Research On The Cell Wall Related Proteins Of Acidithiobacillus Ferrooxidans

At present,Acidithiobacillus ferrooxidans is the most widely applied and investigated bacteria in biometallurgy.The interaction at the interface between microbe and mineral plays an important role in this process.In this thesis,the cell wall related proteins of A.ferrooxidans have been studied.The surface structures researched at the aspect of Proteomics would be helpful to clarify the mechanisms of A.ferrooxidans attaching to minerals.In this study,the cell wall related proteins of A.ferrooxidans were extracted and separated by SDS-PAGE.Liquid chromatography coupled with tandem mass spectrometry was applied to identify the proteins. Firstly,the proteins which were subjected to in-gel digestion with trypsin, were separated by liquid chromatography,and then were online analyzed by the coupled tandem mass spectrometry.As the peptide mass fingerprint(PMF)database has not been consummated,we construct our local putative A.ferrooxidans protein PMF database to be used in the mass spectrometry proteomisc analysis.The acquired 245 MS and MS/MS spectras were sent for searching in the proteins database with Mascot search engine for protein identification.The export signal and subcellular location of each obtained protein was also predicted.Portion of the aimed proteins were obtained.Among the identified proteins, 42.3%of them possess export of the Sec type,7.2%of them show a twin arginine translocation(Tat)signal,18.6%have lipoprotein signal peptides. 21.6%are non-classically secreted proteins,and 19.6%could be exported with their subcellular location prediction.According to their functional categories,43.3%of them belong to Cell Envelope,16.5%to Hypothetical Proeteins,6.2%to Conserved Hypothetical Proteins,and 6.2%belonged to Unknown Functions.Also,there are a few other functional proteins.Furthermore,the molecular weights of these proteins are distributed in the range of 4000 to 118000Da.Their isoelectric points are between 4 and 12,among which 31.96%of which show the pH values below 7,68.04%higher than 7,and 10.31%over pH values of 10.This work provides fundamental research of the cell wall related protein of A. ferrooxidans by revealing the total expressed proteins.We also studied the comparative proteomics of A.ferrooxidans grown under different energy resources(Fe²⁺or sulphur).The cell wall related proteins of A.ferrooxidans were separated by the two-dimensional gel electrophoresis(2-DE)and then identified by the matrix-assisted laser-desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS).The resulted Images were quantitatively analyzed with the PDQuest 2D analysis software.The image analysis indicates 104 differentially-expressed protein spots,18 of which have been identified.