Study On The Extracellular Protein Related To Sulfur Activation By Acidithiobacillus Ferrooxidans ATCC23270

Elemental sulfur may accumulate in the course of metal sulfide dissolution and can form a layer on the metal sulfide surface, which consequently reduces diffusion rates for ions. Acidophilic sulfur-oxidizing bacteria can efficiently eliminate the insert sulfur and replenish the supply of the protons required for bioleaching processes, and so improve the leaching rate of iron. In natural acidic environment, the pathway for element sulfur dissolution is sulfur biooxidation. The sulfur biooxidation is a complex process that involves the attachment/activation of cells to sulfur particles, the transportation of sulfur through the outer membrane and the oxidation of sulfur in the periplasmic space. Sulfur-activated is the principal step of the sulfur biooxidation, However, the exact mechanism underlying sulfur-activated is not well understood.The present work focuses on isolation, identification and functional investigation of extracellular proteins and genes of Acidithiobacillus ferrooxidans, in order to provide basic data on sulfur biooxidation. The primary subjects of which include three parts as follows:(1) Research on comparative extracellular proteomics of A. ferrooxidans grown under different energy (Fe²⁺/S⁰)The extracellular proteins were extracted by stearming method and two-dimensional electrophoresis profiles was established. By using Matrix-assisted laser desorption/ ionization time-of-flight/time-of-flight mass spectroscopy (MALDI-TOF/MS) analysis, we identified a total of 18 extracellular proteins with apparent higher abundance grown in element sulfur than in ferrous sulfate. 12 of these proteins are unknown functions. In addition, six hypothetical proteins (or peptides) of which contain abundant of the cysteine residues.(2) Validate the sulfur-activated extracellular proteins and genes of A. ferrooxidansAccording to the information of A. ferrooxidans genome and the published research results, we chose 10 extracellular proteins genes responding to sulfur-activated and examine their expression analysis by real-time PCR. The results showed that the constitutive expression of all those genes was highly upregulated by growth on element sulfur compounds (and downregulated by growth on ferrous iron). What's more, Genes AFE₂537 and AFE₀927 were upgradulated 141.1 and 214.3-fold, respectively.(3) Expression, purification and functional investigation of the sulfur-activated extracellular proteins from A. ferrooxidansThe gene AFE₂621 encoding putative pilin from A. ferrooxidans was cloned and expressed in E. coli. The extracellular proteins of A. ferrooxidans grown on elemental sulfur and the purified AFE₂621 (pinlin) were characterized with sulfur K-edge XANES spectroscopy. The results showed that the thiol-containing amino acid mainly cysteine play an important role in elemental sulfur activation and oxidation.