| Abstract |
A number of strains which belong to the Pseudomonas species have been widely used to elucidate the adaptive mechanisms underlying increased tolerance against toxic concentrations of aromatic compound organic solvents in Gram-negative bacteria, using them as sole carbon source. A strain with high phenol-removal efficiency is Pseudomonas sp. strain phDV1, which was isolated from enriched mixed culture from samples of petroleum contaminated soil in Denmark. Previous proteomic analysis considering this particular strain have shown that the phenol biodegradation is being via the meta cleavage pathway, when it is present as a sole source of carbon and energy. Furthermore, recent studies have been done considering the membrane fraction of this strain which allowed the identification of the phenol, sucrose and lysogeny broth medium (LB) inducible outer membrane proteins using two-dimensional electrophoresis under denaturating conditions, giving a two-dimensional map that indicates only name of the proteins of a cell compartment. Nevertheless, only little information concerning the protein function can be obtained. An interesting way is to be able to point out the interactions between the proteins in order to have access to their functions and to understand the molecular mechanisms. Particularly, in cellular membranes many well characterized proteins assemble into complexes that carry out important tasks in energy generation, protein trafficking, and small molecule transport.
The interactions between proteins are important for very numerous, if not all, biological functions, and the interacted proteins might form part of a protein complex. To understand the protein complexes of a cell, complexome, is essential for a better understanding of cell functions. Pseudomonas sp. strain phDV1, a Gram-negative bacterium selected for its ability to degrade aromatic compounds. In the present study, we have performed a systematic analysis of Pseudomonas sp. strain phDV1 membrane protein complexes under different growth substrate conditions, using lysogeny broth, sucrose, and phenol as sole carbon source. In order to study protein complexes, an approach allowing the extraction and the analysis in native conditions is needed. In this study the sequential steps that were followed were: (a) obtaining purified preparations of the membranes from Pseudomonas sp. strain phDV1 by two washing steps with 5 mM EDTA and 0.1% lauryl sarcosinate, which removed membrane associated soluble proteins; (b) separation of membrane protein complexes using 1-D Blue-native polyacrylamide gel electrophoresis (BN-PAGE) after solubilization with n-dodecyl-β-maltoside (DDM); (c) combination of the BN with Tricine-SDS-PAGE (d) protein identification of tryptic peptides by mass spectrometry using MALDI TOF/TOF. Conclusively, a number of complexes were identified for the three different carbon sources and differences between the three sources have been mostly detected for proteins that are localized at the outer membrane of the strain Pseudomonas phDV1.
|
Μελέτη του μεμβρανικού κλάσματος του βακτηρίου Pseudomonas sp. phDV1 με μεθόδους πρωτεομικής ανάλυσης
