【作者】 张成桂；

【导师】 夏金兰；

【作者基本信息】 中南大学 ， 微生物学， 2008， 博士

【摘要】 在生物浸出过程中,金属硫化矿在三价铁离子和质子作用下分解,可能产生元素硫累积,进而在金属硫化矿表面形成疏水元素硫层,阻碍金属离子的进一步浸出。浸矿环境中的嗜酸硫氧化细菌能有效消解和利用硫化矿分解过程中的元素硫,同时不断补充在浸出过程中消耗的质子。这表明硫生物氧化在生物浸出中扮演着十分重要的角色,研究和了解这种角色的作用机制、实现途径及其影响因素是十分必要的;另一方面,有关硫生物氧化的分子机制一直是硫生物氧化系统中的关键问题,至今未得到清晰的阐明。本论文旨在更进一步拓展对硫生物氧化过程的了解,研究嗜酸氧化亚铁硫杆菌（Acidithiobacillus ferrooxidans）适应与活化元素硫的分子机制。研究工作主要包括如下三个方面:（1）嗜酸氧化亚铁硫杆菌硫氧化的环境适应性:通过研究几种常见阴离子（氯离子、硝酸根离子、硫酸根离子和磷酸根离子等）对A.ferrooxidans生长和硫氧化活性的影响,揭示环境中阴离子对A.ferrooxidans生长和硫氧化活性影响的一般性规律。结果表明:一般情况下随着环境中阴离子浓度升高,表现出延缓或抑制细菌生长。但磷酸根离子对细菌的生长和硫氧化活性的抑制在浓度相当高的情况下才表现出来,100 mmol/L的磷酸根离子甚至对细菌的生长和硫氧化活性具有促进作用。正常环境下细菌体内参与细胞生理生化反应的蛋白质表达数量相对较多;在高浓度PO₄^3-胁迫下细菌表达蛋白的总数量相对减少,但同时表达一些特异性蛋白来调整和适应对无机阴离子胁迫效应。通过比较研究A.ferrooxidans在元素硫和硫代硫酸盐中细胞形态和生长特性、元素硫在A.ferrooxidans作用前后表面性质变化、细菌自身细胞表面化学性质的变化、以及A.ferrooxidans对元素硫的吸附行为等,了解A.ferrooxidans在元素硫和硫代硫酸盐基质中的细菌细胞适应性。结果表明:细菌能够优先利用可溶性能源基质硫代硫酸盐,生长在硫代硫酸盐中的细菌呈细长形态且细胞浓度较高,细胞亲水性较强;而生长在元素硫中的细菌呈短而粗的形态。细胞表面分泌大量附属物或蛋白质,使元素硫表面的疏水性质改变为亲水性,便于细菌对元素硫的吸附,或者使细菌随机胶结和堆集在硫粒上,这种吸附与解吸附呈一种动态变化过程;同时,硫粒表面因细菌的作用而形成腐蚀小坑。（2）硫基质中嗜酸氧化亚铁硫杆菌胞外蛋白质组的初步研究:研究A.ferrooxidans特化细胞空间（胞外、细胞周质）蛋白质组学,更易于发现在特定功能区域内行驶特定功能的蛋白质。本论文建立了选择性分离和双向电泳展示A.ferrooxidans特化细胞空间蛋白质的方法体系,明确了具体实验参数。研究在亚铁和元素硫基质中的A.ferrooxidans胞外蛋白表达差异,鉴定和分析在元素硫基质中A.ferrooxidans胞外高表达蛋白质。通过对在元素硫基质中A.ferrooxidans胞外高表达蛋白质的鉴定和分析,发现除少数蛋白质（或多肽）有预测功能外,大部分都属于未知功能的蛋白（或多肽）。鉴定的已有功能预测的蛋白（或多肽）包括:接合转移的蛋白质（AFE₁391:conjugal transfer protein）、吸附相关的菌毛蛋白（AFE₂621:pilin,putative）、脂蛋白（AFE₁847:lipoprotein,putative;AFE₀982:vacJ lipoprotein）、多聚糖脱乙酰酶家族蛋白（AFE₀927:polysaccharide deacetylase family protein）和丝氨酸/苏氨酸磷酸水解酶家族蛋白（AFE₁932:Ser/Thr phosphoric acid hydrolasefamily proteins）。生物信息学分析还发现有6个功能未知胞外蛋白质序列中含有较高丰度的半胱氨酸残基,有四个多肽序列中含有CXXC功能模体。由此推测,在元素硫中A.ferrooxidans表达相对较高的胞外蛋白质（或多肽）,推测它们的功能与元素硫的吸附和活化相关。据此提出了多巯基胞外蛋白在元素硫吸附活化过程中可能的作用方式,但其具体功能还需要深入而细致的验证和分析。（3）分析与鉴定硫活化/氧化相关蛋白质和基因的特征:还原性谷胱苷肽在硫活化与氧化过程中发挥重要作用,通过对维持A.ferrooxidans细胞体内的还原性谷胱苷肽水平的谷胱苷肽还原酶（glutathionereductase,GR）基因进行克隆与原核表达,以及对表达产物进行酶学性质的研究,发现该独特的GR不仅能以NADPH为电子供体,也能以NADH作为电子供体将氧化性谷胱苷肽（GSSG）转变为还原性谷胱苷肽（GSH）;序列比对发现该酶NADPH结合模体中的一个保守精氨酸残基位点被天冬酰胺残基所取代。巯基蛋白参与细胞周质空间还原性硫化合物的氧化,二硫键形成蛋白对维持周质空间高水平游离巯基的存在、修复和维持二硫键的正确连接起着十分重要的作用。通过对A.ferrooxidans中二硫键形成蛋白基因dsbG进行克隆与原核表达,以及对表达产物的酶学性质的研究,发现表达产物有二硫键异构酶活性;点突变结果表明其催化模体CXXC中的C119和C122位点是二硫建异构酶活性所需的关键氨基酸残基。结合已有的研究结果和A.ferrooxidans全基因组基因信息,利用RT-PCR方法从转录水平上分别对A.ferrooxidans ATCC 23270基因组中可能编码硫酸盐-硫代硫酸盐结合蛋白基因sbp、膜结合硫代硫酸盐-辅酶Q氧化还原酶基因doxDA以及类硫氰酸酶基因p21等开放阅读框所在的基因座之间的联系进行了鉴定和分析,结果表明它们分别从属于预测的doxDA-1操纵子和doxDA-2操纵子。在此基础上,利用生物信息学的方法对doxDA操纵子的可能启动子序列也进行了预测和分析。更多还原

【Abstract】 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.更多还原