The Study On Molecular Mechanism Of Cellular Sulfur Acclimation And Sulfur Activation By Acidithiobacillus Ferrooxidans

Metal sulfides are chemically attacked by Fe³⁺ and H⁺,resulting in the formation of elemental sulfur,which may aggregate and even form a layer on the metal sulfides' surface and inhibit leaching of metals from the sulfides minerals.The inert elemental sulfur in a typical acidic environment can exclusively be eliminated by biooxidation of acidophilic sulfur-oxidizing bacteria(ASOB),in which the ASOB oxidize the elemental sulfur to the intermediary reductive sulfur compounds and finally to sulfuric acid that replenish the supply of the protons required at for the leaching processes.It is obvious that the sulfur biooxidation of sulfur by ASOB takes an important role in bioleaching,suggesting the necessity to research and then understand the mechanism and realization of that role as well as its influential factors. On the other hand,the molecular mechanism of elemental sulfur oxidation,the most important part of the sulfur biooxidation system of ASOB,has been lack in researching and kept unresolved,indicating also the particularly academic meaningful of research on sulfur biooxidation of ASOB.In order to enlarge the knowledge on sulfur biooxidation of ASOB,the present study focuses on elucidating the molecular mechanism of Acidithiobacillus ferrooxidans cellular sulfur acclimation and sulfur activation.The primary subjects of which are as follows:(1) Cellular acclimation of A.ferrooxidans to sulfur biooxidation This part was dedicated researching on the specific effects of typical anions including Cl^-,NO₃^-,SO₄^2- and PO₄^3-,and then elucidating the general effects of environmental anions on the growth and sulfur-oxidation activity of A.ferrooxidans. The results showed that the relatively high concentrations of the typical anions make delay and inhibit the growth and sulfur oxidation of the cells.Different from any other typical anions,phosphate ions show inhibition at much higher concentration,and they even stimulate growth and sulfur oxidation ofA.ferrooxidans at concentration of 100 mmol/L.2-DE(two-dimential electrophoresis) showed clearly the differential expression of the total proteins of A.ferrooxidans under stress condition of high concentration of PO₄^3- from the normal condition.The higher expressed proteins at each case may take part in the biochemical reaction(including sulfur biooxidation) at normal condition or response to and adapt to the environmental stress condition, respectively.The cellular acclimation of A.ferrooxidans to elemental sulfur and thiosulfate were elucidated in terms of growth and morphology of cells grown on S⁰ or S₂O₃^2-, changes in surface physicochemical properties of S⁰ particles and cells themselves after interaction of bacterial cells and the S⁰ particles,and the adsorption behavior of the cells to S⁰ particles.The results showed that A.ferrooxidans cells were grown faster and demonstrated higher concentration on soluble thiosulfate substrate than on non-soluble hydrophobic sulfur substrate.A.ferrooxidans cells acclimated to the sulphur bio-oxidation by actively expressing some extracellular polymers substance, maybe some proteins,which modified the surface properties of both sulfur particles and the cells themselves.The cells aggregating onto the sulfur surface enhanced the use of sulfur substrate.The A.ferrooxidans cells were present the characteristics to dynamically adsorb to or desorb from the surface of sulfur particles where there were apparently some pits.(2) The extracellular proteomies of A.ferrooxidansIn order to more facily uncover the sulfur-oxidizing related/functional proteins, the proteomics of the specific cellular compartments,including extracellular and periplasmic space,rather than the proteomics of the total cells of A.ferrooxidans was particularly adopted.Experiments were dedicated to development of fitter methods for selectively isolating the extracellular proteins and periplasmic space proteins for their role to modify sulfur particles and to oxidize sulfur respectively,and optimized the two-dimensional electrophoresis parameters.The 2-D electrophoregrams clearly exhibited a few of extracellular proteins of A. ferrooxidans in sulfur and ferrous sulfate energy substrates.The results showed that there were about eighteen protein spots with relatively high abundance in cells grown on sulfur substrate but absent in cells grown on ferrous sulfate substrate.These protein spots were analyzed by MALDI-TOF/TOF.The identification and predication of the extracellular proteins showed them affiliated to different categories according to their potential biological functions,i.e.,AFE₁391:conjugal transfer protein; AFE₂621:pilin,putative;AFE₁847:lipoprotein,putative;AFE₀982:vacJ lipoprotein;AFE 0927:polysaccharide deacetylase family protein;AFE₁932: Ser/Thr protein phosphatase family protein,and the hypothetical proteins. Additionally,six hypothetical proteins(or peptides) which contain abundant of the cysteine residues,indicates that four of them even have one or two CXXC functional motifs.Base on this fact,a putative function mode in sulfur activation of these proteins was proposed.These relatively high expression proteins(or peptide) of cells on sulfur energy substrate should have a relationship with the attachment/activation process,but their specific functions need a further research.(3) The characterization of sulfur aetivation/oxidiation related proteins or genesThe reduced form of glutathione(GSH) was identified to play a catalytic role in accelerating the activation/oxidiation of the elemental sulfur in Acidithiobacillus.The gene of gr from A.ferrooxidans was cloned and expressed in E.coli.It was found that the purified gr expressed product was able to use both NADPH and NADH as the electron donors,which was different from most of previously studied homologous glutathione reductase(GR).One arginine residue of the highly conserved NADPH binding motif,which was critical for NADPH binding,was found to be replaced by an asparagine residue.It was proposed that some thiol group-containing proteins(R-SH) in periplasmic space are employed for sulfur compounds oxidiation in periplasmic space,and the thiol-disulfide interchange proteins are responsible for maintaining high level of free thiol groups and the formation and rearrangement of disulfide bonds in periplasmic space.The gene dsbG,a disulfide bonding gene from A.ferrooxidans was cloned and expressed in E.coli.The enzymatical study showed that the purified DsbG of A. ferrooxidans have the disulfide isomerization activity.Site-directed mutagenesis of the DsbG protein revealed that Cys119 and Cys122 were crucial for DsbG disulfide isomerization activity.On the integration of bioinformative analysis of the A.ferrooxidans genome annotations and the published research results,two clusters of genes that are potentially involved in the oxidation of reduced sulfur compounds,including the sulfate:thiosulfate binding protein coding gene sbp,membrane bound thlosulfate:quinone oxidoreductase coding gene doxDA,and rhodanese-like protein coding gene p21,were chosen and further validated by reverse transcriptase-PCR (RT-PCR).It was found that these two clusters of genes are organized as operons doxDA-1 and doxDA-2 in A.ferrooxidans.The possible promoter sequences of these doxDA operons were finally predicted based on bioinformatic analyses.