A widely conserved bacterial cell division protein that promotes assembly of the tubulin-like protein FtsZ

Abstract

Cell division in bacteria is mediated by the tubulin-like protein FtsZ, which assembles into a structure known as the Z ring at the future site of cytokinesis. We report the discovery of a Z-ring-associated protein in Bacillus subtilis called ZapA. ZapA was found to colocalize with the Z ring in vivo and was capable of binding to FtsZ and stimulating the formation of higher-order assemblies of the cytokinetic protein in vitro. The absence of ZapA alone did not impair cell viability, but the absence of ZapA in combination with the absence of a second, dispensable division protein EzrA caused a severe block in cytokinesis. The absence of ZapA also caused lethality in cells producing lower than normal levels of FtsZ or lacking the division-site-selection protein DivIVA. Conversely, overproduction of ZapA reversed the toxicity of excess levels of the division inhibitor MinD. In toto, the evidence indicates that ZapA is part of the cytokinetic machinery of the cell and acts by promoting Z-ring formation. Finally, ZapA is widely conserved among bacteria with apparent orthologs in many species, including Escherichia coli, in which the orthologous protein exhibited a strikingly similar pattern of subcellular localization to that of ZapA. Members of the ZapA family of proteins are likely to be a common feature of the cytokinetic machinery in bacteria.

Figure 1

Identification of the yshA (zapA) gene. (A) Sau3AI restriction map of the yshAB region of the B. subtilis chromosome. Arrows correspond to predicted genes as annotated by the B. subtilis genome consortium (http://genolist.pasteur.fr/SubtiList). Black bars correspond to Sau3AI fragments present in library clones that were capable of suppressing MinD toxicity or to the yshA (zapA) ORF. (B–E) Colony formation (B,D) and division phenotype (C,E) of cells overexpressing minD alone (B,C) or both minD and yshA (zapA; D,E). Strain FG332 was streaked on LB plates containing 250 μM IPTG (B) or 250 μM IPTG plus 1% xylose (D) and grown overnight at 37°C. For microscopy, the cells were grown in liquid LB medium to the mid-exponential phase of growth and treated with IPTG (C) or with IPTG and xylose (E) for 2 h before being harvested and stained with the membrane dye TMA-DPH. For details see Materials and Methods. (F) Multiple sequence alignment of representative ZapA orthologs. Arrow points to the start of the predicted coiled-coil region in the B. subtilis sequence, as determined by the program Coils. Alignment was made with Clustal-W and BoxShade programs. Sequence accession nos.: Agrobacterium tumefaciens, NP_356887; Bacillus subtilis, NP_390739; Caulobacter vibrioides, NP_422041; Escherichia coli, NP_289478; Haemophilus influenzae, NP_439017; Listeria monocytogenes, NP_464754; Neisseria meningitides, NP_275048; Pseudomonas aeruginosa, NP_253914; Ralstonia solanacearum, NP_520523; Rickettsia prowazekii, NP_221108; Treponema pallidum, NP_219282; Staphylococcus aureus, NP_371665; Streptococcus pneumoniae, NP_344927. The A. tumefaciens sequence has been truncated and only the first 108 amino acids of 125 are shown. Bar, 5 μm.

Figure 2

Subcellular localization of GFP–ZapA and GFP–YgfE. Cells were grown, stained, and subjected to fluorescence microscopy as described in Materials and Methods. Fluorescence from GFP is in green and that from TMA-DPH is false colored in red. (A) Growing cells of the GFP–ZapA-producing B. subtilis strain FG347. The arrows point to cells in which the cytokinetic ring is constricting. (B) Sporulating cells of the GFP–ZapA-producing strain FG347. The arrows point to a polar GFP–ZapA band. GFP–ZapA can also be seen in V-like structures and membrane-associated dots, which we interpret as indicating an association of ZapA–GFP with the FtsZ spiral-like structures observed in cells undergoing sporulation (Ben Yehuda and Losick 2002). (C) ZapA localization in cells depleted of FtsZ. Cells of strain FG369 were grown to OD ∼0.6 in the presence of 1 mM IPTG, harvested, suspended in medium without IPTG, and incubated for 1.5 h at 37°C. (D) GFP–YgfE-producing cells of E. coli strain DH5α harboring pFG53 and growing in LB medium with ampicillin. The arrows point to a cell in which the cytokinetic ring is constricting. Bars, 5 μm.

Figure 3

Division phenotypes of zapA mutation. (A) Failure of a zapA mutant to form colonies at lowered FtsZ levels. All of the strains harbored a construct in which ftsZ was under the control of an IPTG-inducible promoter and, in addition, were zapA⁺ (RL861), mutant for zapA (FG367), mutant for zapA, and harboring a P_xyl–gfp–zapA construct (FG371), or mutant for zapA and mutant for minCD (FG373). The cells were grown to stationary phase in the presence of 500 μM IPTG, serially diluted (10-fold steps), and then 10 μl of each dilution was spotted onto LB plates containing the indicated concentrations of IPTG. For strain FG371 (mutant for zapA and containing P_xyl–gfp–zapA), the plates additionally contained 0.1% xylose. The plates were incubated ∼12 h at 37°C before being photographed. (B–F) Synergy of the zapA mutation with mutations of diviIVA and ezrA. (B) Colonies of strain FG449, which is mutant for zapA and divIVA and contains a P_xyl–divIVA construct, and the congenic zapA⁺ strain FG94, grown in LB medium containing 1% xylose or lacking xylose. (C) Wild-type strain PY79. (D) zapA mutant strain FG356. (E,F) Strain FG381, which is mutant for ezrA and zapA and contains a P_xyl–zapA construct. In E, cells of strain FG381 were grown in the presence of 0.1% xylose, whereas in F, the cells were depleted for ZapA by growth in LB medium in the absence of xylose. Cells in C–F were stained with TMA-DPH.

Figure 4

Biochemical properties of ZapA. (A) SDS-PAGE of FtsZ and His₆–ZapA. The following were subjected to SDS-PAGE: an extract from cells of the FtsZ-overproducing strain W3110(pBS58)(pCXZ) (lane 1); purified FtsZ (lane 2); an extract from cells of the His₆–ZapA-overproducing strain FG443 (lane 3); and purified His₆–ZapA (lane 4). (B) ZapA binds to FtsZ. Ni⁺⁺–NTA affinity chromatography of FtsZ in the presence or absence of His₆–ZapA. Bands correspond to FtsZ and are from a Coomasie stained SDS–polyacrylamide gel. Note that the eluate fractions were more concentrated than the input or the flowthrough (see the Materials and Methods). (C) ZapA promotes assembly of FtsZ in the presence of GTP (left) or in the absence of added nucleotide (right). Light scattering traces of FtsZ polymerization reactions containing 5 μM FtsZ in PEM buffer (50 mM PIPES at pH 6.5, 1 mM EDTA, 5 mM MgCl₂). The arrows indicate the time of addition of GTP (1 mM) or ZapA (5 μM). Light scattering is expressed in arbitrary units. (D) Effect of ZapA on the GTPase activity of FtsZ. GTPase activity was measured as described in Materials and Methods. FtsZ concentration was 5 μM and ZapA, when present, was at 5 μM. Error bars, the standard deviation of three replicates. Similar results were obtained in three experiments. (E) FtsZ polymerization as a function of ZapA concentration. The maximum light scattering signal of individual reactions containing 5 μM FtsZ in PEM buffer and various concentrations of ZapA is plotted. Light scattering expressed in arbitrary units.

Figure 5

Electron microscopy of the products of FtsZ polymerization. (A) Unpolymerized FtsZ (5 μM FtsZ in polymerization buffer); (B) 5 μM FtsZ in the presence of 1 mM GTP; (C) 5 μM FtsZ in the presence of 1 mM GTP; and 0.25 μM His₆–ZapA. (D) Low magnification of reaction products similar to those of C to show the extent of FtsZ bundling; (E) 5 μM FtsZ in the presence of 0.25 μM His₆–ZapA. The arrow points to a mini-ring formed by a single protofilament; (F) 5 μM FtsZ in the presence of 1.25 μM His₆–ZapA. The arrowheads point to mini-rings formed by more than one protofilament. Bars, A–C,E,F, 100 nm; D, 200 nm.

Figure 6

Model for the function of ZapA in Z-ring formation. For simplicity, the figure depicts FtsZ polymerization as occurring by the assembly of FtsZ monomers (gray spheres) into protofilaments, which then associate with each other laterally to form bundles. We do not mean to exclude the possibility that monomers are also directly added to bundles. The model proposes that dimers of ZapA (black Y-shapes) cross-link FtsZ monomers in adjacent protofilaments, thereby stabilizing the lateral interactions. Alternatively, ZapA could promote bundling indirectly by stabilizing the longitudinal bonds along a protofilament. See Discussion for details.