scholarly journals Helical Tubes of FtsZ from Methanococcus jannaschii

2000 ◽  
Vol 381 (9-10) ◽  
pp. 993-999 ◽  
Author(s):  
Jan Löwe ◽  
Linda A. Amos
Keyword(s):  
Ring Structure ◽  
High Yield ◽  
Bacterial Cell Division ◽  
Filament Axis ◽  
Thick Filaments ◽  
Z Ring ◽  
Protein Density ◽  
Helical Tubes

AbstractBacterial cell division depends on the formation of a cytokinetic ring structure, the Z-ring. The bacterial tubulin homologue FtsZ is required for Z-ring formation. FtsZ assembles into various polymeric formsin vitro, indicating a structural role in the septum of bacteria. We have used recombinant FtsZ1 protein fromM. jannaschiito produce helical tubes and sheets with high yield using the GTP analogue GMPCPP [guanylyl-(α,β)-methylene-diphosphate]. The sheets appear identical to the previously reported Ca++-induced sheets of FtsZ fromM. jannaschiithat were shown to consist of ‘thick’-filaments in which two protofilaments run in parallel. Tubes assembled either in Ca++or in GMPCPP contain filaments whose dimensions indicate that they could be equivalent to the ‘thick’-filaments in sheets. Some tubes are hollow but others are filled by additional protein density. Helical FtsZ tubes differ from eukaryotic microtubules in that the filaments curve around the filament axis with a pitch of ~ 430 Å for Ca++-induced tubes or 590–620 Å for GMPCPP. However, their assemblyin vitroas well-ordered polymers over distances comparable to the inner circumference of a bacterium may indicate a rolein vivo. Their size and stability make them suitable for use in motility assays.

scholarly journals Probing for Binding Regions of the FtsZ Protein Surface through Site-Directed Insertions: Discovery of Fully Functional FtsZ-Fluorescent Proteins

2016 ◽  
Vol 199 (1) ◽  
Author(s):  
Desmond A. Moore ◽  
Zakiya N. Whatley ◽  
Chandra P. Joshi ◽  
Masaki Osawa ◽  
Harold P. Erickson
Keyword(s):  
Cell Division ◽  
Fluorescent Proteins ◽  
Sole Source ◽  
Ring Structure ◽  
Content Type ◽  
Z Ring ◽  
Complete Cell ◽  
The Right

ABSTRACT FtsZ, a bacterial tubulin homologue, is a cytoskeletal protein that assembles into protofilaments that are one subunit thick. These protofilaments assemble further to form a “Z ring” at the center of prokaryotic cells. The Z ring generates a constriction force on the inner membrane and also serves as a scaffold to recruit cell wall remodeling proteins for complete cell division in vivo. One model of the Z ring proposes that protofilaments associate via lateral bonds to form ribbons; however, lateral bonds are still only hypothetical. To explore potential lateral bonding sites, we probed the surface of Escherichia coli FtsZ by inserting either small peptides or whole fluorescent proteins (FPs). Among the four lateral surfaces on FtsZ protofilaments, we obtained inserts on the front and back surfaces that were functional for cell division. We concluded that these faces are not sites of essential interactions. Inserts at two sites, G124 and R174, located on the left and right surfaces, completely blocked function, and these sites were identified as possible sites for essential lateral interactions. However, the insert at R174 did not interfere with association of protofilaments into sheets and bundles in vitro. Another goal was to find a location within FtsZ that supported insertion of FP reporter proteins while allowing the FtsZ-FPs to function as the sole source of FtsZ. We discovered one internal site, G55-Q56, where several different FPs could be inserted without impairing function. These FtsZ-FPs may provide advances for imaging Z-ring structure by superresolution techniques. IMPORTANCE One model for the Z-ring structure proposes that protofilaments are assembled into ribbons by lateral bonds between FtsZ subunits. Our study excluded the involvement of the front and back faces of the protofilament in essential interactions in vivo but pointed to two potential lateral bond sites, on the right and left sides. We also identified an FtsZ loop where various fluorescent proteins could be inserted without blocking function; these FtsZ-FPs functioned as the sole source of FtsZ. This advance provides improved tools for all fluorescence imaging of the Z ring and may be especially important for superresolution imaging.

scholarly journals Architecture of the ring formed by the tubulin homologue FtsZ in bacterial cell division

eLife ◽  
2014 ◽  
Vol 3 ◽  
Author(s):  
Piotr Szwedziak ◽  
Qing Wang ◽  
Tanmay A M Bharat ◽  
Matthew Tsim ◽  
Jan Löwe
Keyword(s):  
Cell Division ◽  
Molecular Model ◽  
Three Dimensional ◽  
Bacterial Cell Division ◽  
Electron Cryomicroscopy ◽  
E Coli ◽  
Ftsz Ring ◽  
Z Ring

Membrane constriction is a prerequisite for cell division. The most common membrane constriction system in prokaryotes is based on the tubulin homologue FtsZ, whose filaments in E. coli are anchored to the membrane by FtsA and enable the formation of the Z-ring and divisome. The precise architecture of the FtsZ ring has remained enigmatic. In this study, we report three-dimensional arrangements of FtsZ and FtsA filaments in C. crescentus and E. coli cells and inside constricting liposomes by means of electron cryomicroscopy and cryotomography. In vivo and in vitro, the Z-ring is composed of a small, single-layered band of filaments parallel to the membrane, creating a continuous ring through lateral filament contacts. Visualisation of the in vitro reconstituted constrictions as well as a complete tracing of the helical paths of the filaments with a molecular model favour a mechanism of FtsZ-based membrane constriction that is likely to be accompanied by filament sliding.

scholarly journals A Novel Z-Ring Associated Protein ZapA-Like Protein (PA5407) From Pseudomonas aeruginosa Promotes FtsZ to Form Double Filaments

2021 ◽  
Vol 12 ◽  
Author(s):  
Xiaoyu Wang ◽  
Xueqin Ma ◽  
Zhe Li ◽  
Mingyue Niu ◽  
Meiting Zhai ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Cell Division ◽  
Bacterial Cell ◽  
Regulatory Protein ◽  
Ring Structure ◽  
Bacterial Cell Division ◽  
Bacterial Division ◽  
Z Ring ◽  
Almost All

Bacterial cell division is initiated by the assembly of the contraction ring (Z-ring), which consists of the self-assembled FtsZ protofilaments and dozens of other associate proteins. ZapA, a regulatory protein found in almost all bacteria, stabilizes FtsZ protofilaments to form bundles and enhances the Z-ring condensation. Here, we reported that another small protein from Pseudomonas aeruginosa, ZapA-Like protein (ZapAL; PA5407), is a new FtsZ associated protein. ZapAL exists in many Pseudomonas species and shares only 20% sequence identity to ZapA. ZapAL interacts with FtsZ and induces FtsZ to form long straight double filaments; in comparison, ZapA promotes long bundles with multiple FtsZ filaments. ZapAL has only a mild effect on GTPase activity of FtsZ, which is reduced by around 26% when 10 μM ZapAL is added in the solution. However, to study their assembly dynamics using light-scattering assay, we found that FtsZ-ZapAL double filament is stable and no depolymerization process is observed, which is different from ZapA. Further research found that ZapA and ZapL are likely to form heterodimers. The bundles formed by the mixture of FtsZ-ZapA-ZapAL will depolymerize after GTP is hydrolyzed. Consistent with ZapAL interaction with FtsZ in vitro, the expression of ZapAL-GFP was observed as a narrow band or spots in the middle of the cells, suggesting that it is a component of bacterial division machinery. Similar to ZapA, ZapAL is also not essential for bacterial cell division. Little changes were observed when zapAL gene was deleted, or overexpressed under normal conditions; however, overexpression of ZapAL caused zapA-deficient cells to grow approximately two times longer, showing a mild bacterial division defect. Although we still do not know the exact physiological roles of ZapAL, our results suggest that ZapAL is a novel Z-ring associate protein, which may work together with ZapA to stabilize the FtsZ protofilament and Z-ring structure.

scholarly journals A conserved cell division protein directly regulates FtsZ dynamics in filamentous and unicellular actinobacteria

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Félix Ramos-León ◽  
Matthew J Bush ◽  
Joseph W Sallmen ◽  
Govind Chandra ◽  
Jake Richardson ◽  
...  
Keyword(s):  
Cell Division ◽  
Bacterial Cell ◽  
Mycobacterium Smegmatis ◽  
Bacterial Cell Division ◽  
Z Ring ◽  
Cell Division Protein ◽  
Ring Architecture ◽  
Contractile Structure

Bacterial cell division is driven by the polymerization of the GTPase FtsZ into a contractile structure, the so-called Z-ring. This essential process involves proteins that modulate FtsZ dynamics and hence the overall Z-ring architecture. Actinobacteria like Streptomyces and Mycobacterium lack known key FtsZ-regulators. Here we report the identification of SepH, a conserved actinobacterial protein that directly regulates FtsZ dynamics. We show that SepH is crucially involved in cell division in Streptomyces venezuelae and that it binds FtsZ via a conserved helix-turn-helix motif, stimulating the assembly of FtsZ protofilaments. Comparative in vitro studies using the SepH homolog from Mycobacterium smegmatis further reveal that SepH can also bundle FtsZ protofilaments, indicating an additional Z-ring stabilizing function in vivo. We propose that SepH plays a crucial role at the onset of cytokinesis in actinobacteria by promoting the assembly of FtsZ filaments into division-competent Z-rings that can go on to mediate septum synthesis.

scholarly journals Investigation of Regulation of FtsZ Assembly by SulA and Development of a Model for FtsZ Polymerization

2008 ◽  
Vol 190 (7) ◽  
pp. 2513-2526 ◽  
Author(s):  
Alex Dajkovic ◽  
Amit Mukherjee ◽  
Joe Lutkenhaus
Keyword(s):  
Kinetic Scheme ◽  
Sos Response ◽  
Ring Structure ◽  
Linear Polymers ◽  
Gtpase Activity ◽  
Gtp Hydrolysis ◽  
Z Ring ◽  
Ftsz Assembly

ABSTRACT In Escherichia coli FtsZ organizes into a cytoskeletal ring structure, the Z ring, which effects cell division. FtsZ is a GTPase, but the free energy of GTP hydrolysis does not appear to be used for generation of the constriction force, leaving open the question of the function of the GTPase activity of FtsZ. Here we study the mechanism by which SulA, an inhibitor of FtsZ induced during the SOS response, inhibits FtsZ function. We studied the effects of SulA on the in vitro activities of FtsZ, on Z rings in vivo, and on a kinetic model for FtsZ polymerization in silico. We found that the binding of SulA to FtsZ is necessary but not sufficient for inhibition of polymerization, since the assembly of FtsZ polymers in the absence of the GTPase activity was not inhibited by SulA. We developed a new model for FtsZ polymerization that accounts for the cooperativity of FtsZ and could account for cooperativity observed in other linear polymers. When SulA was included in the kinetic scheme, simulations revealed that SulA with strong affinity for FtsZ delayed, but did not prevent, the assembly of polymers when they were not hydrolyzing GTP. Furthermore, the simulations indicated that SulA controls the assembly of FtsZ by binding to a polymerization-competent form of the FtsZ molecule and preventing it from participating in assembly. In vivo stoichiometry of the disruption of Z rings by SulA suggests that FtsZ may undergo two cooperative transitions in forming the Z ring.

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

Microbiology ◽  
2003 ◽  
Vol 149 (8) ◽  
pp. 2235-2242 ◽  
Author(s):  
Elaine Small ◽  
Stephen G. Addinall
Keyword(s):  
Steady State ◽  
Cell Division ◽  
Dynamic Properties ◽  
Bacterial Cell Division ◽  
Regeneration System ◽  
Z Ring ◽  
Cell Division Protein ◽  
Hydrolysis Of

In vitro polymerization of the essential bacterial cell division protein FtsZ, in the presence of GTP, is rapid and transient due to its efficient binding and hydrolysis of GTP. In contrast, the in vivo polymeric FtsZ structure which drives cell division – the Z-ring – is present in cells for extended periods of time whilst undergoing constant turnover of FtsZ. It is demonstrated that dynamic polymerization of Escherichia coli FtsZ in vitro is sensitive to the ratio of GTP to GDP concentration. Increase of GDP concentration in the presence of a constant GTP concentration reduces both the duration of FtsZ polymerization and the initial light-scattering maximum which occurs upon addition of GTP. It is also demonstrated that by use of a GTP-regeneration system, polymers of FtsZ can be maintained in a steady state for up to 85 min, while preserving their dynamic properties. The authors therefore present the use of a GTP-regeneration system for FtsZ polymerization as an assay more representative of the in vivo situation, where FtsZ polymers are subject to a constant, relatively high GTP to GDP ratio.

scholarly journals A conserved cell division protein directly regulates FtsZ dynamics in filamentous and unicellular actinobacteria

2020 ◽  
Author(s):  
Felix Ramos-Léon ◽  
Matthew J. Bush ◽  
Joseph W. Sallmen ◽  
Govind Chandra ◽  
Jake Richardson ◽  
...  
Keyword(s):  
Cell Division ◽  
Bacterial Cell ◽  
Crucial Role ◽  
Bacterial Cell Division ◽  
Z Ring ◽  
Cell Division Protein ◽  
Ring Architecture ◽  
Contractile Structure

AbstractBacterial cell division is driven by the polymerization of the GTPase FtsZ into a contractile structure, the so-called Z-ring. This essential process involves proteins that modulate FtsZ dynamics and hence the overall Z-ring architecture. Actinobacteria, like Streptomyces and Mycobacterium lack known key FtsZ-regulators. Here we report the identification of SepH, a conserved actinobacterial protein that directly regulates FtsZ dynamics. We show that SepH is crucially involved in cell division in Streptomyces and that it binds FtsZ via a conserved helix-turn-helix motif, stimulating the assembly of FtsZ protofilaments. Comparative in vitro studies using the SepH homolog from Mycobacterium further reveal that SepH can also bundle FtsZ protofilaments, indicating an additional Z-ring stabilizing function in vivo. We propose that SepH plays a crucial role at the onset of cytokinesis in actinobacteria by promoting the rapid assembly of FtsZ filaments into division-competent Z-rings that can go on to mediate septum synthesis.

scholarly journals Species- and C-terminal linker-dependent variations in the dynamic behavior of FtsZ on membranesin vitro

2018 ◽  
Author(s):  
Kousik Sundararajan ◽  
Anthony Vecchiarelli ◽  
Kiyoshi Mizuuchi ◽  
Erin D. Goley
Keyword(s):  
Cell Wall ◽  
Lipid Bilayers ◽  
Caulobacter Crescentus ◽  
Dynamic Structure ◽  
Bacterial Cell Division ◽  
Filament Bundles ◽  
Z Ring ◽  
Local Cell

SummaryBacterial cell division requires the assembly of FtsZ protofilaments into a dynamic structure called the ‘Z-ring’. The Z-ring recruits the division machinery and directs local cell wall remodeling for constriction. The organization and dynamics of protofilaments within the Z-ring coordinate local cell wall synthesis during cell constriction, but their regulation is largely unknown. The disordered C-terminal linker (CTL) region ofCaulobacter crescentusFtsZ (CcFtsZ) regulates polymer structure and turnover in solutionin vitro, and regulates Z-ring structure and activity of cell wall enzymesin vivo. To investigate the contributions of the CTL to the polymerization properties of FtsZ on its physiological platform, the cell membrane, we reconstitutedCcFtsZ polymerization on supported lipid bilayers (SLB) and visualized polymer dynamics and structure using total internal reflection fluorescence microscopy. UnlikeE. coliFtsZ protofilaments that organized into large, bundled patterns,CcFtsZ protofilaments assembled into small, dynamic clusters on SLBs. Moreover,CcFtsZ lacking its CTL formed large networks of straight filament bundles that underwent slower turnover than the dynamic clusters of wildtype FtsZ. Ourin vitrocharacterization provides novel insights into species- and CTL-dependent differences between FtsZ assembly properties that are relevant to Z-ring assembly and function on membranesin vivo.

A Simple Silicon Based Nitric Oxide Sensor

1999 ◽  
Author(s):  
Marcelo Bariatto ◽  
Rogerio Furlan ◽  
Koiti Arakai ◽  
Jorge J. Santiago-Aviles
Keyword(s):  
Nitric Oxide ◽  
Amperometric Detection ◽  
Cellular Level ◽  
High Yield ◽  
Sensor Calibration ◽  
Nitric Oxide Sensor ◽  
Sensor Configuration

Abstract Nitric oxide (NO) is known to mediate many beneficial physiology processes, motivating its detection in vivo as well as in vitro. Electrochemical detection provides the required cellular level determination of NO among several other techniques. In this work, electrochemical micro-sensors for both types of detection, in vivo and in vitro, were developed, exploring the silicon planar technology, which presents high yield and reliability and also permits batch fabrication. The developed in vitro sensor features eight detection sites (10 μm × 10 μm microelectrodes), for determination of nitric oxide spatial distribution or multi-species analysis. Different electrochemical methods were applied to provide sensor calibration and chemical reproducibility. For in vivo analysis, the designed structures have a needle shape (40 μm thick) and they were silicon micro-machined by using plasma etching or etch stop techniques. Different configurations were designed and implemented, containing a number of detection microelectrodes that vary from 2 to 10. The amperometric detection of both nitric oxide and nitride (NO2−) — a molecule that causes an interference — were investigated by using the in vitro micro-sensor configuration. The need of a cationic exchanger (Nafion) was demonstrated in order to provide selectivity to NO for low concentrations. Also, the developed sensor has a sensitivity of 500 A/M.cm2 and a detection limit of 10 μM.

scholarly journals Uncovering and Engineering a Mini-Regulatory Network of the TetR-Family Regulator SACE_0303 for Yield Improvement of Erythromycin in Saccharopolyspora erythraea

2021 ◽  
Vol 9 ◽  
Author(s):  
Ying Liu ◽  
Sabir Khan ◽  
Panpan Wu ◽  
Bowen Li ◽  
Lanlan Liu ◽  
...  
Keyword(s):  
Regulatory Network ◽  
Target Genes ◽  
Antibacterial Activities ◽  
Saccharopolyspora Erythraea ◽  
High Yield ◽  
Yield Strain ◽  
Yield Improvement ◽  
Erythromycin Production

Erythromycins produced by Saccharopolyspora erythraea have broad-spectrum antibacterial activities. Recently, several TetR-family transcriptional regulators (TFRs) were identified to control erythromycin production by multiplex control modes; however, their regulatory network remains poorly understood. In this study, we report a novel TFR, SACE_0303, positively correlated with erythromycin production in Sac. erythraea. It directly represses its adjacent gene SACE_0304 encoding a MarR-family regulator and indirectly stimulates the erythromycin biosynthetic gene eryAI and resistance gene ermE. SACE_0304 negatively regulates erythromycin biosynthesis by directly inhibiting SACE_0303 as well as eryAI and indirectly repressing ermE. Then, the SACE_0303 binding site within the SACE_0303-SACE_0304 intergenic region was defined. Through genome scanning combined with in vivo and in vitro experiments, three additional SACE_0303 target genes (SACE_2467 encoding cation-transporting ATPase, SACE_3156 encoding a large transcriptional regulator, SACE_5222 encoding α-ketoglutarate permease) were identified and proved to negatively affect erythromycin production. Finally, by coupling CRISPRi-based repression of those three targets with SACE_0304 deletion and SACE_0303 overexpression, we performed stepwise engineering of the SACE_0303-mediated mini-regulatory network in a high-yield strain, resulting in enhanced erythromycin production by 67%. In conclusion, the present study uncovered the regulatory network of a novel TFR for control of erythromycin production and provides a multiplex tactic to facilitate the engineering of industrial actinomycetes for yield improvement of antibiotics.

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