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1.
J Proteome Res ; 6(6): 2168-75, 2007 Jun.
Article in English | MEDLINE | ID: mdl-17488104

ABSTRACT

We are confronted daily to unknown microorganisms that have yet to be characterized, detected, and/or analyzed. We propose, in this study, a multidimensional strategy using polyclonal antibodies, consisting of a novel proteomic tool, the ProteomeLab PF2D, coupled to immunological techniques and mass spectrometry (i-PF2D-MS/MS). To evaluate this strategy, we have applied it to Bacillus subtilis, considered here as our unknown bacterial model.


Subject(s)
Antigens, Bacterial/analysis , Bacillus subtilis/immunology , Mass Spectrometry/methods , Proteomics/methods , Amino Acid Sequence , Antibodies, Bacterial/immunology , Antibodies, Bacterial/ultrastructure , Bacillus subtilis/ultrastructure , Blotting, Western , Hydrophobic and Hydrophilic Interactions , Immune Sera , Isoelectric Point , Microscopy, Electron , Molecular Sequence Data
2.
J Microbiol Methods ; 67(3): 593-6, 2006 Dec.
Article in English | MEDLINE | ID: mdl-16822569

ABSTRACT

We propose a multi-dimensional strategy, associating immunodetection to a protein fractionating two-dimensional liquid chromatography tool, for serological characterization of microbial antigens. The originality of such immunoproteomic approaches resides in their application in large-scale studies for rapid serotyping of micro-organisms, evaluation of immunomes and could be proposed in the development and monitoring of vaccines.


Subject(s)
Antigens, Bacterial/analysis , Antigens, Bacterial/immunology , Bacillus subtilis/immunology , Bacterial Proteins/analysis , Bacterial Proteins/immunology , Proteomics , Blotting, Western , Chromatography, Liquid , Electrophoresis, Polyacrylamide Gel , Immunologic Techniques , Microscopy/methods , Proteome/analysis , Serotyping , Spectrum Analysis , Tandem Mass Spectrometry
3.
Rapid Commun Mass Spectrom ; 18(18): 2013-9, 2004.
Article in English | MEDLINE | ID: mdl-15378711

ABSTRACT

In recent years, various mass spectrometry procedures have been developed for bacterial identification. The accuracy and speed with which data can be obtained by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) could make this a powerful tool for environmental monitoring. However, minor variations in the sample preparation can influence the mass spectra significantly. Therefore, the first objectives of this study were the adjustment and the optimization of experimental parameters allowing a rapid identification of whole bacterial cells without laborious sample preparation. The tested experimental parameters were matrix, extraction solvent, salt content, deposition method, culture medium and incubation time. This standardized protocol was applied to identify reference and environmental bacterial strains of Escherichia coli, Salmonella and Acinetobacter. The environmental bacterial strains were isolated from sewage sludge using an original microextraction procedure based on repeated sonications and enzymatic treatments. The bacterial identification was realized by the observation of the respective genus-, species- and strain-specific biomarkers. This bacterial taxonomy could be completed within one hour, with minimal sample preparation, provided that sufficient bacteria had been collected prior to MALDI-TOF analysis.


Subject(s)
Bacteria/classification , Bacteria/isolation & purification , Cell Separation/methods , Environmental Pollutants/isolation & purification , Sewage/analysis , Sewage/microbiology , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization/methods , Actinobacteria/classification , Actinobacteria/cytology , Actinobacteria/isolation & purification , Bacteria/cytology , Cell Culture Techniques/methods , Cell Survival , Colony Count, Microbial/methods , Environmental Monitoring/methods , Escherichia coli/classification , Escherichia coli/cytology , Escherichia coli/isolation & purification , Salmonella/classification , Salmonella/cytology , Salmonella/isolation & purification , Species Specificity
4.
Protein Sci ; 13(5): 1209-18, 2004 May.
Article in English | MEDLINE | ID: mdl-15096627

ABSTRACT

Xyl1 from Streptomyces sp. S38 belongs to the low molecular mass family 11 of endo-beta-1,4-xylanases. Its three-dimensional structure has been solved at 2.0 A and its optimum temperature and pH for enzymatic activity are 60 degrees C and 6.0, respectively. Aspergillus kawachii xylanase XynC belongs to the same family but is an acidophilic enzyme with an optimum pH of 2.0. Structural comparison of Xyl1 and XynC showed differences in residues surrounding the two glutamic acid side chains involved in the catalysis that could be responsible for the acidophilic adaptation of XynC. Mutations W20Y, N48D, A134E, and Y193W were introduced by site-directed mutagenesis and combined in multiple mutants. Trp 20 and Tyr 193 are involved in substrate binding. The Y193W mutation inactivated Xyl1 whereas W20Y decreased the optimum pH of Xyl1 to 5.0 and slightly increased its specific activity. The N48D mutation also decreased the optimum pH of Xyl1 by one unit. The A134E substitution did not induce any change, but when combined with N48D, a synergistic effect was observed with a 1.4 unit decrease in the optimum pH. Modeling showed that the orientations of residue 193 and of the fully conserved Arg 131 are different in acidophilic and "alkaline" xylanases whereas the introduced Tyr 20 probably modifies the pKa of the acid-base catalyst via residue Asn 48. Docking of a substrate analog in the catalytic site highlighted striking differences between Xyl1 and XynC in substrate binding. Hydrophobicity calculations showed a correlation between acidophilic adaptation and a decreased hydrophobicity around the two glutamic acid side chains involved in catalysis.


Subject(s)
Endo-1,4-beta Xylanases/chemistry , Endo-1,4-beta Xylanases/genetics , Models, Molecular , Amino Acid Sequence , Binding Sites , DNA Mutational Analysis , Enzyme Stability , Hydrogen-Ion Concentration , Molecular Sequence Data , Mutagenesis, Site-Directed , Sequence Alignment , Streptomyces/enzymology , Substrate Specificity , Temperature
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