Proteomic analysis of cell envelope from Staphylococcus xylosus C2a, a coagulase-negative staphylococcus.
Staphylococcus xylosus is a saprophytic bacterium commonly found on skin of mammals but also used for its organoleptic properties in manufacturing of fermented meat products. This bacterium is able to form biofilms and to colonize biotic or abiotic surfaces, processes which are mediated, to a certain extent, by cell-envelope proteins. Thus, the present investigation aimed at evaluating and adapting different existing methods for cell-envelope subproteome analyses of the strain S. xylosus C2a. The protocol selected consisted initially of a lysostaphin treatment producing protoplasts and giving a fraction I enriched in cell wall proteins. A second fraction enriched in membrane proteins was then efficiently recovered by a procedure involving delipidation with a mixture of tributyl phosphate, methanol, and acetone and solubilization with a buffer containing ASB14. Proteins were separated using two-dimensional gel electrophoresis (2-DE) and identified using matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF MS). A total of 168 protein spots was identified corresponding to 90 distinct proteins. To categorize and analyze these proteomic data, a rational bioinformatic approach was carried out on proteins identified within cell envelope of S. xylosus C2a. Thirty-four proteins were predicted as membrane-associated with 91% present, as expected, within fraction II enriched in membrane proteins: 24 proteins were predicted as membranal, 3 as lipoproteins, and 7 as components of membrane protein complex. Eighteen out of 25 (72%) proteins predicted as secreted were indeed identified in fraction I enriched in cell wall proteins: 6 proteins were predicted as secreted via Sec translocon, and the remaining 19 proteins were predicted as secreted via unknown secretion system. Eighty-one percent (25/31) of proteins predicted as cytoplasmic were found in fraction II: 8 were clearly predicted as interacting temporarily with membrane components. By coupling conventional 2-DE and bioinformatic analysis, the approach developed allows fractionating, resolving, and analyzing a significant and important set of cell envelope proteins from a coagulase-negative staphylococcus, that is, S. xylosus C2a.
Planchon
Stella
S
INRA Centre de Clermont-Ferrand/Theix, UR454 Microbiologie, Equipe Qualité et Sécurité des Aliments, and Plate-Forme Protéomique, 63122 Saint-Genès Champanelle, France.
Chambon
Christophe
C
Desvaux
Mickaël
M
Chafsey
Ingrid
I
Leroy
Sabine
S
Talon
Régine
R
Hébraud
Michel
M
D020816:Amino Acid Motifs; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism; D003030:Coagulase / Q000737:chemistry*; D019295:Computational Biology / Q000379:methods; D015180:Electrophoresis, Gel, Two-Dimensional; D016680:Genome, Bacterial; D006801:Humans; D007091:Image Processing, Computer-Assisted; D021382:Protein Sorting Signals; D020543:Proteome; D040901:Proteomics / Q000379:methods*; D012867:Skin / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013210:Staphylococcus / Q000378:metabolism*
D001426:Bacterial Proteins; D003030:Coagulase; D021382:Protein Sorting Signals; D020543:Proteome