嗜酸氧化亚铁硫杆菌ISC铁硫簇系统的合成

【目的】铁硫簇是最古老的一种氧化还原中心,它普遍存在于所有生命体内,在光合作用、呼吸作用和固氮作用这三个地球生命最基本的代谢途径中扮演着重要的角色。【方法】以嗜酸氧化亚铁硫杆菌(A.ferrooxidans ATCC 23270)基因组为模板,克隆表达其ISC铁硫簇组装的3个核心蛋白,IscS(半胱氨酸脱硫酶蛋白)、IscU(支架蛋白)和IscA(铁供体蛋白)。【结果】研究发现IscS能催化半胱氨酸脱硫,为铁硫簇的组装提供硫,支架蛋白IscU不具备结合铁的能力,IscA具有较强的铁结合能力。【结论】铁硫簇体外组装证明Fe-IscA在体外能将结合的铁传递给IscS,并在IscU上进行铁硫簇的组装。     

嗜酸氧化亚铁硫杆菌细胞色素C家族afe_0378基因转录表达差异及生物信息学挖掘

嗜酸氧化亚铁硫杆菌是一种极为重要的浸矿微生物,具有氧化各种还原性含硫物及亚铁等功能。采用实时荧光定量PCR技术及生物信息学等手段对该菌一个涉及铁硫氧化由afe_0378基因编码的细胞色素C家族蛋白CycA2进行了研究。结果表明,afe_0378基因在单质硫培养条件下转录水平是亚铁培养条件下的2.67倍。生物信息学分析表明afe_0378编码蛋白CycA2是分子质量为22.959 ku,pI为9.75的结合内膜的周质蛋白,二级结构为全α-螺旋,无β-折叠,包含2个相似结构域及N端一段跨膜疏水螺旋,属于细胞色素C4型蛋白家族。CycA2蛋白分子三维结构模建表明,双血红素与CycA2蛋白序列片段域C48-X-X-C51-H52及C149-X-X-C152-H153中的半胱氨酸共价连接。结合该菌硫代谢背景知识,推测CycA2蛋白的功能是介导细胞色素bc1复合体及终端氧化酶细胞色素aa3复合体之间的电子传递而参与硫代谢。     

嗜酸氧化亚铁硫杆菌基因组学研究进展

嗜酸氧化亚铁硫杆菌(Acidithiobacillus ferrooxidan,A.ferrooxidans)广泛存在于酸性矿物废水中,与生物冶金和环境净化紧密相关。不同来源嗜酸氧化亚铁硫杆菌全基因组的测序,为我们利用比较基因组学和功能基因组学的方法去洞察嗜酸氧化亚铁硫杆菌功能基因,提供了坚实的研究基础和丰富的科研信息。简述了嗜酸氧化亚铁硫杆菌基因组学的基本特征;从比较基因组学和功能基因组学发现了嗜酸氧化亚铁硫杆菌菌株基因组水平的差异;通过生物信息学概述了该菌的铁和硫代谢机制,并从细菌的功能基因组学对其在生物冶金与环境治理等应用进行了展望。     

正交法优化嗜酸氧化亚铁硫杆菌冷冻干燥保护剂

利用正交实验方法,以甘油、海藻糖、蔗糖和牛血清蛋白为因素,对嗜酸氧化亚铁硫杆菌（Acididfiobacillus ferrooxidans,A．ferrooxidans）冷冻干燥保护剂的最优化配比进行了研究。直观分析、因素指标分析和方差分析的结果表明：由甘油、海藻糖、蔗糖和牛血清蛋白组成的冷冻干燥保护剂中,对存活率影响的主次顺序依次为：甘油〉海藻糖〉牛血清蛋白〉蔗糖。保护剂的最优化组合为甘油5％、海藻糖15％、蔗糖18％、牛血清蛋白10％。经过验证,该组合的保护剂可使冷冻干燥嗜酸氧化亚铁硫杆菌的存活率达到94％。     

嗜酸氧化亚铁硫杆菌硫氰酸酶的表达、纯化及其性质鉴定

目的:嗜酸氧化亚铁硫杆菌(Acidithiobacillus ferrooxidans)硫氰酸酶是硫代谢中极其重要的酶,其作用主要包括氰化物的解毒,铁-硫蛋白的合成,以及硫胺、硫尿苷或烟碱乙酸胆碱的生物合成,硫氰酸酶的研究对揭示生物冶金机理具有重要的推动作用.方法:以A.ferrooxidans ATCC23270基因组为模板设计引物,通过PCR扩增得到编码硫氰酸酶的基因,目的基因片段与原核表达载体PLM1构建重组体,然后转入大肠杆菌(Eschcrichia coli,E.coli)DH5a感受态中,基因测序正确后,重组质粒再转入E.coli BL21感受态中,加IPTG诱导蛋白表达,用一步亲和层析法纯化出浓度和纯度都较高的硫氰酸酶.结果:SDS-PAGE分析证实蛋白分子量为21kD,紫外可见光分析,确定硫氰酸酶中含有铁硫簇,酶活测定发现重组硫氰酸酶在体外不具有酶活性,可能与酶反应条件及信号肽的切断有关.结论:体外成功克隆.表达,纯化出重组体硫氰酸酶,其基本性质也得到阐述.     

嗜酸氧化亚铁硫杆菌亚铁氧化酶基因分子多态性研究

嗜酸氧化亚铁硫杆菌（Acidithiobacillus ferrooxidans, A.f）属于化能自养、革兰氏阴性细菌,好氧嗜酸,以氧化亚铁离子或还原态硫化物（S0&S2-）获得能量生长。在其Fe2+氧化系统中,亚铁氧化酶起重要作用。对不同硫化矿区分离得到的5株A.f进行生长动力学和对亚铁氧化活性的对比研究,结果显示不同菌株之间存在表型差异。提取A.f菌株的基因组DNA,设计引物,对亚铁氧化酶基因进行PCR扩增,同时对扩增产物直接进行测序,并同GenBank的参考序列进行比对分析,发现序列相似性在99%～97%之间,分析编码区域发现在第187～195位可能存在一个高变异区：在第187～189位,菌株YTW编码的氨基酸Thr→Pro;而在第193～195位,菌株TK是Met→Asn; 菌株BY则是Met→Ile。另外在位点第219位,所有菌株都是T→C,但编码的氨基酸未发生变化,属于同义密码子。对于编码区上游序列,比对后没有差异;而对于编码区下游序列,经比对发现存在一些差异位点。     

抗Cu2+嗜酸氧化亚铁硫杆菌的驯化及诱变育种

从我国三大铜矿的酸性矿坑水中富集分离出9个具有较强活性的嗜酸氧化亚铁硫杆菌菌株,经过Cu~(2 )的系列浓度梯度的培养,选出其中天然抗铜能力最强的菌株26~#,在Cu~(2 )浓度为0．20mol/L的9K培养基中能在72h内完全氧化培养基中的Fe~(2 ),在含0．22mol/L Cu2~(2 )的9K培养基中能在192h内完全氧化培养基中的Fe~(2 )。以CuSO_4·5H_2O为单变量驯化介质驯化该26~#抗铜菌株,26~#驯化菌株的Fe~(2 )氧化能力明显增强:在含0．25mol/LCu~(2 )的9K培养基中能在84h内完全氧化其中的Fe~(2 )。为了提高驯化菌的稳定性,将驯化后的26~#菌株用紫外线进行诱变。研究结果表明:驯化诱变对菌种的改良有重要的作用,诱变后菌株的生长性能稳定,氧化活性进一步提高,26~#驯化诱变菌在0．25mol/LCu~(2 )存在的条件下完全氧化9K培养基中Fe~(2 )的时间约为60h,对Fe~(2 )氧化能力明显强于驯化菌及野生菌。     

Ni2+胁迫下嗜酸氧化亚铁硫杆菌亚铁氧化应激效应

【目的】嗜酸氧化亚铁硫杆菌(Acidithiobacillus ferrooxidans)作为湿法冶金的主要功能菌,其在重金属Ni²⁺胁迫下的亚铁氧化应激机制还不清楚。【方法】在两步法培养体系中,设计由低到高Ni²⁺胁迫浓度,利用分光光度法、电化学法和实时荧光定量PCR法等从宏观到微观探究Ni²⁺胁迫下A.ferrooxidans菌亚铁氧化应激效应。【结果】两步培养法体系下A.ferrooxidans菌能够耐受较高浓度的Ni²⁺毒性(≤30 g/L Ni²⁺)。当Ni²⁺胁迫浓度增加至40 g/L时,与对照组(不添加Ni²⁺胁迫)相比,A.ferrooxidans亚铁氧化率和速率显著降低(24 h的亚铁氧化抑制率约为59.4%),亚铁氧化代谢相关的rus操纵子各功能基因的表达量也显著下调,尤其是基因cyc1(最高下调了16倍)和coxBACD(4个不同亚基最高下调2.7–7.4倍);同时Fe²⁺氧化相关的胞外电子传递能力也降低,对照组(0 g/L Ni²⁺,29.0µA)最大还原峰电流值高于实验组(40 g/L Ni²⁺,24.5µA)。【结论】超高浓度Ni²⁺胁迫环境对A.ferrooxidans菌有毒性,导致该菌rus操纵子上各功能基因表达量下调,同时造成胞外电子传递速率降低,最终致使A.ferrooxidans菌亚铁氧化能力降低。研究结果将为含Ni固废高效生物浸出提供理论支持。     

氧化亚铁硫杆菌铁氧化系统分子生物学研究进展

氧化亚铁硫杆菌（Thiobacillus ferrooxidans,简称T.f）是目前研究最多、最具经济价值的浸矿微生物。由于该菌的能量代谢对于生物浸矿起决定作用,因此其机制的研究必然能促进对该菌生理特性的认识及其遗传改造。氧化亚铁硫杆菌生长方式代表了迄今所知的能够进行生长的热力学极限,可供该菌生长利用的△Eh仅有340mV,氧化Fe2+所能得到的能量很少。在 Fe2+氧化过程中,电子通过电子传递链最终传递给氧,     

嗜酸氧化亚铁硫杆菌生长动力学方程的应用

基于Monod模型推导出了A．f的生长动力学方程模型,采用Gauss-Newton算法确定了在不同初始条件下细菌生长的动力学参数,即最大比生长速率‰、Monod常数K及R0。通过在不同初始条件下细菌生长特性的研究,得到了相应初始生长条件下以限制性底物亚铁离子浓度为表征的生长动力学方程,理论上揭示了动力学参数变化对细菌生长的影响规律,其中生长动力学方程的数值模拟与实验数据相吻合。     

Immobilization of arsenite and ferric iron by Acidithiobacillus ferrooxidans and its relevance to acid mine drainage

Weathering of the As-rich pyrite-rich tailings of the abandoned mining site of Carnoulès (southeastern France) results in the formation of acid waters heavily loaded with arsenic. Dissolved arsenic present in the seepage waters precipitates within a few meters from the bottom of the tailing dam in the presence of microorganisms. An Acidithiobacillus ferrooxidans strain, referred to as CC1, was isolated from the effluents. This strain was able to remove arsenic from a defined synthetic medium only when grown on ferrous iron. This A. ferrooxidans strain did not oxidize arsenite to arsenate directly or indirectly. Strain CC1 precipitated arsenic unexpectedly as arsenite but not arsenate, with ferric iron produced by its energy metabolism. Furthermore, arsenite was almost not found adsorbed on jarosite but associated with a poorly ordered schwertmannite. Arsenate is known to efficiently precipitate with ferric iron and sulfate in the form of more or less ordered schwertmannite, depending on the sulfur-to-arsenic ratio. Our data demonstrate that the coprecipitation of arsenite with schwertmannite also appears as a potential mechanism of arsenite removal in heavily contaminated acid waters. The removal of arsenite by coprecipitation with ferric iron appears to be a common property of the A. ferrooxidans species, as such a feature was observed with one private and three collection strains, one of which was the type strain.     

The pyrite iron cycle catalyzed by Acidithiobacillus ferrooxidans

In this paper, we study a model of the biotic pyrite iron cycle catalyzed by bacteria Acidithiobacillus ferrooxidans, in mining activity sites waste dumps. Chemical reactions, reaction rates and the population growth model are mostly taken from the existing literature. Analysis of the corresponding dynamical system shows the existence of up to four non-trivial steady state solutions. The stability of the equilibria solutions is determined, finding up to two coexisting stable solutions. Two Hopf bifurcations and a region of parameter space in which there are stable periodic solutions are found. In addition, we find a homoclinic bifurcation which gives rise to a stable periodic orbit of large period. The existence of these stable oscillatory solutions gives a possible explanation for the oscillations of bacteria concentration and pH for the iron cycle, described in Jaynes et al. (Water Resour Res 20:233–242, 1984).     

Effects of electron transport inhibitors and uncouplers on the oxidation of ferrous iron and compounds interacting with ferric iron in Acidithiobacillus ferrooxidans

Oxidation of Fe2+, ascorbic acid, propyl gallate, tiron, L-cysteine, and glutathione by Acidithiobacillus ferrooxidans was studied with respect to the effect of electron transport inhibitors and uncouplers on the rate of oxidation. All the oxidations were sensitive to inhibitors of cytochrome c oxidase, KCN, and NaN3. They were also partially inhibited by inhibitors of complex I and complex III of the electron transport system. Uncouplers at low concentrations stimulated the oxidation and inhibited it at higher concentrations. The oxidation rates of Fe2+ and L-cysteine inhibited by complex I and complex III inhibitors (amytal, rotenone, antimycin A, myxothiazol, and HQNO) were stimulated more extensively by uncouplers than the control rates. Atabrine, a flavin antagonist, was an exception, and atabrine-inhibited oxidation activities of all these compounds were further inhibited by uncouplers. A model for the electron transport pathways of A. ferrooxidans is proposed to account for these results. In the model these organic substrates reduce ferric iron on the surface of cells to ferrous iron, which is oxidized back to ferric iron through the Fe2+ oxidation pathway, leading to cytochrome oxidase to O2. Some of electrons enter the uphill (energy-requiring) electron transport pathway to reduce NAD+. Uncouplers at low concentrations stimulate Fe2+ oxidation by stimulating cytochrome oxidase by uncoupling. Higher concentrations lower deltap to the level insufficient to overcome the potentially uphill reaction at rusticyanin-cytochrome c4. Inhibition of uphill reactions at complex I and complex III leads to deltap accumulation and inhibition of cytochrome oxidase. Uncouplers remove the inhibition of deltap and stimulate the oxidation. Atabrine inhibition is not released by uncouplers, which implies a possibility of atabrine inhibition at a site other than complex I, but a site somehow involved in the Fe2+ oxidation pathway.     

固定化Acidithiobacillus ferrooxidans对Fe2+氧化及其生物反应器研究进展

Acidithiobacillus ferrooxidans 对Fe2+的生物氧化是一个非常重要的反应过程, 在生物浸矿、H2S等废气的脱硫、含重金属污泥和酸性矿坑废水的处理等领域有着重要的应用。近些年来,大量的研究主要集中A. ferrooxidans及其反应过程等方面,然而,A. ferrooxidans对Fe2+的催化氧化速率缓慢和稳定性欠佳等问题仍然限制了其商业应用。因此,对A. ferrooxidans的固定化及其生物反应器研究是该技术进一步发展的关键。本文评述了A. ferrooxidans最新应用、存在的问题和解决办法,重点比较了目前文献中报道的各种A. ferrooxidans固定材料、方法,并对目前采用的各种固定化A. ferrooxidans生物反应系统的效率和结构等方面进行了讨论和分析。