Structure and cell envelope associations of flagellar basal complexes of Vibrio cholerae and Campylobacter fetus

1984 ◽  
Vol 30 (3) ◽  
pp. 322-333 ◽  
Author(s):  
F. G. Ferris ◽  
T. J. Beveridge ◽  
M. L. Marceau-Day ◽  
A. D. Larson
Keyword(s):  
Outer Membrane ◽  
Vibrio Cholerae ◽  
Cell Envelope ◽  
Sodium Dodecyl ◽  
Three Dimensions ◽  
Thin Sections ◽  
Campylobacter Fetus ◽  
Basal Complex ◽  
Nonionic Detergent ◽  
Triton X

To isolate intact flagella with basal complexes from Vibrio cholerae, a rhamnolipid hemolysin from Pseudomonas aeruginosa was used to disrupt the cell envelope and flagellar sheath. The nonionic detergent, Triton X-100, provided similar results for Campylobacter fetus. Each of these basal complexes possessed, in addition to the four classical rings, concentric membrane rings (CMR's) similar to those found in Aquaspirillum serpens. Through the use of stereo imaging (which allows structures to be visualized in three dimensions) of thin sections of cells which had been sequentially treated with a number of envelope perturbants (i.e., ethylenediaminetetraacetate, lysozyme, Triton X-100, rhamnolipid hemolysin, and sodium dodecyl sulfate), we have progressively exposed the component parts of the basal organelles in V. cholerae and C. fetus. Since the action of these envelope perturbants has been well documented, we have been able to determine the associations of the exposed portions of the flagellar basal complex and the layer of the cell envelope in which they would normally reside. From our observations we have concluded that in both V. cholerae and C. fetus the L ring is embedded in the outer membrane and the P ring is associated with the peptidoglycan. The CMR's are bracketed by the L and P rings and are sandwiched between the outer membrane and the peptidoglycan. Elements of both the S and M rings appear to be associated with the plasma membrane.

scholarly journals Direct Interaction of the EpsL and EpsM Proteins of the General Secretion Apparatus in Vibrio cholerae

1999 ◽  
Vol 181 (10) ◽  
pp. 3129-3135 ◽  
Author(s):  
Maria Sandkvist ◽  
Lloyd P. Hough ◽  
Mira M. Bagdasarian ◽  
Michael Bagdasarian
Keyword(s):  
Outer Membrane ◽  
Vibrio Cholerae ◽  
Cytoplasmic Membrane ◽  
Cell Envelope ◽  
Gel Filtration ◽  
Direct Interaction ◽  
Stable Complex ◽  
Extracellular Secretion ◽  
Triton X

ABSTRACT The general secretion pathway of gram-negative bacteria is responsible for extracellular secretion of a number of different proteins, including proteases and toxins. This pathway supports secretion of proteins across the cell envelope in two distinct steps, in which the second step, involving translocation through the outer membrane, is assisted by at least 13 different gene products. Two of these components, the cytoplasmic membrane proteins EpsL and EpsM ofVibrio cholerae, have been purified and characterized. Based on gel filtration analysis, both purified EpsM(His)6 and wild-type EpsL present in anEscherichia coli Triton X-100 extract are dimeric proteins. EpsL and EpsM were also found to interact directly and form a Triton X-100 stable complex that could be precipitated with either anti-EpsL or anti-EpsM antibodies. In addition, when the L and M proteins were coexpressed in E. coli, they formed a stable complex and protected each other from proteolytic degradation, indicating that these two proteins interact in vivo and that no other Eps protein is required for their association. Since EpsL is predicted to contain a large cytoplasmic domain, while EpsM is predominantly exposed on the periplasmic side, we speculate that these components might be part of a structure that is involved in bridging the inner and outer membranes. Furthermore, since EpsL has previously been shown to interact with the autophosphorylating cytoplasmic membrane protein EpsE, we hypothesize that this trimolecular complex might be involved in regulating the opening and closing of the secretion pore and/or transducing energy to the site of outer membrane translocation.

Amikacin disrupts the cell envelope of Pseudomonas aeruginosa ATCC 9027

1988 ◽  
Vol 34 (1) ◽  
pp. 12-18 ◽  
Author(s):  
S. G. Walker ◽  
T. J. Beveridge
Keyword(s):  
Electron Microscopy ◽  
Pseudomonas Aeruginosa ◽  
Outer Membrane ◽  
Cell Envelope ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Atomic Absorption Spectrophotometry ◽  
Fracture Pattern ◽  
Thin Sections ◽  
Biochemical Analyses

Amikacin, an aminoglycoside known to inhibit protein synthesis, was found to perturb the outer membrane of a sensitive Pseudomonas aeruginosa strain (ATCC 9027). This perturbation was monitored using electron microscopy and biochemical analyses. Following exposure to 20 μg amikacin/mL for 15 min, the outer membrane of exponentially growing cells lost 15% of its protein, 18% of its lipopolysaccharide, and 18% of its phosphate. Sodium dodecyl sulphate – polyacrylamide gel electrophoresis showed that the whole spectrum of outer membrane protein and lipopolysaccharide was affected. Similarly, atomic absorption spectrophotometry revealed that magnesium and calcium were also lost. When cells were treated with amikacin, electron microscopy of negative stains showed a substantial increase in outer membrane blebbing. Freeze fractures revealed changes in membrane fracture pattern and particle distribution, and thin sections revealed a sequential disruption of the cell envelope beginning at the outer membrane and ending at the plasma membrane. This study supports the proposal that aminoglycoside antibiotics cross the outer membrane of Pseudomonas aeruginosa by displacing metal cations necessary to stabilize the organic constituents of the membrane. Their removal results in loss of the outer membrane and the formation of transient small holes which permit the antibiotic access to the cytoplasmic membrane where it is transported into the cytoplasm.

Subunit composition of goblet-shaped particles from the cell wall of Flexibacter polymorphus

1983 ◽  
Vol 29 (12) ◽  
pp. 1689-1693 ◽  
Author(s):  
H. F. Ridgway ◽  
R. A. Lewin
Keyword(s):  
Cell Envelope ◽  
Gradient Centrifugation ◽  
Polyacrylamide Gel Electrophoresis ◽  
Buoyant Density ◽  
Sodium Dodecyl ◽  
Cesium Chloride ◽  
Protein Subunits ◽  
Nonionic Detergent ◽  
Gliding Bacterium ◽  
Triton X

Submicroscopic goblet-shaped particles (goblets) were released from the cell envelope of the marine gliding bacterium Flexibacter polymorphus when treated with the nonionic detergent Triton X-100 followed by sonication. The goblets were purified by cesium chloride density gradient centrifugation and exhibited an equilibrium buoyant density of 1.30 g/mL at 23 °C. They were composed of protein and a small amount of carbohydrate (approximately 3.4% by weight). Aqueous suspensions exhibited an absorption maximum in the ultraviolet at a wavelength of 276 nm and a smaller shoulder at 281 nm. Phospholipids were not detected in purified preparations of goblets, though they are known to be prominent constituents of the intact membranes of this microbe. Polyacrylamide gel electrophoresis of goblets solubilized in sodium dodecyl sulfate and 2-mercaptoethanol indicated four major polypeptide components ranging in molecular weight from 13 000 to 80 000. This number of different protein subunits corroborates earlier ultrastructural observations indicating a multisubunit composition.

The outer membrane of Haemophilus influenzae type b: cell envelope associations of major proteins

1983 ◽  
Vol 29 (2) ◽  
pp. 280-287 ◽  
Author(s):  
J. W. Coulton ◽  
D. T. F. Wan
Keyword(s):  
Molecular Weight ◽  
Sodium Dodecyl Sulphate ◽  
Cell Envelope ◽  
Sodium Dodecyl ◽  
Haemophilus Influenzae Type ◽  
Major Protein ◽  
Haemophilus Influenzae Type B ◽  
Type B ◽  
Molecular Weights ◽  
Triton X

Membrane proteins fom the cell envelope of Haemophilus influenzae type b ATCC 9795 were examined by sodium dodecyl sulphate – polyacrylamide gel electrophoresis. When envelopes were extracted with a phosphate-based buffer containing 2% Triton X-100, a major protein of molecular weight 43 000 was detected in fractions containing cytoplasmic membrane proteins. The cell wall material which was Triton X-100 insoluble contained six major proteins of molecular weights 46 000, 40 000, 36 000, 30 000, 27 000, and 16 000. One of these proteins showed a shift in molecular weight from 27 000 to 36 000 when it was heated over a temperature range from 50 °C to 100 °C in buffer containing 2% sodium dodecyl sulphate, 5% 2-mercaptoethanol. This alteration in mobility could be demonstrated either by the membrane-bound form of the protein or by a detergent-soluble form of the protein. Enriched preparations of the 36 000 molecular weight form were obtained by a series of purification steps. Extraction of the Triton X-100 insoluble material with buffer containing 2% Triton X-100, 5.0 mM EDTA yielded chiefly one major protein molecular weight 30 000 and many minor protein species. Pretreatment of the Triton X-100 insoluble fraction with lysozyme followed by extraction with buffer containing 2% Triton X-100, 5.0 mM EDTA released two proteins of molecular weights 16 000 and 27 000 and few minor proteins. By these operational manipulations, the proteins of molecular weights 16 000 and 27 000 may be considered as peptidoglycan-associated proteins.

scholarly journals Rcs Phosphorelay Responses to Truncated Lipopolysaccharide-Induced Cell Envelope Stress in Yersinia enterocolitica

Molecules ◽  
2020 ◽  
Vol 25 (23) ◽  
pp. 5718
Author(s):  
Jiao Meng ◽  
Junhong Xu ◽  
Can Huang ◽  
Jingyu Chen
Keyword(s):  
Stress Response ◽  
Outer Membrane ◽  
Yersinia Enterocolitica ◽  
Antimicrobial Agents ◽  
Cell Envelope ◽  
Sodium Dodecyl ◽  
Cell Surface Hydrophobicity ◽  
Membrane Function ◽  
Cell Functions ◽  
Envelope Stress

Lipopolysaccharide (LPS) is the major component of the outer membrane of Gram-negative bacteria, and its integrity is monitored by various stress response systems. Although the Rcs system is involved in the envelope stress response and regulates genes controlling numerous bacterial cell functions of Yersinia enterocolitica, whether it can sense the truncated LPS in Y. enterocolitica remains unclear. In this study, the deletion of the Y. enterocolitica waaF gene truncated the structure of LPS and produced a deep rough LPS. The truncated LPS increased the cell surface hydrophobicity and outer membrane permeability, generating cell envelope stress. The truncated LPS also directly exposed the smooth outer membrane to the external environment and attenuated the resistance to adverse conditions, such as impaired survival under polymyxin B and sodium dodecyl sulfate (SDS) exposure. Further phenotypic experiment and gene expression analysis indicated that the truncated LPS was correlated with the activation of the Rcs phosphorelay, thereby repressing cell motility and biofilm formation. Our findings highlight the importance of LPS integrity in maintaining membrane function and broaden the understanding of Rcs phosphorelay signaling in response to cell envelope stress, thus opening new avenues to develop effective antimicrobial agents for combating Y. enterocolitica infections.

scholarly journals Cell Envelope Perturbation Induces Oxidative Stress and Changes in Iron Homeostasis in Vibrio cholerae

2009 ◽  
Vol 191 (17) ◽  
pp. 5398-5408 ◽  
Author(s):  
Aleksandra E. Sikora ◽  
Sinem Beyhan ◽  
Michael Bagdasarian ◽  
Fitnat H. Yildiz ◽  
Maria Sandkvist
Keyword(s):  
Oxidative Stress ◽  
Gene Expression ◽  
Outer Membrane ◽  
Vibrio Cholerae ◽  
Iron Homeostasis ◽  
Cell Envelope ◽  
Membrane Integrity ◽  
Genetic Alterations ◽  
Iron Storage ◽  
Study Gene Expression

ABSTRACT The Vibrio cholerae type II secretion (T2S) machinery is a multiprotein complex that spans the cell envelope. When the T2S system is inactivated, cholera toxin and other exoproteins accumulate in the periplasmic compartment. Additionally, loss of secretion via the T2S system leads to a reduced growth rate, compromised outer membrane integrity, and induction of the extracytoplasmic stress factor RpoE (A. E. Sikora, S. R. Lybarger, and M. Sandkvist, J. Bacteriol. 189:8484-8495, 2007). In this study, gene expression profiling reveals that inactivation of the T2S system alters the expression of genes encoding cell envelope components and proteins involved in central metabolism, chemotaxis, motility, oxidative stress, and iron storage and acquisition. Consistent with the gene expression data, molecular and biochemical analyses indicate that the T2S mutants suffer from internal oxidative stress and increased levels of intracellular ferrous iron. By using a tolA mutant of V. cholerae that shares a similar compromised membrane phenotype but maintains a functional T2S machinery, we show that the formation of radical oxygen species, induction of oxidative stress, and changes in iron physiology are likely general responses to cell envelope damage and are not unique to T2S mutants. Finally, we demonstrate that disruption of the V. cholerae cell envelope by chemical treatment with polymyxin B similarly results in induction of the RpoE-mediated stress response, increased sensitivity to oxidants, and a change in iron metabolism. We propose that many types of extracytoplasmic stresses, caused either by genetic alterations of outer membrane constituents or by chemical or physical damage to the cell envelope, induce common signaling pathways that ultimately lead to internal oxidative stress and misregulation of iron homeostasis.

Erratum: Structure and cell envelope associations of flagellar basal complexes of Vibrio cholerae and Campylobacter fetus

1984 ◽  
Vol 30 (8) ◽  
pp. 1092-1092
Author(s):  
F. G. Ferris ◽  
T. J. Beveridge ◽  
M. L. Marceau-Day ◽  
A. D. Larson
Keyword(s):  
Vibrio Cholerae ◽  
Cell Envelope ◽  
Campylobacter Fetus

scholarly journals Comparative studies of Yersinia pestis outer membrane isolation techniques and their potential use in plague epidemiology

1990 ◽  
Vol 32 (2) ◽  
pp. 78-83 ◽  
Author(s):  
Frederico Guilherme Coutinho Abath ◽  
Luís Carlos de Sousa Ferreira
Keyword(s):  
Yersinia Pestis ◽  
Outer Membrane ◽  
Outer Membrane Proteins ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Protein Patterns ◽  
Isolation Techniques ◽  
Sds Page ◽  
Triton X ◽  
Potential Use

In the present study three techniques for obtaining outer membrane enriched fractions from Yersinia pestis were evaluated. The techniques analysed were: differential solubilization of the cytoplasmic membrane with Sarkosyl or Triton X-100, and centrifugation in sucrose density gradients. The sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of outer membrane isolated by the different methods resulted in similar protein patterns. The measurement of NADH-dehydrogenase and succinate dehydrogenase (inner membrane enzymes) indicated that the outer membrane preparations obtained by the three methods were pure enough for analytical studies. In addition, preliminary evidences on the potential use of outer membrane proteins for the identification of geographic variants of Y. pestis wild isolates are presented.

Description of two regular structures associated to the cell envelope of Thermus thermophilus: a structural transformation?

1990 ◽  
Vol 48 (1) ◽  
pp. 268-269
Author(s):  
J.L. Carrascosa ◽  
José R. Castón ◽  
M.M. Faraldo ◽  
J. Berenguer ◽  
M.A. de Pedro
Keyword(s):  
Outer Membrane ◽  
Cell Envelope ◽  
Thermus Thermophilus ◽  
Freeze Fracture ◽  
Two Dimensional ◽  
Regular Structures ◽  
Main Component ◽  
Unit Cell Dimensions ◽  
20 Nm ◽  
Triton X

Thermus thermophilus HB8 is a Gram-negative thermophilic eubacteria, with a well-defined outer membrane and a thin peptidoglycan layer around the cytoplasmic membrane. There is a S-layer on the cell envelope, whose main component is a protein of Mr around 100.000, masked by a lipopolysaccharide-like outer surface that can be revealed by freeze-fracture and shadowing of the native surface of the cells of T. thermophilus.When membrane fractions of this bacteria are treated with Triton X-100 in presence of EDTA, two-dimensional crystals with a hexagonal ordering could be observed by negative staining (figures 1a, 1b). This structure, called S1, had an unit cell dimensions a = b = 20 nm. Nevertheless, if the treatment is developed without the quelant agent, a structure with p2 symmetry is observed, S2, (figures 1d, 1e) with a = 24 and b = 21 nm. We have characterized both crystals by electron microscopy and two-dimensional analysis by Fourier methods. The resulting images showed very different characteristics; while the p6 structure is a typical S-layer, fig. 1c, the other one is a porin-like or regular outer membrane protein structure, fig. 1f, (2).This is surprising because both fractions contains the same ˝P100˝ protein (revealed by biochemical analysis).

scholarly journals Characterization of Outer Membrane Protein OmpU of Vibrio cholerae O1

1998 ◽  
Vol 66 (10) ◽  
pp. 4726-4728 ◽  
Author(s):  
Noboru Nakasone ◽  
Masaaki Iwanaga
Keyword(s):  
Membrane Protein ◽  
Outer Membrane ◽  
Vibrio Cholerae ◽  
Outer Membrane Protein ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Rabbit Small Intestine ◽  
Vibrio Cholerae O1

ABSTRACT The outer membrane protein OmpU of Vibrio cholerae O1 strain 86B3 was characterized with reference to colonization of the intestine by the organism. The purified OmpU exhibited a pI of 3.6. Upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis, it migrated to 38, 32, and 110 kDa when the sample was heated at 100°C for 2 min, 50°C for 15 min, and room temperature for 30 min, respectively. The purified OmpU was not hemagglutinative. Anti-OmpU serum did not agglutinate strain 86B3 or other V. cholerae organisms. OmpU adhered to the brush border of the rabbit small intestine; adhesion of the organisms to the intestine treated in advance with OmpU was not inhibited. Treating the organisms in advance with anti-OmpU Fab did not inhibit adhesion to the intestine. These results obtained in vitro suggest that OmpU is not involved in the adhesion of V. cholerae to the intestinal epithelium.

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