scholarly journals Export of Virulence Genes and Shiga Toxin by Membrane Vesicles of Escherichia coli O157:H7

1999 ◽  
Vol 65 (5) ◽  
pp. 1843-1848 ◽  
Author(s):  
Glynis L. Kolling ◽  
Karl R. Matthews
Keyword(s):  
Escherichia Coli ◽  
Virulence Genes ◽  
Outer Membrane Proteins ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Genetic Material ◽  
Membrane Vesicles ◽  
Immunoblot Analysis ◽  
Proteinase K ◽  
Dna Encoding

ABSTRACT Membrane vesicles released by Escherichia coli O157:H7 into culture medium were purified and analyzed for protein and DNA content. Electron micrographs revealed vesicles that are spherical, range in size from 20 to 100 nm, and have a complete bilayer. Analysis of vesicle protein by sodium dodecyl sulfate-polyacrylamide gel electrophoresis demonstrates vesicles that contain many proteins with molecular sizes similar to outer membrane proteins and a number of cellular proteins. Immunoblot (Western) analysis of vesicles suggests the presence of cell antigens. Treatment of vesicles with exogenous DNase hydrolyzed surface-associated DNA; PCR demonstrated that vesicles contain DNA encoding the virulence genes eae,stx1 and stx2, and uidA, which encodes for β-galactosidase. Immunoblot analysis of intact and lysed, proteinase K-treated vesicles demonstrate that Shiga toxins 1 and 2 are contained within vesicles. These results suggest that vesicles contain toxic material and transfer experiments demonstrate that vesicles can deliver genetic material to other gram-negative organisms.

scholarly journals Proteomic Analysis of Outer Membranes and Vesicles from Wild-Type Serogroup B Neisseria meningitidis and a Lipopolysaccharide-Deficient Mutant

2006 ◽  
Vol 75 (3) ◽  
pp. 1364-1372 ◽  
Author(s):  
Jeannette N. Williams ◽  
Paul J. Skipp ◽  
Holly E. Humphries ◽  
Myron Christodoulides ◽  
C. David O'Connor ◽  
...  
Keyword(s):  
Membrane Proteins ◽  
Neisseria Meningitidis ◽  
Outer Membrane ◽  
Tricarboxylic Acid Cycle ◽  
Outer Membrane Proteins ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Membrane Vesicles ◽  
Outer Membrane Vesicles ◽  
Deficient Mutant

ABSTRACT Current experimental vaccines against serogroup B Neisseria meningitidis are based on meningococcal outer membrane (OM) proteins present in outer membrane vesicles (OMV) in which toxic lipopolysaccharide is depleted by detergent extraction. Knowledge of the composition of OM and OMV is essential for developing new meningococcal vaccines based on defined antigens. In the current study, sodium dodecyl sulfate-polyacrylamide gel electrophoresis and nanocapillary liquid chromatography-tandem mass spectrometry were used to investigate the proteomes of OM and OMV from meningococcal strain MC58 and OM from a lipopolysaccharide-deficient mutant. The analysis of OM revealed a composition that was much more complex than the composition that has been reported previously; a total of 236 proteins were identified, only 6.4% of which were predicted to be located in the outer membrane. The most abundant proteins included not only the well-established major OM proteins (PorA, PorB, Opc, Rmp, and Opa) but also other proteins, such as pilus-associated protein Q (PilQ) and a putative macrophage infectivity protein. All of these proteins were also present in OMV obtained by extraction of the OM with deoxycholate. There were markedly increased levels of some additional proteins in OM from the lipopolysaccharide-deficient mutant, including enzymes that contribute to the tricarboxylic acid cycle. In all the preparations, the proteins not predicted to have an OM location were predominantly periplasmic or cytoplasmic or had an unknown location, and relatively few cytoplasmic membrane proteins were detected. However, several proteins that have previously been identified as potential vaccine candidates were not detected in either OM preparations or in OMV. These results have important implications for the development and use of vaccines based on outer membrane proteins.

Structure of the O-chain polysaccharide of the phenol-phase soluble lipopolysaccharide of Escherichia coli 0:157:H7

1986 ◽  
Vol 64 (1) ◽  
pp. 21-28 ◽  
Author(s):  
Malcolm B. Perry ◽  
Leann MacLean ◽  
Douglas W. Griffith
Keyword(s):  
Escherichia Coli ◽  
Brucella Abortus ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Extraction Procedure ◽  
Periodate Oxidation ◽  
Cross Reactivity ◽  
E Coli ◽  
Structure Formula ◽  
Phenol Water

The phenol-phase soluble lipopolysaccharide isolated from Escherichia coli 0:157 by the hot phenol–water extraction procedure was shown by sodium dodecyl sulfate–polyacrylamide gel electrophoresis, periodate oxidation, methylation, and 13C and 1H nuclear magnetic resonance studies to be an unbranched linear polysaccharide with a tetrasaccharide repeating unit having the structure:[Formula: see text]The serological cross-reactivity of E. coli 0:157 with Brucella abortus, Yersinia enterocolitica (serotype 0:9), group N Salmonella, and some other E. coli species can be related immunochemically to the presence of 1,2-glycosylated N-acylated 4-amino-4,6-dideoxy-α-D-mannopyranosyl residues in the O-chains of their respective lipopolysaccharides.

scholarly journals Weissellicin 110, a Newly Discovered Bacteriocin from Weissella cibaria 110, Isolated from Plaa-Som, a Fermented Fish Product from Thailand

2007 ◽  
Vol 73 (7) ◽  
pp. 2247-2250 ◽  
Author(s):  
Sirinat Srionnual ◽  
Fujitoshi Yanagida ◽  
Li-Hsiu Lin ◽  
Kuang-Nan Hsiao ◽  
Yi-sheng Chen
Keyword(s):  
Proteolytic Enzymes ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Protein Band ◽  
Proteinase K ◽  
Mass Spectrometry Analysis ◽  
Fermented Fish ◽  
Spectrometry Analysis ◽  
Weissella Cibaria ◽  
Fish Product

ABSTRACT Weissella cibaria 110, isolated from the Thai fermented fish product plaa-som, was found to produce a bacteriocin active against some gram-positive bacteria. Bacteriocin activity was not eliminated by exposure to high temperatures or catalase but was destroyed by exposure to the proteolytic enzymes proteinase K and trypsin. The bacteriocin from W. cibaria 110 was purified, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the purified bacteriocin contained one protein band that was approximately 2.5 kDa in size. Mass spectrometry analysis showed the mass of the peptide to be approximately 3,487.8 Da. N-terminal amino acid sequence analysis was performed, and 27 amino acids were identified. Because it has no similarity to other known bacteriocins, this bacteriocin was defined as a new bacteriocin and termed weissellicin 110.

scholarly journals Acetate and Formate Stress: Opposite Responses in the Proteome of Escherichia coli

2001 ◽  
Vol 183 (21) ◽  
pp. 6466-6477 ◽  
Author(s):  
Christopher Kirkpatrick ◽  
Lisa M. Maurer ◽  
Nikki E. Oyelakin ◽  
Yuliya N. Yoncheva ◽  
Russell Maurer ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Opposite Effect ◽  
Stress Proteins ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Acid Resistance ◽  
Luria Broth ◽  
Acetyl Coa ◽  
Fermentation Products ◽  
E Coli

ABSTRACT Acetate and formate are major fermentation products ofEscherichia coli. Below pH 7, the balance shifts to lactate; an oversupply of acetate or formate retards growth. E. coli W3110 was grown with aeration in potassium-modified Luria broth buffered at pH 6.7 in the presence or absence of added acetate or formate, and the protein profiles were compared by two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Acetate increased the steady-state expression levels of 37 proteins, including periplasmic transporters for amino acids and peptides (ArtI, FliY, OppA, and ProX), metabolic enzymes (YfiD and GatY), the RpoS growth phase regulon, and the autoinducer synthesis protein LuxS. Acetate repressed 17 proteins, among them phosphotransferase (Pta). An ackA-pta deletion, which nearly eliminates interconversion between acetate and acetyl-coenzyme A (acetyl-CoA), led to elevated basal levels of 16 of the acetate-inducible proteins, including the RpoS regulon. Consistent with RpoS activation, the ackA-pta strain also showed constitutive extreme-acid resistance. Formate, however, repressed 10 of the acetate-inducible proteins, including the RpoS regulon. Ten of the proteins with elevated basal levels in the ackA-ptastrain were repressed by growth of the mutant with formate; thus, the formate response took precedence over the loss of theackA-pta pathway. The similar effects of exogenous acetate and the ackA-pta deletion, and the opposite effect of formate, could have several causes; one possibility is that the excess buildup of acetyl-CoA upregulates stress proteins but excess formate depletes acetyl-CoA and downregulates these proteins.

scholarly journals Absence of the Outer Membrane Phospholipase A Suppresses the Temperature-Sensitive Phenotype of Escherichia coli degPMutants and Induces the Cpx and ςE Extracytoplasmic Stress Responses

2001 ◽  
Vol 183 (18) ◽  
pp. 5230-5238 ◽  
Author(s):  
Geoffrey R. Langen ◽  
Jill R. Harper ◽  
Thomas J. Silhavy ◽  
S. Peter Howard
Keyword(s):  
Escherichia Coli ◽  
Outer Membrane ◽  
Stress Responses ◽  
Elevated Temperatures ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Phospholipase A ◽  
Temperature Sensitive ◽  
Outer Membrane Phospholipase A ◽  
Temperature Sensitive Phenotype

ABSTRACT DegP is a periplasmic protease that is a member of both the ςE and Cpx extracytoplasmic stress regulons ofEscherichia coli and is essential for viability at temperatures above 42°C. [U-14C]acetate labeling experiments demonstrated that phospholipids were degraded indegP mutants at elevated temperatures. In addition, chloramphenicol acetyltransferase, β-lactamase, and β-galactosidase assays as well as sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis indicated that large amounts of cellular proteins are released from degP cells at the nonpermissive temperature. A mutation in pldA, which encodes outer membrane phospholipase A (OMPLA), was found to rescue degPcells from the temperature-sensitive phenotype. pldA degP mutants had a normal plating efficiency at 42°C, displayed increased viability at 44°C, showed no degradation of phospholipids, and released far lower amounts of cellular protein to culture supernatants. degP and pldA degP mutants containing chromosomal lacZ fusions to Cpx and ςE regulon promoters indicated that both regulons were activated in the pldA mutants. The overexpression of the envelope lipoprotein, NlpE, which induces the Cpx regulon, was also found to suppress the temperature-sensitive phenotype ofdegP mutants but did not prevent the degradation of phospholipids. These results suggest that the absence of OMPLA corrects the degP temperature-sensitive phenotype by inducing the Cpx and ςE regulons rather than by inactivating the phospholipase per se.

Isolation and Preliminary Characterization of a Bacteriocin Produced by Lactobacillus plantarum N014 Isolated from Nham, a Traditional Thai Fermented Pork

2006 ◽  
Vol 69 (8) ◽  
pp. 1937-1943 ◽  
Author(s):  
PONGSAK RATTANACHAIKUNSOPON ◽  
PARICHAT PHUMKHACHORN
Keyword(s):  
Lactobacillus Plantarum ◽  
Proteolytic Enzymes ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Proteinase K ◽  
Exponential Growth Phase ◽  
Plasmid Isolation ◽  
Inhibitory Spectrum ◽  
Lipase A

Lactobacillus plantarum N014 was isolated from nham, a traditional Thai fermented pork, and exhibited antimicrobial activity against Listeria monocytogenes. Its bacteriocin had a broad inhibitory spectrum toward both gram-positive and gram-negative bacteria. The bacteriocin activity was sensitive to all proteolytic enzymes used in this study, including papain, pepsin, pronase E, proteinase K, and trypsin, but was resistant to the other enzymes, such as α-amylase, lipase A, and lysozyme. Furthermore, activity was stable over various heat treatments and pH values. The bacteriocin exerted a bacteriolytic mode of action. It was produced during the exponential growth phase and reached its highest level as producer cells entered the stationary phase. Adsorption of the bacteriocin onto producer cells was pH-dependent. No bacteriocin adsorption was detected at pH 1 to 3, whereas 100% bacteriocin adsorption was found at pH 7. Plasmid isolation revealed that L. plantarum N014 contained no plasmids. From Tricine–sodium dodecyl sulfate–polyacrylamide gel electrophoresis and growth inhibition testing against L. monocytogenes, the estimated molecular mass of L. plantarum N014 bacteriocin was 8 kDa.

scholarly journals Plasma thrombomodulin in health and diseases

Blood ◽  
1990 ◽  
Vol 76 (10) ◽  
pp. 2024-2029 ◽  
Author(s):  
S Takano ◽  
S Kimura ◽  
S Ohdama ◽  
N Aoki
Keyword(s):  
Distress Syndrome ◽  
Pulmonary Thromboembolism ◽  
Polyacrylamide Gel Electrophoresis ◽  
Plasma Concentrations ◽  
Sodium Dodecyl ◽  
Acute Hepatic Failure ◽  
Immunoblot Analysis ◽  
Enzyme Linked Immunosorbent Assay ◽  
Molecular Weights ◽  
Disease States

Abstract Sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by immunoblot analysis of plasma thrombomodulin concentrate revealed that four degraded forms of thrombomodulin with different molecular weights are present in plasma. Plasma concentrations of thrombomodulin in patients with various diseases were measured by two methods of enzyme- linked immunosorbent assay using monoclonal antibodies. One method measures intact thrombomodulin and degraded forms of thrombomodulin; the other does not detect the two smaller degraded forms of thrombomodulin present in plasma. The results indicated that thrombomodulin was increased in the circulating blood of patients with disseminated intravascular coagulation syndrome, pulmonary thromboembolism, adult respiratory distress syndrome, chronic renal failure, or acute hepatic failure. The different values obtained by the two methods indicate that the increase of plasma thrombomodulin found in these patients was mainly due to an increase of the smaller fragments of degraded forms, suggesting that the release of thrombomodulin from endothelial cells was accelerated in various disease states by proteolytic activity generated on the surface of the endothelium and may be removed from the circulation mostly by the kidneys and liver.

scholarly journals Expression of Aleutian Mink Disease Parvovirus Capsid Proteins in a Baculovirus Expression System for Potential Diagnostic Use

1994 ◽  
Vol 6 (1) ◽  
pp. 23-29 ◽  
Author(s):  
Wai-Hong Wu ◽  
Marshall E. Bloom ◽  
Bradley D. Berry ◽  
Michael J. McGinley ◽  
Kenneth B. Platt
Keyword(s):  
Microscopic Examination ◽  
Expression System ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Electron Microscopic Examination ◽  
Baculovirus Expression System ◽  
Immunoblot Analysis ◽  
Capsid Proteins ◽  
Electron Microscopic ◽  
Nuclear Polyhedrosis

A 2.3-kb cDNA clone encoding Aleutian mink disease parvovirus (ADV) structural proteins VP1 and VP2 was inserted into the polyhedron gene of Autographa calijbmica nuclear polyhedrosis virus (AcNPV) and expressed by the recombinant virus, AcADV-1, in Spodoptera frugiperda-9 cells. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and western immunoblot analysis (WIA) indicated that synthesis of both VP1 and VP2 was being directed by AcADV-1. Fluorescence microscopic examination of AcADV-1 -infected S. frugiperda-9 cells indicated that the recombinant protein was present within the nucleus of the cells, and electron microscopic examination of these cells revealed the presence of small particles 23–25 nm in diameter. Structures resembling empty ADV capsids could be purified on CsCl density gradients, thus indicating that the ADV proteins were self-assembling. The antigenicity of recombinant VP1 and VP2 was evaluated by WIA. Sera collected from 16 mink prior to infection with ADV did not react with VP1 and VP2. Ten sera collected from mink with counter current immunoelectrophoresis (CIE) titers greater than 4 (log2) reacted with VP1 and VP2 in WIA. Two of 6 sera with CIE titers of 4 and 1 of 14 sera with CIE titers <4 reacted with the recombinant proteins. These results suggest that baculovirus recombinant ADV capsid proteins may be useful as diagnostic antigens.

Mechanism of regulation of calcium-pumping activity in chick intestine

1992 ◽  
Vol 262 (5) ◽  
pp. G797-G805
Author(s):  
J. Takito ◽  
T. Shinki ◽  
H. Tanaka ◽  
T. Suda
Keyword(s):  
Vitamin D ◽  
Calcium Transport ◽  
Calcium Uptake ◽  
Early Stage ◽  
Basolateral Membrane ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Membrane Vesicles ◽  
Intestinal Calcium Transport ◽  
Pumping Activity

The role of the calcium pump in the stimulation of intestinal calcium transport activity by 1 alpha,25-dihydroxyvitamin D3 [1 alpha,25(OH)2D3] was examined in chicks. The in situ intestinal absorption of calcium increased approximately threefold in the duodenum, jejunum, and ileum 6 h after a single injection of 625 ng of 1 alpha,25(OH)2D3 into vitamin D-deficient chicks. The same treatment also increased approximately twofold the rate of ATP-dependent calcium uptake by the basolateral membrane vesicles (BL) isolated from those three sites. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that a Mg(2+)-dependent calcium-stimulated phosphorylated intermediate with an apparent molecular mass of 105 kDa appeared in the BL. The 1 alpha,25(OH)2D3 treatment gave no change in the levels of the intermediate. Pretreatment of the BL with alkaline phosphatase decreased the calcium uptake by the BL isolated from 1 alpha,25(OH)2D3-treated chicks, but it had little effect on the uptake by the BL from vitamin D-deficient chicks. These results suggest that at an early stage of the 1 alpha,25(OH)2D3-induced intestinal calcium transport process, the vitamin regulates the calcium-pumping activity of chick intestinal BL by phosphorylation and dephosphorylation but not by a stoichiometric change in the pump.

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