doi: 10.1128/JB.188.5.1680-1690.2006.
Trapping of a spiral-like intermediate of the bacterial cytokinetic protein FtsZ
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- PMID:16484179
- PMCID: PMC1426551
- DOI: 10.1128/JB.188.5.1680-1690.2006
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Trapping of a spiral-like intermediate of the bacterial cytokinetic protein FtsZ
Katherine A Michie et al. J Bacteriol.2006 Mar.
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Abstract
The earliest stage in bacterial cell division is the formation of a ring, composed of the tubulin-like protein FtsZ, at the division site. Tight spatial and temporal regulation of Z-ring formation is required to ensure that division occurs precisely at midcell between two replicated chromosomes. However, the mechanism of Z-ring formation and its regulation in vivo remain unresolved. Here we identify the defect of an interesting temperature-sensitive ftsZ mutant (ts1) of Bacillus subtilis. At the nonpermissive temperature, the mutant protein, FtsZ(Ts1), assembles into spiral-like structures between chromosomes. When shifted back down to the permissive temperature, functional Z rings form and division resumes. Our observations support a model in which Z-ring formation at the division site arises from reorganization of a long cytoskeletal spiral form of FtsZ and suggest that the FtsZ(Ts1) protein is captured as a shorter spiral-forming intermediate that is unable to complete this reorganization step. The ts1 mutant is likely to be very valuable in revealing how FtsZ assembles into a ring and how this occurs precisely at the division site.
Figures
Localization of FtsZ in wild-type andts1 strains at the nonpermissive temperature. IFM was performed on wild-type (short cell with arrow) andts1 (long filament) cells grown at 49°C for 1 h. The section of filament bound by white lines is magnified in insets. (A) Phase-contrast image. (B) FtsZ localization. (C) DAPI staining of DNA. (D) Overlay of B and C. Scale bar, 5 μm.
IEM analysis of FtsZ(Ts1) localization. Wild-type andts1 cells growing exponentially at 34°C were shifted to 49°C. Representative immunoelectron micrographs of thin-sectioned wild-type (A) andts1 (B) cells after 1 h of growth at 49°C are shown. Note the strong labeling immediately to the inside of the cell wall (where we assume the plasma membrane lies) in the wild-type cells, in comparison to thets1 cells. The cell wall shows different widths depending on the plane of section. Scale bar, 500 nm. (C) Graph of the distribution of anti-FtsZ gold particles in both wild-type andts1 cells at 34°C and 49°C as a proportion of the population versus distance (in micrometers) from the inside of the cell wall. The numbers in parentheses indicate the number of gold particles counted for each sample.
FtsZ-YFP and FtsZ(Ts1)-YFP localization after 1 h at 49°C. (A) FtsZ-YFP localization in a wild-type background (SU488). (B) FtsZ(Ts1)-YFP localization in thets1 background (SU489). The filled arrow points to a spiral-like structure, whereas the open arrows show dot patterns that would be consistent with spiral formation. (C to E) Enlarged spiral localizations of FtsZ(Ts1)-YFP in SU489. The localization in C is the same as that in B with the filled arrow. (F) Time-lapse images obtained of FtsZ(Ts1)-YFP localization in SU489 during a shift down to room temperature after 1 h at 49°C: (i) 0 min, (ii) 3 min, (iii) 6 min, and (iv) 9 min. (G) FtsZ(Ts1)-YFP localization in SU475 (wild-type background) when 0.02% xylose is added to induce production of this fusion protein. (H) As in G, except 0.1% xylose has been added. Scale bar, 5 μm for A, B, G, and H.
(A) Multiple sequence alignments of FtsZ homologs from various organisms. Arrows and boxed regions indicate the residues (equivalent to 240 and 278 of theB. subtilis sequence) substituted in thets1 strain. Alignment was made using ClustalW as described in Materials and Methods. Below the alignments is a schematic representation of the secondary structure predicted for this region based on the crystal structure of FtsZ fromM. jannaschii. (B) Predicted structure ofB. subtilis FtsZ modeled on theM. jannaschii FtsZ crystal structure using the SWISS-MODEL server, showing the locations of residues 240 and 278 (see Materials and Methods).
Effect of temperature on mean cell length of wild-type (WT),ts1, and single-mutantftsZ(A240V) andftsZ(A278V)B. subtilis strains. Error bars denote SEM. Some errors are too small to be shown on the graph.
(A) CD spectra of purifiedB. subtilis FtsZ and FtsZ(A240V) proteins. Spectra were obtained over 184 to 260 nm at 18°C for FtsZ and FtsZ(A240V), both at 1.25 mM, in 20 mM Tris (pH 7.5), 50 mM sodium fluoride, and 10% glycerol. (B) Equilibrium unfolding curve of the same solutions of FtsZ and FtsZ(A240V) over a temperature range of 20 to 80°C, monitored at 222 nm. Note the biphasic unfolding behavior of the wild-type protein.
Negative stain electron microscopy of FtsZ and FtsZ(A240V). (A to E) FtsZ in the absence of GTP and Ca2+ (A), with GTP only (B), and in the presence of GTP and Ca2+ (C to E). Note the high number of rings and bundled protofilaments. (F to H) FtsZ(A240V) in the absence of GTP and Ca2+ (F), with GTP only (G), and with GTP and Ca2+ (H). All experiments were performed at room temperature. The protein concentration was 0.6 mg/ml. Scale bars represent 100 nm in all panels except C, where the scale bar represents 200 nm.
GTPase activity of wild-type and A240V FtsZ (both at 10 μM at 30°C in 50 mM MES, 5 mM magnesium chloride, and 10% glycerol [pH 6.5]), measured as inorganic phosphate (Pi) release over 20 min after the addition of GTP (300 μM). Error bars denote SEM.
Schematic diagram proposing how midcell Z rings arise from spirals in wild-type, vegetatively growingB. subtilis cells during the cell cycle and the nature of the defect in the FtsZ(Ts1) protein in thets1 mutant. At the nonpermissive temperature, the FtsZ(Ts1) protein is unable to complete reorganization into a ring at the division site. Instead, it is trapped as a short spiral close to the cell center between two replicated nucleoids (large ovals). The polymerized FtsZ structures are illustrated as small circles. Individual circles do not necessarily represent single FtsZ molecules. Also, it is not known whether the pitch of the shorterts1 spiral differs from that of the wild type.
References
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