Pore-Forming Activity of Outer Membrane Extracts from the Unicellular Cyanobacterium Synechocystis sp. PCC 6714

1989 ◽  
Vol 44 (1-2) ◽  
pp. 165-169 ◽  
Author(s):  
Uwe J. Jürgens ◽  
Roland Benz
Keyword(s):  
Outer Membrane ◽  
Cell Walls ◽  
Pore Diameter ◽  
Single Channel ◽  
Outer Membrane Proteins ◽  
Lipid Bilayer Membranes ◽  
Bilayer Membranes ◽  
Unicellular Cyanobacterium ◽  
Sds Page ◽  
Synechocystis Sp

Abstract Cell walls of the unicellular cyanobacterium Synechocystis sp. PCC 6714, isolated from cell homogenates, were found to be unusually resistant against extraction with various detergents, organic solvents, chaotropic agents, and proteases. The major outer membrane proteins (M r 67,000; 61,000; 94,000) were solubilized by differential SDS-extraction and purified by preparative SDS-PAGE. The extracted proteins, reconstituted into lipid bilayer membranes, formed two types of pores with single-channel conductances of 2.2 nS (pore diameter of 1.4 nm) and 0.3 nS (pore diameter not determined), respectively. Carotenoids and lipopolysaccharide were found to be associated with the extracted major proteins.

scholarly journals The C-Terminal Domain of the Bordetella pertussisAutotransporter BrkA Forms a Pore in Lipid Bilayer Membranes

1999 ◽  
Vol 181 (18) ◽  
pp. 5838-5842 ◽  
Author(s):  
Jennifer L. Shannon ◽  
Rachel C. Fernandez
Keyword(s):  
Outer Membrane ◽  
Lipid Bilayer ◽  
Single Channel ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Lipid Bilayer Membranes ◽  
Channel Conductance ◽  
Terminal Protein ◽  
Bilayer Membranes ◽  
Terminal Domain

ABSTRACT BrkA is a 103-kDa outer membrane protein of Bordetella pertussis that mediates resistance to antibody-dependent killing by complement. It is proteolytically processed into a 73-kDa N-terminal domain and a 30-kDa C-terminal domain as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. BrkA is also a member of the autotransporter family of proteins. Translocation of the N-terminal domain of the protein across the outer membrane is hypothesized to occur through a pore formed by the C-terminal domain. To test this hypothesis, we performed black lipid bilayer experiments with purified recombinant protein. The BrkA C-terminal protein showed an average single-channel conductance of 3.0 nS in 1 M KCl. This result strongly suggests that the C-terminal autotransporter domain of BrkA is indeed capable of forming a pore.

scholarly journals Reconstitution of bacterial outer membrane TonB-dependent transporters in planar lipid bilayer membranes

2009 ◽  
Vol 106 (51) ◽  
pp. 21990-21995 ◽  
Author(s):  
E. Udho ◽  
K. S. Jakes ◽  
S. K. Buchanan ◽  
K. J. James ◽  
X. Jiang ◽  
...  
Keyword(s):  
Outer Membrane ◽  
Lipid Bilayer ◽  
Planar Lipid Bilayer ◽  
Lipid Bilayer Membranes ◽  
Bilayer Membranes ◽  
Bacterial Outer Membrane

scholarly journals N-Acetylcysteine Selectively Antagonizes the Activity of Imipenem in Pseudomonas aeruginosa by an OprD-Mediated Mechanism

2015 ◽  
Vol 59 (6) ◽  
pp. 3246-3251 ◽  
Author(s):  
Jerónimo Rodríguez-Beltrán ◽  
Gabriel Cabot ◽  
Estela Ynés Valencia ◽  
Coloma Costas ◽  
German Bou ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Pseudomonas Aeruginosa ◽  
Acinetobacter Baumannii ◽  
Outer Membrane ◽  
Bacterial Species ◽  
Outer Membrane Proteins ◽  
Antibiotic Activity ◽  
Simultaneous Treatment ◽  
Content Type ◽  
Sds Page

ABSTRACTThe modulating effect ofN-acetylcysteine (NAC) on the activity of different antibiotics has been studied inPseudomonas aeruginosa. Our results demonstrate that, in contrast to previous reports, only the activity of imipenem is clearly affected by NAC. MIC and checkerboard determinations indicate that the NAC-based modulation of imipenem activity is dependent mainly on OprD. SDS-PAGE of outer membrane proteins (OMPs) after NAC treatments demonstrates that NAC does not modify the expression of OprD, suggesting that NAC competitively inhibits the uptake of imipenem through OprD. Similar effects on imipenem activity were obtained withP. aeruginosaclinical isolates. Our results indicate that imipenem-susceptibleP. aeruginosastrains become resistant upon simultaneous treatment with NAC and imipenem. Moreover, the generality of the observed effects of NAC on antibiotic activity was assessed with two additional bacterial species,Escherichia coliandAcinetobacter baumannii. Caution should be taken during treatments, as the activity of imipenem may be modified by physiologically attainable concentrations of NAC, particularly during intravenous and nebulized regimes.

scholarly journals Establishing a Direct Role for the Bartonella bacilliformis Invasion-Associated Locus B (IalB) Protein in Human Erythrocyte Parasitism

2001 ◽  
Vol 69 (7) ◽  
pp. 4373-4381 ◽  
Author(s):  
Sherry A. Coleman ◽  
Michael F. Minnick
Keyword(s):  
Membrane Proteins ◽  
Outer Membrane ◽  
Protein Localization ◽  
Outer Membrane Proteins ◽  
Polyacrylamide Gel Electrophoresis ◽  
Buoyant Density ◽  
Sodium Dodecyl ◽  
Amino Acid Sequence Similarity ◽  
Bartonella Bacilliformis ◽  
Sds Page

ABSTRACT The invasion-associated locus A and B genes (ialAB) ofBartonella bacilliformis were previously shown to confer an erythrocyte-invasive phenotype upon Escherichia coli, indirectly implicating their role in virulence. We report the first direct demonstration of a role for ialB as a virulence factor in B. bacilliformis. The presence of a secretory signal sequence and amino acid sequence similarity to two known outer membrane proteins involved in virulence suggested that IalB was an outer membrane protein. To develop an antiserum for protein localization, the ialB gene was cloned in frame into an expression vector with a six-histidine tag and under control of thelacZ promoter. The IalB fusion protein was purified by nickel affinity chromatography and used to raise polyclonal antibodies. IalB was initially localized to the bacterial membrane fraction. To further localize IalB, B. bacilliformis inner and outer membranes were fractionated by sucrose density gradient centrifugation and identified by appearance, buoyant density (ρ), and cytochromeb content. Inner and outer membrane proteins were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and IalB was positively identified by Western blot. Contrary to expectations, IalB was localized to the inner membrane of the pathogen. To directly demonstrate a role for IalB in erythrocyte parasitism, the B. bacilliformis ialB gene was disrupted by insertional mutagenesis. The resulting ialB mutant strain was complemented in trans with a replicative plasmid encoding the full-length ialB gene. PCR and high-stringency DNA hybridization confirmed mutagenesis and transcomplementation events. Abrogation and restoration of ialB expression was verified by SDS-PAGE and immunoblotting. In vitro virulence assays showed that mutagenesis of ialB decreased bacterial association and invasion of human erythrocytes by 47 to 53% relative to controls. Transcomplementation of ialB restored erythrocyte association and invasion rates to levels observed in the parental strain. These data provide direct evidence for IalB's role in erythrocyte parasitism and represent the first demonstration of molecular Koch's postulates for a Bartonella species.

scholarly journals Cloning, expression and purification of outer membrane protein PorA of Neisseria meningitidis serogroup B

2011 ◽  
Vol 5 (12) ◽  
pp. 856-862 ◽  
Author(s):  
Fakhri Haghi ◽  
Shahin Najar Peerayeh ◽  
Seyed Davar Siadat ◽  
Mehran Montajabiniat
Keyword(s):  
Recombinant Protein ◽  
Neisseria Meningitidis ◽  
Outer Membrane ◽  
Prokaryotic Expression ◽  
Membrane Vesicle ◽  
Outer Membrane Proteins ◽  
Expression System ◽  
Sds Page ◽  
Prokaryotic Expression System ◽  
Prokaryotic Expression Vector

Introduction: Neisseria meningitidis is a major causative agent of bacterial septicemia and meningitis in humans. Currently, there are no vaccines to prevent disease caused by strains of N. meningitidis serogroup B. PorA is a major component of the outer membrane of N. meningitidis and functions as a cationic porin. This study aimed to clone and determine the expression of PorA. Methodology: A 1200 bp fragment of porA gene was amplified by PCR from serogroup B N. meningitidis and then cloned into prokaryotic expression vector pET-32a. For expression of recombinant protein, pET32a-porA plasmid was transformed into competent Origami B (DE3) cells. Recombinant protein was overexpressed with isopropythio-beta-D-galctoside (IPTG) and affinity purified by Ni-NTA agarose. SDS-PAGE and western blotting were performed for protein determination and verification. Results: Cloning of porA was confirmed by colony-PCR and enzymatic digestion. In comparison with the corresponding sequences of original genes, the nucleotide sequence homology of the cloned porA gene was 97%. IPTG with a dosage of 1.0 mmol/L could efficiently induce protein expression. SDS-PAGE analysis showed that our constructed prokaryotic expression system pET32a-PorA-Origami efficiently produces a target recombinant protein with a molecular weight of 65 kDa. The recombinant PorA was overexpressed as inclusion bodies and reacted with the serum from a rabbit previously immunized with native outer membrane vesicle. Conclusion: This prokaryotic expression system provides an easy method for producing recombinant PorA and may also be useful for the production of other bacterial outer membrane proteins for vaccine studies.

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.

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