共查询到20条相似文献,搜索用时 15 毫秒
1.
Joshua Cambridge Alexandra Blinkova David Magnan David Bates James R. Walker 《Journal of bacteriology》2014,196(1):36-49
Chromosome replication and cell division of Escherichia coli are coordinated with growth such that wild-type cells divide once and only once after each replication cycle. To investigate the nature of this coordination, the effects of inhibiting replication on Z-ring formation and cell division were tested in both synchronized and exponentially growing cells with only one replicating chromosome. When replication elongation was blocked by hydroxyurea or nalidixic acid, arrested cells contained one partially replicated, compact nucleoid located mid-cell. Cell division was strongly inhibited at or before the level of Z-ring formation. DNA cross-linking by mitomycin C delayed segregation, and the accumulation of about two chromosome equivalents at mid-cell also blocked Z-ring formation and cell division. Z-ring inhibition occurred independently of SOS, SlmA-mediated nucleoid occlusion, and MinCDE proteins and did not result from a decreased FtsZ protein concentration. We propose that the presence of a compact, incompletely replicated nucleoid or unsegregated chromosome masses at the normal mid-cell division site inhibits Z-ring formation and that the SOS system, SlmA, and MinC are not required for this inhibition. 相似文献
2.
Estefanía Salvarelli Marcin Krupka Germán Rivas Jesus Mingorance Paulino Gómez-Puertas Carlos Alfonso Ana Isabel Rico 《The Journal of biological chemistry》2015,290(41):25081-25089
The cell division protein FtsZ assembles in vitro by a mechanism of cooperative association dependent on GTP, monovalent cations, and Mg2+. We have analyzed the GTPase activity and assembly dynamics of Streptococcus pneumoniae FtsZ (SpnFtsZ). SpnFtsZ assembled in an apparently cooperative process, with a higher critical concentration than values reported for other FtsZ proteins. It sedimented in the presence of GTP as a high molecular mass polymer with a well defined size and tended to form double-stranded filaments in electron microscope preparations. GTPase activity depended on K+ and Mg2+ and was inhibited by Na+. GTP hydrolysis exhibited a delay that included a lag phase followed by a GTP hydrolysis activation step, until reaction reached the GTPase rate. The lag phase was not found in polymer assembly, suggesting a transition from an initial non-GTP-hydrolyzing polymer that switches to a GTP-hydrolyzing polymer, supporting models that explain FtsZ polymer cooperativity. 相似文献
3.
Rubén Ahijado-Guzmán Carlos Alfonso Belén Reija Estefanía Salvarelli Jesús Mingorance Silvia Zorrilla Bego?a Monterroso Germán Rivas 《The Journal of biological chemistry》2013,288(38):27358-27365
The influence of potassium content (at neutral pH and millimolar Mg2+) on the size distribution of FtsZ polymers formed in the presence of constantly replenished GTP under steady-state conditions was studied by a combination of biophysical methods. The size of the GTP-FtsZ polymers decreased with lower potassium concentration, in contrast with the increase in the mass of the GDP-FtsZ oligomers, whereas no effect was observed on FtsZ GTPase activity and critical concentration of polymerization. Remarkably, the concerted formation of a narrow size distribution of GTP-FtsZ polymers previously observed at high salt concentration was maintained in all KCl concentrations tested. Polymers induced with guanosine 5′-(α,β-methylene)triphosphate, a slowly hydrolyzable analog of GTP, became larger and polydisperse as the potassium concentration was decreased. Our results suggest that the potassium dependence of the GTP-FtsZ polymer size may be related to changes in the subunit turnover rate that are independent of the GTP hydrolysis rate. The formation of a narrow size distribution of FtsZ polymers under very different solution conditions indicates that it is an inherent feature of FtsZ, not observed in other filament-forming proteins, with potential implications in the structural organization of the functional Z-ring. 相似文献
4.
Characterization of the Porphyromonas gingivalis FtsZ Containing a Novel GTPase Activity 总被引:5,自引:0,他引:5
The FtsZ protein is a GTPase that is essential for cell division. We have cloned, sequenced, and expressed the FtsZ (PgFtsZ)
gene from the Porphyromonas gingivalis, an oral, anaerobic, rod-shaped bacterium implicated in progressive periodontal disease. The PgFtsZ gene consisted of 1374
bp and coded for an acidic protein with a calculated molecular mass of 50,253 Da. The deduced amino acid sequence exhibited
a significant homology with E. coli FtsZ (54% identical residues). Like other prokaryotic FtsZs, PgFtsZ possessed the clear motifs for GTP binding (GGGTGTG)
and hydrolysis (NLDFADV). When PgFtsZ was overexpressed in E. coli, cell division was inhibited. Recombinant PgFtsZ was purified to homogeneity and characterized. The purified PgFtsZ exhibited
GTPase activity even in the absence of Mg2+, and completely retained its activity with EDTA. Furthermore, Na+ and K+ ions inhibited its GTPase activity in a dose-dependent manner. These results suggest that PgFtsZ contains an atypical GTPase
activity that has not been previously described.
Received: 25 May 2001 / Accepted: 8 August 2001 相似文献
5.
Ashwini K. Mishra Claudia M. Del Campo Robert E. Collins Craig R. Roy David G. Lambright 《The Journal of biological chemistry》2013,288(33):24000-24011
GTPase activating proteins (GAPs) from pathogenic bacteria and eukaryotic host organisms deactivate Rab GTPases by supplying catalytic arginine and glutamine fingers in trans and utilizing the cis-glutamine in the DXXGQ motif of the GTPase for binding rather than catalysis. Here, we report the transition state mimetic structure of the Legionella pneumophila GAP LepB in complex with Rab1 and describe a comprehensive structure-based mutational analysis of potential catalytic and recognition determinants. The results demonstrate that LepB does not simply mimic other GAPs but instead deploys an expected arginine finger in conjunction with a novel glutamic acid finger, which forms a salt bridge with an indispensible switch II arginine that effectively locks the cis-glutamine in the DXXGQ motif of Rab1 in a catalytically competent though unprecedented transition state configuration. Surprisingly, a heretofore universal transition state interaction with the cis-glutamine is supplanted by an elaborate polar network involving critical P-loop and switch I serines. LepB further employs an unusual tandem domain architecture to clamp a switch I tyrosine in an open conformation that facilitates access of the arginine finger to the hydrolytic site. Intriguingly, the critical P-loop serine corresponds to an oncogenic substitution in Ras and replaces a conserved glycine essential for the canonical transition state stereochemistry. In addition to expanding GTP hydrolytic paradigms, these observations reveal the unconventional dual finger and non-canonical catalytic network mechanisms of Rab GAPs as necessary alternative solutions to a major impediment imposed by substitution of the conserved P-loop glycine. 相似文献
6.
Assembly of an FtsZ Mutant Deficient in GTPase Activity Has Implications for FtsZ Assembly and the Role of the Z Ring in Cell Division
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FtsZ, the ancestral homologue of eukaryotic tubulins, assembles into the Z ring, which is required for cytokinesis in prokaryotic cells. Both FtsZ and tubulin have a GTPase activity associated with polymerization. Interestingly, the ftsZ2 mutant is viable, although the FtsZ2 mutant protein has dramatically reduced GTPase activity due to a glycine-for-aspartic acid substitution within the synergy loop. In this study, we have examined the properties of FtsZ2 and found that the reduced GTPase activity is not enhanced by DEAE-dextran-induced assembly, indicating it has a defective catalytic site. In the absence of DEAE-dextran, FtsZ2 fails to assemble unless supplemented with wild-type FtsZ. FtsZ has to be at or above the critical concentration for copolymerization to occur, indicating that FtsZ is nucleating the copolymers. The copolymers formed are relatively stable and appear to be stabilized by a GTP-cap. These results indicate that FtsZ2 cannot nucleate assembly in vitro, although it must in vivo. Furthermore, the stability of FtsZ-FtsZ2 copolymers argues that FtsZ2 polymers would be stable, suggesting that stable FtsZ polymers are able to support cell division. 相似文献
7.
A perturbation of FtsZ assembly dynamics has been shown to inhibit bacterial cytokinesis. In this study, the antibacterial activity of 151 rhodanine compounds was assayed using Bacillus subtilis cells. Of 151 compounds, eight strongly inhibited bacterial proliferation at 2 μM. Subsequently, we used the elongation of B. subtilis cells as a secondary screen to identify potential FtsZ-targeted antibacterial agents. We found that three compounds significantly increased bacterial cell length. One of the three compounds, namely, CCR-11 [(E)-2-thioxo-5-({[3-(trifluoromethyl)phenyl]furan-2-yl}methylene)thiazolidin-4-one], inhibited the assembly and GTPase activity of FtsZ in vitro. CCR-11 bound to FtsZ with a dissociation constant of 1.5 ± 0.3 μM. A docking analysis indicated that CCR-11 may bind to FtsZ in a cavity adjacent to the T7 loop and that short halogen-oxygen, H-bonding, and hydrophobic interactions might be important for the binding of CCR-11 with FtsZ. CCR-11 inhibited the proliferation of B. subtilis cells with a half-maximal inhibitory concentration (IC(50)) of 1.2 ± 0.2 μM and a minimal inhibitory concentration of 3 μM. It also potently inhibited proliferation of Mycobacterium smegmatis cells. Further, CCR-11 perturbed Z-ring formation in B. subtilis cells; however, it neither visibly affected nucleoid segregation nor altered the membrane integrity of the cells. CCR-11 inhibited HeLa cell proliferation with an IC(50) value of 18.1 ± 0.2 μM (~15 × IC(50) of B. subtilis cell proliferation). The results suggested that CCR-11 inhibits bacterial cytokinesis by inhibiting FtsZ assembly, and it can be used as a lead molecule to develop FtsZ-targeted antibacterial agents. 相似文献
8.
GTP analogue inhibits polymerization and GTPase activity of the bacterial protein FtsZ without affecting its eukaryotic homologue tubulin 总被引:1,自引:0,他引:1
The prokaryotic tubulin homologue FtsZ plays a key role in bacterial cell division. Selective inhibitors of the GTP-dependent polymerization of FtsZ are expected to result in a new class of antibacterial agents. One of the challenges is to identify compounds which do not affect the function of tubulin and various other GTPases in eukaryotic cells. We have designed a novel inhibitor of FtsZ polymerization based on the structure of the natural substrate GTP. The inhibitory activity of 8-bromoguanosine 5'-triphosphate (BrGTP) was characterized by a coupled assay, which allows simultaneous detection of the extent of polymerization (via light scattering) and GTPase activity (via release of inorganic phosphate). We found that BrGTP acts as a competitive inhibitor of both FtsZ polymerization and GTPase activity with a Ki for GTPase activity of 31.8 +/- 4.1 microM. The observation that BrGTP seems not to inhibit tubulin assembly suggests a structural difference of the GTP-binding pockets of FtsZ and tubulin. 相似文献
9.
The FtsZ protein of Bacillus subtilis is localized at the division site and has GTPase activity that is dependent upon FtsZ concentration 总被引:16,自引:3,他引:13
The ftsZ gene is essential for cell division in both Escherichia coli and Bacillus subtilis. In E. coli FtsZ forms a cytokinetic ring at the division site whose formation is under cell-cycle control. In addition, the FtsZ from E. coli has a GTPase activity that shows an unusual lag in vitro. In this study we show that FtsZ in Bacillus subtilis forms a ring that is at the tip of the invaginating septum. The FtsZ ring is dynamic since it is formed as division is initiated, changes diameter during septation, and disperses upon completion of septation. In vitro the purified FtsZ from B. subtilis exhibits a GTPase activity without a demonstrable lag, but the GTPase activity is markedly dependent upon the FtsZ concentration, suggesting that the FtsZ protein must oligomerize to express the GTPase activity. 相似文献
10.
11.
Oligomerization of FtsZ converts the FtsZ tail motif (conserved carboxy‐terminal peptide) into a multivalent ligand with high avidity for partners ZipA and SlmA
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A short conserved motif located at the carboxy terminus of FtsZ, referred to here as the CCTP (c onserved c arboxy‐t erminal p eptide), is required for the interaction of FtsZ with many of its partners. In Escherichia coli interaction of FtsZ with its membrane anchors, ZipA and FtsA, as well as the spatial regulators of Z‐ring formation, MinC and SlmA, requires the CCTP. ZipA interacts with FtsZ with high affinity and interacts with the CCTP with low affinity, but the reason for this difference is not clear. In this study, we show that this difference is due to the oligomerization of FtsZ converting the CCTP to a multivalent ligand that binds multiple ZipAs bound to a surface with high avidity. Artificial dimerization of the CCTP is sufficient to increase the affinity for ZipA in vitro. Similar principles apply to the interaction of FtsZ with SlmA. Although done in vitro, these results have implications for the recruitment of FtsZ to the membrane in vivo, the interaction of FtsZ with spatial regulators and the reconstitution of FtsZ systems in vitro. 相似文献
12.
13.
Ndd, the Bacteriophage T4 Protein That Disrupts the Escherichia coli Nucleoid, Has a DNA Binding Activity
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Early in a bacteriophage T4 infection, the phage ndd gene causes the rapid destruction of the structure of the Escherichia coli nucleoid. Even at very low levels, the Ndd protein is extremely toxic to cells. In uninfected E. coli, overexpression of the cloned ndd gene induces disruption of the nucleoid that is indistinguishable from that observed after T4 infection. A preliminary characterization of this protein indicates that it has a double-stranded DNA binding activity with a preference for bacterial DNA rather than phage T4 DNA. The targets of Ndd action may be the chromosomal sequences that determine the structure of the nucleoid. 相似文献
14.
Martín-García F Salvarelli E Mendieta-Moreno JI Vicente M Mingorance J Mendieta J Gómez-Puertas P 《FEBS letters》2012,586(8):1236-1239
FtsZ, the prokaryotic ortholog of tubulin, assembles into polymers in the bacterial division ring. The interfaces between monomers contain a GTP molecule, but the relationship between polymerization and GTPase activity is not unequivocally proven. A set of short FtsZ polymers were modelled and the formation of active GTPase structures was monitored using molecular dynamics. Only the interfaces nearest the polymer ends exhibited an adequate geometry for GTP hydrolysis. Simulated conversion of interfaces from close-to-end to internal position and vice versa resulted in their spontaneous rearrangement between active and inactive conformations. This predicted behavior of FtsZ polymer ends was supported by in vitro experiments. 相似文献
15.
Alexej Dick Laura Graf Daniel Olal Alexander von der Malsburg Song Gao Georg Kochs Oliver Daumke 《The Journal of biological chemistry》2015,290(20):12779-12792
Myxovirus resistance (Mx) GTPases are induced by interferon and inhibit multiple viruses, including influenza and human immunodeficiency viruses. They have the characteristic domain architecture of dynamin-related proteins with an N-terminal GTPase (G) domain, a bundle signaling element, and a C-terminal stalk responsible for self-assembly and effector functions. Human MxA (also called MX1) is expressed in the cytoplasm and is partly associated with membranes of the smooth endoplasmic reticulum. It shows a protein concentration-dependent increase in GTPase activity, indicating regulation of GTP hydrolysis via G domain dimerization. Here, we characterized a panel of G domain mutants in MxA to clarify the role of GTP binding and the importance of the G domain interface for the catalytic and antiviral function of MxA. Residues in the catalytic center of MxA and the nucleotide itself were essential for G domain dimerization and catalytic activation. In pulldown experiments, MxA recognized Thogoto virus nucleocapsid proteins independently of nucleotide binding. However, both nucleotide binding and hydrolysis were required for the antiviral activity against Thogoto, influenza, and La Crosse viruses. We further demonstrate that GTP binding facilitates formation of stable MxA assemblies associated with endoplasmic reticulum membranes, whereas nucleotide hydrolysis promotes dynamic redistribution of MxA from cellular membranes to viral targets. Our study highlights the role of nucleotide binding and hydrolysis for the intracellular dynamics of MxA during its antiviral action. 相似文献
16.
Richa Jaiswal Ronak Y. Patel Jayant Asthana Bhavya Jindal Petety V. Balaji Dulal Panda 《The Journal of biological chemistry》2010,285(41):31796-31805
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 Arg93-Glu138 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. 相似文献
17.
18.
Mutants of FtsZ targeting the protofilament interface: effects on cell division and GTPase activity
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The bacterial cell division protein FtsZ assembles into straight protofilaments, one subunit thick, in which subunits appear to be connected by identical bonds or interfaces. These bonds involve the top surface of one subunit making extensive contact with the bottom surface of the subunit above it. We have investigated this interface by site-directed mutagenesis. We found nine bottom and eight top mutants that were unable to function for cell division. We had expected that some of the mutants might poison cell division substoichiometrically, but this was not found for any mutant. Eight of the bottom mutants exhibited dominant negative effects (reduced colony size) and four completely blocked colony formation, but this required expression of the mutant protein at four to five times the wild-type FtsZ level. Remarkably, the top mutants were even weaker, most showing no effect at the highest expression level. This suggests a directional assembly or treadmilling, where subunit addition is primarily to the bottom end of the protofilament. Selected pairs of top and bottom mutants showed no GTPase activity up to 10 to 20 microM, in contrast to the high GTPase activity of wild-type FtsZ above 1 muM. Overall, these results suggest that in order for a subunit to bind a protofilament at the 1 microM K(d) for elongation, it must have functional interfaces at both the top and bottom. This is inconsistent with the present model of the protofilament, as a simple stack of subunits one on top of the other, and may require a new structural model. 相似文献
19.
蛋白质合成过程一般被归纳为由合成的起始、肽链的延伸和合成的终止组成的三步曲 . 然而,随着对核糖体再循环因子 (ribosome recycling factor , RRF) 在蛋白质合成过程中作用的深入研究,人们提出了蛋白质生物合成应是四步曲, 这第四步就是翻译终止后核糖体复合物的解体 , 也就是通常说的核糖体循环再利用 . 简要地介绍了翻译终止后复合物解体的可能机制:核糖体再循环因子和蛋白质合成延伸因子 G 在核糖体上协同作用催化这一过程的完成 . 相似文献
20.