喜温硫杆菌蛋白质组学研究技术建立及抗砷机制初步探讨
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摘要
喜温硫杆菌(Acidthiobacillus caldus)是一类专性自养极端嗜酸性硫杆菌,分布于硫化矿床、酸性矿水及土壤中。喜温硫杆菌在生物浸矿、煤的脱硫、含硫废水的处理等方面及在自然界的硫循环中都占有重要地位,尤其在生物冶金中有广泛应用,是浸矿复合菌系中的主要组分之一。它可以利用附着在矿物表面的固体硫,暴露出矿物表面,从而使浸矿细菌充分接触到矿物并起作用;其代谢产物还可被其它浸矿细菌所利用,并有助于硫的溶解。通过这些机理,喜温硫杆菌大大提高了浸矿效率,在生物冶金中发挥着重要的作用。
在生物冶金的应用过程中,喜温硫杆菌对存在于矿石中的重金属如砷、汞、银等缺乏抗性或抗性不强,使得菌体活性降低,浸矿效率下降。已有研究证明,喜温硫杆菌通过系列驯化可以提高对重金属(如砷)的抗性,但是这种抗性随着砷胁迫压力的消失会很快丧失。这表明喜温硫杆菌胞内存在着对重金属胁迫的适应机制。利用蛋白质组学技术对其进行研究,对阐明该菌耐重金属机制以及构建高效抗重金属工程菌并用于生产应用具有重要意义。
目前蛋白质组学研究主要有组成性蛋白质组学研究和差异比较蛋白质组学研究两种策略。本研究采用差异比较蛋白质组学研究策略,以喜温硫杆菌的砷抗性为研究突破口,比较了喜温硫杆菌野生株(耐受10mM亚砷酸钠)和驯化株(耐受30mM亚砷酸钠)的蛋白表达差异,对差异蛋白进行质谱鉴定,试图揭示喜温硫杆菌的耐砷机制。
喜温硫杆菌蛋白质组学研究方面,国内外还未见报道。蛋白质二维电泳技术结合质谱鉴定是进行蛋白质组学研究的主要手段。其中二维电泳技术又因材料的不同而使技术流程和分离效果呈现较大的差别,因此建立喜温硫杆菌蛋白质二维电泳技术方法是进行该菌蛋白质组学研究的基础。本实验建立了喜温硫杆菌胞内全蛋白二维电泳技术,并获得了高重现性的喜温硫杆菌蛋白图谱。在pH4-7的驯化菌株蛋白电泳图谱中解析出了1188个蛋白点,共有335个蛋白的表达发生了差异。与野生株相比,驯化菌株中,新产生60个蛋白点,49个蛋白点消失。
利用基质辅助激光解吸离子化-飞行时间质谱及其串联质谱分析,有6个点得到了鉴定,包括D11、D14,D9、D13、D18、D19。根据数据库比对及聚类分析,我们推测该几类蛋白分别属于蛋白合成因子、尿嘧啶DNA糖基酶、鞭毛收缩ATP酶Pi1T、氨基转移酶,并可能在喜温硫杆菌抗砷过程中发挥着重要作用。
除了这6个蛋白外,我们还对另外14个蛋白点进行了鉴定,由于这些蛋白点可能是新蛋白或者与现有数据库中的已知蛋白的序列相似性太低,这些蛋白的性质以及它们在耐砷过程中所起的作用有待于进一步的研究。
综上所述,本研究首先建立了喜温硫杆菌蛋白质组的研究技术方法,研究了野生型菌株与抗砷菌株的蛋白质表达差异,并对差异蛋白进行了质谱鉴定和数据库检索分析。通过分析,我们推测了喜温硫杆菌中参与抗砷的功能蛋白和可能的调控机制,为进一步研究喜温硫杆菌的抗砷性能奠定了基础。
Acidithiobacillus caldus,a new strain isolated in 1994,is a gram-negative, extremely acidophilic obligately autotrophic bacterium,which has been used industrially in metal leaching of mineral ores and in the microbial desulfurization of coal in combination with Acidithiooxidans ferrooxidans.Acidithiobacillus caldus can utilize the sulfur compounds covering on the surface of the ores as energy source,and expose the inner part of ores.The metabolites of A.caldus can be the nutritions of other leaching bacteria and even can solubilize the solid sulfur.With the above unique characters,A.caldus is worth to be investigated on its important functional genes and proteins.Proteomics has been put forward for many years,and has been shown to be a good way to isolate and identify the functional proteins.However,the methods to study Acidithiobacillus caldus` proteomics have not been established,which needs to be investigated.
Even if with its wide applications in bioleaching,the sensitivity to heavy matals, such as mercury,chromium and arsenic,has limited its further applications.Arsenic, one of the heavy metals,has been known as a virulent element for a long time. Enviromental arsenic species can enter into cells by different ways and kill the organisms by interrupting the synthesis of ATP or binding to sulphydryl groups of some proteins and enzymes.Under the arsenical lethiferous stress,many microorganisms including prokaryotes and eukaryotes have evolved to acquire certain types of arsenic resistance mechanism and survive in the arsenical containing niche. Previous researches have demonstrated that Acidithiobacillus caldus can be induced to resistant to arsenic.But the induction character tends to degenerate and disappear, when the arsenic pressure is withdrawn.Therefore,it is necessary to investigate the arsenic resistance mechanism of A.caldus.
In this research,we established a A.caldus proteomic research technique at first, and then studied the arsenic resistant mechanism.We compared the response of A. caldus to the absence and presence of arsenic in medium at the proteome level. Two-demensional polyacrylamide gel electrophoresi,(2D-PAGE)was used to quantify the protein expression levels after the tota intercellular proteins were acquired.Upregulated proteins and downregulated proteins were identified using statistical analysis with PDQuest.About 1188 proteirs were displayed on pH4-7 electrophoresis maps of the arsenic resistant strains.Compared with the sensitive strains,60 proteins appeared and 49 proteins disappeared.
Then Mass Spectrometry(MS)was utilized to evaluate the mass of peptides derived from trypsin digested discrepant proteins.Peptide mass fingerprinting(PMF) and MS/MS data obtained were used to search in the SWISS-PROT and NCBInr database using the Mascot search engine.Finally,we identified 6 proteins,which included D9,D11,D13,D14,D18,D19,and predicted their potential roles in arsenic resistant process.According to our research,D11 could be a ribosomal protein,S4 and D14 could be the translation elongation factor Tu.D11 and D14 both exist in the arsenic resistant bacterium and are responsible for prote(?)n synthesis.D9,a conserved hypothetical protein,also exists in arsenic resistant bacterium,which shows that the hypothetical protein plays a role in arsenic resistantance.D13 is a Uracil DNA glycosylase and reponsible for DNA repair.It exists in the arsenic resistant strains, and its real role in arsenic resistantance is still unknown.D18 is a Tfp pilus assembly protein,also called pilus retraction ATPase(PilT).D19 is Aspartate/tyrosine/aromatic aminotransferase.They both exist in the arsenic resistant organism.Besides,we also indentified other 14 proteins.For the inadequacy of the database,their roles of them were not yet elucidated.Anyway,the basic protei(?)mics methods and arsenic resistance related peoteins of A.caldus were studied in this thesis.
在生物冶金的应用过程中,喜温硫杆菌对存在于矿石中的重金属如砷、汞、银等缺乏抗性或抗性不强,使得菌体活性降低,浸矿效率下降。已有研究证明,喜温硫杆菌通过系列驯化可以提高对重金属(如砷)的抗性,但是这种抗性随着砷胁迫压力的消失会很快丧失。这表明喜温硫杆菌胞内存在着对重金属胁迫的适应机制。利用蛋白质组学技术对其进行研究,对阐明该菌耐重金属机制以及构建高效抗重金属工程菌并用于生产应用具有重要意义。
目前蛋白质组学研究主要有组成性蛋白质组学研究和差异比较蛋白质组学研究两种策略。本研究采用差异比较蛋白质组学研究策略,以喜温硫杆菌的砷抗性为研究突破口,比较了喜温硫杆菌野生株(耐受10mM亚砷酸钠)和驯化株(耐受30mM亚砷酸钠)的蛋白表达差异,对差异蛋白进行质谱鉴定,试图揭示喜温硫杆菌的耐砷机制。
喜温硫杆菌蛋白质组学研究方面,国内外还未见报道。蛋白质二维电泳技术结合质谱鉴定是进行蛋白质组学研究的主要手段。其中二维电泳技术又因材料的不同而使技术流程和分离效果呈现较大的差别,因此建立喜温硫杆菌蛋白质二维电泳技术方法是进行该菌蛋白质组学研究的基础。本实验建立了喜温硫杆菌胞内全蛋白二维电泳技术,并获得了高重现性的喜温硫杆菌蛋白图谱。在pH4-7的驯化菌株蛋白电泳图谱中解析出了1188个蛋白点,共有335个蛋白的表达发生了差异。与野生株相比,驯化菌株中,新产生60个蛋白点,49个蛋白点消失。
利用基质辅助激光解吸离子化-飞行时间质谱及其串联质谱分析,有6个点得到了鉴定,包括D11、D14,D9、D13、D18、D19。根据数据库比对及聚类分析,我们推测该几类蛋白分别属于蛋白合成因子、尿嘧啶DNA糖基酶、鞭毛收缩ATP酶Pi1T、氨基转移酶,并可能在喜温硫杆菌抗砷过程中发挥着重要作用。
除了这6个蛋白外,我们还对另外14个蛋白点进行了鉴定,由于这些蛋白点可能是新蛋白或者与现有数据库中的已知蛋白的序列相似性太低,这些蛋白的性质以及它们在耐砷过程中所起的作用有待于进一步的研究。
综上所述,本研究首先建立了喜温硫杆菌蛋白质组的研究技术方法,研究了野生型菌株与抗砷菌株的蛋白质表达差异,并对差异蛋白进行了质谱鉴定和数据库检索分析。通过分析,我们推测了喜温硫杆菌中参与抗砷的功能蛋白和可能的调控机制,为进一步研究喜温硫杆菌的抗砷性能奠定了基础。
Acidithiobacillus caldus,a new strain isolated in 1994,is a gram-negative, extremely acidophilic obligately autotrophic bacterium,which has been used industrially in metal leaching of mineral ores and in the microbial desulfurization of coal in combination with Acidithiooxidans ferrooxidans.Acidithiobacillus caldus can utilize the sulfur compounds covering on the surface of the ores as energy source,and expose the inner part of ores.The metabolites of A.caldus can be the nutritions of other leaching bacteria and even can solubilize the solid sulfur.With the above unique characters,A.caldus is worth to be investigated on its important functional genes and proteins.Proteomics has been put forward for many years,and has been shown to be a good way to isolate and identify the functional proteins.However,the methods to study Acidithiobacillus caldus` proteomics have not been established,which needs to be investigated.
Even if with its wide applications in bioleaching,the sensitivity to heavy matals, such as mercury,chromium and arsenic,has limited its further applications.Arsenic, one of the heavy metals,has been known as a virulent element for a long time. Enviromental arsenic species can enter into cells by different ways and kill the organisms by interrupting the synthesis of ATP or binding to sulphydryl groups of some proteins and enzymes.Under the arsenical lethiferous stress,many microorganisms including prokaryotes and eukaryotes have evolved to acquire certain types of arsenic resistance mechanism and survive in the arsenical containing niche. Previous researches have demonstrated that Acidithiobacillus caldus can be induced to resistant to arsenic.But the induction character tends to degenerate and disappear, when the arsenic pressure is withdrawn.Therefore,it is necessary to investigate the arsenic resistance mechanism of A.caldus.
In this research,we established a A.caldus proteomic research technique at first, and then studied the arsenic resistant mechanism.We compared the response of A. caldus to the absence and presence of arsenic in medium at the proteome level. Two-demensional polyacrylamide gel electrophoresi,(2D-PAGE)was used to quantify the protein expression levels after the tota intercellular proteins were acquired.Upregulated proteins and downregulated proteins were identified using statistical analysis with PDQuest.About 1188 proteirs were displayed on pH4-7 electrophoresis maps of the arsenic resistant strains.Compared with the sensitive strains,60 proteins appeared and 49 proteins disappeared.
Then Mass Spectrometry(MS)was utilized to evaluate the mass of peptides derived from trypsin digested discrepant proteins.Peptide mass fingerprinting(PMF) and MS/MS data obtained were used to search in the SWISS-PROT and NCBInr database using the Mascot search engine.Finally,we identified 6 proteins,which included D9,D11,D13,D14,D18,D19,and predicted their potential roles in arsenic resistant process.According to our research,D11 could be a ribosomal protein,S4 and D14 could be the translation elongation factor Tu.D11 and D14 both exist in the arsenic resistant bacterium and are responsible for prote(?)n synthesis.D9,a conserved hypothetical protein,also exists in arsenic resistant bacterium,which shows that the hypothetical protein plays a role in arsenic resistantance.D13 is a Uracil DNA glycosylase and reponsible for DNA repair.It exists in the arsenic resistant strains, and its real role in arsenic resistantance is still unknown.D18 is a Tfp pilus assembly protein,also called pilus retraction ATPase(PilT).D19 is Aspartate/tyrosine/aromatic aminotransferase.They both exist in the arsenic resistant organism.Besides,we also indentified other 14 proteins.For the inadequacy of the database,their roles of them were not yet elucidated.Anyway,the basic protei(?)mics methods and arsenic resistance related peoteins of A.caldus were studied in this thesis.
引文
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