scholarly journals Slow Polymerization of Mycobacterium tuberculosis FtsZ

2000 ◽  
Vol 182 (14) ◽  
pp. 4028-4034 ◽  
Author(s):  
E. Lucile White ◽  
Larry J. Ross ◽  
Robert C. Reynolds ◽  
Lainne E. Seitz ◽  
Georgia D. Moore ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Escherichia Coli ◽  
Mycobacterium Tuberculosis ◽  
Tubulin Polymerization ◽  
Gtpase Activity ◽  
Gtp Hydrolysis ◽  
Minimum Concentration ◽  
E Coli ◽  
Cell Division Protein ◽  
Mycobacterial Protein

ABSTRACT The essential cell division protein, FtsZ, from Mycobacterium tuberculosis has been expressed in Escherichia coliand purified. The recombinant protein has GTPase activity typical of tubulin and other FtsZs. FtsZ polymerization was studied using 90° light scattering. The mycobacterial protein reaches maximum polymerization much more slowly (∼10 min) than E. coliFtsZ. Depolymerization also occurs slowly, taking 1 h or longer under most conditions. Polymerization requires both Mg2+and GTP. The minimum concentration of FtsZ needed for polymerization is 3 μM. Electron microscopy shows that polymerized M. tuberculosis FtsZ consists of strands that associate to form ordered aggregates of parallel protofilaments. Ethyl 6-amino-2,3-dihydro-4-phenyl-1H-pyrido[4,3-b][1,4]diazepin-8-ylcarbamate (SRI 7614), an inhibitor of tubulin polymerization synthesized at Southern Research Institute, inhibits M. tuberculosis FtsZ polymerization, inhibits GTP hydrolysis, and reduces the number and sizes of FtsZ polymers.

In Vitro Antibacterial Activity and Mechanism of Monocaprylin against Escherichia coli and Staphylococcus aureus

2018 ◽  
Vol 81 (12) ◽  
pp. 1988-1996 ◽  
Author(s):  
JIANYU WANG ◽  
MAOMAO MA ◽  
JUN YANG ◽  
LONG CHEN ◽  
PING YU ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Escherichia Coli ◽  
Staphylococcus Aureus ◽  
Antibacterial Activity ◽  
Membrane Potential ◽  
Cell Membrane ◽  
Sodium Benzoate ◽  
Rapid Change ◽  
Potassium Sorbate ◽  
E Coli

ABSTRACT In the present study, the antibacterial activity of monocaprylin in comparison with sodium benzoate and potassium sorbate against Staphylococcus aureus and Escherichia coli was assessed by measuring MIC, MBC, effect of pH on MIC, and incubation temperature on bactericidal efficacy. Results showed that monocaprylin exhibited an excellent antibacterial activity against both strains, with the lowest MIC and MBC of 1.28 mg/mL. A MIC of monocaprylin remained unchanged despite the pH values of culture medium, ranging from 5 to 9, unlike that of potassium sorbate or sodium benzoate. Furthermore, monocaprylin at MBC effectively reduced the population of E. coli and S. aureus by >5.5 log CFU/mL at 25°C within 6 h and decreased E. coli by approximately 5.0 log CFU/mL and S. aureus by 2.9 log CFU/mL at 12 h. The underlying mechanism of monocaprylin was then investigated by measuring β-galactosidase activity, membrane potential, release of cellular contents, scanning electron microscopy, and transmission electron microscopy observations. Results indicated that monocaprylin killed E. coli by the rapid change in permeability and integrity of cell membrane, leading to decline of membrane potential, leakage of nucleic acids and proteins, and ultimately cell membrane disintegration and lysis. On the other hand, monocaprylin might exert its antibacterial activity against S. aureus mainly by diffusing across the cell wall, collapsing the cell membrane, and disturbing the order of intracellular contents. These findings indicated that monocaprylin had better antibacterial ability compared with traditional synthetic preservatives and might be a potential antibacterial additive independent of pH.

scholarly journals Homologous bd oxidases share the same architecture but differ in mechanism

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Alexander Theßeling ◽  
Tim Rasmussen ◽  
Sabrina Burschel ◽  
Daniel Wohlwend ◽  
Jan Kägi ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Escherichia Coli ◽  
Specific Inhibitor ◽  
Protonmotive Force ◽  
Geobacillus Thermodenitrificans ◽  
E Coli ◽  
Cryo Electron Microscopy ◽  
Respiratory Chains

Abstract Cytochrome bd oxidases are terminal reductases of bacterial and archaeal respiratory chains. The enzyme couples the oxidation of ubiquinol or menaquinol with the reduction of dioxygen to water, thus contributing to the generation of the protonmotive force. Here, we determine the structure of the Escherichia coli bd oxidase treated with the specific inhibitor aurachin by cryo-electron microscopy (cryo-EM). The major subunits CydA and CydB are related by a pseudo two fold symmetry. The heme b and d cofactors are found in CydA, while ubiquinone-8 is bound at the homologous positions in CydB to stabilize its structure. The architecture of the E. coli enzyme is highly similar to that of Geobacillus thermodenitrificans, however, the positions of heme b595 and d are interchanged, and a common oxygen channel is blocked by a fourth subunit and substituted by a more narrow, alternative channel. Thus, with the same overall fold, the homologous enzymes exhibit a different mechanism.

scholarly journals Association of ω with the C-Terminal Region of the β′ Subunit Is Essential for Assembly of RNA Polymerase in Mycobacterium tuberculosis

2018 ◽  
Vol 200 (12) ◽  
Author(s):  
Chunyou Mao ◽  
Yan Zhu ◽  
Pei Lu ◽  
Lipeng Feng ◽  
Shiyun Chen ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Mycobacterium Tuberculosis ◽  
Rna Polymerase ◽  
Essential Role ◽  
Β Subunit ◽  
Content Type ◽  
E Coli ◽  
Core Enzyme ◽  
Loop Region

ABSTRACT The ω subunit is the smallest subunit of bacterial RNA polymerase (RNAP). Although homologs of ω are essential in both eukaryotes and archaea, this subunit has been known to be dispensable for RNAP in Escherichia coli and in other bacteria. In this study, we characterized an indispensable role of the ω subunit in Mycobacterium tuberculosis . Unlike the well-studied E. coli RNAP, the M. tuberculosis RNAP core enzyme cannot be functionally assembled in the absence of the ω subunit. Importantly, substitution of M. tuberculosis ω with ω subunits from E. coli or Thermus thermophilus cannot restore the assembly of M. tuberculosis RNAP. Furthermore, by replacing different regions in M. tuberculosis ω with the corresponding regions from E. coli ω, we found a nonconserved loop region in M. tuberculosis ω essential for its function in RNAP assembly. From RNAP structures, we noticed that the location of the C-terminal region of the β′ subunit (β′CTD) in M. tuberculosis RNAP but not in E. coli or T. thermophilus RNAP is close to the ω loop region. Deletion of this β′CTD in M. tuberculosis RNAP destabilized the binding of M. tuberculosis ω on RNAP and compromised M. tuberculosis core assembly, suggesting that these two regions may function together to play a role in ω-dependent RNAP assembly in M. tuberculosis . Sequence alignment of the ω loop and the β′CTD regions suggests that the essential role of ω is probably restricted to mycobacteria. Together, our study characterized an essential role of M. tuberculosis ω and highlighted the importance of the ω loop region in M. tuberculosis RNAP assembly. IMPORTANCE DNA-dependent RNA polymerase (RNAP), which consists of a multisubunit core enzyme (α 2 ββ′ω) and a dissociable σ subunit, is the only enzyme in charge of transcription in bacteria. As the smallest subunit, the roles of ω remain the least well studied. In Escherichia coli and some other bacteria, the ω subunit is known to be nonessential for RNAP. In this study, we revealed an essential role of the ω subunit for RNAP assembly in the human pathogen Mycobacterium tuberculosis , and a mycobacterium-specific ω loop that plays a role in this function was also characterized. Our study provides fresh insights for further characterizing the roles of bacterial ω subunit.

scholarly journals Studies of the Interaction of Escherichia coli YjeQ with the Ribosome In Vitro

2004 ◽  
Vol 186 (5) ◽  
pp. 1381-1387 ◽  
Author(s):  
Denis M. Daigle ◽  
Eric D. Brown
Keyword(s):  
Escherichia Coli ◽  
Ribosomal Subunit ◽  
Gtp Hydrolysis ◽  
E Coli ◽  
Cell Extracts ◽  
30S Subunit ◽  
Ob Fold ◽  
Nucleotide Binding Proteins ◽  
Dependent Interaction

ABSTRACT Escherichia coli YjeQ represents a conserved group of bacteria-specific nucleotide-binding proteins of unknown physiological function that have been shown to be essential to the growth of E. coli and Bacillus subtilis. The protein has previously been characterized as possessing a slow steady-state GTP hydrolysis activity (8 h−1) (D. M. Daigle, L. Rossi, A. M. Berghuis, L. Aravind, E. V. Koonin, and E. D. Brown, Biochemistry 41: 11109-11117, 2002). In the work reported here, YjeQ from E. coli was found to copurify with ribosomes from cell extracts. The copy number of the protein per cell was nevertheless low relative to the number of ribosomes (ratio of YjeQ copies to ribosomes, 1:200). In vitro, recombinant YjeQ protein interacted strongly with the 30S ribosomal subunit, and the stringency of that interaction, revealed with salt washes, was highest in the presence of the nonhydrolyzable GTP analog 5′-guanylylimidodiphosphate (GMP-PNP). Likewise, association with the 30S subunit resulted in a 160-fold stimulation of YjeQ GTPase activity, which reached a maximum with stoichiometric amounts of ribosomes. N-terminal truncation variants of YjeQ revealed that the predicted OB-fold region was essential for ribosome binding and GTPase stimulation, and they showed that an N-terminal peptide (amino acids 1 to 20 in YjeQ) was necessary for the GMP-PNP-dependent interaction of YjeQ with the 30S subunit. Taken together, these data indicate that the YjeQ protein participates in a guanine nucleotide-dependent interaction with the ribosome and implicate this conserved, essential GTPase as a novel factor in ribosome function.

scholarly journals Efeito de Óleos Essenciais Sobre Bactérias Gram-Negativas Isoladas de Alimentos

2019 ◽  
Vol 23 (3) ◽  
pp. 180
Author(s):  
Bruno Antunes Contrucci ◽  
Rosimeire Silva ◽  
Roberto Andreani Junior ◽  
Dora Inés Kozusny-Andreani
Keyword(s):  
Escherichia Coli ◽  
Pseudomonas Aeruginosa ◽  
Essential Oils ◽  
Rosmarinus Officinalis ◽  
Citrus Limon ◽  
Cymbopogon Citratus ◽  
Prunus Amygdalus ◽  
Cinnamomum Zeylanicum ◽  
Minimum Concentration ◽  
E Coli

Os óleos essenciais são produtos do metabolismo secundário de plantas e são conhecidos por possuir diferentes propriedades biológicas, incluindo atividades antimicrobianas, podendo agir como antibacteriano, antifúngico e antiviral. Objetivou-se nesta pesquisa avaliar a atividade antibacteriana de óleos essenciais sobre cepas de Escherichia coli e Pseudomonas aeruginosa isoladas de alimentos. Foram utilizados os óleos de Eucalyptus globolus (eucalipto comum), Prunus amygdalus (amêndoa), Cymbopongon nardus (citronela), Rosmarinus officinalis (alecrim), Cinnamomum zeylanicum (caneleira), Cymbopogon citratus (capim limão), Citrus limon (limão), Caryophyllus aromaticus (cravo). Foram utilizadas dez linhagens de E. coli e dez de P. aeuriginosa. Para determinação da Concentração Inibitória Mínima (CIM) dos óleos essenciais foi utilizado o método de microdiluição em placas de 96 poços. As concentrações bactericidas mínimas (CBM) foram determinadas a partir dos resultados da CIM. Designou-se como CBM a concentração mínima em que não ocorreu crescimento bacteriano. Verificou-se  que todos os óleos essenciais apresentaram atividade antibacteriana, no entanto os óleos de E. gobulus e R. officinalis foram mais ativos frente a E. coli (CBM=3,13%), e menos eficazes frente a P. aeruginosa (CBM=25%), enquanto que o de C. nardus apresentou atividade biológica frente a P. aeruginosa na concentração de 6,25%. A atividade antimicrobiana dos óleos essenciais testados aponta a possibilidade de desenvolver agentes antimicrobianos eficientes e de baixo custo no controle de E. coli e P. aeruginosa.  Palavras-chave: Escherichia coli.  Pseudomonas aeruginosa. Plantas Medicinais. Controle.AbstractEssential oils are secondary plant metabolism produtcts and are known to have different biological properties, including antimicrobial activities,which may act as antibacterial, antifungal and antiviral. The objective of this research was to evaluate the essential oils antibacterial activityon strains of Escherichia coli and Pseudomonas aeruginosa isolated from food. Essential oils Eucalyptus globolus, Prunus amygdalus, Cymbopongon nardus, Rosmarinus officinalis, Cinnamomum zeylanicum, Cymbopogon citratus, Citrus limon, Caryophyllus aromaticus wereused. Ten strains of E. coli and ten of P. aeuriginosa were used. To determine the Minimum Inhibitory Concentration (MIC) of the essentialoils, the 96-well plate microdilution method was used. Minimum bactericidal concentrations (MBC) were determined from MIC results. CBM was the minimum concentration at which no bacterial growth occurred. It was verified that all the essential oils presented antibacterial activity, however the oils of Eucaliptus gobulus and Rosmarinus officinalis were more active against E. coli (MBC = 3.13%), and less effective against P. aeruginosa (CBM = 25 %), while that of Cymbopongon nardus showed biological activity against P. aeruginosa at 6.25% concentration. The antimicrobial activity of the tested essential oils indicates the possibility of developing efficient and low cost antimicrobial agents in the control of E. coli and P. aeruginosa.Keywords: Escherichia coli. Pseudomonas aeruginosa. Medicinal Plants. Control.

scholarly journals Bacterial SOS Checkpoint Protein SulA Inhibits Polymerization of Purified FtsZ Cell Division Protein

1998 ◽  
Vol 180 (15) ◽  
pp. 3946-3953 ◽  
Author(s):  
Dorina Trusca ◽  
Solomon Scott ◽  
Chris Thompson ◽  
David Bramhill
Keyword(s):  
Escherichia Coli ◽  
Dna Damage ◽  
Control System ◽  
Cell Division ◽  
Gtpase Activity ◽  
Checkpoint Control ◽  
Checkpoint Protein ◽  
Point Mutant ◽  
Reversible Inhibition ◽  
Cell Division Protein

ABSTRACT Cell division of Escherichia coli is inhibited when the SulA protein is induced in response to DNA damage as part of the SOS checkpoint control system. The SulA protein interacts with the tubulin-like FtsZ division protein. We investigated the effects of purified SulA upon FtsZ. SulA protein inhibits the polymerization and the GTPase activity of FtsZ, while point mutant SulA proteins show little effect on either of these FtsZ activities. SulA did not inhibit the polymerization of purified FtsZ2 mutant protein, which was originally isolated as insensitive to SulA. These studies define polymerization assays for FtsZ which respond to an authentic cellular regulator. The observations presented here support the notion that polymerization of FtsZ is central to its cellular role and that direct, reversible inhibition of FtsZ polymerization by SulA may account for division inhibition.

scholarly journals GTPase activity-coupled treadmilling of the bacterial tubulin FtsZ organizes septal cell-wall synthesis

2016 ◽  
Author(s):  
Xinxing Yang ◽  
Zhixin Lyu ◽  
Amanda Miguel ◽  
Ryan McQuillen ◽  
Kerwyn Casey Huang ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Cell Wall ◽  
Spatial Distribution ◽  
Molecular Composition ◽  
Central Component ◽  
Gtpase Activity ◽  
Cell Wall Synthesis ◽  
Gtp Hydrolysis

AbstractThe bacterial tubulin FtsZ is the central component of the division machinery, coordinating an ensemble of proteins involved in septal cell-wall synthesis to ensure successful constriction. How cells achieve this coordination is unknown. We used a combination of imaging, genetic and biochemical approaches to demonstrate that in Escherichia coli cells FtsZ exhibits dynamic treadmilling predominantly determined by its GTPase activity, and that the treadmilling dynamics directs processive movement of the septal cell-wall synthesis machinery. In FtsZ mutants with severely reduced treadmilling, the spatial distribution of septal synthesis and the molecular composition and ultrastructure of the septal cell wall are substantially altered. Thus, the treadmilling of FtsZ provides a novel and robust mechanism for achieving uniform septal cell-wall synthesis to enable correct new pole morphology.One-sentence summaryThe bacterial tubulin FtsZ uses GTP hydrolysis to power treadmilling, driving processive synthesis of the septal cell wall.

scholarly journals Assembly principles of a unique cage formed by hexameric and decameric E. coli proteins

eLife ◽  
2014 ◽  
Vol 3 ◽  
Author(s):  
Hélène Malet ◽  
Kaiyin Liu ◽  
Majida El Bakkouri ◽  
Sze Wah Samuel Chan ◽  
Gregory Effantin ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Escherichia Coli ◽  
Lysine Decarboxylase ◽  
Lateral Interactions ◽  
E Coli ◽  
Aaa Atpase ◽  
Cryo Electron Microscopy ◽  
Conformational Rearrangements ◽  
The Individual ◽  
Assembly Principles

A 3.3 MDa macromolecular cage between two Escherichia coli proteins with seemingly incompatible symmetries–the hexameric AAA+ ATPase RavA and the decameric inducible lysine decarboxylase LdcI–is reconstructed by cryo-electron microscopy to 11 Å resolution. Combined with a 7.5 Å resolution reconstruction of the minimal complex between LdcI and the LdcI-binding domain of RavA, and the previously solved crystal structures of the individual components, this work enables to build a reliable pseudoatomic model of this unusual architecture and to identify conformational rearrangements and specific elements essential for complex formation. The design of the cage created via lateral interactions between five RavA rings is unique for the diverse AAA+ ATPase superfamily.

scholarly journals Mutagenesis in the α3α4 GyrA Helix and in the Toprim Domain of GyrB Refines the Contribution of Mycobacterium tuberculosis DNA Gyrase to Intrinsic Resistance to Quinolones

2008 ◽  
Vol 52 (8) ◽  
pp. 2909-2914 ◽  
Author(s):  
Stéphanie Matrat ◽  
Alexandra Aubry ◽  
Claudine Mayer ◽  
Vincent Jarlier ◽  
Emmanuelle Cambau
Keyword(s):  
Escherichia Coli ◽  
Mycobacterium Tuberculosis ◽  
Primary Structure ◽  
Three Dimensional ◽  
Dna Gyrase ◽  
Quinolone Resistance ◽  
Dimensional Model ◽  
Three Dimensional Model ◽  
Intrinsic Resistance ◽  
E Coli

ABSTRACT The replacement of M74 in GyrA, A83 in GyrA, and R447 in GyrB of Mycobacterium tuberculosis gyrase by their Escherichia coli homologs resulted in active enzymes as quinolone susceptible as the E. coli gyrase. This demonstrates that the primary structure of gyrase determines intrinsic quinolone resistance and was supported by a three-dimensional model of N-terminal GyrA.

scholarly journals Shape Changes and Interaction Mechanism of Escherichia coli Cells Treated with Sericin and Use of a Sericin-Based Hydrogel for Wound Healing

2016 ◽  
Vol 82 (15) ◽  
pp. 4663-4672 ◽  
Author(s):  
Rui Xue ◽  
Yalong Liu ◽  
Qingsong Zhang ◽  
Congcong Liang ◽  
Huazhen Qin ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Escherichia Coli ◽  
Cell Membrane ◽  
Interaction Mechanism ◽  
Bacterial Cells ◽  
Content Type ◽  
E Coli ◽  
Membrane Blebbing ◽  
Sterile Gauze

ABSTRACTTo verify the interaction mechanism between sericin andEscherichia coli, especially the morphological and structural changes in the bacterial cells, the antimicrobial activity of sericin againstE. colias a model for Gram-negative bacteria was investigated. The antibacterial activity of sericin onE. coliand the interaction mechanism were investigated in this study by analyzing the growth, integrity, and morphology of the bacterial cells following treatment with sericin. The changes in morphology and cellular compositions of bacterial cells treated with sericin were observed by an inverted fluorescence microscope, scanning electron microscopy, and transmission electron microscopy. Changes in electrical conductivity, total sugar concentration of the broth for the bacteria, and protein expression of the bacteria were determined to investigate the permeability of the cell membrane. A sericin-based hydrogel was prepared for anin vivostudy of wound dressing. The results showed that the antibacterial activity of the hydrogel increased with the increase in the concentration of sericin from 10 g/liter to 40 g/liter. The introduction of sericin induces membrane blebbing ofE. colicells caused by antibiotic action on the cell membrane. The cytoplasm shrinkage phenomenon was accompanied by blurring of the membrane wall boundaries. WhenE. colicells were treated with sericin, release of intracellular components quickly increased. The electrical conductivity assay indicated that the charged ions are reduced after exposure to sericin so that the integrity of the cell membrane is weakened and metabolism is blocked. In addition, sodium dodecyl sulfate-polyacrylamide gel electrophoresis demonstrated that sericin hinders the expression of bacterial protein. Sericin may damage the integrity of the bacterial cell membrane, thereby eventually inhibiting the growth and reproduction ofE. coli. Compared to sterile gauze, the sericin-based hydrogel promoted fibroblast cell proliferation and accelerated the formation of granulation tissues and neovessels.IMPORTANCEThe specific relationship and interaction mechanism between sericin andE. colicells were investigated and elucidated. The results show that after 12 h of treatment, sericin molecules induce membrane blebbing ofE. colicells, and the bacteria show decreases in liquidity and permeability of biological membrane, resulting in alterations in the conductivity of the culture medium and the integrity of the outer membrane. The subsequentin vivoresults demonstrate that the sericin-poly(N-isopropylacrylamide-N,N′-methylene-bis-acrylamide [NIPAm-MBA]) hydrogel accelerated wound healing compared to that with sterile gauze, which is a beneficial result for future applications in clinical medicine and the textile, food, and coating industries.

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