The Experts below are selected from a list of 261 Experts worldwide ranked by ideXlab platform
Terry J Beveridge - One of the best experts on this subject based on the ideXlab platform.
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Native Cell Wall Organization Shown by Cryo-Electron Microscopy Confirms the Existence of a Periplasmic Space in Staphylococcus aureus
Journal of bacteriology, 2006Co-Authors: Valerio R F Matias, Terry J BeveridgeAbstract:The current perception of the ultrastructure of gram-positive cell envelopes relies mainly on electron microscopy of thin sections and on sample preparation. Freezing of cells into a matrix of amorphous ice (i.e., vitrification) results in optimal specimen preservation and allows the observation of cell envelope boundary layers in their (frozen) hydrated state. In this report, cryo-transmission electron microscopy of frozen-hydrated sections of Staphylococcus aureus D2C was used to examine cell envelope organization. A bipartite wall was positioned above the plasma membrane and consisted of a 16-nm low-density inner wall zone (IWZ), followed by a 19-nm high-density outer wall zone (OWZ). Observation of plasmolyzed cells, which were used to artificially separate the membrane from the wall, showed membrane vesicles within the Space associated with the IWZ in native cells and a large gap between the membrane and OWZ, suggesting that the IWZ was devoid of a cross-linked polymeric cell wall network. Isolated wall fragments possessed only one zone of high density, with a constant level of density throughout their thickness, as was previously seen with the OWZs of intact cells. These results strongly indicate that the IWZ represents a Periplasmic Space, composed mostly of soluble low-density constituents confined between the plasma membrane and OWZ, and that the OWZ represents the peptidoglycan-teichoic acid cell wall network with its associated proteins. Cell wall differentiation was also seen at the septum of dividing cells. Here, two high-density zones were sandwiched between three low-density zones. It appeared that the septum consisted of an extension of the IWZ and OWZ from the outside peripheral wall, plus a low-density middle zone that separated adjacent septal cross walls, which could contribute to cell separation during division.
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cryo electron microscopy reveals native polymeric cell wall structure in bacillus subtilis 168 and the existence of a Periplasmic Space
Molecular Microbiology, 2005Co-Authors: Valerio R F Matias, Terry J BeveridgeAbstract:Summary Ultrarapid freezing of bacteria (i.e. vitrification) results in optimal preservation of native structure. In this study, cryo-transmission electron microscopy of frozen-hydrated sections was used to gain insight into the organization of the Bacillus subtilis 168 cell envelope. A bipartite structure was seen above the plasma membrane consisting of a low-density 22 nm region above which a higher-density 33 nm region or outer wall zone (OWZ) resided. The interface between these two regions appeared to possess the most mass. In intact and in teichoic acid-extracted wall fragments, only a single region was seen but the mass distribution varied from being dense on the inside to less dense on the outside (i.e. similar to the OWZ). In plasmolysed cells, the inner wall zone (IWZ)'s thickness expanded in size but the OWZ's thickness remained constant. As the IWZ expanded it became filled with plasma membrane vesicles indicating that the IWZ had little substance and was empty of the wall's polymeric network of peptidoglycan and teichoic acid. Together these results strongly suggest that the inner zone actually represents a Periplasmic Space confined between the plasma membrane and the wall matrix and that the OWZ is the peptidoglycan-teichoic acid polymeric network of the wall.
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cryo transmission electron microscopy of frozen hydrated sections of escherichia coli and pseudomonas aeruginosa
Journal of Bacteriology, 2003Co-Authors: Valerio R F Matias, Jacques Dubochet, Ashraf Alamoudi, Terry J BeveridgeAbstract:High-pressure freezing of Escherichia coli K-12 and Pseudomonas aeruginosa PAO1 in the presence of cryoprotectants provided consistent vitrification of cells so that frozen-hydrated sections could be cut, providing ∼2-nm resolution of structure. The size and shape of the bacteria, as well as their surface and cytoplasmic constituents, were nicely preserved and compared well with other published high-resolution techniques. Cells possessed a rich cytoplasm containing a diffuse dispersion of ribosomes and genetic material. Close examination of cells revealed that the Periplasmic Space was compressed during cryosectioning, a finding which provided supporting evidence that this Space is filled by a compressible gel. Since the outer membrane and peptidoglycan layer are bonded together via lipoproteins, the Space between them (although still part of the Periplasmic Space) was not as compacted. Even when this cryosectioning compression was taken into account, there was still substantial variability in the width of the Periplasmic Space. It is possible that the protoplast has some capacity to float freely within the periplasm.
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Cryo-Transmission Electron Microscopy of Frozen-Hydrated Sections of Escherichia coli and Pseudomonas aeruginosa
Journal of bacteriology, 2003Co-Authors: Valerio R F Matias, Jacques Dubochet, Ashraf Al-amoudi, Terry J BeveridgeAbstract:High-pressure freezing of Escherichia coli K-12 and Pseudomonas aeruginosa PAO1 in the presence of cryoprotectants provided consistent vitrification of cells so that frozen-hydrated sections could be cut, providing approximately 2-nm resolution of structure. The size and shape of the bacteria, as well as their surface and cytoplasmic constituents, were nicely preserved and compared well with other published high-resolution techniques. Cells possessed a rich cytoplasm containing a diffuse dispersion of ribosomes and genetic material. Close examination of cells revealed that the Periplasmic Space was compressed during cryosectioning, a finding which provided supporting evidence that this Space is filled by a compressible gel. Since the outer membrane and peptidoglycan layer are bonded together via lipoproteins, the Space between them (although still part of the Periplasmic Space) was not as compacted. Even when this cryosectioning compression was taken into account, there was still substantial variability in the width of the Periplasmic Space. It is possible that the protoplast has some capacity to float freely within the periplasm.
Valerio R F Matias - One of the best experts on this subject based on the ideXlab platform.
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Native Cell Wall Organization Shown by Cryo-Electron Microscopy Confirms the Existence of a Periplasmic Space in Staphylococcus aureus
Journal of bacteriology, 2006Co-Authors: Valerio R F Matias, Terry J BeveridgeAbstract:The current perception of the ultrastructure of gram-positive cell envelopes relies mainly on electron microscopy of thin sections and on sample preparation. Freezing of cells into a matrix of amorphous ice (i.e., vitrification) results in optimal specimen preservation and allows the observation of cell envelope boundary layers in their (frozen) hydrated state. In this report, cryo-transmission electron microscopy of frozen-hydrated sections of Staphylococcus aureus D2C was used to examine cell envelope organization. A bipartite wall was positioned above the plasma membrane and consisted of a 16-nm low-density inner wall zone (IWZ), followed by a 19-nm high-density outer wall zone (OWZ). Observation of plasmolyzed cells, which were used to artificially separate the membrane from the wall, showed membrane vesicles within the Space associated with the IWZ in native cells and a large gap between the membrane and OWZ, suggesting that the IWZ was devoid of a cross-linked polymeric cell wall network. Isolated wall fragments possessed only one zone of high density, with a constant level of density throughout their thickness, as was previously seen with the OWZs of intact cells. These results strongly indicate that the IWZ represents a Periplasmic Space, composed mostly of soluble low-density constituents confined between the plasma membrane and OWZ, and that the OWZ represents the peptidoglycan-teichoic acid cell wall network with its associated proteins. Cell wall differentiation was also seen at the septum of dividing cells. Here, two high-density zones were sandwiched between three low-density zones. It appeared that the septum consisted of an extension of the IWZ and OWZ from the outside peripheral wall, plus a low-density middle zone that separated adjacent septal cross walls, which could contribute to cell separation during division.
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cryo electron microscopy reveals native polymeric cell wall structure in bacillus subtilis 168 and the existence of a Periplasmic Space
Molecular Microbiology, 2005Co-Authors: Valerio R F Matias, Terry J BeveridgeAbstract:Summary Ultrarapid freezing of bacteria (i.e. vitrification) results in optimal preservation of native structure. In this study, cryo-transmission electron microscopy of frozen-hydrated sections was used to gain insight into the organization of the Bacillus subtilis 168 cell envelope. A bipartite structure was seen above the plasma membrane consisting of a low-density 22 nm region above which a higher-density 33 nm region or outer wall zone (OWZ) resided. The interface between these two regions appeared to possess the most mass. In intact and in teichoic acid-extracted wall fragments, only a single region was seen but the mass distribution varied from being dense on the inside to less dense on the outside (i.e. similar to the OWZ). In plasmolysed cells, the inner wall zone (IWZ)'s thickness expanded in size but the OWZ's thickness remained constant. As the IWZ expanded it became filled with plasma membrane vesicles indicating that the IWZ had little substance and was empty of the wall's polymeric network of peptidoglycan and teichoic acid. Together these results strongly suggest that the inner zone actually represents a Periplasmic Space confined between the plasma membrane and the wall matrix and that the OWZ is the peptidoglycan-teichoic acid polymeric network of the wall.
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cryo transmission electron microscopy of frozen hydrated sections of escherichia coli and pseudomonas aeruginosa
Journal of Bacteriology, 2003Co-Authors: Valerio R F Matias, Jacques Dubochet, Ashraf Alamoudi, Terry J BeveridgeAbstract:High-pressure freezing of Escherichia coli K-12 and Pseudomonas aeruginosa PAO1 in the presence of cryoprotectants provided consistent vitrification of cells so that frozen-hydrated sections could be cut, providing ∼2-nm resolution of structure. The size and shape of the bacteria, as well as their surface and cytoplasmic constituents, were nicely preserved and compared well with other published high-resolution techniques. Cells possessed a rich cytoplasm containing a diffuse dispersion of ribosomes and genetic material. Close examination of cells revealed that the Periplasmic Space was compressed during cryosectioning, a finding which provided supporting evidence that this Space is filled by a compressible gel. Since the outer membrane and peptidoglycan layer are bonded together via lipoproteins, the Space between them (although still part of the Periplasmic Space) was not as compacted. Even when this cryosectioning compression was taken into account, there was still substantial variability in the width of the Periplasmic Space. It is possible that the protoplast has some capacity to float freely within the periplasm.
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Cryo-Transmission Electron Microscopy of Frozen-Hydrated Sections of Escherichia coli and Pseudomonas aeruginosa
Journal of bacteriology, 2003Co-Authors: Valerio R F Matias, Jacques Dubochet, Ashraf Al-amoudi, Terry J BeveridgeAbstract:High-pressure freezing of Escherichia coli K-12 and Pseudomonas aeruginosa PAO1 in the presence of cryoprotectants provided consistent vitrification of cells so that frozen-hydrated sections could be cut, providing approximately 2-nm resolution of structure. The size and shape of the bacteria, as well as their surface and cytoplasmic constituents, were nicely preserved and compared well with other published high-resolution techniques. Cells possessed a rich cytoplasm containing a diffuse dispersion of ribosomes and genetic material. Close examination of cells revealed that the Periplasmic Space was compressed during cryosectioning, a finding which provided supporting evidence that this Space is filled by a compressible gel. Since the outer membrane and peptidoglycan layer are bonded together via lipoproteins, the Space between them (although still part of the Periplasmic Space) was not as compacted. Even when this cryosectioning compression was taken into account, there was still substantial variability in the width of the Periplasmic Space. It is possible that the protoplast has some capacity to float freely within the periplasm.
Philippe Leone - One of the best experts on this subject based on the ideXlab platform.
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type ix secretion system porm and gliding machinery gldm form arches spanning the Periplasmic Space
Nature Communications, 2018Co-Authors: Philippe Leone, Jennifer Roche, Maxence S Vincent, Quang Hieu Tran, Aline Desmyter, Eric CascalesAbstract:Type IX secretion system (T9SS), exclusively present in the Bacteroidetes phylum, has been studied mainly in Flavobacterium johnsoniae and Porphyromonas gingivalis. Among the 18 genes, essential for T9SS function, a group of four, porK-N (P. gingivalis) or gldK-N (F. johnsoniae) belongs to a co-transcribed operon that expresses the T9SS core membrane complex. The central component of this complex, PorM (or GldM), is anchored in the inner membrane by a trans-membrane helix and interacts through the outer membrane PorK-N complex. There is a complete lack of available atomic structures for any component of T9SS, including the PorKLMN complex. Here we report the crystal structure of the GldM and PorM Periplasmic domains. Dimeric GldM and PorM, each contain four domains of ~180-A length that span most of the Periplasmic Space. These and previously reported results allow us to propose a model of the T9SS core membrane complex as well as its functional behavior. No structural data for the bacterial type IX secretion system (T9SS) are available so far. Here, the authors present the crystal structures of the Periplasmic domains from two major T9SS components PorM and GldM, which span most of the Periplasmic Space, and propose a putative model of the T9SS core membrane complex.
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Type IX secretion system PorM and gliding machinery GldM form arches spanning the Periplasmic Space.
Nature communications, 2018Co-Authors: Philippe Leone, Jennifer Roche, Maxence S Vincent, Quang Hieu Tran, Aline Desmyter, Eric Cascales, Christine Kellenberger, Christian Cambillau, Alain RousselAbstract:Type IX secretion system (T9SS), exclusively present in the Bacteroidetes phylum, has been studied mainly in Flavobacterium johnsoniae and Porphyromonas gingivalis. Among the 18 genes, essential for T9SS function, a group of four, porK-N (P. gingivalis) or gldK-N (F. johnsoniae) belongs to a co-transcribed operon that expresses the T9SS core membrane complex. The central component of this complex, PorM (or GldM), is anchored in the inner membrane by a trans-membrane helix and interacts through the outer membrane PorK-N complex. There is a complete lack of available atomic structures for any component of T9SS, including the PorKLMN complex. Here we report the crystal structure of the GldM and PorM Periplasmic domains. Dimeric GldM and PorM, each contain four domains of ~180-A length that span most of the Periplasmic Space. These and previously reported results allow us to propose a model of the T9SS core membrane complex as well as its functional behavior.
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Type IX secretion system PorM and gliding machinery GldM form arches spanning the Periplasmic Space
Nature Publishing Group, 2018Co-Authors: Philippe Leone, Jennifer Roche, Maxence S Vincent, Quang Hieu Tran, Aline Desmyter, Eric Cascales, Christine Kellenberger, Christian Cambillau, Alain RousselAbstract:No structural data for the bacterial type IX secretion system (T9SS) are available so far. Here, the authors present the crystal structures of the Periplasmic domains from two major T9SS components PorM and GldM, which span most of the Periplasmic Space, and propose a putative model of the T9SS core membrane complex
Jacques Dubochet - One of the best experts on this subject based on the ideXlab platform.
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Granular Layer in the Periplasmic Space of Gram-Positive Bacteria and Fine Structures of Enterococcus gallinarum and Streptococcus gordonii Septa Revealed by Cryo-Electron Microscopy of Vitreous Sections
Journal of bacteriology, 2006Co-Authors: Benoît Zuber, Marisa Haenni, Tânia Ribeiro, Kathrin Minnig, Fátima Lopes, Philippe Moreillon, Jacques DubochetAbstract:High-resolution structural information on optimally preserved bacterial cells can be obtained with cryo-electron microscopy of vitreous sections. With the help of this technique, the existence of a Periplasmic Space between the plasma membrane and the thick peptidoglycan layer of the gram-positive bacteria Bacillus subtilis and Staphylococcus aureus was recently shown. This raises questions about the mode of polymerization of peptidoglycan. In the present study, we report the structure of the cell envelope of three gram-positive bacteria (B. subtilis, Streptococcus gordonii, and Enterococcus gallinarum). In the three cases, a previously undescribed granular layer adjacent to the plasma membrane is found in the Periplasmic Space. In order to better understand how nascent peptidoglycan is incorporated into the mature peptidoglycan, we investigated cellular regions known to represent the sites of cell wall production. Each of these sites possesses a specific structure. We propose a hypothetic model of peptidoglycan polymerization that accommodates these differences: peptidoglycan precursors could be exported from the cytoplasm to the Periplasmic Space, where they could diffuse until they would interact with the interface between the granular layer and the thick peptidoglycan layer. They could then polymerize with mature peptidoglycan. We report cytoplasmic structures at the E. gallinarum septum that could be interpreted as cytoskeletal elements driving cell division (FtsZ ring). Although immunoelectron microscopy and fluorescence microscopy studies have demonstrated the septal and cytoplasmic localization of FtsZ, direct visualization of in situ FtsZ filaments has not been obtained in any electron microscopy study of fixed and dehydrated bacteria.
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cryo transmission electron microscopy of frozen hydrated sections of escherichia coli and pseudomonas aeruginosa
Journal of Bacteriology, 2003Co-Authors: Valerio R F Matias, Jacques Dubochet, Ashraf Alamoudi, Terry J BeveridgeAbstract:High-pressure freezing of Escherichia coli K-12 and Pseudomonas aeruginosa PAO1 in the presence of cryoprotectants provided consistent vitrification of cells so that frozen-hydrated sections could be cut, providing ∼2-nm resolution of structure. The size and shape of the bacteria, as well as their surface and cytoplasmic constituents, were nicely preserved and compared well with other published high-resolution techniques. Cells possessed a rich cytoplasm containing a diffuse dispersion of ribosomes and genetic material. Close examination of cells revealed that the Periplasmic Space was compressed during cryosectioning, a finding which provided supporting evidence that this Space is filled by a compressible gel. Since the outer membrane and peptidoglycan layer are bonded together via lipoproteins, the Space between them (although still part of the Periplasmic Space) was not as compacted. Even when this cryosectioning compression was taken into account, there was still substantial variability in the width of the Periplasmic Space. It is possible that the protoplast has some capacity to float freely within the periplasm.
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Cryo-Transmission Electron Microscopy of Frozen-Hydrated Sections of Escherichia coli and Pseudomonas aeruginosa
Journal of bacteriology, 2003Co-Authors: Valerio R F Matias, Jacques Dubochet, Ashraf Al-amoudi, Terry J BeveridgeAbstract:High-pressure freezing of Escherichia coli K-12 and Pseudomonas aeruginosa PAO1 in the presence of cryoprotectants provided consistent vitrification of cells so that frozen-hydrated sections could be cut, providing approximately 2-nm resolution of structure. The size and shape of the bacteria, as well as their surface and cytoplasmic constituents, were nicely preserved and compared well with other published high-resolution techniques. Cells possessed a rich cytoplasm containing a diffuse dispersion of ribosomes and genetic material. Close examination of cells revealed that the Periplasmic Space was compressed during cryosectioning, a finding which provided supporting evidence that this Space is filled by a compressible gel. Since the outer membrane and peptidoglycan layer are bonded together via lipoproteins, the Space between them (although still part of the Periplasmic Space) was not as compacted. Even when this cryosectioning compression was taken into account, there was still substantial variability in the width of the Periplasmic Space. It is possible that the protoplast has some capacity to float freely within the periplasm.
Ilhan Kim - One of the best experts on this subject based on the ideXlab platform.
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thioredoxin in the Periplasmic Space of escherichia coli as a physiological electron donor to Periplasmic thiol peroxidase p20
Journal of Biochemistry and Molecular Biology, 1999Co-Authors: Meekyung Cha, Ilhan KimAbstract:We previously reported that a novel thiol peroxidase (p20) from Escherichia coli is a distinct Periplasmic peroxidase that detoxifies hydroperoxides together with glutathione or thioredoxin. Until now, there was no experimental evidence for the presence of thioredoxin (Trx) in the Periplasmic Space. In an attempt to confirm the physiological function of p20 as a thiol peroxidase supported by Trx in the Periplasmic Space, we have purified a Trx activity from the Periplasmic Space of Escherichia coli and identified the Trx as the same protein as the cytoplasmic Trx. The presence of Trx in the Periplasmic Space of Escherichia coli suggests that p20 is a unique extracellular Trx-linked thiol peroxidase.
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overexpression of escherichia coli thiol peroxidase in the Periplasmic Space
Journal of Microbiology and Biotechnology, 1998Co-Authors: Sungjin Kim, Meekyung Cha, Ilhan Kim, Hakun KimAbstract:Overproduction of Escherichia coli thiol peroxidase in the Periplasmic Space was achieved by locating the appropriate gene on a downstream region of the strong T7 promoter. E. coli strain BL21 carrying the recombinant plasmid pSK-TPX was induced by IPTG, lysed, and analyzed by SDS-polyacrylamide gel electrophoresis. A large amount of the overexpressed thiol peroxidase was located in the Periplasmic Space. A homogeneous thiol peroxidase was obtained from E. coli osmotic shock fluid by simple one-step gel permeation chromatography.
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Thioredoxin-linked "Thiol Peroxidase" from Periplasmic Space of Escherichia coli
The Journal of biological chemistry, 1995Co-Authors: Meekyung Cha, Hakun Kim, Ilhan KimAbstract:Three different molecular masses (24, 22, and 20 kDa) of antioxidant proteins were purified in Escherichia coli. These proteins exhibited the preventive effects against the inactivation of glutamine synthetase activity and the cleavage of DNA by a metal-catalyzed oxidation system capable of generating reactive oxygen species. Their antioxidant activities were supported by a thiol-reducing equivalent such as dithiothreitol. Analysis of the amino-terminal amino acid sequences and the immunoblots between 24- and 22-kDa proteins indicates that the 24-kDa protein is an intact form of the 22-kDa protein that was previously identified 22-kDa subunit (AhpC) of E. coli alkyl hydroperoxide reductase (AhpC/AhpF). We isolated and sequenced an E. coli genomic DNA fragment that encodes 20-kDa protein. Comparison of the deduced amino acid sequence of the 20-kDa protein with that of AhpC revealed no sequence homology. A search of a data bank showed that the 20-kDa protein is a new type of antioxidant enzyme. The synthesis of this novel 20-kDa protein was increased in response to oxygen stress during growth. The 20-kDa protein resides mainly in the Periplasmic Space of E. coli, whereas the 24-kDa AhpC resides mainly in the matrix. The 20-kDa protein was functionally linked to the thioredoxin as an in vivo thiol-regenerating system and exerted a peroxidase activity. This 20-kDa protein is thus named "thiol peroxidase," which could act as an antioxidant enzyme removing peroxides or H2O2 within the catalase- and peroxidase-deficient Periplasmic Space of E. coli.