scholarly journals Deletion of the ftsZ-Like Gene Results in the Production of Superparamagnetic Magnetite Magnetosomes in Magnetospirillum gryphiswaldense

2009 ◽  
Vol 192 (4) ◽  
pp. 1097-1105 ◽  
Author(s):  
Yao Ding ◽  
Jinhua Li ◽  
Jiangning Liu ◽  
Jing Yang ◽  
Wei Jiang ◽  
...  
Keyword(s):  
Cell Division ◽  
Transcription Unit ◽  
Activity Levels ◽  
Wild Type ◽  
Electron Microscopic ◽  
Magnetospirillum Gryphiswaldense ◽  
Polycistronic Transcription ◽  
Field Lines ◽  
A Chain

ABSTRACT Magnetotactic bacteria (MTB) synthesize unique organelles termed “magnetosomes,” which are membrane-enclosed structures containing crystals of magnetite or greigite. Magnetosomes form a chain around MamK cytoskeletal filaments and provide the basis for the ability of MTB to navigate along geomagnetic field lines in order to find optimal microaerobic habitats. Genomes of species of the MTB genus Magnetospirillum, in addition to a gene encoding the tubulin-like FtsZ protein (involved in cell division), contain a second gene termed “ftsZ-like,” whose function is unknown. In the present study, we found that the ftsZ-like gene of Magnetospirillum gryphiswaldense strain MSR-1 belongs to a 4.9-kb mamXY polycistronic transcription unit. We then purified the recombinant FtsZ-like protein to homogeneity. The FtsZ-like protein efficiently hydrolyzed ATP and GTP, with ATPase and GTPase activity levels of 2.17 and 5.56 μmol phosphorus per mol protein per min, respectively. The FtsZ-like protein underwent GTP-dependent polymerization into long filamentous bundles in vitro. To determine the role of the ftsZ-like gene, we constructed a ftsZ-like mutant (ΔftsZ-like mutant) and its complementation strain (ΔftsZ-like_C strain). Growth of ΔftsZ-like cells was similar to that of the wild type, indicating that the ΔftsZ-like gene is not involved in cell division. Transmission electron microscopic observations indicated that the ΔftsZ-like cells, in comparison to wild-type cells, produced smaller magnetosomes, with poorly defined morphology and irregular alignment, including large gaps. Magnetic analyses showed that ΔftsZ-like produced mainly superparamagnetic (SP) magnetite particles, whereas wild-type and ΔftsZ-like_C cells produced mainly single-domain (SD) particles. Our findings suggest that the FtsZ-like protein is required for synthesis of SD particles and magnetosomes in M. gryphiswaldense.

scholarly journals Analysis of MinC Reveals Two Independent Domains Involved in Interaction with MinD and FtsZ

2000 ◽  
Vol 182 (14) ◽  
pp. 3965-3971 ◽  
Author(s):  
Zonglin Hu ◽  
Joe Lutkenhaus
Keyword(s):  
Escherichia Coli ◽  
Cell Division ◽  
Functional Domains ◽  
Wild Type ◽  
Terminal Domain ◽  
Ring Assembly ◽  
Z Ring ◽  
Ftsz Assembly

ABSTRACT In Escherichia coli FtsZ assembles into a Z ring at midcell while assembly at polar sites is prevented by themin system. MinC, a component of this system, is an inhibitor of FtsZ assembly that is positioned within the cell by interaction with MinDE. In this study we found that MinC consists of two functional domains connected by a short linker. When fused to MalE the N-terminal domain is able to inhibit cell division and prevent FtsZ assembly in vitro. The C-terminal domain interacts with MinD, and expression in wild-type cells as a MalE fusion disrupts minfunction, resulting in a minicell phenotype. We also find that MinC is an oligomer, probably a dimer. Although the C-terminal domain is clearly sufficient for oligomerization, the N-terminal domain also promotes oligomerization. These results demonstrate that MinC consists of two independently functioning domains: an N-terminal domain capable of inhibiting FtsZ assembly and a C-terminal domain responsible for localization of MinC through interaction with MinD. The fusion of these two independent domains is required to achieve topological regulation of Z ring assembly.

scholarly journals Plk is a functional homolog of Saccharomyces cerevisiae Cdc5, and elevated Plk activity induces multiple septation structures.

1997 ◽  
Vol 17 (6) ◽  
pp. 3408-3417 ◽  
Author(s):  
K S Lee ◽  
R L Erikson
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Cycle ◽  
Cell Division ◽  
Specific Activity ◽  
Ectopic Expression ◽  
Sf9 Cells ◽  
Wild Type ◽  
C Terminus ◽  
M Phase

Plk is a mammalian serine/threonine protein kinase whose activity peaks at the onset of M phase. It is closely related to other mammalian kinases, Snk, Fnk, and Prk, as well as to Xenopus laevis Plx1, Drosophila melanogaster polo, Schizosaccharomyces pombe Plo1, and Saccharomyces cerevisiae Cdc5. The M phase of the cell cycle is a highly coordinated process which insures the equipartition of genetic and cellular materials during cell division. To enable understanding of the function of Plk during M phase progression, various Plk mutants were generated and expressed in Sf9 cells and budding yeast. In vitro kinase assays with Plk immunoprecipitates prepared from Sf9 cells indicate that Glu206 and Thr210 play equally important roles for Plk activity and that replacement of Thr210 with a negatively charged residue elevates Plk specific activity. Ectopic expression of wild-type Plk (Plk WT) complements the cell division defect associated with the cdc5-1 mutation in S. cerevisiae. The degree of complementation correlates closely with the Plk activity measured in vitro, as it is enhanced by a mutationally activated Plk, T210D, but is not observed with the inactive forms K82M, D194N, and D194R. In a CDC5 wild-type background, expression of Plk WT or T210D, but not of inactive forms, induced a sharp accumulation of cells in G1. Consistent with elevated Plk activity, this phenomenon was enhanced by the C-terminally deleted forms WT deltaC and T210D deltaC. Expression of T210D also induced a class of cells with unusually elongated buds which developed multiple septal structures. This was not observed with the C-terminally deleted form T210D deltaC, however. It appears that the C terminus of Plk is not required for the observed cell cycle influence but may be important for polarized cell growth and septal structure formation.

scholarly journals The sep1 mutant of Saccharomyces cerevisiae arrests in pachytene and is deficient in meiotic recombination.

Genetics ◽  
1995 ◽  
Vol 139 (2) ◽  
pp. 495-509 ◽  
Author(s):  
D X Tishkoff ◽  
B Rockmill ◽  
G S Roeder ◽  
R D Kolodner
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Meiotic Recombination ◽  
Microscopic Analysis ◽  
Mutant Gene ◽  
Strand Break ◽  
Strand Exchange ◽  
Wild Type ◽  
Electron Microscopic ◽  
Pathways Analysis

Abstract Strand exchange protein 1 (Sep1) from Saccharomyces cerevisiae promotes homologous pairing of DNA in vitro and sep1 mutants display pleiotropic phenotypes in both vegetative and meiotic cells. In this study, we examined in detail the ability of the sep1 mutant to progress through meiosis I prophase and to undergo meiotic recombination. In meiotic return-to-growth experiments, commitment to meiotic recombination began at the same time in wild type and mutant; however, recombinants accumulated at decreased rates in the mutant. Gene conversion eventually reached nearly wild-type levels, whereas crossing over reached 15-50% of wild type. In an assay of intrachromosomal pop-out recombination, the sep1, dmc1 and rad51 single mutations had only small effects; however, pop-out recombination was virtually eliminated in the sep1 dmc1 and sep1 rad51 double mutants, providing evidence for multiple recombination pathways. Analysis of meiotic recombination intermediates indicates that the sep1 mutant is deficient in meiotic double-strand break repair. In a physical assay, the formation of mature reciprocal recombinants in the sep1 mutant was delayed relative to wild type and ultimately reached only 50% of the wild-type level. Electron microscopic analysis of meiotic nuclear spreads indicates that the sep1 delta mutant arrests in pachytene, with apparently normal synaptonemal complex. This arrest is RAD9-independent. We hypothesize that the Sep1 protein participates directly in meiotic recombination and that other strand exchange enzymes, acting in parallel recombination pathways, are able to substitute partially for the absence of the Sep1 protein.

Reduced Activation of the Gla19Ala FX Variant via the Extrinsic Coagulation Pathway Results in Symptomatic CRMred FX Deficiency

2002 ◽  
Vol 88 (08) ◽  
pp. 236-241 ◽  
Author(s):  
M. Pinotti ◽  
G. Marchetti ◽  
M. Baroni ◽  
F. Cinotti ◽  
M. Morfini ◽  
...  
Keyword(s):  
Factor Viia ◽  
Activity Levels ◽  
Factor X ◽  
Wild Type ◽  
Rich Domain ◽  
Factor Viiia ◽  
Domain Interactions ◽  
Vitro Model

SummaryWe characterized a symptomatic CRMred factor X (FX) deficiency produced by the Glu19Ala mutation in the γ-carboxyglutamic-rich domain. FX activity levels in plasma were markedly reduced in prothrombin time assays (< 1-5%), whereas in activated partial thromboplastin assays (16%) and in RVV assays (17%) the reduction in activity mirrored that in antigen levels (17%). Activation of recombinant 19Ala-FX by factor IXa/factor VIIIa or RVV, and the activity in thrombin generation assays, were comparable to those of wild-type FX. Differently, complete activation of recombinant 19AlaFX required a factor VIIa/TF concentration 30-fold higher than that of wild-type FX. The recombinant FVIIa significantly reduced PT values in 19Ala-FX reconstituted plasma, thus suggesting an alternative approach for treatment of FX deficiencies characterized by defective FX activation.The study of this FX deficiency provides an “in vivo” and “in vitro” model for the investigation of Gla domain interactions.

scholarly journals Selection for arsenite resistance causes reversible changes in minicircle composition and kinetoplast organization in Leishmania mexicana.

1994 ◽  
Vol 14 (1) ◽  
pp. 587-596 ◽  
Author(s):  
S T Lee ◽  
H Y Liu ◽  
S P Lee ◽  
C Tarn
Keyword(s):  
Nuclear Dna ◽  
Transition Period ◽  
Electron Microscopic Examination ◽  
Leishmania Mexicana ◽  
Wild Type ◽  
Electron Microscopic ◽  
Dot Hybridization ◽  
Arsenite Resistance ◽  
Leishmania Parasites

Certain minor minicircle sequence classes in the kinetoplast DNA (kDNA) networks of arsenite- or tunicamycin-resistant Leishmania mexicana amazonensis variants whose nuclear DNA is amplified appear to be preferentially selected to replicate (S. T. Lee, C. Tarn, and K. P. Chang, Mol. Biochem. Parasitol. 58:187-204, 1993). These sequences replace the predominant wild-type minicircle sequences to become dominant species in the kDNA network. The switch from wild-type-specific to variant-specific minicircles takes place rapidly within the same network, the period of minicircle dominance changes being defined as the transition period. To investigate the structural organization of the kDNA networks during this transition period, we analyzed kDNA from whole arsenite-resistant Leishmania parasites by dot hybridization with sequence-specific DNA probes and by electron-microscopic examination of isolated kDNA networks in vitro. Both analyses concluded that during the switch of dominance the predominant wild-type minicircle class was rapidly lost and that selective replication of variant-specific minicircles subsequently filled the network step by step. There was a time during the transition when few wild-type- or variant-specific minicircles were present, leaving the network almost empty and exposing a species of thick, long, fibrous DNA which seemed to form a skeleton for the network. Both minicircles and maxicircles were found to attach to these long DNA fibrils. The nature of the long DNA fibrils is not clear, but they may be important in providing a framework for the network structure and a support for the replication of minicircles and maxicircles.

scholarly journals Fusion expression of nanobodies specific for the insecticide fipronil on magnetosomes in Magnetospirillum gryphiswaldense MSR-1

2021 ◽  
Vol 19 (1) ◽  
Author(s):  
Sha Wu ◽  
Fengfei Ma ◽  
Jinxin He ◽  
Qing X. Li ◽  
Bruce D. Hammock ◽  
...  
Keyword(s):  
Colloidal Stability ◽  
Reduced Glutathione ◽  
Magnetotactic Bacteria ◽  
Water Soluble ◽  
Enzyme Linked Immunosorbent Assay ◽  
Crystallographic Structure ◽  
Wild Type ◽  
Magnetospirillum Gryphiswaldense ◽  
Thermal Stable

Abstract Background Magnetic nanoparticles such as magnetosomes modified with antibodies allow a high probability of their interaction with targets of interest. Magnetosomes biomineralized by magnetotactic bacteria are in homogeneous nanoscale size and have crystallographic structure, and high thermal and colloidal stability. Camelidae derived nanobodies (Nbs) are small in size, thermal stable, highly water soluble, easy to produce, and fusible with magnetosomes. We aimed to functionalize Nb-magnetosomes for the analysis of the insecticide fipronil. Results Three recombinant magnetotactic bacteria (CF, CF+ , and CFFF) biomineralizing magnetosomes with different abundance of Nbs displayed on the surface were constructed. Compared to magnetosomes from the wild type Magnetospirillum gryphiswaldense MSR-1, all of the Nb-magnetosomes biosynthesized by strains CF, CF+ , and CFFF showed a detectable level of binding capability to fipronil-horseradish peroxidase (H2-HRP), but none of them recognized free fipronil. The Nb-magnetosomes from CFFF were oxidized with H2O2 or a glutathione mixture consisting of reduced glutathione and oxidized glutathione in vitro and their binding affinity to H2-HRP was decreased, whereas that to free fipronil was enhanced. The magnetosomes treated with the glutathione mixture were employed to develop an enzyme-linked immunosorbent assay for the detection of fipronil in water samples, with average recoveries in a range of 78–101%. Conclusions The economical and environmental-friendly Nb-magnetosomes biomineralized by the bacterial strain MSR-1 can be potentially applied to nanobody-based immunoassays for the detection of fipronil or nanobody-based assays in general.

scholarly journals Structure and Mutational Analyses of Escherichia coli ZapD Reveal Charged Residues Involved in FtsZ Filament Bundling

2016 ◽  
Vol 198 (11) ◽  
pp. 1683-1693 ◽  
Author(s):  
Elyse J. Roach ◽  
Charles Wroblewski ◽  
Laura Seidel ◽  
Alison M. Berezuk ◽  
Dyanne Brewer ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Cell Division ◽  
Bacterial Cell ◽  
Site Directed Mutagenesis ◽  
Bacterial Cell Division ◽  
Wild Type ◽  
Content Type ◽  
E Coli ◽  
Z Ring

ABSTRACTBacterial cell division is an essential and highly coordinated process. It requires the polymerization of the tubulin homologue FtsZ to form a dynamic ring (Z-ring) at midcell. Z-ring formation relies on a group of FtsZ-associatedproteins (Zap) for stability throughout the process of division. InEscherichia coli, there are currently five Zap proteins (ZapA through ZapE), of which four (ZapA, ZapB, ZapC, and ZapD) are small soluble proteins that act to bind and bundle FtsZ filaments. In particular, ZapD forms a functional dimer and interacts with the C-terminal tail of FtsZ, but little is known about its structure and mechanism of action. Here, we present the crystal structure ofEscherichia coliZapD and show it forms a symmetrical dimer with centrally located α-helices flanked by β-sheet domains. Based on the structure of ZapD and its chemical cross-linking to FtsZ, we targeted nine charged ZapD residues for modification by site-directed mutagenesis. Usingin vitroFtsZ sedimentation assays, we show that residues R56, R221, and R225 are important for bundling FtsZ filaments, while transmission electron microscopy revealed that altering these residues results in different FtsZ bundle morphology compared to those of filaments bundled with wild-type ZapD. ZapD residue R116 also showed altered FtsZ bundle morphology but levels of FtsZ bundling similar to that of wild-type ZapD. Together, these results reveal that ZapD residues R116, R221, and R225 likely participate in forming a positively charged binding pocket that is critical for bundling FtsZ filaments.IMPORTANCEZ-ring assembly underpins the formation of the essential cell division complex known as the divisome and is required for recruitment of downstream cell division proteins. ZapD is one of several proteins inE. colithat associates with the Z-ring to promote FtsZ bundling and aids in the overall fitness of the division process. In the present study, we describe the dimeric structure ofE. coliZapD and identify residues that are critical for FtsZ bundling. Together, these results advance our understanding about the formation and dynamics of the Z-ring prior to bacterial cell division.

scholarly journals Hsp90 of E. coli modulates assembly of FtsZ, the bacterial tubulin homolog

2019 ◽  
Vol 116 (25) ◽  
pp. 12285-12294 ◽  
Author(s):  
Anuradha Balasubramanian ◽  
Monica Markovski ◽  
Joel R. Hoskins ◽  
Shannon M. Doyle ◽  
Sue Wickner
Keyword(s):  
Cell Division ◽  
Bacterial Species ◽  
Heat Shock Protein 90 ◽  
Client Protein ◽  
Wild Type ◽  
E Coli ◽  
Independent Process ◽  
Client Proteins ◽  
Turn Over

Heat shock protein 90 (Hsp90) is a highly conserved molecular chaperone involved in ATP-dependent client protein remodeling and activation. It also functions as a protein holdase, binding and stabilizing clients in an ATP-independent process. Hsp90 remodels over 300 client proteins and is essential for cell survival in eukaryotes. In bacteria, Hsp90 is a highly abundant protein, although very few clients have been identified and it is not essential for growth in many bacterial species. We previously demonstrated that in Escherichia coli, Hsp90 causes cell filamentation when expressed at high levels. Here, we have explored the cause of filamentation and identified a potentially important client of E. coli Hsp90 (Hsp90Ec), FtsZ. We observed that FtsZ, a bacterial tubulin homolog essential for cell division, fails to assemble into FtsZ rings (divisomes) in cells overexpressing Hsp90Ec. Additionally, Hsp90Ec interacts with FtsZ and inhibits polymerization of FtsZ in vitro, in an ATP-independent holding reaction. The FtsZ–Hsp90Ec interaction involves residues in the client-binding region of Hsp90Ec and in the C-terminal tail of FtsZ, where many cell-division proteins and regulators interact. We observed that E. coli deleted for the Hsp90Ec gene htpG turn over FtsZ more rapidly than wild-type cells. Additionally, the length of ΔhtpG cells is reduced compared to wild-type cells. Altogether, these results suggest that Hsp90Ec is a modulator of cell division, and imply that the polypeptide-holding function of Hsp90 may be a biologically important chaperone activity.

Selection for arsenite resistance causes reversible changes in minicircle composition and kinetoplast organization in Leishmania mexicana

1994 ◽  
Vol 14 (1) ◽  
pp. 587-596
Author(s):  
S T Lee ◽  
H Y Liu ◽  
S P Lee ◽  
C Tarn
Keyword(s):  
Nuclear Dna ◽  
Transition Period ◽  
Electron Microscopic Examination ◽  
Leishmania Mexicana ◽  
Wild Type ◽  
Electron Microscopic ◽  
Dot Hybridization ◽  
Arsenite Resistance ◽  
Leishmania Parasites

Certain minor minicircle sequence classes in the kinetoplast DNA (kDNA) networks of arsenite- or tunicamycin-resistant Leishmania mexicana amazonensis variants whose nuclear DNA is amplified appear to be preferentially selected to replicate (S. T. Lee, C. Tarn, and K. P. Chang, Mol. Biochem. Parasitol. 58:187-204, 1993). These sequences replace the predominant wild-type minicircle sequences to become dominant species in the kDNA network. The switch from wild-type-specific to variant-specific minicircles takes place rapidly within the same network, the period of minicircle dominance changes being defined as the transition period. To investigate the structural organization of the kDNA networks during this transition period, we analyzed kDNA from whole arsenite-resistant Leishmania parasites by dot hybridization with sequence-specific DNA probes and by electron-microscopic examination of isolated kDNA networks in vitro. Both analyses concluded that during the switch of dominance the predominant wild-type minicircle class was rapidly lost and that selective replication of variant-specific minicircles subsequently filled the network step by step. There was a time during the transition when few wild-type- or variant-specific minicircles were present, leaving the network almost empty and exposing a species of thick, long, fibrous DNA which seemed to form a skeleton for the network. Both minicircles and maxicircles were found to attach to these long DNA fibrils. The nature of the long DNA fibrils is not clear, but they may be important in providing a framework for the network structure and a support for the replication of minicircles and maxicircles.

Cytoplasmic Granules in Lymphocytes from Rats Dosed with SK&F 14336-D

1971 ◽  
Vol 29 ◽  
pp. 556-557
Author(s):  
T. G. Merrill ◽  
B. J. Payne ◽  
A. J. Tousimis
Keyword(s):  
Lipid Storage ◽  
Pokeweed Mitogen ◽  
Electron Microscopic ◽  
Cytoplasmic Granules ◽  
Storage Diseases ◽  
Tay Sachs Disease ◽  
Large Numbers ◽  
Microscopic Studies ◽  
Neurovisceral Lipidosis

Rats given SK&F 14336-D (9-[3-Dimethylamino propyl]-2-chloroacridane), a tranquilizing drug, developed an increased number of vacuolated lymphocytes as observed by light microscopy. Vacuoles in peripheral blood of rats and humans apparently are rare and are not usually reported in differential counts. Transforming agents such as phytohemagglutinin and pokeweed mitogen induce similar vacuoles in in vitro cultures of lymphocytes. These vacuoles have also been reported in some of the lipid-storage diseases of humans such as amaurotic familial idiocy, familial neurovisceral lipidosis, lipomucopolysaccharidosis and sphingomyelinosis. Electron microscopic studies of Tay-Sachs' disease and of chloroquine treated swine have demonstrated large numbers of “membranous cytoplasmic granules” in the cytoplasm of neurons, in addition to lymphocytes. The present study was undertaken with the purpose of characterizing the membranous inclusions and developing an experimental animal model which may be used for the study of lipid storage diseases.

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