471; + ZapA: n = 507) is shown. (D–F) Representative kymographs of FtsZ‐mNG (D) and computed FtsZ treadmilling velocity (E) and angles (F) in the absence or presence of ZapA overexpression. Number of filaments analyzed in (E) (WT: n = 203; + ZapA: n = 193) and number of angles analyzed in (F) (WT: n = 94; + ZapA: n = 97) are shown. The box plots (E, F) show the 25th and 75th percentiles as the box limits, with the mean at the center; whiskers extend from −SD to +SD of the mean. ns, not significant; ****p < 0.0001; two‐tailed Student's t test. Kymo, kymograph; PMI, projection of maximum intensity.

Figure 2

Mutations at the junction of FtsZ subunits in the filament confer resistance to ZapA overexpression. (A) Location of FtsZ mutations in the filament structure of Klebsiella pneumoniae FtsZ (PDB#: 8IBN). Residues on the surface of FtsZ are colored yellow (top face) or magenta (bottom face), whereas residues buried inside the FtsZ molecule are colored red. Note that mutations isolated by site‐directed mutagenesis are colored pink. GMPCPP is shown as a stick. Residue numbers are according to Escherichia coli FtsZ. (B) Spot test to assess the resistance of FtsZ mutants to ZapA overexpression. Plasmid expressing ZapA (pSD319) was transformed into strains expressing different FtsZ mutants from pBANG112 or its derivatives, and the transformants were subjected to a spot test on plates with or without IPTG. KE/NA, K141N and E219A; KR/NE, K141N and R258E; ER/AE, E219A and R258E; KER/NAE, K141N, R258E, K141N, E219A, and R258E.

Figure 3

Z rings formed by FtsZ mutants are resistant to ZapA overexpression. (A) Representative images of Z rings (ZipA‐mCherry) in cells expressing wild‐type FtsZ or its variants in the absence or presence of ZapA overexpression. Cells expressing wild‐type FtsZ or its variants were grown in exponential phase, ZapA was induced with 500 μM IPTG, and ZipA‐mCherry was expressed from its native promoter. ZipA‐mCherry was imaged by fluorescence microscopy. PH, phase contrast. (B) Representative images of co‐localization of ZipA‐mCherry with HADA in cells expressing wild‐type FtsZ or its variants in the absence or presence of ZapA overexpression. Strains were grown as in (A); nascent PG was labeled with HADA as described in the Material and Methods section. White arrows and triangles indicate the normal and aberrant Z rings that overlap and do not overlap with a HADA signal, respectively. Note that sPG synthesis was blocked in cells expressing wild‐type FtsZ but not in cells expressing FtsZ mutants in the presence of overexpressed ZapA. HADA, HCC‐amino‐D‐alanine hydrochloride; PG, peptidoglycan. (C) Quantification of the co‐localization of ZipA‐mCherry and the HADA signal in (B). Data shown are the average of three experiments with more than 200 cells. Error bars indicate the SD. ns, not significant; ****p < 0.0001; two‐tailed Student's t test. Scale bars, 5 μm.

Figure 4

FtsZ mutants display resistance to ZapA in vitro. (A) Sedimentation assay to test the influence of FtsZ mutations on the crosslinking of FtsZ filaments by ZapA. FtsZ or its variants (5 μM) were mixed with or without equal molar ZapA in polymerization buffer (50 mM HEPES pH 6.8, 10 mM MgCl₂, and 200 mM KCl) in the presence of GTP (2.5 mM) in a 50 μl reaction volume. The samples were incubated at room temperature for 5 min before being centrifuged. The pellets and supernatants were analyzed by SDS‐PAGE. (B) Negative stain electron microscopy analysis of the effect of the FtsZ mutations on the crosslinking of FtsZ filaments by ZapA. The reactions were performed as in (A), but the final concentration of proteins was lowered to 2.5 µM. GTP was added to a final concentration of 1 mM. Yellow lines show the width of the FtsZ filament bundles. Scale bar, 0.2 μm.

Figure 5

FtsZ mutations weaken the interaction between FtsZ and ZapA. (A) Bacterial two‐hybrid (BTH) assay to determine the domains important for FtsZ interaction with ZapA. Pairs of plasmids expressing the indicated fusions to the T18 and T25 domains of adenine cyclase were transformed into strain BTH101. A single transformant was resuspended in 1 ml of LB solution and spotted on LB plates containing IPTG and X‐gal. Plates were incubated at 30°C for 12 h before imaging. Blue indicates a positive interaction. +, positive control. A representative image from three independent experiments is shown. FtsZ^FL, full length FtsZ. (B) BTH assay to access the impact of FtsZ mutations on the interaction between FtsZ and ZapA. Experiments were performed as in (A). (C) Pull‐down assay to determine the interaction between FtsZ mutants and ZapA. SUMO‐ZapA and FtsZ or its variants were incubated and treated according to the pull‐down assay described in the Materials and Methods section. All fractions were collected during the procedure and analyzed by SDS‐PAGE. Note that since the GTPase defective mutant FtsZ^D212N binds more readily to ZapA in vitro, the mutations were tested in the FtsZ^D212N background. (D) Quantification of the effect of mutations on the pull‐down efficiency of ZapA on FtsZ mutants in (C).

Figure 6

The ZapA dimer head contacts the junction between adjacent FtsZ subunits in the filament, as revealed by in vivo BMOE crosslinking assay. (A) Location of FtsZ mutations in an FtsZ dimer from the filament structure of K. pneumoniae FtsZ (PDB#: 8IBN). Residues affecting FtsZ interaction with ZapA are colored magenta and the cysteine mutation N73C is colored blue. GMPCPP is shown as a stick in brown. Residues' numbers are according to E. coli FtsZ. (B) Location of mutations in E. coli ZapA tetramer structure (PDB#: 4P1M). The cysteine mutation T50C is colored blue, whereas residues important for ZapA interaction with FtsZ are shown as spheres in yellow. (C) BMOE‐crosslinking assay to test the effect of FtsZ mutations on FtsZ's interaction with ZapA in vivo. Cells expressing FtsZ^N73C or its variants carrying the FtsZ mutations and ZapA^C19A,T50C were treated with BMOE or DMF for 15 min. Cells were then harvested by centrifugation and lysed in 1× SDS‐PAGE buffer, boiled for 10 min, and loaded onto SDS‐PAGE gel for western blot as described in the Materials and Methods section. BMOE, bis‐maleimidoethane; CLS, crosslinked species; DMF, dimethylformamide; NCLS, nonspecific crosslinked species.

Figure 7

The N‐terminal motif of FtsZ is important for its interaction with ZapA in vivo. (A) AlphaFold 3 model of the FtsZ–ZapA complex indicates that the N‐terminal segment of FtsZ interacts with ZapA. The dimer head of ZapA is colored gray, while the N‐terminal motif of FtsZ is colored cyan. Residues important for interaction are indicated (FtsZ: F2, E3, P4, and D10; ZapA: R16, N18, R44, Q52, and N60). (B) Sequence logo of the N‐terminal motif of FtsZ across diverse bacterial species using Weblogo3. Alignment of the FtsZ N‐terminal sequences is shown in Figure S12A. (C) Spot test of the effect of mutations in the N‐terminal motif of FtsZ on the resistance to ZapA overexpression. A plasmid expressing ZapA (pSD319) was transformed into strains expressing different FtsZ mutants, and the transformants were subjected to a spot test on plates with or without IPTG. (D) Representative images of Z rings (ZipA‐mCherry) in cells expressing wild‐type FtsZ or FtsZ^F2A in the absence or presence of ZapA overexpression. (E) Representative images of co‐localization of ZipA‐mCherry with HADA in cells expressing wild‐type FtsZ or FtsZ^F2A in the absence or presence of ZapA overexpression. (F) Quantification of the co‐localization of ZipA‐mCherry and the HADA signal in (E). Data shown are the average of three experiments with more than 200 cells for each. Error bars indicate the SD of three experiments. ns, not significant; ****p < 0.0001; two‐tailed Student's t test. Scale bars, 5 μm. (G) BTH assay to test the impact of the F2A mutation of FtsZ on the interaction between FtsZ^1–316 and ZapA. The test was carried out as shown in Figure 5A.

Figure 8

The F2 residue of FtsZ is important for its interaction with ZapA in vitro. (A) Pull‐down assay to assess the effect of the F2A mutation on FtsZ's interaction with ZapA. SUMO‐ZapA and FtsZ^D212N or FtsZ^{D212N, F2A} were incubated and treated according to the pull‐down assay described in the Materials and Methods section. All fractions were collected during the procedure and analyzed by SDS‐PAGE. (B) The effect of the F2A mutation on the pull‐down efficiency of FtsZ by ZapA quantified by measuring the fraction of FtsZ in the elution to the total amount of FtsZ in the input. (C) Sedimentation assay to test the impact of the F2A mutation on FtsZ's interaction with ZapA. The reactions were prepared as described in the Materials and Methods section with or without equal molar of ZapA. The samples were incubated at room temperature for 5 min before being centrifuged and the pellets and supernatants were analyzed by SDS‐PAGE. (D) Negative stain electron microscopy analysis of the effect of the F2A mutation on FtsZ interaction with ZapA. The reactions were performed as in (C), but the final concentration of proteins was lowered to 1 µM. GTP was added to a final concentration of 1 mM. The yellow line shows the width of FtsZ filament bundles. Scale bars, 0.2 μm.

Figure 9

Localization of ZapA depends on its interaction with FtsZ. (A) Representative images of ZapA localization in cells expressing FtsZ mutants. Exponentially growing cultures of CYb1 (W3110, ftsZ ⁰ zapA‐gfp‐cat zapB::kan/pACYC, ftsZ) and derivatives carrying various ftsZ alleles were visualized by fluorescence microscopy to assess ZapA‐GFP localization at 30°C. Scale bar, 5 μm. (B) Quantification of the localization of ZapA‐GFP in cells expressing different FtsZ variants in (A). Data are presented as mean value ± SD. ****p < 0.0001; 0.0008 < **p < 0.0016; two‐tailed Student's t test. Number of cells analyzed (WT: n = 334; F2A: n = 836; K141N: n = 275; E219A: n = 352; R258E: n = 239; V128I: n = 242; KE/NA: n = 289; KR/KE: n = 347; KER/NAE: n = 876; F2A, K141N: n = 475; F2A, E219A: n = 918; F2A, R258E: n = 542; FKER/ANAE: n = 943) is shown.

Figure 10

A proposed model for the mechanism of ZapA‐mediated straightening and crosslinking of FtsZ filaments. (A) A diagram depicting the mechanism of ZapA. ZapA tetramers bind to the N‐terminal tail of FtsZ to associate with curved FtsZ filaments. Subsequently, each dimer head of a ZapA tetramer binds to the junction of FtsZ subunits in a filament, functioning as a staple to straighten the longitudinal interaction between FtsZ subunits. Since a ZapA tetramer is bipolar with a triple twofold symmetry, the two dimer heads can crosslink two adjacent parallel FtsZ filaments that rotate 180° to each other. In this way, ZapA tetramers straighten and crosslink FtsZ filaments simultaneously to facilitate their organization into the Z ring. The linker and the conserved C‐terminal peptide of FtsZ are omitted for simplicity. The treadmilling directions of FtsZ filaments are indicated by the arrows. (B) A docking model for the FtsZ–ZapA complex. FtsZ dimers from the filament structure of K. pneumoniae FtsZ (PDB#: 8IBN) and the ZapA tetramer in complex with the N‐terminal motif of FtsZ (predicted by AlphaFold 3) were used to generate the model by HDOCK. The ZapA tetramer, colored yellow, binds to both the N‐terminal motif and the junction between FtsZ subunits in the filament. Residues important for FtsZ binding to ZapA are colored magenta, whereas those important for ZapA binding to FtsZ are colored pink. GTP is colored brown and shown as a stick.