scholarly journals Repression of the glucose-inducible outer-membrane protein OprB during utilization of aromatic compounds and organic acids in Pseudomonas putida CSV86

Microbiology ◽  
2011 ◽  
Vol 157 (5) ◽  
pp. 1531-1540 ◽  
Author(s):  
Rahul Shrivastava ◽  
Bhakti Basu ◽  
Ashwini Godbole ◽  
M. K. Mathew ◽  
Shree K. Apte ◽  
...  
Keyword(s):  
Membrane Protein ◽  
Organic Acids ◽  
Pseudomonas Putida ◽  
Outer Membrane ◽  
Outer Membrane Protein ◽  
Aromatic Compounds ◽  
Mass Spectrometry Analysis ◽  
Binding Studies ◽  
Spectrometry Analysis ◽  
Glucose Binding

Pseudomonas putida CSV86 shows preferential utilization of aromatic compounds over glucose. Protein analysis and [14C]glucose-binding studies of the outer membrane fraction of cells grown on different carbon sources revealed a 40 kDa protein that was transcriptionally induced by glucose and repressed by aromatics and succinate. Based on 2D gel electrophoresis and liquid chromatography-tandem mass spectrometry analysis, the 40 kDa protein closely resembled the porin B of P. putida KT2440 and carbohydrate-selective porin OprB of various Pseudomonas strains. The purified native protein (i) was estimated to be a homotrimer of 125 kDa with a subunit molecular mass of 40 kDa, (ii) displayed heat modifiability of electrophoretic mobility, (iii) showed channel conductance of 166 pS in 1 M KCl, (iv) permeated various sugars (mono-, di- and tri-saccharides), organic acids, amino acids and aromatic compounds, and (v) harboured a glucose-specific and saturable binding site with a dissociation constant of 1.3 µM. These results identify the glucose-inducible outer-membrane protein of P. putida CSV86 as a carbohydrate-selective protein OprB. Besides modulation of intracellular glucose-metabolizing enzymes and specific glucose-binding periplasmic space protein, the repression of OprB by aromatics and organic acids, even in the presence of glucose, also contributes significantly to the strain’s ability to utilize aromatics and organic acids over glucose.

scholarly journals Modulation of Glucose Transport Causes Preferential Utilization of Aromatic Compounds in Pseudomonas putida CSV86

2007 ◽  
Vol 189 (21) ◽  
pp. 7556-7562 ◽  
Author(s):  
Aditya Basu ◽  
Rahul Shrivastava ◽  
Bhakti Basu ◽  
Shree K. Apte ◽  
Prashant S. Phale
Keyword(s):  
Organic Acids ◽  
Pseudomonas Putida ◽  
Glucose Transport ◽  
Aromatic Compounds ◽  
Carbon Sources ◽  
Mass Spectrometry Analysis ◽  
Periplasmic Space ◽  
Two Dimensional Gel Electrophoresis ◽  
Spectrometry Analysis ◽  
Glucose Binding

ABSTRACT Pseudomonas putida CSV86 utilizes aromatic compounds in preference to glucose and coutilizes aromatics and organic acids. Protein analysis of cells grown on different carbon sources, either alone or in combination, revealed that a 43-kDa periplasmic-space protein was induced by glucose and repressed by aromatics and succinate. Two-dimensional gel electrophoresis and liquid chromatography-tandem mass spectrometry analysis identified this protein as closely resembling the sugar ABC transporter of Pseudomonas putida KT2440. A partially purified 43-kDa protein showed glucose binding activity and was specific for glucose. The results demonstrate that the aromatic- and organic acid-mediated repression of a periplasmic-space glucose binding protein and consequent inhibition of glucose transport are responsible for this strain's ability to utilize aromatics and organic acids in preference to glucose.

scholarly journals Serological Reactivity and Biochemical Characterization of Methylated and Unmethylated Forms of a Recombinant Protein Fragment Derived from Outer Membrane Protein B of Rickettsia typhi

2008 ◽  
Vol 15 (4) ◽  
pp. 684-690 ◽  
Author(s):  
Chien-Chung Chao ◽  
Zhiwen Zhang ◽  
Hui Wang ◽  
Abdulnaser Alkhalil ◽  
Wei-Mei Ching
Keyword(s):  
Membrane Protein ◽  
Outer Membrane ◽  
Outer Membrane Protein ◽  
Mass Spectrometry Analysis ◽  
Enzyme Linked Immunosorbent Assay ◽  
Composition Analysis ◽  
Rickettsia Typhi ◽  
Serological Reactivity ◽  
Lysine Residues ◽  
Protein B

ABSTRACT Rickettsia typhi, an obligate intracellular bacterium that causes murine typhus, possesses a heavily methylated outer membrane protein B (OmpB) antigen. This immunodominant antigen is responsible for serological reactions and is capable of eliciting protective immune responses with a guinea pig model. Western blot analysis of partially digested OmpB with patient sera revealed that most of the reactive fragments are larger than 20 kDa. One of these fragments, which is located at the N terminus (amino acids 33 to 273), fragment A (At), has been expressed in Escherichia coli. The expressed protein (rAt) was purified by chromatography and properly refolded by sequential dialysis. The refolded rAt protein was recognized by at least 87% of the typhus group patient sera as determined by enzyme-linked immunosorbent assay (ELISA). However, the titers were lower than those obtained with OmpB of R. typhi. Since native OmpB is hypermethylated at lysine residues, we chemically methylated the lysine residues in rAt. The methylation was confirmed by amino acid composition analysis, and the methylation pattern of the methylated rAt (mrAt) protein was similar to that of native At from OmpB, as revealed by liquid chromatography-mass spectrometry analysis. Both rAt and mrAt were evaluated in an ELISA for their serological reactivity with patient sera. Among patient sera tested, 83% exhibited higher titers with mrAt than with rAt. These results suggest that rAt, with or without methylation, can potentially replace rickettsia-derived OmpB or whole-cell antigen for the diagnosis of R. typhi infection.

scholarly journals Chlamydia pneumoniae Major Outer Membrane Protein Is a Surface-Exposed Antigen That Elicits Antibodies Primarily Directed against Conformation-Dependent Determinants

2001 ◽  
Vol 69 (5) ◽  
pp. 3082-3091 ◽  
Author(s):  
Katerina Wolf ◽  
Elizabeth Fischer ◽  
David Mead ◽  
Guangming Zhong ◽  
Roseanna Peeling ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Membrane Protein ◽  
Outer Membrane ◽  
Outer Membrane Protein ◽  
Chlamydia Pneumoniae ◽  
Major Outer Membrane Protein ◽  
Mass Spectrometry Analysis ◽  
Antigenic Determinants ◽  
Specific Monoclonal Antibody ◽  
Species Specific

ABSTRACT The major outer membrane protein (MOMP) of Chlamydia trachomatis serovariants is known to be an immunodominant surface antigen. Moreover, it is known that the C. trachomatis MOMP elicits antibodies that recognize both linear and conformational antigenic determinants. In contrast, it has been reported that the MOMP of Chlamydia pneumoniae is not surface exposed and is immunorecessive. We hypothesized that the discrepancies betweenC. trachomatis and C. pneumoniae MOMP exposure on intact chlamydiae and immunogenic properties might be because the focus of the host's immune response is directed to conformational epitopes of the C. pneumoniae MOMP. We therefore conducted studies aimed at defining the surface exposure of MOMP and the conformational dominance of MOMP antibodies. We present here a description of C. pneumoniaespecies-specific monoclonal antibody (MAb), GZD1E8, which recognizes a conformational epitope on the surface of C. pneumoniae. This MAb is potent in the neutralization ofC. pneumoniae infectivity in vitro. Another previously described C. pneumoniaespecies-specific monoclonal antibody, RR-402, displayed very similar characteristics. However, the antigenic determinant recognized by RR-402 has yet to be identified. We show by immunoprecipitation ofC. pneumoniae with GZD1E8 and RR-402 MAbs and by mass spectrometry analysis of immunoprecipitated proteins that both antibodies GZD1E8 and RR-402 recognize the MOMP of C. pneumoniae and that this protein is localized on the surface of the organism. We also show that human sera fromC. pneumoniae-positive donors consistently recognize the MOMP by immunoprecipitation, indicating that the MOMP ofC. pneumoniae is an immunogenic protein. These findings have potential implications for both C. pneumoniae vaccine and diagnostic assay development.

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