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  • American Society for Microbiology  (21)
  • 1
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    Elimination of Channel-Forming Activity by Insertional Inactivation of the p13 Gene in Borrelia burgdorferi
    American Society for Microbiology ; 2002
    In:  Journal of Bacteriology Vol. 184, No. 24 ( 2002-12-15), p. 6811-6819
    Details
    In: Journal of Bacteriology, American Society for Microbiology, Vol. 184, No. 24 ( 2002-12-15), p. 6811-6819
    Kurzfassung: P13 is a chromosomally encoded 13-kDa integral outer membrane protein of the Lyme disease agent, Borrelia burgdorferi . The aim of this study was to investigate the function of the P13 protein. Here, we inactivated the p13 gene by targeted mutagenesis and investigated the porin activities of outer membrane proteins by using lipid bilayer experiments. Channel-forming activity was lost in the p13 mutant compared to wild-type B. burgdorferi , indicating that P13 may function as a porin. We purified native P13 to homogeneity by fast performance liquid chromatography and demonstrated that pure P13 has channel-forming activity with a single-channel conductance in 1 M KCl of 3.5 nS, the same as the porin activity that was lost in the p13 mutant. Further characterization of the channel formed by P13 suggested that it is cation selective and voltage independent. In addition, no major physiological effects of the inactivated p13 gene could be detected under normal growth conditions. The inactivation of p13 is the first reported inactivation of a gene encoding an integral outer membrane protein in B. burgdorferi . Here, we describe both genetic and biophysical experiments indicating that P13 in B. burgdorferi is an outer membrane protein with porin activity.
    Materialart: Online-Ressource
    ISSN: 0021-9193 , 1098-5530
    URL: Article
    DOI: 10.1128/JB.184.24.6811-6819.2002
    Sprache: Englisch
    Verlag: American Society for Microbiology
    Publikationsdatum: 2002
    ZDB Id: 1481988-0
    SSG: 12
    Standort Signatur Einschränkungen Verfügbarkeit
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  • 2
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    Channel-Forming (Porin) Activity in Herpetosiphon aurantiacus Hp a2
    American Society for Microbiology ; 2004
    In:  Journal of Bacteriology Vol. 186, No. 19 ( 2004-10), p. 6667-6670
    Details
    In: Journal of Bacteriology, American Society for Microbiology, Vol. 186, No. 19 ( 2004-10), p. 6667-6670
    Kurzfassung: Detergent extracts of cell envelopes of the gliding bacterium Herpetosiphon aurantiacus formed channels in lipid bilayers. Fast protein liquid chromatography across a HiTrap-Q cation-exchange column demonstrated that a 45-kDa protein forms the channel. The observation of a channel-forming protein suggests that Herpetosiphon aurantiacus Hp a2 has a permeability barrier on its surface.
    Materialart: Online-Ressource
    ISSN: 0021-9193 , 1098-5530
    URL: Article
    DOI: 10.1128/JB.186.19.6667-6670.2004
    Sprache: Englisch
    Verlag: American Society for Microbiology
    Publikationsdatum: 2004
    ZDB Id: 1481988-0
    SSG: 12
    Standort Signatur Einschränkungen Verfügbarkeit
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  • 3
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    The Cell Wall of the Pathogenic Bacterium Rhodococcus equi Contains Two Channel-Forming Proteins with Different Properties
    American Society for Microbiology ; 2003
    In:  Journal of Bacteriology Vol. 185, No. 9 ( 2003-05), p. 2952-2960
    Details
    In: Journal of Bacteriology, American Society for Microbiology, Vol. 185, No. 9 ( 2003-05), p. 2952-2960
    Kurzfassung: 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 PorA Req ( R. equi pore A) is characterized by the formation of cation-selective channels, which are voltage gated. PorA Req 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 PorB Req [ 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 .
    Materialart: Online-Ressource
    ISSN: 0021-9193 , 1098-5530
    URL: Article
    DOI: 10.1128/JB.185.9.2952-2960.2003
    Sprache: Englisch
    Verlag: American Society for Microbiology
    Publikationsdatum: 2003
    ZDB Id: 1481988-0
    SSG: 12
    Standort Signatur Einschränkungen Verfügbarkeit
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  • 4
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    Moraxella catarrhalis M35 Is a General Porin That Is Important for Growth under Nutrient-Limiting Conditions and in the Nasopharynges of Mice
    American Society for Microbiology ; 2008
    In:  Journal of Bacteriology Vol. 190, No. 24 ( 2008-12-15), p. 7994-8002
    Details
    In: Journal of Bacteriology, American Society for Microbiology, Vol. 190, No. 24 ( 2008-12-15), p. 7994-8002
    Kurzfassung: Moraxella catarrhalis is a gram-negative respiratory pathogen that is an important causative agent for otitis media and exacerbations of chronic obstructive pulmonary disease. We have previously predicted the outer membrane protein M35 to be a general porin, and in the current study, we have investigated the function of M35 and its importance for survival of M. catarrhalis in vivo. Lipid bilayer experiments reveal that refolded M35 functions as a channel that is typical of gram-negative bacterial porins. M35 forms wide and water-filled channels with a single-channel conductance of about 1.25 nS in 1 M KCl solution and has only a small selectivity for cations over anions. When the in vitro growth characteristics of two M35 deletion mutant strains of M. catarrhalis were compared to the wild-type parent isolates, the growth of the mutant strains was inhibited only under nutrient-poor conditions. This growth defect could be eliminated by additional glutamic acid, but not additional aspartic acid, glycine, sucrose, or glucose. The mutant strains compensated for the lack of M35 by enhancing their uptake of glutamic acid, and this enhanced rate of glutamic acid uptake was attributed to the compensatory upregulation of a protein of approximately 40 kDa. M35 was also found to be essential for nasal colonization of mice, demonstrating that its presence is essential for survival of M. catarrhalis in vivo. These results suggest that M35 is a general porin that is necessary for the uptake of important energy sources by M. catarrhalis and that it is likely that M35 is an essential functional protein for in vivo colonization.
    Materialart: Online-Ressource
    ISSN: 0021-9193 , 1098-5530
    URL: Article
    DOI: 10.1128/JB.01039-08
    Sprache: Englisch
    Verlag: American Society for Microbiology
    Publikationsdatum: 2008
    ZDB Id: 1481988-0
    SSG: 12
    Standort Signatur Einschränkungen Verfügbarkeit
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  • 5
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    Corynebacterium diphtheriae : Identification and Characterization of a Channel-Forming Protein in the Cell Wall
    American Society for Microbiology ; 2007
    In:  Journal of Bacteriology Vol. 189, No. 21 ( 2007-11), p. 7709-7719
    Details
    In: Journal of Bacteriology, American Society for Microbiology, Vol. 189, No. 21 ( 2007-11), p. 7709-7719
    Kurzfassung: The cell wall fraction of the gram-positive, nontoxic Corynebacterium diphtheriae strain C8 r (−) 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.
    Materialart: Online-Ressource
    ISSN: 0021-9193 , 1098-5530
    URL: Article
    DOI: 10.1128/JB.00864-07
    Sprache: Englisch
    Verlag: American Society for Microbiology
    Publikationsdatum: 2007
    ZDB Id: 1481988-0
    SSG: 12
    Standort Signatur Einschränkungen Verfügbarkeit
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  • 6
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    Reconstitution Experiments and Gene Deletions Reveal the Existence of Two-Component Major Cell Wall Channels in the Genus Corynebacterium
    American Society for Microbiology ; 2010
    In:  Journal of Bacteriology Vol. 192, No. 3 ( 2010-02), p. 786-800
    Details
    In: Journal of Bacteriology, American Society for Microbiology, Vol. 192, No. 3 ( 2010-02), p. 786-800
    Kurzfassung: Two small polypeptides, PorA and PorH, are known to form cell wall channels in Corynebacterium glutamicum and in Corynebacterium efficiens . The genes coding for both polypeptides are localized in close proximity to one another between the genes coding for GroEl2 and a polyphosphate kinase (PKK2). In this study, we investigated the relationship of PorA and PorH to one another. The results suggested that the major cell wall channels of Corynebacterium glutamicum , Corynebacterium efficiens , and Corynebacterium diphtheriae need the obligatory presence of two distinct polypeptides, one of class PorA and one of class PorH, to form an active cell wall channel. Identification of genes coding for homologous proteins in the chromosome of Corynebacterium callunae suggested a similar result for this strain. Contrary to our previous reports on channel-forming proteins in these strains, a heterooligomeric structure composed of PorA and PorH is needed in all of them to form the major cell wall channel. This was concluded from complementation experiments using a porH - and porA -deficient C . glutamicum strain. The stringent necessity of proteins of either class to recover the wild-type channels was demonstrated by black lipid bilayer experiments using detergent or organic solvent extracts of the complemented porH - and porA -deficient C . glutamicum strain. The channel-forming capability of recombinant expressed, affinity-purified PorA and PorH proteins of C . glutamicum revealed that the channels consisted solely of these two components. This agreed with results obtained from a transcript coding for both channel-forming components identified in C . glutamicum by Northern blot analysis and reverse transcription-PCR analysis. The transcription start point of the genes was determined by the rapid amplification of cDNA ends approach, allowing the prediction of the −35 and −10 regions of the promoter. The results demonstrate that the cell wall channels within the genus Corynebacterium may be formed by two-component oligomers.
    Materialart: Online-Ressource
    ISSN: 0021-9193 , 1098-5530
    URL: Article
    DOI: 10.1128/JB.01142-09
    Sprache: Englisch
    Verlag: American Society for Microbiology
    Publikationsdatum: 2010
    ZDB Id: 1481988-0
    SSG: 12
    Standort Signatur Einschränkungen Verfügbarkeit
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  • 7
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    Biochemical Identification and Biophysical Characterization of a Channel-Forming Protein from Rhodococcus erythropolis
    American Society for Microbiology ; 2000
    In:  Journal of Bacteriology Vol. 182, No. 3 ( 2000-02), p. 764-770
    Details
    In: Journal of Bacteriology, American Society for Microbiology, Vol. 182, No. 3 ( 2000-02), p. 764-770
    Kurzfassung: 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 of R. 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.
    Materialart: Online-Ressource
    ISSN: 0021-9193 , 1098-5530
    URL: Article
    DOI: 10.1128/JB.182.3.764-770.2000
    Sprache: Englisch
    Verlag: American Society for Microbiology
    Publikationsdatum: 2000
    ZDB Id: 1481988-0
    SSG: 12
    Standort Signatur Einschränkungen Verfügbarkeit
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  • 8
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    Differences in Purinergic Amplification of Osmotic Cell Lysis by the Pore-Forming RTX Toxins Bordetella pertussis CyaA and Actinobacillus pleuropneumoniae ApxIA: the Role of Pore Size
    American Society for Microbiology ; 2013
    In:  Infection and Immunity Vol. 81, No. 12 ( 2013-12), p. 4571-4582
    Details
    In: Infection and Immunity, American Society for Microbiology, Vol. 81, No. 12 ( 2013-12), p. 4571-4582
    Kurzfassung: A large subgroup of the r epeat in t o x in (RTX) family of leukotoxins of Gram-negative pathogens consists of pore-forming hemolysins. These can permeabilize mammalian erythrocytes (RBCs) and provoke their colloid osmotic lysis (hemolytic activity). Recently, ATP leakage through pannexin channels and P2X receptor-mediated opening of cellular calcium and potassium channels were implicated in cell permeabilization by pore-forming toxins. In the study described here, we examined the role played by purinergic signaling in the cytolytic action of two RTX toxins that form pores of different sizes. The cytolytic potency of ApxIA hemolysin of Actinobacillus pleuropneumoniae , which forms pores about 2.4 nm wide, was clearly reduced in the presence of P2X 7 receptor antagonists or an ATP scavenger, such as pyridoxalphosphate-6-azophenyl-2′,4′-disulfonic acid (PPADS), Brilliant Blue G, ATP oxidized sodium salt, or hexokinase. In contrast, antagonists of purinergic signaling had no impact on the hemolytic potency of the adenylate cyclase toxin-hemolysin (CyaA) of Bordetella pertussis , which forms pores of 0.6 to 0.8 nm in diameter. Moreover, the conductance of pores formed by ApxIA increased with the toxin concentration, while the conductance of the CyaA single pore units was constant at various toxin concentrations. However, the P2X 7 receptor antagonist PPADS inhibited in a concentration-dependent manner the exacerbated hemolytic activity of a CyaA-ΔN489 construct (lacking 489 N-terminal residues of CyaA), which exhibited a strongly enhanced pore-forming propensity ( 〉 20-fold) and also formed severalfold larger conductance units in planar lipid bilayers than intact CyaA. These results point to a pore size threshold of purinergic amplification involvement in cell permeabilization by pore-forming RTX toxins.
    Materialart: Online-Ressource
    ISSN: 0019-9567 , 1098-5522
    URL: Article
    DOI: 10.1128/IAI.00711-13
    RVK:
    XA 10000
    Sprache: Englisch
    Verlag: American Society for Microbiology
    Publikationsdatum: 2013
    ZDB Id: 1483247-1
    Standort Signatur Einschränkungen Verfügbarkeit
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  • 9
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    PorA Represents the Major Cell Wall Channel of the Gram-Positive Bacterium Corynebacterium glutamicum
    American Society for Microbiology ; 2003
    In:  Journal of Bacteriology Vol. 185, No. 16 ( 2003-08-15), p. 4779-4786
    Details
    In: Journal of Bacteriology, American Society for Microbiology, Vol. 185, No. 16 ( 2003-08-15), p. 4779-4786
    Kurzfassung: The cell wall of the gram-positive bacterium Corynebacterium glutamicum contains a channel (porin) for the passage of hydrophilic solutes. The channel-forming polypeptide PorA is a 45-amino-acid acidic polypeptide with an excess of four negatively charged amino acids, which is encoded by the 138-bp gene porA . porA was deleted from the chromosome of C.glutamicum wild-type strain ATCC 13032 to obtain mutant ATCC 13032Δ porA . Southern blot analysis demonstrated that porA was deleted. Lipid bilayer experiments revealed that PorA was not present in the cell wall of the mutant strain. Searches within the known chromosome of C. glutamicum by using National Center for Biotechnology Information BLAST and reverse transcription-PCR showed that no other PorA-like protein is encoded on the chromosome or is expressed in the deletion strain. The porA deletion strain exhibited slower growth and longer growth times than the C. glutamicum wild-type strain. Experiments with different antibiotics revealed that the susceptibility of the mutant strain was much lower than that of the wild-type C. glutamicum strain. The results presented here suggest that PorA represents a major hydrophilic pathway through the cell wall and that C. glutamicum contains cell wall channels which are not related to PorA.
    Materialart: Online-Ressource
    ISSN: 0021-9193 , 1098-5530
    URL: Article
    DOI: 10.1128/JB.185.16.4779-4786.2003
    Sprache: Englisch
    Verlag: American Society for Microbiology
    Publikationsdatum: 2003
    ZDB Id: 1481988-0
    SSG: 12
    Standort Signatur Einschränkungen Verfügbarkeit
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  • 10
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    The BBA01 Protein, a Member of Paralog Family 48 from Borrelia burgdorferi , Is Potentially Interchangeable with the Channel-Forming Protein P13
    American Society for Microbiology ; 2006
    In:  Journal of Bacteriology Vol. 188, No. 12 ( 2006-06-15), p. 4207-4217
    Details
    In: Journal of Bacteriology, American Society for Microbiology, Vol. 188, No. 12 ( 2006-06-15), p. 4207-4217
    Kurzfassung: The Borrelia burgdorferi genome exhibits redundancy, with many plasmid-carried genes belonging to paralogous gene families. It has been suggested that certain paralogs may be necessary in various environments and that they are differentially expressed in response to different conditions. The chromosomally located p13 gene which codes for a channel-forming protein belongs to paralog family 48, which consists of eight additional genes. Of the paralogous genes from family 48, the BBA01 gene has the highest homology to p13 . Herein, we have inactivated the BBA01 gene in B. burgdorferi strain B31-A. This mutant shows no apparent phenotypic difference compared to the wild type. However, analysis of BBA01 in a C-terminal protease A (CtpA)-deficient background revealed that like P13, BBA01 is posttranslationally processed at its C terminus. Elevated BBA01 expression was obtained in strains with the BBA01 gene introduced on the shuttle vector compared to the wild-type strain. We could further demonstrate that BBA01 is a channel-forming protein with properties surprisingly similar to those of P13. The single-channel conductance, of about 3.5 nS, formed by BBA01 is comparable to that of P13, which together with the high degree of sequence similarity suggests that the two proteins may have similar and interchangeable functions. This is further strengthened by the up-regulation of the BBA01 protein and its possible localization in the outer membrane in a p13 knockout strain, thus suggesting that P13 can be replaced by BBA01.
    Materialart: Online-Ressource
    ISSN: 0021-9193 , 1098-5530
    URL: Article
    DOI: 10.1128/JB.00302-06
    Sprache: Englisch
    Verlag: American Society for Microbiology
    Publikationsdatum: 2006
    ZDB Id: 1481988-0
    SSG: 12
    Standort Signatur Einschränkungen Verfügbarkeit
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