Study On Sulfur Oxidation-related Periplasmic Proteins Of Acidithiobacillus Ferrooxidans ATCC 23270

At present, Acidithiobacillus ferrooxidans is the most widely applied and investigated bacterium in biometallurgy. It is a chemolithoautotrophic, Gram-negative bacterium which can get energy for growth from the oxidation of ferrous to ferric iron and elemental sulfur or reduced inorganic sulfur compounds to sulfate using oxygen as electron acceptor. The oxidation of elemental sulfur by A. ferrooxidans is a complex process which involves the attachment of cells to sulfur particles, the transport through the out membrane and the oxidation. The most relevant reactions for sulfur oxidation take place at the periplasmic space. However the mechanism of biological sulfur activation and oxidation in A. ferrooxidans is still unclear because of the lack of related rearches developed all over the world. In this thesis, the sulfur oxidation-related periplasmic proteins of A. ferrooxidans have been studied. The periplasmic proteins researched at the aspect of differentail Proteomics would be helpful to clarify the mechanisms of A. ferrooxidans sulfur oxidation.In the present work, we analyzed and characterized the proteins presented in the periplasmic fraction of A. ferrooxidans ATCC 23270 cultured in two different energy substrates, ferrous sulfate and elemental sulfur by two-dimensional polyacrylamide gel electrophoresis (2-DE), respectively. A total of 17 periplasmic proteins with apparent higher abundance grown on elemental sulfur than on ferrous sulfate were identified with the use of matrix-assisted laser-desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). These identified periplasmic proteins functionally classified into nine categories according to the database. They can mediate functions of binding heavy metals, modifying protein (conformation), tolerating stress, transducting signals, which may occur in the process of sulfur oxidation.In order to validate the findings of two-dimensional gel electrophoresis (2-DE), real-time quantitative RT-PCR was used to analyze changes in transcription levels of the chosen periplasmic protein genes of A.ferrooxidans ATCC 23270 grown on sulfur and ferrous. Expression analysis of the corresponding genes by Real Time-PCR showed the constitutive expression of all those genes were highly upregulated when grown on element sulfur compounds. The researches of the correponding genes of the identified periplasmic proteins by RT-PCR confirmed the results of 2-DE, indicating they may related with sulfur metabolism in A. ferrooxidans, the functions of the differentially expressed genes need to be further researched.