Construction Of Proteomic Technology For Acidithiobacillus Caldus Research And Study On Arsenic Resistant Mechanism

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to resistant to arsenic.But the induction character tends to degenerate and disappear, when the arsenic pressure is withdrawn.Therefore,it is necessary to investigate the arsenic resistance mechanism of A.caldus.In this research,we established a A.caldus proteomic research technique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.