Dynamic FtsZ polymerization is sensitive to the GTP to GDP ratio and can be maintained at steady state using a GTP-regeneration system

Small, Elaine; Addinall, Stephen G.

doi:10.1099/mic.0.26126-0

Cited by 48 publications

(59 citation statements)

References 47 publications

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“…These techniques in combination provide information about the size of FtsZ protofilaments (8) and have been used previously to study other FtsZ inhibitors (21). For these experiments, a stable high GTP/ GDP ratio was achieved by the use of an enzymatic GTP regeneration system (6).…”

Section: Kil Peptide Disrupts Ftsz Protofilaments Triggered By Gtp Lementioning

confidence: 99%

Evidence That Bacteriophage λ Kil Peptide Inhibits Bacterial Cell Division by Disrupting FtsZ Protofilaments and Sequestering Protein Subunits

Hernández-Rocamora

Alfonso

Margolin

et al. 2015

Journal of Biological Chemistry

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Background: Kil peptide from bacteriophage targets FtsZ to prevent host cell division. Results: Kil disrupts FtsZ protofilaments producing shorter oligomers of variable size with reduced GTPase activity. Conclusion: At high concentrations, Kil likely inhibits FtsZ assembly via a subunit sequestration mechanism. Significance: This is the first biophysical study of how a bacteriophage disruptor of bacterial division inhibits FtsZ assembly.

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Section: Kil Peptide Disrupts Ftsz Protofilaments Triggered By Gtp Lementioning

confidence: 99%

Evidence That Bacteriophage λ Kil Peptide Inhibits Bacterial Cell Division by Disrupting FtsZ Protofilaments and Sequestering Protein Subunits

Hernández-Rocamora

Alfonso

Margolin

et al. 2015

Journal of Biological Chemistry

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“…Assembly-GDP-bound FtsZ polymers have been found to be comparatively unstable compared with the GTP-bound polymers (38,39). Because the GTP hydrolysis rate of the mutant FtsZ was found to be slower than the WT-FtsZ, it is logical to think that under GTP-challenged conditions, E93R-FtsZ polymers would disassemble at a rate slower than that of the WT-FtsZ.…”

Section: E93r Mutation Increased the Steady State Duration Of Ftszmentioning

confidence: 99%

E93R Substitution of Escherichia coli FtsZ Induces Bundling of Protofilaments, Reduces GTPase Activity, and Impairs Bacterial Cytokinesis

Jaiswal

Patel

Asthana

et al. 2010

Journal of Biological Chemistry

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Recently, we found that divalent calcium has no detectable effect on the assembly of Mycobacterium tuberculosis FtsZ (MtbFtsZ), whereas it strongly promoted the assembly of Escherichia coli FtsZ (EcFtsZ). While looking for potential calcium binding residues in EcFtsZ, we found a mutation (E93R) that strongly promoted the assembly of EcFtsZ. The mutation increased the stability and bundling of the FtsZ protofilaments and produced a dominating effect on the assembly of the wild type FtsZ (WT-FtsZ). Although E93R-FtsZ was found to bind to GTP similarly to the WT-FtsZ, it displayed lower GTPase activity than the WT-FtsZ. E93R-FtsZ complemented for its wild type counterpart as observed by a complementation test using JKD7-1/pKD3 cells. However, the bacterial cells became elongated upon overexpression of the mutant allele. We modeled the structure of E93R-FtsZ using the structures of MtbFtsZ/Methanococcus jannaschi FtsZ (MjFtsZ) dimers as templates. The MtbFtsZ-based structure suggests that the Arg 93 -Glu 138 salt bridge provides the additional stability, whereas the effect of mutation appears to be indirect (allosteric) if the EcFtsZ dimer is similar to that of MjFtsZ. The data presented in this study suggest that an increase in the stability of the FtsZ protofilaments is detrimental for the bacterial cytokinesis.Cell division in almost all bacteria is primarily driven by FtsZ, a protein considered as the homologue of the eukaryotic cytoskeletal protein tubulin (1, 2). FtsZ is a highly conserved protein in prokaryotes and plays a key role in the spatiotemporal regulation of the divisome assembly (2). Polymerization of FtsZ results in the formation of a ring-like structure, known as the Z-ring. The Z-ring is a highly dynamic structure (3), and the dynamics of the Z-ring is attributed to the assembly and disassembly of FtsZ protofilaments (4). Perturbation of the FtsZ assembly has been reported to impair bacterial cell division (5-9).Spatiotemporal regulation of the Z-ring at the midcell is tightly regulated by several effectors of FtsZ assembly (10 -12). Although the arrangement of FtsZ polymers in the bacterial Z-ring has not yet been visualized, the reconstitution of the FtsZ polymers in vitro has contributed a lot to our understanding of the bacterial cell division. Recently, an attempt was made to reconstitute the Z-ring in a liposome in vitro, and it was found that neither FtsZ assembly nor the generation of the constriction force requires FtsA or other downstream division proteins (13). Further, FtsZ polymers have been found to have intrinsic ability to form cytoplasmic ringlike structures in yeast cells, indicating that FtsZ assembly into the ring may not require any other regulatory protein (14).FtsZ protein of different bacteria exhibit different assembly properties in vitro. For example, the polymerization of Escherichia coli FtsZ (EcFtsZ) 2 has been observed to be more rapid than that of the Mycobacterium tuberculosis FtsZ (MtbFtsZ) (15). Recently, we have shown that divalent calcium strongly influenced ...

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“…The FtsZ9124 protein was expressed from pKD126-9124, as previously described for wild-type FtsZ (41,52). FtsZ6460 was expressed by induction of a culture of BL21(DE3)/pLysS containing plasmid pT73Z-6460 at an A 600 of 0.3 with 0.5 mM isopropyl-␤-Dthiogalactopyranoside (IPTG) for 2 h. FtsZ9124 was initially purified as previously described for wild-type FtsZ protein (52). It was found that a substantial amount of the FtsZ9124 protein was lost during the 20% ammonium sulfate precipitation step; therefore, a second preparation in which the 20% ammonium sulfate precipitate was not discarded was produced.…”

Section: Methodsmentioning

confidence: 99%

“…Negative-stain electron microscopy was performed as described previously (52). Samples were taken from polymerization reaction mixtures before and 1 and 10 min after GTP addition.…”

Section: Methodsmentioning

confidence: 99%

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New Temperature-Sensitive Alleles of ftsZ in Escherichia coli

et al. 2005

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We isolated five new temperature-sensitive alleles of the essential cell division gene ftsZ in Escherichia coli, using P1-mediated, localized mutagenesis. The five resulting single amino acid changes (Gly 109 3Ser 109 for ftsZ6460, Ala 129 3Thr 129 for ftsZ972, Val 157 3Met 157 for ftsZ2066, Pro 203 3Leu 203 for ftsZ9124, and Ala 239 3 Val 239 for ftsZ2863) are distributed throughout the FtsZ core region, and all confer a lethal cell division block at the nonpermissive temperature of 42°C. In each case the division block is associated with loss of Z-ring formation such that fewer than 2% of cells show Z rings at 42°C. The ftsZ9124 and ftsZ6460 mutations are of particular interest since both result in abnormal Z-ring formation at 30°C and therefore cause significant defects in FtsZ polymerization, even at the permissive temperature. Neither purified FtsZ9124 nor purified FtsZ6460 exhibited polymerization when it was assayed by light scattering or electron microscopy, even in the presence of calcium or DEAE-dextran. Hence, both mutations also cause defects in FtsZ polymerization in vitro. Interestingly, FtsZ9124 has detectable GTPase activity, although the activity is significantly reduced compared to that of the wild-type FtsZ protein. We demonstrate here that unlike expression of ftsZ84, multicopy expression of the ftsZ6460, ftsZ972, and ftsZ9124 alleles does not complement the respective lethalities at the nonpermissive temperature. In addition, all five new mutant FtsZ proteins are stable at 42°C. Therefore, the novel isolates carrying single ftsZ(Ts) point mutations, which are the only such strains obtained since isolation of the classical ftsZ84 mutation, offer significant opportunities for further genetic characterization of FtsZ and its role in cell division.The FtsZ protein of Escherichia coli is essential throughout the process of cell division (3,11,29,33,48,49). Following the segregation of daughter chromosomes, FtsZ polymerizes around the inner circumference at midcell to form the Z ring (9). At least nine proteins (all of which are essential for cell division) are recruited to the division site in an FtsZ-dependent fashion (12, 13), and the Z ring then leads this division apparatus inward, ultimately allowing separation of daughter cells. Division proteins ZipA and FtsA are recruited to the Z ring by direct interaction with FtsZ, and the recruitment of at least one of these proteins is essential for productive Z-ring formation (21,23,25,31,32,45,56,59). All other division proteins are thought to localize to the Z ring in a hierarchical fashion (that is, the localization is dependent on the prior presence of other cell division proteins). Therefore, all proteins which make up the divisome ultimately depend on the presence of the Z ring for their localization to the septum.The timing of the arrival at the division site of these proteins is thought to reflect the order in which they are involved during cell division (4,13,22,30). An exception is FtsW, which has been ascribed either an early (10, 24) or ...

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Dynamic FtsZ polymerization is sensitive to the GTP to GDP ratio and can be maintained at steady state using a GTP-regeneration system

Cited by 48 publications

References 47 publications

Evidence That Bacteriophage λ Kil Peptide Inhibits Bacterial Cell Division by Disrupting FtsZ Protofilaments and Sequestering Protein Subunits

Evidence That Bacteriophage λ Kil Peptide Inhibits Bacterial Cell Division by Disrupting FtsZ Protofilaments and Sequestering Protein Subunits

E93R Substitution of Escherichia coli FtsZ Induces Bundling of Protofilaments, Reduces GTPase Activity, and Impairs Bacterial Cytokinesis

New Temperature-Sensitive Alleles of ftsZ in Escherichia coli

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