Non-disruptive release of Pseudomonas putida proteins by in situ electric breakdown of intact cells.
Analysis of the native proteome of bacterial cells typically involves physical procedures (sonication, French press) and/or biochemical methods (treatment with lysozyme, osmotic shock etc.) to break open the bacteria to yield a soluble protein fraction. Such procedures are not only time consuming, but they change bacterial physiology during manipulation and affect labile post-translational modifications such as His-P bonds. In this work, we document the efficacy of the dielectric breakdown of live bacteria for releasing and delivering the protein contents of intact cells directly into a non-denaturing gel system. By means of such an in situ electrophoresis, the protein pool enters the separation medium without any manipulation of the cells other than being exposed to a moderate electric voltage. To validate the method we have followed the fate of the two forms of the PtsN (EIIA(Ntr)) protein of the phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS) of Pseudomonas putida through the various stages of the procedure. Apart of detecting the corresponding polypeptides, we show that this procedure releases the bulk of the proteome while keeping unharmed the phosphorylation state of EIIA(Ntr) as it was present in the cells prior to applying the electric field. The method is applicable to other bacteria as well.
Pflüger
Katharina
K
Centro Nacional de Biotecnología CSIC, Campus de Cantoblanco, 28049 Madrid, Spain.
di Bartolo
Ilaria
I
Velázquez
Francisco
F
de Lorenzo
Víctor
V
D001426:Bacterial Proteins / Q000378:metabolism* / Q000502:physiology; D002462:Cell Membrane / Q000502:physiology; D004560:Electricity; D004563:Electrochemistry; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods; D015964:Gene Expression Regulation, Bacterial; D008855:Microscopy, Electron, Scanning; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System / Q000378:metabolism / Q000502:physiology*; D016958:Pseudomonas putida / Q000201:enzymology / Q000378:metabolism*
D001426:Bacterial Proteins; C423631:PtsN protein, Pseudomonas putida; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System