【作者】 李兵；

【导师】 林建强； 林建群；

【作者基本信息】 山东大学 ， 微生物学， 2010， 博士

【摘要】 生物冶金工艺与其他物理化学冶金方法相比,具有成本低廉、环境友好、适于开采贫矿、难冶矿的特点。提高浸矿微生物的生物活性和适应性,将有效提高生物冶金过程的生产效率。在目前已探明的金矿资源中,含砷金矿占有很大的比例,其中砷金比达到2000：1的金矿资源占总探明储量的5%(刘四清宋焕斌,1998)。砷是一种具有毒性和致癌能力的半金属物质。在生物浸矿过程中经常要处理砷黄铁矿等含砷矿石。砷黄铁矿的生物氧化过程主要由硫氧化细菌喜温硫杆菌(Acidithiobacillus caldus)、铁氧化细菌嗜铁钩端螺旋菌(Leptospirillum ferriphilum)以及铁硫氧化菌氧化亚铁硫杆菌(Acidithiobacillus ferrooxidans)混合进行。嗜铁钩端螺旋菌(Leptospirillum ferriphilum)是生物浸矿反应中主要的铁氧化微生物,特别是在生物氧化处理过程的中后期高氧化还原电位条件下,是处于主导优势地位的铁氧化菌。该菌主要以氧化Fe2+产生Fe3+获得能量,而Fe3+则是生物浸矿过程中重要的氧化剂。嗜铁钩端螺旋菌的氧化活性和生命活力将直接影响含砷矿物的生物浸出效率。如果处理样品中的砷毒离子浓度超过了嗜铁钩端螺旋菌的砷耐受水平,生物浸出也就难以进行。因此,深入了解嗜铁钩端螺旋菌的砷耐受能力,砷抗性机制,将为进一步的微生物育种提供理论指导。但是由于嗜铁钩端螺旋菌的遗传背景,生理代谢等研究甚少,无法直接从该菌现有的生理调节机制及遗传基础入手获得该菌的砷抗性机制。所以,采用比较蛋白质组学的研究策略,引入双向电泳等蛋白质组学研究技术,全局性的研究该菌的砷耐受机制是一种可行的方法。双向电泳技术(Two dimensional electrophoresis,2-DE)可以全局性、高通量研究微生物的蛋白质表达差异,高分辨率,高灵敏度的展示和分离出现差异表达的蛋白,是分析复杂混合物和蛋白质组学研究中的关键技术。微生物与外界环境的相互作用和生物学响应往往是通过多个系统的综合协调来完成的,涉及多种酶系和调控系统的变化,这正是蛋白质组学的着眼点。因此,构建L. ferriphilum的2-DE分析技术平台,并在此基础上采用蛋白质组学研究技术来研究L. ferriphilum的抗砷、耐砷机制是科学合理的选择。L. ferriphilum是极端嗜酸性专性化能自养革兰氏阴性菌。其获能途径决定了该菌培养物中菌体密度小,各种可溶性离子杂质浓度高。而2-DE技术则要求样品中蛋白浓度较高,杂质浓度,尤其是各种可溶性离子杂质浓度尽可能的低。因此,普通的2-DE方案无法获得分离良好,点量丰富的L. ferriphilum 2-DE图谱,目前国内外还没有关于L. ferriphilum全局蛋白2-DE实验的报道。本研究通过对2-DE的样品制备、杂质去除、上样方法、聚焦参数选择、染色方法等关键环节进行对比实验,制定了一套适用于L. ferriphilum的2-DE实验方案,首次获得了点阵清晰,可稳定重现,胶体考马斯染色法获得独立蛋白点达到600个以上的L. ferriphilum 2-DE图谱。在此基础上,为了开展L.f砷胁迫条件下的差异蛋白质组学研究,本研究通过驯化培养获得具有较高耐砷性能的L.f菌株,通过2D电泳技术把耐砷菌株和普通对照菌株的菌体蛋白进行比较,确定差异蛋白,通过MALDI-TOF、MS/MS技术和相关生物信息学检索比对工具对差异蛋白进行分析。从而更全面地了解L.f的耐砷机制。目前已知的微生物耐砷抗砷系统主要是广泛存在的Arsenic Resistance System,即Ars系统。近期还有报道在砷胁迫条件下,某些热激蛋白(HSP蛋白),产能代谢相关蛋白,质膜阴离子通道蛋白的表达量也会提高。显示在砷胁迫条件下,细胞试图通过排出毒性离子、提高能源供给、提高蛋白质的稳定性等途径维持正常的生理生化活动。通过对L. ferriphilum ML-04的耐砷菌株和普通出发菌株的蛋白质2-DE图谱进行数字化比对和分析,发现65个蛋白点出现超过3倍的表达量变化(t-test,p<0.05)。有38种蛋白得到高分匹配和识别,其中,有25种蛋白质匹配确认了具体的生物学功能。其中很多蛋白有助于提高细胞对砷的耐受能力,包括促进三价砷离子的排出以保护一些重要蛋白的稳定性；保护和促进修复染色体,保障染色体的复制过程；避免重要的硫、磷元素的浪费；保持一些重要的砷耐受蛋白的活性等。而且,为了更有效的保护细胞,提高砷耐受能力,L. ferriphilumML-04还通过改变代谢通路中一些关键酶的表达量调整了细胞内一些代谢通路,例如碳水化合物的代谢、能量代谢、氨基酸代谢等。此外,该蛋白质组学研究还检测到了2套Ars砷抗性系统。一套是arsRC-arsB型的LJML04Ars1基因簇,一套是arsRDABC型LfML04Ars2基因簇。与先前报道的arsRDABC基因簇相比,LfML04Ars2基因簇中还多了一个编码框,编码了Pst型磷酸特异性转运系统的PstS蛋白。该PstS蛋白所属的Pst磷酸盐转运系统是细胞应对磷酸饥饿及抗五价砷毒离子相关系统。该系统的组成蛋白出现在砷抗性系统中,暗示在嗜铁钩端螺旋菌ML-04菌株中,砷抗性系统与某些磷酸饥饿响应系统存在联动关系。由于砷毒离子可以造成细胞磷酸饥饿,并可以干扰细胞内的多种磷酸化反应过程,这种砷抗性系统与磷酸饥饿响应系统的联动可以使ML-04获得更强的砷耐受能力。本研究建立的嗜铁钩端螺旋菌2-DE技术平台及相关研究策略,可应用于嗜铁钩端螺旋菌对其他多种环境条件变化的细胞适应性机制的研究,例如与生物浸矿密切相关的其他金属毒性离子的抗性和耐受机制、能量代谢变化、pH扰动、热冲击响应等生理生化过程,对于实际细菌浸出过程的优化,高效浸矿菌的微生物育种都具有重要的参考价值和理论意义。更多还原

【Abstract】 Biomining is a mineral pretreatment approach. Comparing with chemical approaches, the bioleaching method displays great advantages of cost-saving and environmental-friendly, particularly in dealing with the low grade ores and hard mining ores. The enhancement in activity and adaptability of the biomining microorganisms will increase the efficiency of biomining process and biomining industrial.In the certified reserves goldmine all over the world, the arsenic bearing goldmine takes a huge part and there are about 5% goldmine’s arsenic/gold ratio are more than 2000:1 (Liu siqing and Song huanbin,1998). The biooxidation process were dominated by Acidilhiobacillus caldus, Leptospirillum ferriphilum and Acidithiobacillus ferrooxidans. L. ferriphilum is important in bioleaching. In real applications, it is under heavy stresses of heavy metal ions and high oxygen reduction potential (ORP). This bacterial can oxidize the iron(Ⅱ) to iron(Ⅲ) and generate energy. As the metabolism product of L. ferriphilum, iron(Ⅲ) also is a key oxidizer in bioleaching. So the oxidize activity and physiological activity will effect the biooxidize efficiency of arsenic bearing goldore’s process. If the arsenic concentration exceed the arsenic tolerance ability of L. ferriphilum. Biooxidize of this arsenic bearing gold ore will not works. So the study of the detoxicity and tolerance mechanism were needed. The results of these researches will provide some useful guidance for further microbiology strain breeding works.Because the genetic background and physiological mechanisms of L. ferriphilum are still not very clear. The research of this bacteria can not based on the directly genetic analyze and physiological studies. So, the investigation about the arsenic tolerance and detoxification mechanism of L. ferriphilum will be conducted from a global aspect by the differential proteomic research approaches such as Two-dimensional-electrophoresis (2-DE) and matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS).Two-dimensional-electrophoresis (2-DE) and comparative proteomic analysis are useful to investigate the responses of L. ferriphilum to environmental stresses. But,2-DE analysis for L. ferriphilum is not successful as the samples of L. ferriphilum contain low protein concentration, complex composition, high salt concentration, and many other interfering components, which make it difficult for 2-DE analysis. In this research, optimizations on the sample preparation and purification methods, sample volume, sample loading methods for isoelectric focusing (IEF), and gel visualization methods were made. More than 629 Coomassie stained spots in single gel were obtained. The image quality and protein concentration in most of the spots met the requirements for both differential spots analysis and mass-spectrum analysis. The 2-DE protocol for L. ferriphilum was successfully developed for the first time.Based on the 2-DE technic and proteomic research strategy, the response of Leptospirillum ferriphilum ML-04 under arsenic stress was analyzed using two-dimensional-electrophoresis (2-DE), matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS), and bio-informatics research methods.65 differential expressed protein were fond by 2-DE analysis (t-test, p<0.05).38 arsenic response proteins were highly identified. The arsenic resistant or tolerant mechanisms of these arsenic response proteins were studied and elucidated. In addition, some arsenic-resistance-system (ARS) member proteins were identified from these 38 arsenic response proteins. With the help of the genomic analysis, two ars operon like structures were discovered in the chromosome of this strain. One is a arsRC-arsB type LfML04Ars1 cluster. The other one is arsRDABC type cluster named LfFL04Ars2. Comparing with the former reported other arsRDABC type clusters, the LfML04Ars 2 contains a more gene ORF, which encodes a component protein of the Pst phosphate specific transfer system, the PstS like protein. This result indicats that there maybe a linkage relationship between the Ars arsenic resistance system and phosphate specific transfer system in this strain. The gene structure feature and possible function of these ars operon like structures were analyzed.更多还原