浸矿微生物硫酸盐同化与重金属抗性耦合作用机制的研究
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摘要
我国矿产资源的显著特点是品位低、复杂、难处理和中小型矿居多,传统的选冶工艺很难进行经济、绿色、有效地提取。生物冶金具有低成本、短流程、污染小等优点,具有广阔的应用前景。浸矿微生物浸出一段时间之后,其生长的环境中就会富含高浓度的硫酸盐及有毒重金属离子,这是制约其浸出时间、浸出效率的重要的环境因素。而在微生物的解毒机制中,硫酸盐的同化途径可能发挥了重要的作用,因而对于浸矿微生物重金属的抗性机制,以及硫酸盐同化与重金属解毒耦合作用机制的研究,对微生物适应极端环境的分子机制的揭示,对浸矿过程中,重金属的耐受和浸矿效率的提高,以及在环境领域更广阔的应用其抗性机制,都将具有非常重要的意义。
本文旨在对浸矿微生物的重金属抗性机制进行深入的探索,研究浸矿微生物的重金属抗性以及与硫酸盐同化的内在联系,研究工作主要有以下几个方面：
①浸矿微生物重金属抗性机制的初步探索
在本研究给定的重金属浓度下, A. ferooxidans表现出了明显的抑制作用并且随着重金属浓度的增加,抑制作用越明显。适合研究的Cd2+、Zn2+、Pb2+三种重金属耐受浓度分别为Cd2+(5mM、15mM、30mM), Zn2+(150mM、300mM、600mM), Pb2+(2.5mM、5mM、10mM)。在Cd2+胁迫下,通过测定镉离子的亚细胞结构镉离子含量、细菌细胞体内GSH的含量、硫酸根离子浓度、硫酸盐同化途径的相关酶类的酶活,结果表明其可能存在的抗性机制有细胞累积、排出系统、以及生物转化或螯合等几种方式。
在Zn2+胁迫下,通过测定菌体内GSH的含量、Cys含量、硫酸盐同化途径的相关酶类酶活,结果表明,随着Pb2+浓度的增加,其体内GSH含量有所增加,Cys含量的变化不明显,硫酸盐同化途径的相关酶类也有所变化,但是却是表现为先增加后降低,说明硫酸盐同化途径可能对锌离子的抗性有一定的作用,但是在浓度更大的情况下,硫酸盐同化途径可能已经不能满足细菌菌体的需要,其可能启动了更多的重金属抗性机制。
在Pb2+的胁迫下,通过测定菌体内GSH的含量、Cys含量、硫酸盐同化途径的相关酶类的酶活,结果表明,谷胱甘肽、半胱氨酸的含量,随着Pb2+浓度的增大而降低,硫酸盐同化相关酶类的活性有所变化但是却与Cd2+的情况相反,说明Pb2+胁迫菌体情况下硫酸盐同化对抗性的贡献可能不明显,或是与Cd2+相反。
②浸矿微生物重金属抗性基因的差异表达分析
通过生物信息学的分析,发现了十个可能与重金属抗性相关的基因。利用Real-time PCR技术,对Cd2+胁迫下重金属抗性基因的差异表达情况进行了考查,结果表明,在不同浓度的Cd2+刺激下,重金属抗性基因在转录水平上的表达量,均呈上调趋势,相对对照组大约上调了1-8倍,且随着Cd2+浓度的增加,上调倍数也随之增加。预测Cd2+的转运模型为Czc操纵子是由H+离子梯度驱动,由位于内膜上的QcA1,2A将重金属Cd(Ⅱ)泵出到细胞质,然后由CzcB1A,f(?)将重金属穿梭至外膜,并预防重金属进入周质,CzcB1A,f也可以提供其他重金属的特异性。CzcC1A,f则完成将重金属排出到外部环境的全过程。CmtR是重金属的转录调控因子,位于胞内,作为识别的信号。CadB1A,f和CadB2A,f是转运体,帮助CzcB1A,f将重金属运出体外。
在Zn2+胁迫下,重金属抗性基因的表达差异情况结果表明,在不同浓度的Zn2+胁迫下,重金属抗性基因在转录水平上的表达量也是呈上调趋势,大约是对照组的1.2到630倍,且随着Zn2+浓度的增加,上调倍数也随之增加,说明这些基因的表达情况,大部分与Cd2+胁迫下情况相似。但是CzcABCAf重金属排出系统的基因(AFE-0671CzcA1A,f、AFE-1144CzcA2A,f、AFE-1142CzcC1A,f和AFE-1143CzcB1A,f)变化不大,说明对于Zn2+的抗性的贡献可能不是很大,而基因AFE-2437CadB1A,f、AFE-2438CadB2A,f、AFE-2920CadB3A,f这三个转运蛋白,在Zn2+胁迫下,却是随着重金属浓度的增大而逐渐增加的,说明转运蛋白对于锌离子的抗性贡献较大。
在Pb2+胁迫下,重金属抗性基因的表达差异情况研究发现,在不同浓度的Pb2+胁迫下,重金属抗性基因在转录水平上的表达量均呈下调趋势,且随着Pb2+浓度的增加,并没有明显的调控趋势,说明在Pb2+胁迫下大多数抗性基因,没有表现出明显的调控或者为负调控。
③重金属胁迫下硫酸盐同化途径相关基因的表达差异分析
通过生物信息学分析,找寻到硫酸盐同化的相关的关键基因,对其进行体外重组表达和酶活分析,确定了硫酸盐同化的基本代谢路径。被运输到体内的硫酸盐被cysDN编码的ATP硫酸化酶激活为APS(adenosine-5'-phosphosulfate),随后被APS还原酶还原为亚硫酸盐,亚硫酸盐又被cysJI编码的亚硫酸盐还原酶还原为硫化物,最后硫化物与O-乙酰丝氨酸由cysM编码的半胱氨酸合成酶同化入半胱氨酸。
通过对重金属胁迫下硫酸盐同化途径相关基因的表达差异分析,结果表明在Cd2+胁迫下,硫酸盐同化的相关基因的表达,表现为上调趋势,大约是对照组的1到3倍,随着离子浓度的增大,上调倍数越大。但是锌离子胁迫下只有少数几个基因表达上调,而铅离子胁迫下,则没有明显的上调。表明硫酸盐同化基因对于镉离子的抗性的较大,而对其他两种金属离子则较小
④浸矿微生物重金属抗性关键基因的过表达分析
研究发现,CysE和CmtR基因是转录调控作用的关键的抗性基因,为了进一步的了解其功能,采用过表达分析的方法考查了其转入受体菌之后的抗性的变化。结果表明：在不同浓度Cd2+的胁迫下,BL21导入重金属抗性基因后,相对于对照组,菌体的生物量,没有明显的变化,但是其体内Cys和GSH的含量却有所增加,同时体内的重金属含量也是有所增加,说明导入抗性基因后其重金属的抗性明显增强；不同浓度Zn2+的胁迫下,BL21导入重金属抗性基因后,其相应的结果与Cd2+相似,说明导入抗性基因后其重金属抗性增强；不同浓度Pb2+的作用下,BL21导入重金属抗性基因后,无论是菌体的生物量、Cys和GSH的含量,还是其体内的重金属含量,相对于对照组均是降低的,说明导入抗性基因后其重金属抗性没有明显的改善或者是下降的。
⑤重金属浸出及其种群优势度的分析
16S rDNA-RFLP分析结果表明,所选取的三个AMD(酸性矿坑水)采样点中,均含有Acidithiobacillus菌属的微生物,每个样点均有部分菌株独立于已知菌株存在,独立存在的菌株数目占所考查菌株数目的一半左右；并且三个样点之间浸矿微生物的种类及数量相似度较大,说明这三个考查点其中的地球化学环境相似。用AMD对四种不同组成的矿石浸出后,发现其对Ag、Pb及Cu有一定的浸出能力,而对As和Pb几乎没有浸出效果。微生物群落分析得出：H71和F11群落结构差异较大,而J72和S71几乎一样。从系统发育分析可知,所考查的序列可分为两大分支：Acidithiobacillus菌属和相对独立的菌属,不同矿石样品处理后均存在这两支,且发育距离较近。
The significant feature of the mineral resources of our state is low grade, and complex, intractable, ores of small and medium-sized. If we use mostly traditional method to smelt and extract, it is difficulty to extract economy, green effectively. Compared to traditional metallurgical processes, bioleaching shows a low-cost, short process, small pollution and so on. When we use bacterial bioleaching in time, the system will be rich in high concentrations of sulfate, and other toxic heavy metal ions, which are the important environmental factors which restricted the leaching time, leaching efficiency. Sulfate assimilation may play an important role in metal resistance mechanism. Therefore, we discuss the coupling mechanism of sulfate assimilation and heavy metal resistance, which will reveal molecular mechanisms of tolerance such an extreme environment. It will may improve tolerance of heavy metals, and improve efficiency of leaching.
In order to enlarge the knowledge on metal resistance mechanism of Acidithiobacillus ferrooxidans, the present study focuses on elucidating the molecular mechanism of metal resistance and sulfate assimilation. The primary subjects of which are as follows:
①Preliminary exploration of the mechanism of heavy metal resistance about A. ferrooxidans
When bacteria were incubated in medium containing metal ions, the growth rates decreased significantly compared to those of bacteria grown in medium without cadmium, the growth rates suppression is more obvious than metal concentration increasing. It is appropriate to study that metal tolerance concentration separately is Cd2+(5mM,15mM,30mM), Zn2+(150mM,300mM,600mM), Pb2+(2.5mM,5mM,10mM). According to measure metal content of subcellular structures, GSH and SO42-content, enzymes activity of sulfate assimilation pathway, the result showed that the metal resistance mechanism may be bioaccumulation, metal ion efflux, bioconversion or biochelation et al.
When bacteria were incubated in medium containing zinc, according to measure GSH and Cys content, enzymes activity of sulfate assimilation pathway, the result showed that glutathione content is increased, sulfate assimilation pathway enzymes also change, but Cys content did not change significantly. It is indicated that sulfate assimilation pathway may have a certain role in the resistance to zinc ions.
When bacteria were incubated in medium containing zinc, according to measure GSH and Cys content, enzymes activity of sulfate assimilation pathway, the result showed that cell growth was inhibited, glutathione, Cys content was decreased with increasing concentration Pb2+. It is indicated that contribution of sulfate assimilation may not be obvious, or contrary to the cadmium ion.
②Analysis of metal resistance genes expression differences
A bioinformatic search of its genome showed the presence of at least10genes that are possibly related to cadmium homeostasis. The expression of most of these open reading frames was studied by real-time reverse transcriptase PCR using A. ferrooxidans cells adapted for growth in the presence of high concentrations of Cd2+The putative A.ferrooxidans Cd2+resistance determinants were found to be upregulated when this bacterium was exposed to Cd2+in the range of5to30mM and the fold has increased along with the increase of the concentration of Cd2+. Scheme was summarized the possible role of Cd2+resistance determinants in A. ferrooxidans. This proposal is based on the expression results presented in this work. This complex reaction allows transport of substrates across the complete envelope of A. ferrooxidans that is composed of outer membrane (OM), periplasmic space (PP) and cytoplasmic membrane (CPM). The czc operon is driven by a H+ion gradient that allows czcA2A.f (located in the inner membrane) to pump heavy metals Cd (Ⅱ) out from cytoplasm. czcB1A.f then shuttles the metals from the inner to the outer membrane, preventing contact with the periplasm. czcB1A.f may also provide specificity for other heavy metals. czcC1A.f finishes the process by exporting metals to the external environment. CmtR of heavy metals transcription factors, located in the intracellular, as the identification of the signal. CadB1A.f and CadB2A.f are transporter factors, to help czcBA.f transported heavy metals from the body.
The expression of most of these open reading frames was studied by real-time reverse transcriptase PCR, when bacteria were incubated in medium containing zinc. The results indicated that the putative A. ferrooxidans most resistance determinants were found to be upregulated and the fold has increased along with the increase of the concentration of Pb2+. The efflux system genes of CzcABCA.f(AFE-0671CzcA1A.f, AFE-1144CzcA2A.f, AFE-1142CzcC1A.f and AFE-1143CzcB1A.f) is not obviously change, which indicated contribution of the efflux system genes may not be big. However, the expression of the transporter genes of AFE-2437CadB1A.f, AFE-2438CadB2A.f, AFE-2920CadB3A.f were found to be upregulated and the fold has obviously increased along with the increase of the concentration of Zn2+. It is indicated that contribution of the transporter genes may be bigger to the zinc resistance.
The expression of most of these open reading frames was studied by real-time reverse transcriptase PCR, when bacteria were incubated in medium containing plumbum. The results showed that the putative A.ferrooxidans most resistance determinants were found to be downregulated and most of the resistance gene did not show an obviously regulation or negative regulation.
③Analysis of sulfate assimilation genes expression differences
According to analysis and characterization of sulfate assimilation genes in A. ferrooxidans, we concluded sulfate assimilation possible pathway. Sulfate which is transported into intracellular, firstly, it is assimilated in the form of adenosine phosphosulfate (APS), by an ATP sulfurylase (encoded by genes cysD and cysN). Then this reaction, which yields APS, reduced to sulfite by APS reductase (encoded by genes cysH). The sulfite is reduced to sulfide by NADPH-sulfite reductase (encoded by genes cysIJ), the last step, Sulfur, reduced as H2S, reacts with O-acetylserine by O-acetylserinelyase (encoded by genes cysM) gene to give cysteine, OAS is synthesized by acetylation of serine with acetyl-CoenzymeA catalyzed by serine acetyltransferase (encoded by genes cysE).
The expression of sulfate assimilation genes was studied by real-time reverse transcriptase PCR using A. ferrooxidans cells adapted for growth in the presence of high concentrations of metal. The results showed that the genes were found to be upregulated when this bacterium was exposed to Cd2+and Pb2+and the fold has increased along with the increase of the concentration of Cd2+and Pb2+, on the contrary to Pb2+
④Analysis of overexpression to the metal resistance genes
Base on the experiment results, we found that these two genes, CysE and CmtR, were key transcriptional regulation factors. In order to further examine these two genes to metal resistance, we used the method of overexpression to determine the change of metal resistance after transferred to the receptor bacteria. The result is as follows:
When this bacterium was exposed to Cd2+, relative to the control group, the cell biomass no significant change, but intracellular Cys and GSH content was increased, at the same time intracellular heavy metals content is also increased, after metal resistance genes transferred to the receptor bacterial BL21. The result indicated that resistance of the receptor BL21significantly enhanced, after metal resistance genes transferred to the receptor BL21. When this bacterium was exposed to Cd2+, the result is similar to Pb2+. But when this bacterium was exposed to Pb2+, the result is opposite to Cd2+and Pb2+
⑤Metal bioleaching and population analysis
16s rDNA-RFLP analysis results showed that part of three sampling points are the independent Acidithiobacillus bacteria genera of microbes, which accounts for about half the number of test strains, three sample point between ore leaching microbial species and quantity larger similarity. Explain the three test point of geochemical environment and microbial structure relative change are not big, also showed that microbial diversity is survival system and surrounding environment of exchange degree of influence. There are a lot of related research Acidithiobacillus bacteria is already as immersion ore research model bacteria genera, Acidithiobacillus bacteria genera of microbes was found in AMD of the spring mountain gold mine, this chapter research AMD has certain immersion ore ability. To investigate four ore sample microbial community analysis:H71and F11community structure are diverse, J72and S71are almost the same. From the system development analysis, we can conclude that the test sequences can be divided into two branches:Acidithiobacillus bacterial genera and relatively independent bacteria genera; Different ore sample treatment after contain the two bacterial generas, and development at close range. AMD has certain leaching ability to Ag、Pb and Cu, but As and Pb almost no leaching effect.
本文旨在对浸矿微生物的重金属抗性机制进行深入的探索,研究浸矿微生物的重金属抗性以及与硫酸盐同化的内在联系,研究工作主要有以下几个方面：
①浸矿微生物重金属抗性机制的初步探索
在本研究给定的重金属浓度下, A. ferooxidans表现出了明显的抑制作用并且随着重金属浓度的增加,抑制作用越明显。适合研究的Cd2+、Zn2+、Pb2+三种重金属耐受浓度分别为Cd2+(5mM、15mM、30mM), Zn2+(150mM、300mM、600mM), Pb2+(2.5mM、5mM、10mM)。在Cd2+胁迫下,通过测定镉离子的亚细胞结构镉离子含量、细菌细胞体内GSH的含量、硫酸根离子浓度、硫酸盐同化途径的相关酶类的酶活,结果表明其可能存在的抗性机制有细胞累积、排出系统、以及生物转化或螯合等几种方式。
在Zn2+胁迫下,通过测定菌体内GSH的含量、Cys含量、硫酸盐同化途径的相关酶类酶活,结果表明,随着Pb2+浓度的增加,其体内GSH含量有所增加,Cys含量的变化不明显,硫酸盐同化途径的相关酶类也有所变化,但是却是表现为先增加后降低,说明硫酸盐同化途径可能对锌离子的抗性有一定的作用,但是在浓度更大的情况下,硫酸盐同化途径可能已经不能满足细菌菌体的需要,其可能启动了更多的重金属抗性机制。
在Pb2+的胁迫下,通过测定菌体内GSH的含量、Cys含量、硫酸盐同化途径的相关酶类的酶活,结果表明,谷胱甘肽、半胱氨酸的含量,随着Pb2+浓度的增大而降低,硫酸盐同化相关酶类的活性有所变化但是却与Cd2+的情况相反,说明Pb2+胁迫菌体情况下硫酸盐同化对抗性的贡献可能不明显,或是与Cd2+相反。
②浸矿微生物重金属抗性基因的差异表达分析
通过生物信息学的分析,发现了十个可能与重金属抗性相关的基因。利用Real-time PCR技术,对Cd2+胁迫下重金属抗性基因的差异表达情况进行了考查,结果表明,在不同浓度的Cd2+刺激下,重金属抗性基因在转录水平上的表达量,均呈上调趋势,相对对照组大约上调了1-8倍,且随着Cd2+浓度的增加,上调倍数也随之增加。预测Cd2+的转运模型为Czc操纵子是由H+离子梯度驱动,由位于内膜上的QcA1,2A将重金属Cd(Ⅱ)泵出到细胞质,然后由CzcB1A,f(?)将重金属穿梭至外膜,并预防重金属进入周质,CzcB1A,f也可以提供其他重金属的特异性。CzcC1A,f则完成将重金属排出到外部环境的全过程。CmtR是重金属的转录调控因子,位于胞内,作为识别的信号。CadB1A,f和CadB2A,f是转运体,帮助CzcB1A,f将重金属运出体外。
在Zn2+胁迫下,重金属抗性基因的表达差异情况结果表明,在不同浓度的Zn2+胁迫下,重金属抗性基因在转录水平上的表达量也是呈上调趋势,大约是对照组的1.2到630倍,且随着Zn2+浓度的增加,上调倍数也随之增加,说明这些基因的表达情况,大部分与Cd2+胁迫下情况相似。但是CzcABCAf重金属排出系统的基因(AFE-0671CzcA1A,f、AFE-1144CzcA2A,f、AFE-1142CzcC1A,f和AFE-1143CzcB1A,f)变化不大,说明对于Zn2+的抗性的贡献可能不是很大,而基因AFE-2437CadB1A,f、AFE-2438CadB2A,f、AFE-2920CadB3A,f这三个转运蛋白,在Zn2+胁迫下,却是随着重金属浓度的增大而逐渐增加的,说明转运蛋白对于锌离子的抗性贡献较大。
在Pb2+胁迫下,重金属抗性基因的表达差异情况研究发现,在不同浓度的Pb2+胁迫下,重金属抗性基因在转录水平上的表达量均呈下调趋势,且随着Pb2+浓度的增加,并没有明显的调控趋势,说明在Pb2+胁迫下大多数抗性基因,没有表现出明显的调控或者为负调控。
③重金属胁迫下硫酸盐同化途径相关基因的表达差异分析
通过生物信息学分析,找寻到硫酸盐同化的相关的关键基因,对其进行体外重组表达和酶活分析,确定了硫酸盐同化的基本代谢路径。被运输到体内的硫酸盐被cysDN编码的ATP硫酸化酶激活为APS(adenosine-5'-phosphosulfate),随后被APS还原酶还原为亚硫酸盐,亚硫酸盐又被cysJI编码的亚硫酸盐还原酶还原为硫化物,最后硫化物与O-乙酰丝氨酸由cysM编码的半胱氨酸合成酶同化入半胱氨酸。
通过对重金属胁迫下硫酸盐同化途径相关基因的表达差异分析,结果表明在Cd2+胁迫下,硫酸盐同化的相关基因的表达,表现为上调趋势,大约是对照组的1到3倍,随着离子浓度的增大,上调倍数越大。但是锌离子胁迫下只有少数几个基因表达上调,而铅离子胁迫下,则没有明显的上调。表明硫酸盐同化基因对于镉离子的抗性的较大,而对其他两种金属离子则较小
④浸矿微生物重金属抗性关键基因的过表达分析
研究发现,CysE和CmtR基因是转录调控作用的关键的抗性基因,为了进一步的了解其功能,采用过表达分析的方法考查了其转入受体菌之后的抗性的变化。结果表明：在不同浓度Cd2+的胁迫下,BL21导入重金属抗性基因后,相对于对照组,菌体的生物量,没有明显的变化,但是其体内Cys和GSH的含量却有所增加,同时体内的重金属含量也是有所增加,说明导入抗性基因后其重金属的抗性明显增强；不同浓度Zn2+的胁迫下,BL21导入重金属抗性基因后,其相应的结果与Cd2+相似,说明导入抗性基因后其重金属抗性增强；不同浓度Pb2+的作用下,BL21导入重金属抗性基因后,无论是菌体的生物量、Cys和GSH的含量,还是其体内的重金属含量,相对于对照组均是降低的,说明导入抗性基因后其重金属抗性没有明显的改善或者是下降的。
⑤重金属浸出及其种群优势度的分析
16S rDNA-RFLP分析结果表明,所选取的三个AMD(酸性矿坑水)采样点中,均含有Acidithiobacillus菌属的微生物,每个样点均有部分菌株独立于已知菌株存在,独立存在的菌株数目占所考查菌株数目的一半左右；并且三个样点之间浸矿微生物的种类及数量相似度较大,说明这三个考查点其中的地球化学环境相似。用AMD对四种不同组成的矿石浸出后,发现其对Ag、Pb及Cu有一定的浸出能力,而对As和Pb几乎没有浸出效果。微生物群落分析得出：H71和F11群落结构差异较大,而J72和S71几乎一样。从系统发育分析可知,所考查的序列可分为两大分支：Acidithiobacillus菌属和相对独立的菌属,不同矿石样品处理后均存在这两支,且发育距离较近。
The significant feature of the mineral resources of our state is low grade, and complex, intractable, ores of small and medium-sized. If we use mostly traditional method to smelt and extract, it is difficulty to extract economy, green effectively. Compared to traditional metallurgical processes, bioleaching shows a low-cost, short process, small pollution and so on. When we use bacterial bioleaching in time, the system will be rich in high concentrations of sulfate, and other toxic heavy metal ions, which are the important environmental factors which restricted the leaching time, leaching efficiency. Sulfate assimilation may play an important role in metal resistance mechanism. Therefore, we discuss the coupling mechanism of sulfate assimilation and heavy metal resistance, which will reveal molecular mechanisms of tolerance such an extreme environment. It will may improve tolerance of heavy metals, and improve efficiency of leaching.
In order to enlarge the knowledge on metal resistance mechanism of Acidithiobacillus ferrooxidans, the present study focuses on elucidating the molecular mechanism of metal resistance and sulfate assimilation. The primary subjects of which are as follows:
①Preliminary exploration of the mechanism of heavy metal resistance about A. ferrooxidans
When bacteria were incubated in medium containing metal ions, the growth rates decreased significantly compared to those of bacteria grown in medium without cadmium, the growth rates suppression is more obvious than metal concentration increasing. It is appropriate to study that metal tolerance concentration separately is Cd2+(5mM,15mM,30mM), Zn2+(150mM,300mM,600mM), Pb2+(2.5mM,5mM,10mM). According to measure metal content of subcellular structures, GSH and SO42-content, enzymes activity of sulfate assimilation pathway, the result showed that the metal resistance mechanism may be bioaccumulation, metal ion efflux, bioconversion or biochelation et al.
When bacteria were incubated in medium containing zinc, according to measure GSH and Cys content, enzymes activity of sulfate assimilation pathway, the result showed that glutathione content is increased, sulfate assimilation pathway enzymes also change, but Cys content did not change significantly. It is indicated that sulfate assimilation pathway may have a certain role in the resistance to zinc ions.
When bacteria were incubated in medium containing zinc, according to measure GSH and Cys content, enzymes activity of sulfate assimilation pathway, the result showed that cell growth was inhibited, glutathione, Cys content was decreased with increasing concentration Pb2+. It is indicated that contribution of sulfate assimilation may not be obvious, or contrary to the cadmium ion.
②Analysis of metal resistance genes expression differences
A bioinformatic search of its genome showed the presence of at least10genes that are possibly related to cadmium homeostasis. The expression of most of these open reading frames was studied by real-time reverse transcriptase PCR using A. ferrooxidans cells adapted for growth in the presence of high concentrations of Cd2+The putative A.ferrooxidans Cd2+resistance determinants were found to be upregulated when this bacterium was exposed to Cd2+in the range of5to30mM and the fold has increased along with the increase of the concentration of Cd2+. Scheme was summarized the possible role of Cd2+resistance determinants in A. ferrooxidans. This proposal is based on the expression results presented in this work. This complex reaction allows transport of substrates across the complete envelope of A. ferrooxidans that is composed of outer membrane (OM), periplasmic space (PP) and cytoplasmic membrane (CPM). The czc operon is driven by a H+ion gradient that allows czcA2A.f (located in the inner membrane) to pump heavy metals Cd (Ⅱ) out from cytoplasm. czcB1A.f then shuttles the metals from the inner to the outer membrane, preventing contact with the periplasm. czcB1A.f may also provide specificity for other heavy metals. czcC1A.f finishes the process by exporting metals to the external environment. CmtR of heavy metals transcription factors, located in the intracellular, as the identification of the signal. CadB1A.f and CadB2A.f are transporter factors, to help czcBA.f transported heavy metals from the body.
The expression of most of these open reading frames was studied by real-time reverse transcriptase PCR, when bacteria were incubated in medium containing zinc. The results indicated that the putative A. ferrooxidans most resistance determinants were found to be upregulated and the fold has increased along with the increase of the concentration of Pb2+. The efflux system genes of CzcABCA.f(AFE-0671CzcA1A.f, AFE-1144CzcA2A.f, AFE-1142CzcC1A.f and AFE-1143CzcB1A.f) is not obviously change, which indicated contribution of the efflux system genes may not be big. However, the expression of the transporter genes of AFE-2437CadB1A.f, AFE-2438CadB2A.f, AFE-2920CadB3A.f were found to be upregulated and the fold has obviously increased along with the increase of the concentration of Zn2+. It is indicated that contribution of the transporter genes may be bigger to the zinc resistance.
The expression of most of these open reading frames was studied by real-time reverse transcriptase PCR, when bacteria were incubated in medium containing plumbum. The results showed that the putative A.ferrooxidans most resistance determinants were found to be downregulated and most of the resistance gene did not show an obviously regulation or negative regulation.
③Analysis of sulfate assimilation genes expression differences
According to analysis and characterization of sulfate assimilation genes in A. ferrooxidans, we concluded sulfate assimilation possible pathway. Sulfate which is transported into intracellular, firstly, it is assimilated in the form of adenosine phosphosulfate (APS), by an ATP sulfurylase (encoded by genes cysD and cysN). Then this reaction, which yields APS, reduced to sulfite by APS reductase (encoded by genes cysH). The sulfite is reduced to sulfide by NADPH-sulfite reductase (encoded by genes cysIJ), the last step, Sulfur, reduced as H2S, reacts with O-acetylserine by O-acetylserinelyase (encoded by genes cysM) gene to give cysteine, OAS is synthesized by acetylation of serine with acetyl-CoenzymeA catalyzed by serine acetyltransferase (encoded by genes cysE).
The expression of sulfate assimilation genes was studied by real-time reverse transcriptase PCR using A. ferrooxidans cells adapted for growth in the presence of high concentrations of metal. The results showed that the genes were found to be upregulated when this bacterium was exposed to Cd2+and Pb2+and the fold has increased along with the increase of the concentration of Cd2+and Pb2+, on the contrary to Pb2+
④Analysis of overexpression to the metal resistance genes
Base on the experiment results, we found that these two genes, CysE and CmtR, were key transcriptional regulation factors. In order to further examine these two genes to metal resistance, we used the method of overexpression to determine the change of metal resistance after transferred to the receptor bacteria. The result is as follows:
When this bacterium was exposed to Cd2+, relative to the control group, the cell biomass no significant change, but intracellular Cys and GSH content was increased, at the same time intracellular heavy metals content is also increased, after metal resistance genes transferred to the receptor bacterial BL21. The result indicated that resistance of the receptor BL21significantly enhanced, after metal resistance genes transferred to the receptor BL21. When this bacterium was exposed to Cd2+, the result is similar to Pb2+. But when this bacterium was exposed to Pb2+, the result is opposite to Cd2+and Pb2+
⑤Metal bioleaching and population analysis
16s rDNA-RFLP analysis results showed that part of three sampling points are the independent Acidithiobacillus bacteria genera of microbes, which accounts for about half the number of test strains, three sample point between ore leaching microbial species and quantity larger similarity. Explain the three test point of geochemical environment and microbial structure relative change are not big, also showed that microbial diversity is survival system and surrounding environment of exchange degree of influence. There are a lot of related research Acidithiobacillus bacteria is already as immersion ore research model bacteria genera, Acidithiobacillus bacteria genera of microbes was found in AMD of the spring mountain gold mine, this chapter research AMD has certain immersion ore ability. To investigate four ore sample microbial community analysis:H71and F11community structure are diverse, J72and S71are almost the same. From the system development analysis, we can conclude that the test sequences can be divided into two branches:Acidithiobacillus bacterial genera and relatively independent bacteria genera; Different ore sample treatment after contain the two bacterial generas, and development at close range. AMD has certain leaching ability to Ag、Pb and Cu, but As and Pb almost no leaching effect.
引文
[1]杨显万,沈庆峰,郭玉霞.微生物湿法冶金.北京:冶金工业出版社,2003.
[2]Rawling D E, Johnson D B. The microbiology of biomining:development and optimization of mineral-oxidizing microbial consortia. Microbiology. 2007,153:315-324.
[3]Brierley C L. Biohydrometallurgy prospects. Hydrometallurgy.2006, 104(3-4):324-328.
[4]Rudolfs W, Helbronner A. Oxidation of Zinc Sulfides by Microorganism. Soil Sci.1922,14:459-464.
[5]Colmer A R, Hinckle M E. The Role of Microorganism in Acid Mine Drainage. A Preliminary Report, Science.1947,106:253-256.
[6]Faramarzi M A, Stagars M, Pensini E, et al. Metal solubilization from metal-containing solid materials by cyanogenic Chromobacterim violaceum. J Biotechnol.2004,113:321-326.
[7]毕银丽,胡瑜,苏高华,等.微生物对煤矿固体废弃物的脱硫效应.环境工程学报.2008,2(1)：92-96.
[8]Gonez C, Bosecker K. Leaching of heavy metals from contamined soil by using Thiobacillus ferrooxidans or Thiobacillus thiooxidans. Geomicrobiology.1999,16(3):233-244.
[9]刘俊,龚文琪,申求实,等.低品位磷矿的生物浸出研究.金属矿山.2008,385：54-58.
[10]Rawlings D E:Heavy metal mining using microbes. Annu Rev. Microbiol. 2002,56:65-91.
[11]Gonzalez T E, Llobet B E, Casamayor E O, et al. Microbial ecology of an extreme acidic environment, the Tinto River. Appl Environ Microbiol.2003, 69(8):4853-4865.
[12]Norris P R, Clark D A, Owen J P, et al. Characteristics of Sulfobacillus acidophilus sp. nov. and other moderately thermophilic mineral-sulphide-oxidizing bacteria. Microbiology.1996,142(4):775-783.
[13]Waiting H R. The bioleaching of sulphide minerals with emphasis on copper sulphide-A review. Hydrometallurgy.2006,84:81-108.
[14]Demergasso C S, Galleiguillos P A, Escudero G, et al. Molecular characterization of microbial population in low-grade copper ore bioleaching test heap. Hydrometallurgy.2005,80:241-253.
[15]Robbins E I. Bacteria and archaea in acidic environments and a key to morphological identification. Hydrobiologia.2000,433:61-89.
[16]Groudev, Stoyan N, Groudeva V I. Microbial communities in four industrial copper dump leaching operations in Bulgaria. FEMS microbiological reviews.1993,11:261-268.
[17]吴学玲.抗性浸矿细菌的选育及抗性机理的研究.中南大学博士学位论文.2007,7-8.
[18]Claudio A. Navarro, Luis H, et al. Transcriptional and Functional Studies of Acidithiobacillus ferrooxidans Genes Related to Survival in the Presence of Copper. Applied and environmental microbiology.2009,75(19):6102-6109.
[19]Orell A, Navarro A, et al. Copper resistance mechanisms of biomining bacteria and archaea living under extremely high concentrations of metals. Biohydrometallurgy.2009,71-73:279-282.
[20]徐柳,宋琴,茆灿泉.金属结合蛋白(肽)与环境重金属生物修复.生物工程杂志.2004,24(4)：39-43.
[21]曾景海,齐鸿雁,杨建州,等.重金属抗性菌Bacillus cereus HQ-1对银离子的生物吸附-微沉淀成品作用.环境科学与技术.2008,29(1):225-230.
[22]杨峰,尹华,彭辉,等.融合菌-活性污泥联合曝气吸附处理重金属铬.环境科学与技术.2006,26(5):3 18-322.
[23]White C, Gadd G M. Accumulation and effects of cadmium on sulphate-reducing bacterial biofilms. Microbiology.1998,44:1407-1415.
[24]Gharieb M M, Gadd G M. Role of glutathione in detoxification of metal(loid)s by Saccharomyces cerevisiae. BioMetals.2004,17:183-188.
[25]张玉刚,龙新宪,陈雪梅.微生物处理重金属废水的研究进展.环境科学与技术.2008,31(6)：58-63.
[26]Goulhen F, Bruschi M. Cr (VI) detoxification by Desulfovibrio vulgaris strain Hildenborough:microbe-metal interactions studies. Appl Microbiol Biotechnol.2006,71:892-897.
[27]高小朋,张欠欠,冯进辉.微生物还原Cr(VI)的研究进展.微生物通报.2008,35(5)：820-824.
[28]Stahler F N, Odenbreit S, Haas R, et al. The novel Helicobacter pylori CPbABC metal efflux pump is required for cadmium, zinc, and nickel resistance, urease modulation, and gastric colonization. Infect Immun.2006, 74(7):3845-3852.
[29]Paulsen I T, Park J H, Choi P S. A family of Gram-negative bacterial outer membrane factors that function in the export of proteins, carbohydrates, drugs and heavy metals from Gram-negative bacteria. FEMS Microbiol Lett. 1997,156:1-8.
[30]Nies D H. Efflux-mediated heavy metal resistance in prokaryotes. FEMS Microbiology.2003,27:313-339.
[31]Saier J, Tam R, Reizer A, et al. Two novel families of bacterial membrane proteins concerned with nodulation, cell division and transport. Mol. Microbiol.1994,11:841-847.
[32]seng T T, Gratwick K S, Kollman J, et al. The RND superfamily:an ancient, ubiquitous and diverse family that includes human disease and development proteins. J. Mol. Microbiol. Biotechnol.1999,1:107-125.
[33]Johnson J M, Church G M. Alignment and structure prediction of divergent protein families:periplasmic and outer membrane proteins of bacterial efflux pumps. J. Mol. Biol.1999,287:695-715.
[34]Andersen C, Hughes C, Koronakis V. Protein export and drug efflux through bacterial channel-tunnels. Curr. Opin. Cell Biol.2001,13:412-416.
[35]Paulsen I T, Park J H, Choi P S, et al. A family of Gram-negative bacterial outer membrane factors that function in the export of proteins, carbohydrates, drugs and heavy metals from Gram-negative bacteria. FEMS Microbiol. Lett. 1997,156:1-8.
[36]Milton H, Saier J. A functional-phylogenetic classification system for transmembrane solute transporters. Microbiol Mol Biol Rev.2000,64: 354-411.
[37]Koronakis V, Shar A, Koronakis E, et al. Crystal structure of the bacterial membrane protein TolC central to multidrug efflux and protein export. Nature 2000.405:914-919.
[38]FaganM J, Saier M H J. P-type ATPases of eukaryotes and bacteria: Sequence analyses and construction of phylogenetic trees. Journal of Molecular Evolution.1994,38(1):57-99.
[39]Borsetti F, Martelli P L, Casadio R, et al. Metals and metalloids in photosynthetic bacteria:interactions, resistance and putative home-ostasis Revealed by genome analysis. In The Purple Phototrophic Bacteria. Netherlands:Springer Science Business Media BV.2009,28:655-689.
[40]Rensing C, Fan B, Sharma R, et al. CopA:An Escherichia coli Cu(I)-translocating P-type ATPase. Proc Natl Acad. Sci. USA.2000,97: 652-656.
[41]Guerinot M L. The ZIP family of metal transporters. Biochim. Biophys. Acta. 2000,1465:190-199.
[42]Christopher J H, Gregor G, Sylvia F, et al. New developments in the understanding of the cation diffusion facilitator family. J Ind Microbiol Biotechnol.2005,32(6):215-26.
[43]Mario G, Luis L, et al. A gene cluster involved in metal homeostasis in the cyanobacterium Synechocystisspstrain PCC 6803. Journal of Bacteriology. 2000,182(6):1507-1514.
[44]Wu C C, Gardarina, Catty P, et al. The Cadmium Transport Sites of CadA, the Cd2+-ATPase from Listeria monocytogenes. Journal of Biological Chemistry.2006,281(40):29533-29541.
[45]周围,李友国,杨凯,等.1株抗重金属铬细菌的分离鉴定及抗性基因型的初步研究.华中农业大学学报.2008,27(2):248-250.
[46]赵肖为,李清彪,卢英华,等.高选择性基因工程菌E. coli SE 5000生物富集水体中的镍离子.环境科学学报.2004,24(2):231-236.
[47]Borremans, Hobman, Provoost, et al. Cloning and Functional Analysis of the pbr Lead Resistance Determinant of Ralstonia metallidurans CH34. Journal of Bacteriology.2001,183(19):5651-5658.
[48]Jonathn J. Caguiat, Alice L. et al. Cd(Ⅱ)-Responsive and Constitutive Mutants Implicate a Novel Domain in MerR. Journal of Bacteriology.1999, 181(11):3462-3471.
[49]洪斌.微生物对砷的地球化学行为的影响-暨地下水砷污染机制的最新研究进展.地球科学进展.2006,21(1):77-82.
[50]Freeman J L, Salt D E. The metal tolerance profile of Thlaspi goesingense is mimicked in Arabidopsis thaliana heterologously expressing serine acetyl-transferase. BMC Plant Biology.2007,7(63):1471-1481.
[51]Stoyanov J V, Hobman J L & Brown N L. CueR (YbbI) of Escherichia coli is a MerRfamily regulator controlling expression of the copper exporter CopA. Mol. Microbiol.2001,39:502-511.
[52]Brown N L, Stoyanov J V, Kidd S P, et al. The MerR family of transcriptional regulators. FEMS Microbiol Rev.2003,27:145-163.
[53]Gaballa A, Cao M & Helmann J D. Two MerR homologues that affect copper induction of the Bacillus subtilis copZA operon. Microbiology.2003, 149:3413-3421.
[54]Cavet J S, Graham A I, Meng W. A cadmium-lead-sensing ArsR-SmtB repressor with novel sensory sites. Complementary metal discriminationby NmtR and CmtR in a common cytosol. J. Biol. Chem.2003,278: 44560-44566.
[55]Malin M, Leif B. Metal-binding proteins and peptidesin bioremediation and phytoremediation of heavy metals. Trends in Biotechnology.2001,19(2): 67-74.
[56]Hatzios S K, Bertozzil C R. The regulation of sulfur metabolism in Mycobacterium Tuberculosis. PLoS Pathogens.2011,7(7):1-8.
[57]Saharan R K, Sharma S C. Effects of Glutathione Modulation on Oxidative Stress and Enzymatic Antioxidant Defence in Yeast. Curr Microbiol.2011, 62:944-949.
[58]Torricelli E, Gorbi G, Skowronska P B. Cadmium tolerance, cysteine and thiol peptide levels in wild type and chromium-tolerant strains of Scenedesmus acutus (Chlorophyceae). Aquatic Toxicology.2004,68: 315-323.
[59]Gorbi G, Zanni C, Corradi M G. Sulfur starvation and chromium tolerance in Scenedesmus acutus:A possible link between metal tolerance and the regulation of sulfur uptake/assimilation processes. Aquatic Toxicology.2007, 84:457-464.
[60]Saito K. Regulation of sulfate transport and synthesis of sulfurcontaining amino acids. Curr Opin Plant Biol,2000,3:188-195.
[61]Kredich, N M. Biosynthesis of cysteine in Escherichia coli and Salmonella. Cellular and molecular biology. Neidhardt, F.C. (ed) Washington, D.C,1996, 514-527.
[62]Pereira Y, Lagniel G, Godat E, et al. Chromate causes sulfur starvation in yeast. Toxicological sciences.2008,106(2):400-412.
[63]Momose Y, Iwahashi H. Bioassay of cadmium using a DNA microarray: genome-wide expression patterns of Saccharomyces cerevisiae response to cadmium. Environ Toxicol Chem.2003,20:2353-2360.
[64]Nocito F F, Pirovano L, Cocucci M, et al. Cadmium-induced sulfate uptake in maize roots. Plant Physiol.2002,129:1872-1879.
[65]Vido K, Spector D, Lagniel G, et al. A proteome analysis of the cadmium response in Saccharomyces cerevisiae. J Biol Chem.2001,276:8469-8474.
[66]Xia J L, Wu S, Zhang R Y, et al. Effects of Copper Exposure on Expression of Glutathione-Relate Genes in Acidithiobacillus ferrooxidans. Curr Microbiol.2011,62(5):1460-1466.
[67]Xia L X, Yin C, Cai L, et al. Metabolic changes of Acidithiobacillus caldus under Cu2+ stress. Journal of Basic Microbiology.2010,50(6):591-598.
[68]Leyh T S, Taylor J C, Markham G D. The sulfate activation locus of Escherichia coli K-12:cloning, genetic, and enzymatic characterization. Journal of Biological Chemistry.1988,263(5):2409-2416.
[69]Hell R. Molecular physiology of plant sulfur metabolism. Planta.1997,202: 138-148.
[70]Takahashi H, Asanuma W, Saito K. Cloning of an Arabidopsis cDNA encoding a chloroplast localizing sulphate transporter isoform. J. Exp. Bot. 1999,50(340):1713-1714.
[71]Li H, Deyrup A, Mensch J R, et al. The isolation and characterization of cDNA encoding the mouse bifunctional ATP sulfurylase-adenosine 5'-phosphosulfate kinase. J. Biol. Chem.1995,270:29453-29459.
[72]Venkatachalam KV, Fuda H, Koonin E V, et al. Siteselected mutagenesis of a conserved nucleotide binding HXGH motif located in the ATP sulfurylase domain of human bifunctional 3'-phosphoadenosine 5'-phosphosulfate synthase. Biochim. Biophys. Acta.1998,1429:284-291.
[73]Tobias C U, Michael B, Robert H. Crystal structure of ATP sulfurylase from Saccharomyces cerevisiae, a key enzyme in sulfate activation. The EMBO Journal.2001,20(3):316-329.
[74]Mougous J D, Lee D H, Hubbard S C. Molecular Basis for G Protein Control of the Prokaryotic ATP Sulfurylase. Molecular Cell.2006,21: 109-122.
[75]Savage H, Montoya G, Svensson C, et al. Crystal structure of phosphoadenylyl sulphate (PAPS) reductase:a new family of adenine nucleotide alpha hydrolases. Structure.1997,5:895-906.
[76]Liu C, Martin E, and Leyh T S. GTPase activation of ATP sulfurylase:the mechanism. Biochemistry.1994,33:2042-2047.
[77]Liu C, Suo Y, and Leyh T S. The energetic linkage of GTP hydrolysis and the synthesis of activated sulfate. Biochemistry.1994b,33:7309-7314.
[78]Zheng C L, Zhang Y F, Liu Y D, et al. Characterization and reconstitute of a [Fe4S4] Adenosine 5'-phosphosulfate reductase from Acidithiobacillus ferrooxidans. Current Microbiology.2009,58(6):586-592.
[79]Ostrowski J, Wu J.Y, Rueger D C, et al. Characterization of the cysJIH regions of Salmonella typhimurium and Escherichia coli B. DNA sequences of cysl and cysH and a model for the siroheme Fe4S4 active center of sulfite reductase hemoprotein based on amino acid homology with spinach nitrite redutase. Journal of Biological Chemistry.1989,264(26):15726-15737.
[80]Bick J A, Dennis J J, Zylstra G J, et al. Identification of a new class of 5'-adenylylsulfate (APS) reductases from sulfate-assimilating bacteria. J. Bacteriol.2000,182:135-142.
[81]Kim S K, Rahman A, Bick J A, et al. Properties of the cysteine residues and iron-sulfur cluster of the assimilatory 5'-adenylyl sulfate reductase from Pseudomonas aeruginosa. Biochemistry.2004,43:13478-13486.
[82]Kopriva S, Buchert T, Fritz G, et al. The presence of an iron-sulfur cluster in adenosine 5'-phosphosulfate reductase separates organisms utilizing adenosine 5'-phosphosulfate and phosphoadenosine 5'-phosphosulfate for sulfate assimilation. J. Biol. Chem.2002,277:21786-21791.
[83]Kanno N, Nagahisa E, Sato M, et al. Adenosine 5'-phosphosulfate sulfotransferase from the marine macroalga Porphyra yezoensis Ueda (Rhodophyta):Stabilization, purification, and properties, Planta.1996,198: 440-446.
[84]Fritz G, Roth A, Schiffer A, et al. Structure of adenylylsulfate reductase from the hyperthermophilic Archaeoglobus fulgidus at 1.6-A resolution. Proc Natl Acad Sci. USA.2002,99:1836-1841.
[85]Fritz G, Buchert T, Kroneck P M. The function of the [Fe4S4] clusters and FAD in bacterial and archaeal adenylysulfate reductases. J Biol Chem.2002, 277:26066-26073.
[86]Carroll K S, Gao H, Chen H, et al. Investigation of the iron-sulfur cluster in Mycobacterium tuberculosis APS reductase:implications for substrate binding and catalysis. Biochemistry.2005,44:14647-14657.
[87]Siegel L M, Murphy J M, Kamin H. The Eseherichia coli hemoflavoprotein: molecular parameters and prosthetic groups. Journal of Biological Chemistry. 1973,248:251-261.
[88]Ostrowski J, Barber M J, Rueger D C, et al. Characterization of the flavoprotein moieties of NADPH-sulfite reductase from Salmonella typhimurium and Escherichia coli. Physicochemical and catalytic properties, amino acid sequencededuced from the DNA sequence of cysJ, and comparison with NADPH-cytochrome P-450 reductase. Journal of Biological Chemistry.1989,264(27):15796-15808.
[89]Byrne C R, Monroe R S, Ward K A & Kredich N M. DNA sequences of the cysK regions of Salmonella typhimurium and Escherichia coli and linkage of the cysK region to ptsH. J. Bacteriol.1988,170:3150-3158.
[90]Becker M A, Kredich N M & Tomkins G M. Purification and characterization of cysteine synthetase, a bifunctional protein complex, from Salmonella typhimurium. J. Biol. Chem.1969,244:2418-2427.
[91]Schnackerz K D, Tai C H, Simmons J W, et al. Identification and spectral characterization of the external aldimine intermediate of the O-acetylserine sulfhydrylase reaction. Biochemistry.1995,34:12152-12160.
[92]Cook P F, Hara S, Nalabolu S & Schnackerz K D. PH dependence of the absorbance and 31P NMR spectra of O-acetylserine sulfhydrylase in the absence and presence of O-acetyl-L-serine. Biochemistry.1992,31: 2298-2303.
[93]Rege V D, Kredich N M, Tai C H, et al. A change in the internal aldimine lysine (K42) in O-acetylserine sulfhyrylase to alanine indicates its importance in transamination and as general base cactalyst. Biochemistry. 1996,35:13485-13493.
[94]McClure G D & Cook P F. Product binding to the a-carboxyl subsite results in a conformational change at the active site of O-acetylserine sulfhydrylase-A:evidence from fluorescence spectroscopy. Biochemistry.1994,33: 1674-1683.
[95]Strambini G, Cioni P & Cook P F. Tryptophan and coenzyme luminescence as a probe of conformation along the O-acetylserine sulfhydrylase reaction pathway. Biochemistry.1996,35:8392-8400.
[96]Peter G S. Jagannatha R, Hohenester E, et al. Three-dimensional Structure of O-acetylserine Sulfhydrylase from Salmonella typhimurium. J. Mol. Biol. 1998,283:121-133.
[97]Guedon E, Verstraete I M. Cysteine Metabolismand Its Regulation in Bacteria. Microbiol Monographs.2007,5:195-218.
[98]Thomas D, Surdin K Y. Metabolism of sulphur amino acids in Saccharomyces cerevisiae. Microbiol. Mol. Biol.1997,61(4):503-532.
[99]Pye V E, Tingey A P, Robson R L. The Structure and Mechanism of Serine Acetyltransferase from Escherichia coli. The journal of biological chemistry. 2004,279(39):40729-40736.
[100]Haas F H, Heeg C, Queiroz R, et al. Mitochondrial Serine Acetyltransferase Functions as a Pacemaker of Cysteine Synthesis in Plant Cells. Plant Physiology.2008,148:1055-1067.
[101]Tuovinen O H, Kelly D P. Biology of Thiobacillus ferrooxidans in relation to the microbiological leaching of sulphide ores. Z Allg Mikrobiol.1972, 12(4):311-346.
[102]Wu X L, Ding J N, Gao J, et al. Isolation and identification of metal-resistant iron-oxidizing bacteria. Minerals & Metallurgical Processing. 2007,24(1):57-60.
[103]王雪峰,林建群,韩梅等.细菌铜抗性机制研究进展.湖北农业科学.2009,48(1)：218-224.
[104]武顺.铜离子对嗜酸氧化亚铁硫杆菌ATCC 23270谷胱甘肽相关酶基因表达水平的影响.中南大学硕士论文.2009,52-62.
[105]Truper H G, Lynne A R. Purification and properties of adenylyl sulfate reductase from the phototrophic sulfur bacterium, Thiocapsa roseopersicina. J Bacteriol.1971,108:1112-1121.
[106]Droux M, Ruffet M L, Douce R, et al. Interactions between serine acetyltransferase and O-acetylserine (thiol) lyase in higher plantsstructural and kinetic properties of the free and bound enzymes. Eur JBiochem.1998, 1:235-245.
[107]Warrilow A G S and Hawkesford M J. Separation, subcellular location and influence of sulphur nutrition isoforms of cysteine synthase in spinach. J. Exp. Bot.1998,49(327):1625-1636.
[108]Nies D H. The cobalt, zinc, cadmium efflux system czcABC from alcaligenes eutrophus functions as a cation-proton antiporter in Escherichia coli. J. Bacteriol.1995,177:2707-2712.
[109]Nies D H, Silver S. Ion efflux systems involved in bacterial metal resistances. J Ind Microbiol.1995,14:189-199.
[110]Nies D H. Efflux-mediated heavy metal resistance in prokaryotes. FEMS Microbiol.2003,27:313-339.
[111]Nies D H. Resistance to cadmium, cobalt, zinc, and nickel in microbes. Plasmid.1992,27:17-28.
[112]Notico F F, Lancilli C, Crema B, et al. Heavy metal stress and sulfate uptake in maize roots. Plant physiology.2006,141:1138-1148.
[113]Phartiyal P, Kim W S, Cahoon R E, et al. The role 5-adenylysulfate reductase in the sulfur assimilation pathway of soybean:molecular cloning, kinetic characterization, and gene expression. Phytochemistry.2008,69: 356-364.
[114]Kim S D, Bae J E, Park H S, et al. Bioleaching of cadmium and nickel from synthetic sediments by Acidithiobacillus ferrooxidans. Environ Geochem Health.2005.27(3):229-235.
[115]Baillet F, Magnin J P, Cheruy A, et al. Chromium precipitation by the acidophilic bacterium Thiobacillus ferrooxidans. Biotech letters.2004,20: 95-99.
[116]Van Z, Munste L J, Rawlings D E, et al. Construction of arsB and tetH mutants of the sulfur-oxidizing bacterium Acidithiobacillus caldus by marker exchange. Appl Environ Microbiol.2008.74(18):5686-5694.
[117]Alvarez S, Jerez C A. Copper ions stimulate polyphosphate degradation and phosphate efflux in Acidithiobacillus ferrooxidans. Appl Environ Microbiol. 2004,70(9):5177-5182.
[118]Ramirez P, Guiliani N, Valenzuela L, et al. Differential protein expression during growth of Acidithiobacillus ferrooxidans on ferrous iron, sulfur compounds, or metal sulfides. Appl Environ Microbiol.2004,70(8): 4491-4498.
[119]相丽,姜毓君,刘伟,等.金黄色葡萄球菌基因表达的DNA扣除法Real-time RT-PCR相对定量分析.微生物学报.2008,48(4):526-531.
[120]Livak K J, Schmittgen T D. Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔCT Method. Methods.2001,25(4): 402-408.
[121]戴艳霞.氧化亚铁硫杆菌与隐性嗜酸杆菌共培养体系构建及铁代谢基因差异表达研究.中南大学硕士论文.2009,80-100.
[122]Mergeay M, Nies D, Schlegel H G, et al. Alcaligenes eutrophus CH34 is a facultative chemolithotroph with plasmid-bound resistance to heavy metals. J. Bacteriol.1985,162:328-334.
[123]Collard J M, Corbisier P, Diels L, et al. Plamids for heavy metal resistance in Alcaligenes eutrophus CH34:mechanisms and applications. FEMS Microbiol Rev,1994,14(4):405-414.
[124]Nies D, Mergeay M, Friedrich B, et al. Cloning of plasmid genes encoding resistance to cadmium, zinc, and cobalt in Alcaligenes eutrophus CH34. J. Bacteriol.1987,169:4865-4868.
[125]Kunito T, Kusano T, Oyaizu H, et al. Cloning and sequence analysis of czc genes in Alcaligenes sp. strain CT14. Biosci. Biotechnol. Biochem.1996,60: 699-704.
[126]Lelie D, Schwuchow T, Schwidetzky U, et al. Two component regulatory system involved in transcriptional control of heavy metal homoeostasis in Alcaligenes eutrophus. Mol. Microbiol.1997,23:493-503.
[127]GroMe C, Grass G, Anton A, et al. Transcriptional organization of the czc heavy metal homoeostasis determinant from Alcaligenes eutrophus. J. Bacteriol.1999,181:2385-2393.
[128]Nies D H and Brown N L. Two-component systems in theregulation of heavy metal resistance. In:Metal Ions in Gene Regu-lation (Silver, S. and Walden, W, Eds).1998,77-103.
[129]Nies D H. CzcR and CzcD, gene products a(?)ecting regulationof resistance to cobalt, zinc and cadmium (czc system) in Alcaligeneseutrophus. J. Bacteriol.1992,174:8102-8110.
[130]Nies D H, Silver S. Plasmid-determined inducible effluxis responsible for resistance to cadmium, zinc, and cobalt in Alcali-genes eutrophus. J. Bacteriol.1989,171:896-900.
[131]Munson G P, Lam D L, Outten F W, et al. Identification of a copper-responsive two-component system on the chromosome of Escherichia coli K-12. J. Bacteriol.2000,182:5864-5871.
[132]Outten F W, Hu(?)man D L, Hale J A, et al. The independent cue and cus systems confer copper tolerance during aerobic and anaerobic growth in Escherichia coli. J. Biol.Chem.2001,276:30670-30677.
[133]Grass G. and Rensing C. Genes involved in copper homeostasis in Escherichia coli. J. Bacteriol.2001,183:2145-2147.
[134]Grass G. and Rensing C. CueO is a multi-copper oxidase that confers copper tolerance in Escherichia coli. Biochem. Biophys Res Commun.2001, 286:902-908.
[135]Hu(?)man D L, Huyett J, Outten F W, et al. Spectroscopy of Cu(Ⅱ)-PcoC and the multicopper oxidase function of PcoA, twoessential components of Escherichia coli pco copper resistance operon. Biochemistry.2002,41: 10046-10055.
[136]Franke S, Grass G, Rensing C, et al. Molecular analysis of the copper-transporting CusCFBA efflux system from Escherichia coli. J. Bacteriol.2003,277(35):32195-32201.
[137]侯冬梅,苗博,王洋洋,等.嗜酸氧化亚铁硫杆菌中锌离子转运基因的鉴定与分析.中国有色金属学报.2012,22(5)：1497-1520.
[138]Magnet S, Courvalin P and Lambert T. Resistance-nodulation-cell division-type efflux pump involved in aminoglycoside resistance in Acinetobacter baumannii strain BM4454. Antimicrob.Agents Chemother. 2001,45:3375-3380.
[139]Murakami S, Nakashima R, Yamashita R, et al. A Crystal structure of bacterial multidrug efflux transporter AcrB. Nature,2002,419:587-593.
[140]Srikumar R, Li X Z, Poole K. Inner membrane efflux components are responsible for beta-lactam speciccity of multidrug efflux pumps in Pseudomonas aeruginosa. J. Bacteriol.1997,179:7875-7881.
[141]Murata T, Kuwagaki M, Shin T, et al. The substrate specifcity of tripartite efflux systems of Pseudomonas aeruginosa is determined by the RND component. Biochem. Biophys. Res. Commun.2002,299:247-251.
[142]Brzywczy J, Sienko M, Kucharska A, et al. Sulphur amino acid synthesis in Schizosaccharomyces pombe represents a specific variant of sulphur metabolism in fungi. Yeast.2002,19:2-35.
[143]Patron N J, Durnford D G, Kopriva S. Sulfate assimilation in eukaryotes: fusions, relocations and lateral transfers. BioMed Central BMC Evolutionary Biology.2008,8(39):1471-2148.
[144]Ono B, Hazu T, Yoshida S, et al. Cysteine biosynthesis in Saccharomyces cerevisiae:a new outlook on pathway andregulation. Yeast.1999,15: 1365-1375.
[145]王敏.嗜酸氧化亚铁硫杆菌亚硫酸还原酶的α,β亚基的表达纯化、定点突变及相互作用.中南大学硕士学位论文.2008,60-70.
[146]戴云杰.嗜酸氧化亚铁硫杆菌中硫氰酸酶和丝氨酸乙酰转移酶的表达、纯化及性质研究.中南大学硕士学位论文.2009,60-80.
[147]郑春丽.嗜酸氧化亚铁硫杆菌硫同化途径蛋白ATP硫酸化酶、APS还原酶、半胱氨酸合成酶的研究.中南大学硕士学位论文.2009,50-70.
[148]Mansilla M C, de Mendoza D.1-cysteine biosynthesis in Bacillus subtilis: identification, sequencing, and functional characterization of the gene coding for phosphoadenylylsulfate sulfotransferase. J Bacteriol.1997,179: 976-981.
[149]Pinto R, Tang Q X, Britton W J, et al. The Mycobacterium tuberculosis cysD and cysNC genes form a stress-induced operon that encodes a tri-functional sulfate-activating complex. Microbiology.2004,150: 1681-1686.
[150]Berndt C, Lillig C H, Wollenberg M, et al. Characterization and reconstitution of a 4Fe-4S adenylylsulfate/phosphoadenylyl sulfate reductase from Bacillus subtilis. J Biol Chem.2004,279:7850-7855.
[151]Bick J A, Dennis J J, Zylstra G J, et al. Identification of a new class of 5'-adenylylsulfate (APS) reductases from sulfate-assimilating bacteria. J Bacteriol.2000,182:135-142.
[152]Kelley BC, Tuovinen O H, Donald D J. Utilization of 35S-Thiosulphate and an Appraisal of the Role of ATP-Sulphurylase in Chemolithotrophic Thiobacillus ferrooxidans. Arch Microbiol.1976,109:205-208.
[153]Denk D, Bock A.1-cysteine biosynthesis in Escherichia coli:nucleotide sequence and expression of the serine acetyltransferase (cysE) gene from the wild-type and a cysteine-excreting mutant. J Gen Microbiol.1987,133: 515-525.
[154]Campanini B, Speroni F, Salsi E, et al. Interaction of serine acetyltransferase with O-acetylserine sulfhydrylase active site:evidence from fluorescence spectroscopy. Protein Sci.2005,14:2115-2124.
[155]Johnson C M, Huang B, Roderick S L, et al. Kinetic mechanism of the serine acetyltransferase from Haemophilus influenzae. Arch Biochem Biophys.2004,429:115-122.
[156]Zhang C H, Baichen L M. A Nucleotide Metabolite Controls Stress-Responsive Gene Expression and Plant Development. PLoS ONE. 2011,6(10):358-429.
[157]Sarvajeet S G, Narendra T. Cadmium stress tolerance in crop plants Probeing the role of sulfur. Plant Signaling & Behavior.2011,6(2): 192-201.
[158]Torricelli E, Gorbi G, Pawlik S B, et al. Cadmium tolerance, cysteine and thiol peptide levels in wild type and chromium-tolerant strains of Scenedesmus acutus (Chlorophyceae). Aquat Toxicol.2004,68(4):315-323.
[159]Heiss S, Schafer H J, Haag-Kerwer A, et al. Cloning sulfur assimilation genes of Brassica juncea L:cadmium differentially affects the expression of a putative low-affinity sulfate transporter and isoforms of ATP sulfurylase and APS reductase. Plant Mol Biol.1999,39(4):847-857.
[160]Quig D. Cysteine metabolism and metal toxicity. Altern Med Rev.1998, 3(4):262-270.
[161]Wu Y C, Roje S, Sandoval F J, et al. Repression of sulfate assimilation is an adaptive response of yeast to the oxidative stress of zinc deficiency. The journal of biological chemistry.2009,284(40):27544-27556.
[162]Magda M, Helmut S.Cytotoxicity of metals in isolated fish cells:important of the cellular glutathione status.Comparative Biochemistry and Physiology Part A.1998,120(1):83-88.
[163]马纪,孙奋勇,张越,等.miR-122过表达转基因小鼠质粒构建及其功能验证.中国生物工程杂志.2011,31(9):28-34.
[164]陈卫,葛佳佳,张灏,等.半乳糖苷酶基因在大肠杆菌中过量表达及IPTG诱导条件.无锡轻工大学学报.2002,21(5)：492-495.
[165]童贞珍.大肠杆菌IscA和SufA在铁硫簇生物合成中的功能研究.温州医学院硕士学位论文.45-50.
[166]赵楠,林璨瑜,王淑芳,等.过表达B基因的转基因微型番茄的获得.南 开大学学报.2006,39(4)：103-107.
[167]梁智万,林秋红,肖启华,等.口服谷胱甘肽(GSH)治疗慢性铅中毒临床疗效观察.中国工业医学杂志.1997,10(6):352-353.
[168]Prashant M, Nisha K R, Sudesh K Y. Cadmium induced oxidative stress influence on glutathione metabolic genes of Camellia sinensis(L.) O.Kuntze. Environmental Toxicology.2007,22(4):368-374.
[169]刘慧,王晓蓉,王为本,等.不同形态锌离子对鲫鱼谷胱甘肽系统的影响.中国环境学报.2005,25(2):169-173.
[170]孙琴,王超.土壤外源Cd和Pb复合污染对小麦(Tritioum asetivum L.)根系植物络合素和谷胱甘肽的影响.生态环境.2008,17(5):1833-1838.
[171]孙琴,王超.土壤Cd和Pb复合污染对小麦根系植物络合素和谷胱甘肽的影响.农业环境科学学报.2008,27(5)：1913-1918.
[172]Abdin M, Akmal M, Ram M, et al. Constitutive expression of high-affinity sulfate transporter (HAST) gene in Indian mustard showed enhanced sulfur uptake and assimilation. Protoplasma.2011,248(3):591.
[173]Giovannoni S J, MullinsT D, Field K G. Microbial diversity in oceanicsystems:rRNA approaches to the study of unculturable microbes. In: molecular ecology of aquatic microbes.1995,38:217-248.
[174]曹琳辉.银山铅锌矿酸性矿坑水微生物群落结构分析.中南大学硕士学位论文,2007.
[2]Rawling D E, Johnson D B. The microbiology of biomining:development and optimization of mineral-oxidizing microbial consortia. Microbiology. 2007,153:315-324.
[3]Brierley C L. Biohydrometallurgy prospects. Hydrometallurgy.2006, 104(3-4):324-328.
[4]Rudolfs W, Helbronner A. Oxidation of Zinc Sulfides by Microorganism. Soil Sci.1922,14:459-464.
[5]Colmer A R, Hinckle M E. The Role of Microorganism in Acid Mine Drainage. A Preliminary Report, Science.1947,106:253-256.
[6]Faramarzi M A, Stagars M, Pensini E, et al. Metal solubilization from metal-containing solid materials by cyanogenic Chromobacterim violaceum. J Biotechnol.2004,113:321-326.
[7]毕银丽,胡瑜,苏高华,等.微生物对煤矿固体废弃物的脱硫效应.环境工程学报.2008,2(1)：92-96.
[8]Gonez C, Bosecker K. Leaching of heavy metals from contamined soil by using Thiobacillus ferrooxidans or Thiobacillus thiooxidans. Geomicrobiology.1999,16(3):233-244.
[9]刘俊,龚文琪,申求实,等.低品位磷矿的生物浸出研究.金属矿山.2008,385：54-58.
[10]Rawlings D E:Heavy metal mining using microbes. Annu Rev. Microbiol. 2002,56:65-91.
[11]Gonzalez T E, Llobet B E, Casamayor E O, et al. Microbial ecology of an extreme acidic environment, the Tinto River. Appl Environ Microbiol.2003, 69(8):4853-4865.
[12]Norris P R, Clark D A, Owen J P, et al. Characteristics of Sulfobacillus acidophilus sp. nov. and other moderately thermophilic mineral-sulphide-oxidizing bacteria. Microbiology.1996,142(4):775-783.
[13]Waiting H R. The bioleaching of sulphide minerals with emphasis on copper sulphide-A review. Hydrometallurgy.2006,84:81-108.
[14]Demergasso C S, Galleiguillos P A, Escudero G, et al. Molecular characterization of microbial population in low-grade copper ore bioleaching test heap. Hydrometallurgy.2005,80:241-253.
[15]Robbins E I. Bacteria and archaea in acidic environments and a key to morphological identification. Hydrobiologia.2000,433:61-89.
[16]Groudev, Stoyan N, Groudeva V I. Microbial communities in four industrial copper dump leaching operations in Bulgaria. FEMS microbiological reviews.1993,11:261-268.
[17]吴学玲.抗性浸矿细菌的选育及抗性机理的研究.中南大学博士学位论文.2007,7-8.
[18]Claudio A. Navarro, Luis H, et al. Transcriptional and Functional Studies of Acidithiobacillus ferrooxidans Genes Related to Survival in the Presence of Copper. Applied and environmental microbiology.2009,75(19):6102-6109.
[19]Orell A, Navarro A, et al. Copper resistance mechanisms of biomining bacteria and archaea living under extremely high concentrations of metals. Biohydrometallurgy.2009,71-73:279-282.
[20]徐柳,宋琴,茆灿泉.金属结合蛋白(肽)与环境重金属生物修复.生物工程杂志.2004,24(4)：39-43.
[21]曾景海,齐鸿雁,杨建州,等.重金属抗性菌Bacillus cereus HQ-1对银离子的生物吸附-微沉淀成品作用.环境科学与技术.2008,29(1):225-230.
[22]杨峰,尹华,彭辉,等.融合菌-活性污泥联合曝气吸附处理重金属铬.环境科学与技术.2006,26(5):3 18-322.
[23]White C, Gadd G M. Accumulation and effects of cadmium on sulphate-reducing bacterial biofilms. Microbiology.1998,44:1407-1415.
[24]Gharieb M M, Gadd G M. Role of glutathione in detoxification of metal(loid)s by Saccharomyces cerevisiae. BioMetals.2004,17:183-188.
[25]张玉刚,龙新宪,陈雪梅.微生物处理重金属废水的研究进展.环境科学与技术.2008,31(6)：58-63.
[26]Goulhen F, Bruschi M. Cr (VI) detoxification by Desulfovibrio vulgaris strain Hildenborough:microbe-metal interactions studies. Appl Microbiol Biotechnol.2006,71:892-897.
[27]高小朋,张欠欠,冯进辉.微生物还原Cr(VI)的研究进展.微生物通报.2008,35(5)：820-824.
[28]Stahler F N, Odenbreit S, Haas R, et al. The novel Helicobacter pylori CPbABC metal efflux pump is required for cadmium, zinc, and nickel resistance, urease modulation, and gastric colonization. Infect Immun.2006, 74(7):3845-3852.
[29]Paulsen I T, Park J H, Choi P S. A family of Gram-negative bacterial outer membrane factors that function in the export of proteins, carbohydrates, drugs and heavy metals from Gram-negative bacteria. FEMS Microbiol Lett. 1997,156:1-8.
[30]Nies D H. Efflux-mediated heavy metal resistance in prokaryotes. FEMS Microbiology.2003,27:313-339.
[31]Saier J, Tam R, Reizer A, et al. Two novel families of bacterial membrane proteins concerned with nodulation, cell division and transport. Mol. Microbiol.1994,11:841-847.
[32]seng T T, Gratwick K S, Kollman J, et al. The RND superfamily:an ancient, ubiquitous and diverse family that includes human disease and development proteins. J. Mol. Microbiol. Biotechnol.1999,1:107-125.
[33]Johnson J M, Church G M. Alignment and structure prediction of divergent protein families:periplasmic and outer membrane proteins of bacterial efflux pumps. J. Mol. Biol.1999,287:695-715.
[34]Andersen C, Hughes C, Koronakis V. Protein export and drug efflux through bacterial channel-tunnels. Curr. Opin. Cell Biol.2001,13:412-416.
[35]Paulsen I T, Park J H, Choi P S, et al. A family of Gram-negative bacterial outer membrane factors that function in the export of proteins, carbohydrates, drugs and heavy metals from Gram-negative bacteria. FEMS Microbiol. Lett. 1997,156:1-8.
[36]Milton H, Saier J. A functional-phylogenetic classification system for transmembrane solute transporters. Microbiol Mol Biol Rev.2000,64: 354-411.
[37]Koronakis V, Shar A, Koronakis E, et al. Crystal structure of the bacterial membrane protein TolC central to multidrug efflux and protein export. Nature 2000.405:914-919.
[38]FaganM J, Saier M H J. P-type ATPases of eukaryotes and bacteria: Sequence analyses and construction of phylogenetic trees. Journal of Molecular Evolution.1994,38(1):57-99.
[39]Borsetti F, Martelli P L, Casadio R, et al. Metals and metalloids in photosynthetic bacteria:interactions, resistance and putative home-ostasis Revealed by genome analysis. In The Purple Phototrophic Bacteria. Netherlands:Springer Science Business Media BV.2009,28:655-689.
[40]Rensing C, Fan B, Sharma R, et al. CopA:An Escherichia coli Cu(I)-translocating P-type ATPase. Proc Natl Acad. Sci. USA.2000,97: 652-656.
[41]Guerinot M L. The ZIP family of metal transporters. Biochim. Biophys. Acta. 2000,1465:190-199.
[42]Christopher J H, Gregor G, Sylvia F, et al. New developments in the understanding of the cation diffusion facilitator family. J Ind Microbiol Biotechnol.2005,32(6):215-26.
[43]Mario G, Luis L, et al. A gene cluster involved in metal homeostasis in the cyanobacterium Synechocystisspstrain PCC 6803. Journal of Bacteriology. 2000,182(6):1507-1514.
[44]Wu C C, Gardarina, Catty P, et al. The Cadmium Transport Sites of CadA, the Cd2+-ATPase from Listeria monocytogenes. Journal of Biological Chemistry.2006,281(40):29533-29541.
[45]周围,李友国,杨凯,等.1株抗重金属铬细菌的分离鉴定及抗性基因型的初步研究.华中农业大学学报.2008,27(2):248-250.
[46]赵肖为,李清彪,卢英华,等.高选择性基因工程菌E. coli SE 5000生物富集水体中的镍离子.环境科学学报.2004,24(2):231-236.
[47]Borremans, Hobman, Provoost, et al. Cloning and Functional Analysis of the pbr Lead Resistance Determinant of Ralstonia metallidurans CH34. Journal of Bacteriology.2001,183(19):5651-5658.
[48]Jonathn J. Caguiat, Alice L. et al. Cd(Ⅱ)-Responsive and Constitutive Mutants Implicate a Novel Domain in MerR. Journal of Bacteriology.1999, 181(11):3462-3471.
[49]洪斌.微生物对砷的地球化学行为的影响-暨地下水砷污染机制的最新研究进展.地球科学进展.2006,21(1):77-82.
[50]Freeman J L, Salt D E. The metal tolerance profile of Thlaspi goesingense is mimicked in Arabidopsis thaliana heterologously expressing serine acetyl-transferase. BMC Plant Biology.2007,7(63):1471-1481.
[51]Stoyanov J V, Hobman J L & Brown N L. CueR (YbbI) of Escherichia coli is a MerRfamily regulator controlling expression of the copper exporter CopA. Mol. Microbiol.2001,39:502-511.
[52]Brown N L, Stoyanov J V, Kidd S P, et al. The MerR family of transcriptional regulators. FEMS Microbiol Rev.2003,27:145-163.
[53]Gaballa A, Cao M & Helmann J D. Two MerR homologues that affect copper induction of the Bacillus subtilis copZA operon. Microbiology.2003, 149:3413-3421.
[54]Cavet J S, Graham A I, Meng W. A cadmium-lead-sensing ArsR-SmtB repressor with novel sensory sites. Complementary metal discriminationby NmtR and CmtR in a common cytosol. J. Biol. Chem.2003,278: 44560-44566.
[55]Malin M, Leif B. Metal-binding proteins and peptidesin bioremediation and phytoremediation of heavy metals. Trends in Biotechnology.2001,19(2): 67-74.
[56]Hatzios S K, Bertozzil C R. The regulation of sulfur metabolism in Mycobacterium Tuberculosis. PLoS Pathogens.2011,7(7):1-8.
[57]Saharan R K, Sharma S C. Effects of Glutathione Modulation on Oxidative Stress and Enzymatic Antioxidant Defence in Yeast. Curr Microbiol.2011, 62:944-949.
[58]Torricelli E, Gorbi G, Skowronska P B. Cadmium tolerance, cysteine and thiol peptide levels in wild type and chromium-tolerant strains of Scenedesmus acutus (Chlorophyceae). Aquatic Toxicology.2004,68: 315-323.
[59]Gorbi G, Zanni C, Corradi M G. Sulfur starvation and chromium tolerance in Scenedesmus acutus:A possible link between metal tolerance and the regulation of sulfur uptake/assimilation processes. Aquatic Toxicology.2007, 84:457-464.
[60]Saito K. Regulation of sulfate transport and synthesis of sulfurcontaining amino acids. Curr Opin Plant Biol,2000,3:188-195.
[61]Kredich, N M. Biosynthesis of cysteine in Escherichia coli and Salmonella. Cellular and molecular biology. Neidhardt, F.C. (ed) Washington, D.C,1996, 514-527.
[62]Pereira Y, Lagniel G, Godat E, et al. Chromate causes sulfur starvation in yeast. Toxicological sciences.2008,106(2):400-412.
[63]Momose Y, Iwahashi H. Bioassay of cadmium using a DNA microarray: genome-wide expression patterns of Saccharomyces cerevisiae response to cadmium. Environ Toxicol Chem.2003,20:2353-2360.
[64]Nocito F F, Pirovano L, Cocucci M, et al. Cadmium-induced sulfate uptake in maize roots. Plant Physiol.2002,129:1872-1879.
[65]Vido K, Spector D, Lagniel G, et al. A proteome analysis of the cadmium response in Saccharomyces cerevisiae. J Biol Chem.2001,276:8469-8474.
[66]Xia J L, Wu S, Zhang R Y, et al. Effects of Copper Exposure on Expression of Glutathione-Relate Genes in Acidithiobacillus ferrooxidans. Curr Microbiol.2011,62(5):1460-1466.
[67]Xia L X, Yin C, Cai L, et al. Metabolic changes of Acidithiobacillus caldus under Cu2+ stress. Journal of Basic Microbiology.2010,50(6):591-598.
[68]Leyh T S, Taylor J C, Markham G D. The sulfate activation locus of Escherichia coli K-12:cloning, genetic, and enzymatic characterization. Journal of Biological Chemistry.1988,263(5):2409-2416.
[69]Hell R. Molecular physiology of plant sulfur metabolism. Planta.1997,202: 138-148.
[70]Takahashi H, Asanuma W, Saito K. Cloning of an Arabidopsis cDNA encoding a chloroplast localizing sulphate transporter isoform. J. Exp. Bot. 1999,50(340):1713-1714.
[71]Li H, Deyrup A, Mensch J R, et al. The isolation and characterization of cDNA encoding the mouse bifunctional ATP sulfurylase-adenosine 5'-phosphosulfate kinase. J. Biol. Chem.1995,270:29453-29459.
[72]Venkatachalam KV, Fuda H, Koonin E V, et al. Siteselected mutagenesis of a conserved nucleotide binding HXGH motif located in the ATP sulfurylase domain of human bifunctional 3'-phosphoadenosine 5'-phosphosulfate synthase. Biochim. Biophys. Acta.1998,1429:284-291.
[73]Tobias C U, Michael B, Robert H. Crystal structure of ATP sulfurylase from Saccharomyces cerevisiae, a key enzyme in sulfate activation. The EMBO Journal.2001,20(3):316-329.
[74]Mougous J D, Lee D H, Hubbard S C. Molecular Basis for G Protein Control of the Prokaryotic ATP Sulfurylase. Molecular Cell.2006,21: 109-122.
[75]Savage H, Montoya G, Svensson C, et al. Crystal structure of phosphoadenylyl sulphate (PAPS) reductase:a new family of adenine nucleotide alpha hydrolases. Structure.1997,5:895-906.
[76]Liu C, Martin E, and Leyh T S. GTPase activation of ATP sulfurylase:the mechanism. Biochemistry.1994,33:2042-2047.
[77]Liu C, Suo Y, and Leyh T S. The energetic linkage of GTP hydrolysis and the synthesis of activated sulfate. Biochemistry.1994b,33:7309-7314.
[78]Zheng C L, Zhang Y F, Liu Y D, et al. Characterization and reconstitute of a [Fe4S4] Adenosine 5'-phosphosulfate reductase from Acidithiobacillus ferrooxidans. Current Microbiology.2009,58(6):586-592.
[79]Ostrowski J, Wu J.Y, Rueger D C, et al. Characterization of the cysJIH regions of Salmonella typhimurium and Escherichia coli B. DNA sequences of cysl and cysH and a model for the siroheme Fe4S4 active center of sulfite reductase hemoprotein based on amino acid homology with spinach nitrite redutase. Journal of Biological Chemistry.1989,264(26):15726-15737.
[80]Bick J A, Dennis J J, Zylstra G J, et al. Identification of a new class of 5'-adenylylsulfate (APS) reductases from sulfate-assimilating bacteria. J. Bacteriol.2000,182:135-142.
[81]Kim S K, Rahman A, Bick J A, et al. Properties of the cysteine residues and iron-sulfur cluster of the assimilatory 5'-adenylyl sulfate reductase from Pseudomonas aeruginosa. Biochemistry.2004,43:13478-13486.
[82]Kopriva S, Buchert T, Fritz G, et al. The presence of an iron-sulfur cluster in adenosine 5'-phosphosulfate reductase separates organisms utilizing adenosine 5'-phosphosulfate and phosphoadenosine 5'-phosphosulfate for sulfate assimilation. J. Biol. Chem.2002,277:21786-21791.
[83]Kanno N, Nagahisa E, Sato M, et al. Adenosine 5'-phosphosulfate sulfotransferase from the marine macroalga Porphyra yezoensis Ueda (Rhodophyta):Stabilization, purification, and properties, Planta.1996,198: 440-446.
[84]Fritz G, Roth A, Schiffer A, et al. Structure of adenylylsulfate reductase from the hyperthermophilic Archaeoglobus fulgidus at 1.6-A resolution. Proc Natl Acad Sci. USA.2002,99:1836-1841.
[85]Fritz G, Buchert T, Kroneck P M. The function of the [Fe4S4] clusters and FAD in bacterial and archaeal adenylysulfate reductases. J Biol Chem.2002, 277:26066-26073.
[86]Carroll K S, Gao H, Chen H, et al. Investigation of the iron-sulfur cluster in Mycobacterium tuberculosis APS reductase:implications for substrate binding and catalysis. Biochemistry.2005,44:14647-14657.
[87]Siegel L M, Murphy J M, Kamin H. The Eseherichia coli hemoflavoprotein: molecular parameters and prosthetic groups. Journal of Biological Chemistry. 1973,248:251-261.
[88]Ostrowski J, Barber M J, Rueger D C, et al. Characterization of the flavoprotein moieties of NADPH-sulfite reductase from Salmonella typhimurium and Escherichia coli. Physicochemical and catalytic properties, amino acid sequencededuced from the DNA sequence of cysJ, and comparison with NADPH-cytochrome P-450 reductase. Journal of Biological Chemistry.1989,264(27):15796-15808.
[89]Byrne C R, Monroe R S, Ward K A & Kredich N M. DNA sequences of the cysK regions of Salmonella typhimurium and Escherichia coli and linkage of the cysK region to ptsH. J. Bacteriol.1988,170:3150-3158.
[90]Becker M A, Kredich N M & Tomkins G M. Purification and characterization of cysteine synthetase, a bifunctional protein complex, from Salmonella typhimurium. J. Biol. Chem.1969,244:2418-2427.
[91]Schnackerz K D, Tai C H, Simmons J W, et al. Identification and spectral characterization of the external aldimine intermediate of the O-acetylserine sulfhydrylase reaction. Biochemistry.1995,34:12152-12160.
[92]Cook P F, Hara S, Nalabolu S & Schnackerz K D. PH dependence of the absorbance and 31P NMR spectra of O-acetylserine sulfhydrylase in the absence and presence of O-acetyl-L-serine. Biochemistry.1992,31: 2298-2303.
[93]Rege V D, Kredich N M, Tai C H, et al. A change in the internal aldimine lysine (K42) in O-acetylserine sulfhyrylase to alanine indicates its importance in transamination and as general base cactalyst. Biochemistry. 1996,35:13485-13493.
[94]McClure G D & Cook P F. Product binding to the a-carboxyl subsite results in a conformational change at the active site of O-acetylserine sulfhydrylase-A:evidence from fluorescence spectroscopy. Biochemistry.1994,33: 1674-1683.
[95]Strambini G, Cioni P & Cook P F. Tryptophan and coenzyme luminescence as a probe of conformation along the O-acetylserine sulfhydrylase reaction pathway. Biochemistry.1996,35:8392-8400.
[96]Peter G S. Jagannatha R, Hohenester E, et al. Three-dimensional Structure of O-acetylserine Sulfhydrylase from Salmonella typhimurium. J. Mol. Biol. 1998,283:121-133.
[97]Guedon E, Verstraete I M. Cysteine Metabolismand Its Regulation in Bacteria. Microbiol Monographs.2007,5:195-218.
[98]Thomas D, Surdin K Y. Metabolism of sulphur amino acids in Saccharomyces cerevisiae. Microbiol. Mol. Biol.1997,61(4):503-532.
[99]Pye V E, Tingey A P, Robson R L. The Structure and Mechanism of Serine Acetyltransferase from Escherichia coli. The journal of biological chemistry. 2004,279(39):40729-40736.
[100]Haas F H, Heeg C, Queiroz R, et al. Mitochondrial Serine Acetyltransferase Functions as a Pacemaker of Cysteine Synthesis in Plant Cells. Plant Physiology.2008,148:1055-1067.
[101]Tuovinen O H, Kelly D P. Biology of Thiobacillus ferrooxidans in relation to the microbiological leaching of sulphide ores. Z Allg Mikrobiol.1972, 12(4):311-346.
[102]Wu X L, Ding J N, Gao J, et al. Isolation and identification of metal-resistant iron-oxidizing bacteria. Minerals & Metallurgical Processing. 2007,24(1):57-60.
[103]王雪峰,林建群,韩梅等.细菌铜抗性机制研究进展.湖北农业科学.2009,48(1)：218-224.
[104]武顺.铜离子对嗜酸氧化亚铁硫杆菌ATCC 23270谷胱甘肽相关酶基因表达水平的影响.中南大学硕士论文.2009,52-62.
[105]Truper H G, Lynne A R. Purification and properties of adenylyl sulfate reductase from the phototrophic sulfur bacterium, Thiocapsa roseopersicina. J Bacteriol.1971,108:1112-1121.
[106]Droux M, Ruffet M L, Douce R, et al. Interactions between serine acetyltransferase and O-acetylserine (thiol) lyase in higher plantsstructural and kinetic properties of the free and bound enzymes. Eur JBiochem.1998, 1:235-245.
[107]Warrilow A G S and Hawkesford M J. Separation, subcellular location and influence of sulphur nutrition isoforms of cysteine synthase in spinach. J. Exp. Bot.1998,49(327):1625-1636.
[108]Nies D H. The cobalt, zinc, cadmium efflux system czcABC from alcaligenes eutrophus functions as a cation-proton antiporter in Escherichia coli. J. Bacteriol.1995,177:2707-2712.
[109]Nies D H, Silver S. Ion efflux systems involved in bacterial metal resistances. J Ind Microbiol.1995,14:189-199.
[110]Nies D H. Efflux-mediated heavy metal resistance in prokaryotes. FEMS Microbiol.2003,27:313-339.
[111]Nies D H. Resistance to cadmium, cobalt, zinc, and nickel in microbes. Plasmid.1992,27:17-28.
[112]Notico F F, Lancilli C, Crema B, et al. Heavy metal stress and sulfate uptake in maize roots. Plant physiology.2006,141:1138-1148.
[113]Phartiyal P, Kim W S, Cahoon R E, et al. The role 5-adenylysulfate reductase in the sulfur assimilation pathway of soybean:molecular cloning, kinetic characterization, and gene expression. Phytochemistry.2008,69: 356-364.
[114]Kim S D, Bae J E, Park H S, et al. Bioleaching of cadmium and nickel from synthetic sediments by Acidithiobacillus ferrooxidans. Environ Geochem Health.2005.27(3):229-235.
[115]Baillet F, Magnin J P, Cheruy A, et al. Chromium precipitation by the acidophilic bacterium Thiobacillus ferrooxidans. Biotech letters.2004,20: 95-99.
[116]Van Z, Munste L J, Rawlings D E, et al. Construction of arsB and tetH mutants of the sulfur-oxidizing bacterium Acidithiobacillus caldus by marker exchange. Appl Environ Microbiol.2008.74(18):5686-5694.
[117]Alvarez S, Jerez C A. Copper ions stimulate polyphosphate degradation and phosphate efflux in Acidithiobacillus ferrooxidans. Appl Environ Microbiol. 2004,70(9):5177-5182.
[118]Ramirez P, Guiliani N, Valenzuela L, et al. Differential protein expression during growth of Acidithiobacillus ferrooxidans on ferrous iron, sulfur compounds, or metal sulfides. Appl Environ Microbiol.2004,70(8): 4491-4498.
[119]相丽,姜毓君,刘伟,等.金黄色葡萄球菌基因表达的DNA扣除法Real-time RT-PCR相对定量分析.微生物学报.2008,48(4):526-531.
[120]Livak K J, Schmittgen T D. Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔCT Method. Methods.2001,25(4): 402-408.
[121]戴艳霞.氧化亚铁硫杆菌与隐性嗜酸杆菌共培养体系构建及铁代谢基因差异表达研究.中南大学硕士论文.2009,80-100.
[122]Mergeay M, Nies D, Schlegel H G, et al. Alcaligenes eutrophus CH34 is a facultative chemolithotroph with plasmid-bound resistance to heavy metals. J. Bacteriol.1985,162:328-334.
[123]Collard J M, Corbisier P, Diels L, et al. Plamids for heavy metal resistance in Alcaligenes eutrophus CH34:mechanisms and applications. FEMS Microbiol Rev,1994,14(4):405-414.
[124]Nies D, Mergeay M, Friedrich B, et al. Cloning of plasmid genes encoding resistance to cadmium, zinc, and cobalt in Alcaligenes eutrophus CH34. J. Bacteriol.1987,169:4865-4868.
[125]Kunito T, Kusano T, Oyaizu H, et al. Cloning and sequence analysis of czc genes in Alcaligenes sp. strain CT14. Biosci. Biotechnol. Biochem.1996,60: 699-704.
[126]Lelie D, Schwuchow T, Schwidetzky U, et al. Two component regulatory system involved in transcriptional control of heavy metal homoeostasis in Alcaligenes eutrophus. Mol. Microbiol.1997,23:493-503.
[127]GroMe C, Grass G, Anton A, et al. Transcriptional organization of the czc heavy metal homoeostasis determinant from Alcaligenes eutrophus. J. Bacteriol.1999,181:2385-2393.
[128]Nies D H and Brown N L. Two-component systems in theregulation of heavy metal resistance. In:Metal Ions in Gene Regu-lation (Silver, S. and Walden, W, Eds).1998,77-103.
[129]Nies D H. CzcR and CzcD, gene products a(?)ecting regulationof resistance to cobalt, zinc and cadmium (czc system) in Alcaligeneseutrophus. J. Bacteriol.1992,174:8102-8110.
[130]Nies D H, Silver S. Plasmid-determined inducible effluxis responsible for resistance to cadmium, zinc, and cobalt in Alcali-genes eutrophus. J. Bacteriol.1989,171:896-900.
[131]Munson G P, Lam D L, Outten F W, et al. Identification of a copper-responsive two-component system on the chromosome of Escherichia coli K-12. J. Bacteriol.2000,182:5864-5871.
[132]Outten F W, Hu(?)man D L, Hale J A, et al. The independent cue and cus systems confer copper tolerance during aerobic and anaerobic growth in Escherichia coli. J. Biol.Chem.2001,276:30670-30677.
[133]Grass G. and Rensing C. Genes involved in copper homeostasis in Escherichia coli. J. Bacteriol.2001,183:2145-2147.
[134]Grass G. and Rensing C. CueO is a multi-copper oxidase that confers copper tolerance in Escherichia coli. Biochem. Biophys Res Commun.2001, 286:902-908.
[135]Hu(?)man D L, Huyett J, Outten F W, et al. Spectroscopy of Cu(Ⅱ)-PcoC and the multicopper oxidase function of PcoA, twoessential components of Escherichia coli pco copper resistance operon. Biochemistry.2002,41: 10046-10055.
[136]Franke S, Grass G, Rensing C, et al. Molecular analysis of the copper-transporting CusCFBA efflux system from Escherichia coli. J. Bacteriol.2003,277(35):32195-32201.
[137]侯冬梅,苗博,王洋洋,等.嗜酸氧化亚铁硫杆菌中锌离子转运基因的鉴定与分析.中国有色金属学报.2012,22(5)：1497-1520.
[138]Magnet S, Courvalin P and Lambert T. Resistance-nodulation-cell division-type efflux pump involved in aminoglycoside resistance in Acinetobacter baumannii strain BM4454. Antimicrob.Agents Chemother. 2001,45:3375-3380.
[139]Murakami S, Nakashima R, Yamashita R, et al. A Crystal structure of bacterial multidrug efflux transporter AcrB. Nature,2002,419:587-593.
[140]Srikumar R, Li X Z, Poole K. Inner membrane efflux components are responsible for beta-lactam speciccity of multidrug efflux pumps in Pseudomonas aeruginosa. J. Bacteriol.1997,179:7875-7881.
[141]Murata T, Kuwagaki M, Shin T, et al. The substrate specifcity of tripartite efflux systems of Pseudomonas aeruginosa is determined by the RND component. Biochem. Biophys. Res. Commun.2002,299:247-251.
[142]Brzywczy J, Sienko M, Kucharska A, et al. Sulphur amino acid synthesis in Schizosaccharomyces pombe represents a specific variant of sulphur metabolism in fungi. Yeast.2002,19:2-35.
[143]Patron N J, Durnford D G, Kopriva S. Sulfate assimilation in eukaryotes: fusions, relocations and lateral transfers. BioMed Central BMC Evolutionary Biology.2008,8(39):1471-2148.
[144]Ono B, Hazu T, Yoshida S, et al. Cysteine biosynthesis in Saccharomyces cerevisiae:a new outlook on pathway andregulation. Yeast.1999,15: 1365-1375.
[145]王敏.嗜酸氧化亚铁硫杆菌亚硫酸还原酶的α,β亚基的表达纯化、定点突变及相互作用.中南大学硕士学位论文.2008,60-70.
[146]戴云杰.嗜酸氧化亚铁硫杆菌中硫氰酸酶和丝氨酸乙酰转移酶的表达、纯化及性质研究.中南大学硕士学位论文.2009,60-80.
[147]郑春丽.嗜酸氧化亚铁硫杆菌硫同化途径蛋白ATP硫酸化酶、APS还原酶、半胱氨酸合成酶的研究.中南大学硕士学位论文.2009,50-70.
[148]Mansilla M C, de Mendoza D.1-cysteine biosynthesis in Bacillus subtilis: identification, sequencing, and functional characterization of the gene coding for phosphoadenylylsulfate sulfotransferase. J Bacteriol.1997,179: 976-981.
[149]Pinto R, Tang Q X, Britton W J, et al. The Mycobacterium tuberculosis cysD and cysNC genes form a stress-induced operon that encodes a tri-functional sulfate-activating complex. Microbiology.2004,150: 1681-1686.
[150]Berndt C, Lillig C H, Wollenberg M, et al. Characterization and reconstitution of a 4Fe-4S adenylylsulfate/phosphoadenylyl sulfate reductase from Bacillus subtilis. J Biol Chem.2004,279:7850-7855.
[151]Bick J A, Dennis J J, Zylstra G J, et al. Identification of a new class of 5'-adenylylsulfate (APS) reductases from sulfate-assimilating bacteria. J Bacteriol.2000,182:135-142.
[152]Kelley BC, Tuovinen O H, Donald D J. Utilization of 35S-Thiosulphate and an Appraisal of the Role of ATP-Sulphurylase in Chemolithotrophic Thiobacillus ferrooxidans. Arch Microbiol.1976,109:205-208.
[153]Denk D, Bock A.1-cysteine biosynthesis in Escherichia coli:nucleotide sequence and expression of the serine acetyltransferase (cysE) gene from the wild-type and a cysteine-excreting mutant. J Gen Microbiol.1987,133: 515-525.
[154]Campanini B, Speroni F, Salsi E, et al. Interaction of serine acetyltransferase with O-acetylserine sulfhydrylase active site:evidence from fluorescence spectroscopy. Protein Sci.2005,14:2115-2124.
[155]Johnson C M, Huang B, Roderick S L, et al. Kinetic mechanism of the serine acetyltransferase from Haemophilus influenzae. Arch Biochem Biophys.2004,429:115-122.
[156]Zhang C H, Baichen L M. A Nucleotide Metabolite Controls Stress-Responsive Gene Expression and Plant Development. PLoS ONE. 2011,6(10):358-429.
[157]Sarvajeet S G, Narendra T. Cadmium stress tolerance in crop plants Probeing the role of sulfur. Plant Signaling & Behavior.2011,6(2): 192-201.
[158]Torricelli E, Gorbi G, Pawlik S B, et al. Cadmium tolerance, cysteine and thiol peptide levels in wild type and chromium-tolerant strains of Scenedesmus acutus (Chlorophyceae). Aquat Toxicol.2004,68(4):315-323.
[159]Heiss S, Schafer H J, Haag-Kerwer A, et al. Cloning sulfur assimilation genes of Brassica juncea L:cadmium differentially affects the expression of a putative low-affinity sulfate transporter and isoforms of ATP sulfurylase and APS reductase. Plant Mol Biol.1999,39(4):847-857.
[160]Quig D. Cysteine metabolism and metal toxicity. Altern Med Rev.1998, 3(4):262-270.
[161]Wu Y C, Roje S, Sandoval F J, et al. Repression of sulfate assimilation is an adaptive response of yeast to the oxidative stress of zinc deficiency. The journal of biological chemistry.2009,284(40):27544-27556.
[162]Magda M, Helmut S.Cytotoxicity of metals in isolated fish cells:important of the cellular glutathione status.Comparative Biochemistry and Physiology Part A.1998,120(1):83-88.
[163]马纪,孙奋勇,张越,等.miR-122过表达转基因小鼠质粒构建及其功能验证.中国生物工程杂志.2011,31(9):28-34.
[164]陈卫,葛佳佳,张灏,等.半乳糖苷酶基因在大肠杆菌中过量表达及IPTG诱导条件.无锡轻工大学学报.2002,21(5)：492-495.
[165]童贞珍.大肠杆菌IscA和SufA在铁硫簇生物合成中的功能研究.温州医学院硕士学位论文.45-50.
[166]赵楠,林璨瑜,王淑芳,等.过表达B基因的转基因微型番茄的获得.南 开大学学报.2006,39(4)：103-107.
[167]梁智万,林秋红,肖启华,等.口服谷胱甘肽(GSH)治疗慢性铅中毒临床疗效观察.中国工业医学杂志.1997,10(6):352-353.
[168]Prashant M, Nisha K R, Sudesh K Y. Cadmium induced oxidative stress influence on glutathione metabolic genes of Camellia sinensis(L.) O.Kuntze. Environmental Toxicology.2007,22(4):368-374.
[169]刘慧,王晓蓉,王为本,等.不同形态锌离子对鲫鱼谷胱甘肽系统的影响.中国环境学报.2005,25(2):169-173.
[170]孙琴,王超.土壤外源Cd和Pb复合污染对小麦(Tritioum asetivum L.)根系植物络合素和谷胱甘肽的影响.生态环境.2008,17(5):1833-1838.
[171]孙琴,王超.土壤Cd和Pb复合污染对小麦根系植物络合素和谷胱甘肽的影响.农业环境科学学报.2008,27(5)：1913-1918.
[172]Abdin M, Akmal M, Ram M, et al. Constitutive expression of high-affinity sulfate transporter (HAST) gene in Indian mustard showed enhanced sulfur uptake and assimilation. Protoplasma.2011,248(3):591.
[173]Giovannoni S J, MullinsT D, Field K G. Microbial diversity in oceanicsystems:rRNA approaches to the study of unculturable microbes. In: molecular ecology of aquatic microbes.1995,38:217-248.
[174]曹琳辉.银山铅锌矿酸性矿坑水微生物群落结构分析.中南大学硕士学位论文,2007.
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