scholarly journals Biochemical Identification and Biophysical Characterization of a Channel-Forming Protein fromRhodococcus erythropolis

2000 ◽  
Vol 182 (3) ◽  
pp. 764-770 ◽  
Author(s):  
Thomas Lichtinger ◽  
Gila Reiss ◽  
Roland Benz
Keyword(s):  
Cell Wall ◽  
Molecular Mass ◽  
Lipid Bilayer ◽  
Single Channel ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Protein Sequencing ◽  
Cell Wall Fraction ◽  
Channel Conductance ◽  
Single Channel Conductance

ABSTRACT Organic solvent extracts of whole cells of the gram-positive bacterium Rhodococcus erythropolis contain a channel-forming protein. It was identified by lipid bilayer experiments and purified to homogeneity by preparative sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE). The pure protein had a rather low molecular mass of about 8.4 kDa, as judged by SDS-PAGE. SDS-resistant oligomers with a molecular mass of 67 kDa were also observed, suggesting that the channel is formed by a protein oligomer. The monomer was subjected to partial protein sequencing, and 45 amino acids were resolved. According to the partial sequence, the sequence has no significant homology to known protein sequences. To check whether the channel was indeed localized in the cell wall, the cell wall fraction was separated from the cytoplasmic membrane by sucrose step gradient centrifugation. The highest channel-forming activity was found in the cell wall fraction. The purified protein formed large ion-permeable channels in lipid bilayer membranes with a single-channel conductance of 6.0 nS in 1 M KCl. Zero-current membrane potential measurements with different salts suggested that the channel ofR. erythropolis was highly cation selective because of negative charges localized at the channel mouth. The correction of single-channel conductance data for negatively charged point charges and the Renkin correction factor suggested that the diameter of the cell wall channel is about 2.0 nm. The channel-forming properties of the cell wall channel of R. erythropolis were compared with those of other members of the mycolata. These channels have common features because they form large, water-filled channels that contain net point charges.

scholarly journals Corynebacterium diphtheriae: Identification and Characterization of a Channel-Forming Protein in the Cell Wall

2007 ◽  
Vol 189 (21) ◽  
pp. 7709-7719 ◽  
Author(s):  
Bettina Schiffler ◽  
Enrico Barth ◽  
Mamadou Daffé ◽  
Roland Benz
Keyword(s):  
Cell Wall ◽  
Molecular Mass ◽  
Lipid Bilayer ◽  
Single Channel ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Corynebacterium Diphtheriae ◽  
Enzyme Linked Immunosorbent Assay ◽  
Lipid Bilayer Membranes ◽  
Whole Cells

ABSTRACT The cell wall fraction of the gram-positive, nontoxic Corynebacterium diphtheriae strain C8r(−) Tox− (= ATCC 11913) contained a channel-forming protein, as judged from reconstitution experiments with artificial lipid bilayer experiments. The channel-forming protein was present in detergent-treated cell walls and in extracts of whole cells obtained using organic solvents. The protein had an apparent molecular mass of about 66 kDa as determined on Tricine-containing sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels and consisted of subunits having a molecular mass of about 5 kDa. Single-channel experiments with the purified protein suggested that the protein formed channels with a single-channel conductance of 2.25 nS in 1 M KCl. Further single-channel analysis suggested that the cell wall channel is wide and water filled because it has only slight selectivity for cations over anions and its conductance followed the mobility sequence of cations and anions in the aqueous phase. Antibodies raised against PorA, the subunit of the cell wall channel of Corynebacterium glutamicum, detected both monomers and oligomers of the isolated protein, suggesting that there are highly conserved epitopes in the cell wall channels of C. diphtheriae and PorA. Localization of the protein on the cell surface was confirmed by an enzyme-linked immunosorbent assay. The prospective homology of PorA with the cell wall channel of C. diphtheriae was used to identify the cell wall channel gene, cdporA, in the known genome of C. diphtheriae. The gene and its flanking regions were cloned and sequenced. CdporA is a protein that is 43 amino acids long and does not have a leader sequence. cdporA was expressed in a C. glutamicum strain that lacked the major outer membrane channels PorA and PorH. Organic solvent extracts of the transformed cells formed in lipid bilayer membranes the same channels as the purified CdporA protein of C. diphtheriae formed, suggesting that the expressed protein is able to complement the PorA and PorH deficiency of the C. glutamicum strain. The study is the first report of a cell wall channel in a pathogenic Corynebacterium strain.

scholarly journals The Cell Wall of the Pathogenic Bacterium Rhodococcus equi Contains Two Channel-Forming Proteins with Different Properties

2003 ◽  
Vol 185 (9) ◽  
pp. 2952-2960 ◽  
Author(s):  
Franziska G. Rieβ ◽  
Marion Elflein ◽  
Michael Benk ◽  
Bettina Schiffler ◽  
Roland Benz ◽  
...  
Keyword(s):  
Cell Wall ◽  
Molecular Mass ◽  
Single Channel ◽  
Sodium Dodecyl ◽  
Rhodococcus Equi ◽  
Apparent Molecular Mass ◽  
Channel Conductance ◽  
Single Channel Conductance ◽  
Voltage Gated ◽  
Sds Page

ABSTRACT We have identified in organic solvent extracts of whole cells of the gram-positive pathogen Rhodococcus equi two channel-forming proteins with different and complementary properties. The isolated proteins were able to increase the specific conductance of artificial lipid bilayer membranes made from phosphatidylcholine-phosphatidylserine mixtures by the formation of channels able to be permeated by ions. The channel-forming protein PorAReq (R. equi pore A) is characterized by the formation of cation-selective channels, which are voltage gated. PorAReq has a single-channel conductance of 4 nS in 1 M KCl and shows high permeability for positively charged solutes because of the presence of negative point charges. According to the results of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), the protein has an apparent molecular mass of about 67 kDa. The analysis (using the effect of negative charges on channel conductance) of the concentration dependence of the single-channel conductance suggested that the diameter of the cell wall channel is about 2.0 nm. The second channel (formed by PorBReq [R. equi pore B]) shows a preferred movement of anions through the channel and is not voltage gated. This channel shows a single-channel conductance of 300 pS in 1 M KCl and is characterized by the presence of positive point charges in or near the channel mouth. Based on SDS-PAGE, the apparent molecular mass of the channel-forming protein is about 11 kDa. Channel-forming properties of the investigated cell wall porins were compared with those of others isolated from mycolic acid-containing actinomycetes. We present here the first report of a fully characterized anion-selective cell wall channel from a member of the order Actinomycetales.

scholarly journals Identification of the Outer Membrane Porin of Thermus thermophilus HB8: the Channel-Forming Complex Has an Unusually High Molecular Mass and an Extremely Large Single-Channel Conductance

2001 ◽  
Vol 183 (2) ◽  
pp. 800-803 ◽  
Author(s):  
Elke Maier ◽  
Georg Polleichtner ◽  
Birgit Boeck ◽  
Reinhard Schinzel ◽  
Roland Benz
Keyword(s):  
Molecular Mass ◽  
Outer Membrane ◽  
Single Channel ◽  
Gradient Centrifugation ◽  
Thermus Thermophilus ◽  
Sodium Dodecyl ◽  
Thermophilic Bacterium ◽  
High Molecular Mass ◽  
Channel Conductance ◽  
Single Channel Conductance

ABSTRACT The outer membrane of the thermophilic bacterium Thermus thermophilus was isolated using sucrose step gradient centrifugation. Its detergent extracts contained an ion-permeable channel with an extremely high single-channel conductance of 20 nS in 1 M KCl. The channel protein was purified by preparative sodium dodecyl sulfate (SDS)-polyacylamide gel electrophoresis. It has a high molecular mass of 185 kDa, and its channel-forming ability resists boiling in SDS for 10 min.

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.

Cell wall associations of some antigenic proteins of slime-forming, encapsulated Streptococcus cremoris from the fermented milk product "viili"

1986 ◽  
Vol 32 (2) ◽  
pp. 176-178 ◽  
Author(s):  
Raili Forsén ◽  
Teuvo Hentunen ◽  
Kaua Valkonen ◽  
Sirpa Kontusaari
Keyword(s):  
Cell Wall ◽  
Molecular Mass ◽  
Cell Walls ◽  
Gradient Centrifugation ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Fermented Milk ◽  
Milk Product ◽  
Streptococcus Cremoris ◽  
Protein Components

Cell walls were isolated from mechanically disrupted cells of the slime-forming, encapsulated Streptococcus cremoris strains T5 and MLS96 by using sucrose gradient centrifugation as the last purification step. This cell wall isolation procedure was developed to obtain cell wall associated protein components. Sodium dodecyl sulfate – polyacrylamide gel electrophoresis revealed several polypeptide bands; the 50 kiloDalton band was major in strain T5 cell walls and the 26 and 30 kiloDalton bands were major in strain MLS96 cell walls. Both strains contained five antigenic polypeptides with molecular radius (Mr) values of 40, 47, 50, 54, and 70 kiloDaltons as analysed by immunoblotting and autoradiography. The polypeptides of strain MLS96 with molecular mass of 40 and 70 kiloDaltons reacted most strongly with homologous anti-whole cell serum. In addition, antigenic polypeptides with molecular mass of 100 and 160 kiloDaltons were also detected in strain T5.

Comparative analysis of subcellular distribution of protein antigens inMycobacterium bovisbacillus Calmette–Guérin

1997 ◽  
Vol 43 (8) ◽  
pp. 744-750 ◽  
Author(s):  
W. Florio ◽  
G. Freer ◽  
B. Dalla Casa ◽  
G. Batoni ◽  
G. Maisetta ◽  
...  
Keyword(s):  
Cell Wall ◽  
Monoclonal Antibodies ◽  
Cell Membrane ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Cell Wall Fraction ◽  
Bacillus Calmette Guerin ◽  
Cytoplasmic Fraction ◽  
Protein Antigens ◽  
Cell Wall Preparation

The distribution of protein antigens in purified subcellular fractions of Mycobacterium bovis bacillus Calmette–Guérin (BCG) was comparatively analysed by sodium dodecyl sulfate – polyacrylamide gel electrophoresis and immunoblotting with specific monoclonal antibodies and polyclonal sera. The 19- and 38-kDa lipoproteins were mainly detected in the cell wall and cell membrane enriched fractions, and they were extracted from the former by Triton X-114 and Nonidet P-40. The 65-kDa heat-shock protein (hsp) was present in the cytoplasmic fraction and only trace amounts were found in the crude cell wall preparation. In contrast, the 14-kDa hsp was highly represented in the cell wall fraction, besides being present in cytoplasmic fraction. Both superoxide dismutase (SOD) and antigen 85 complex (Ag 85) were abundantly released in culture medium, and to a lower extent, they were present in the cell wall fraction; SOD was present in comparable amounts also in the cytoplasmic fraction, while Ag 85 was far less represented in the same. Sera from mice immunized with culture filtrate (CF) proteins of BCG recognized several antigens in CFs, which were not detectable in cell wall, cell membrane, and cytoplasmic fractions, indicating that CF proteins include secreted antigens which have not yet been identified.Key words: bacillus Calmette–Guérin, monoclonal antibodies, subcellular localization.

scholarly journals Expression of a Gene for a Porin-Like Protein of the OmpA Family from Mycobacterium tuberculosisH37Rv

1998 ◽  
Vol 180 (14) ◽  
pp. 3541-3547 ◽  
Author(s):  
Ryan H. Senaratne ◽  
Hamid Mobasheri ◽  
K. G. Papavinasasundaram ◽  
Peter Jenner ◽  
Edward J. A. Lea ◽  
...  
Keyword(s):  
Molecular Mass ◽  
Lipid Bilayers ◽  
Single Channel ◽  
Specific Activity ◽  
Signal Sequence ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Rabbit Antiserum ◽  
Intact Protein ◽  
Planar Lipid Bilayers

ABSTRACT An open reading frame in the genomic database ofMycobacterium tuberculosis H37Rv was identified as having homology with an outer membrane protein. We found that the gene specified a protein belonging to the OmpA family, which includes some porins of gram-negative organisms. The gene was amplified by PCR and cloned into Escherichia coli. Overexpression of the gene was toxic to the host, but limited amounts could be purified from cells before growth ceased. A truncated gene devoid of the code for a presumed signal sequence was well expressed, but the protein had no pore-forming activity in the liposome swelling assay. However, the intact protein, OmpATb, behaved as a porin of low specific activity, with a pore diameter of 1.4 to 1.8 nm, and was also active in planar lipid bilayers, showing a single-channel conductance of 700 pS. The protein had a molecular mass of about 38 kDa in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A polyclonal rabbit antiserum raised to the truncated protein recognized a protein of similar molecular mass in detergent extracts of broken M. tuberculosis cells. Reverse transcription-PCR confirmed that the gene for OmpATb was expressed in M. tuberculosis cells growing in culture. Comparison of the purified protein with that in the detergent-extracted preparation using liposomes and planar lipid bilayers showed that the two materials had similar pore-forming properties. OmpATb is different from either of the mycobacterial porins described so far. This is the first report of a porin-like molecule from M. tuberculosis; the porin is likely to be important in controlling the access of hydrophilic molecules to the bacterial cell.

Purification and Properties of the F1Fo ATPase of Ilyobacter tartaricus, a Sodium Ion Pump

1998 ◽  
Vol 180 (13) ◽  
pp. 3312-3316 ◽  
Author(s):  
Sandra Neumann ◽  
Ulrich Matthey ◽  
Georg Kaim ◽  
Peter Dimroth
Keyword(s):  
Molecular Mass ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Atp Synthesis ◽  
Apparent Molecular Mass ◽  
Protein Sequencing ◽  
Ion Pump ◽  
Bacterial Membranes ◽  
Dependent Transport ◽  
Ilyobacter Tartaricus

ABSTRACT The ATPase of Ilyobacter tartaricus was solubilized from the bacterial membranes and purified. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified enzyme revealed the usual subunit pattern of a bacterial F1Fo ATPase. The polypeptides with apparent molecular masses of 56, 52, 35, 16.5, and 6.5 kDa were identified as the α, β, γ, ɛ, and c subunits, respectively, by N-terminal protein sequencing and comparison with the sequences of the corresponding subunits from the Na+-translocating ATPase ofPropionigenium modestum. Two overlapping sequences were obtained for the polypeptides moving with an apparent molecular mass of 22 kDa (tentatively assigned as b and δ subunits). No sequence could be determined for the putative a subunit (apparent molecular mass, 25 kDa). The c subunits formed a strong aggregate with the apparent molecular mass of 50 kDa which required treatment with trichloroacetic acid for dissociation. The ATPase was inhibited by dicyclohexyl carbodiimide, and Na+ ions protected the enzyme from this inhibition. The ATPase was specifically activated by Na+ or Li+ ions, markedly at high pH. After reconstitution into proteoliposomes, the enzyme catalyzed the ATP-dependent transport of Na+, Li+, or H+. Proton transport was specifically inhibited by Na+ or Li+ ions, indicating a competition between these alkali ions and protons for binding and translocation across the membrane. These experiments characterize the I. tartaricus ATPase as a new member of the family of FS-ATPases, which use Na+ as the physiological coupling ion for ATP synthesis.

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