Research On The Genetic Diversity And Comparative Proteomics Of Thiobacillus Ferrooxidans

Thiobacillus ferrooxidans is one of the most important leaching bacteria. In order to explore the leaching mechanisms of Thiobacillus ferrooxidans, in this thesis, we have studied Thiobacillus ferrooxidans at the aspects of genetic diversity and comparative Proteomics of Thiobacillus ferrooxidans.We studied the genetic diversity of 7 Thiobacillus ferrooxdians strains from different places by exploring the method of RAPD molecular mark, and the results indicated that there existed obvious diversity. The biggest correlation efficient among different Thiobacillus ferrooxdians strain was 82.93%, and the least was 44.44%. We also experimented the Fe²⁺-oxidation speed of different strains, the result of which indicated that there also exist difference and activity of the strain from Chenmenshan is the most, which was served as experimental bacteria in the latter experiments.We also explored the growth characteristics of Thiobacillus ferrooxidans for the purpose of performing the comparative proteome analysis of the organism. Being one of the fastidious bacteria, Thiobacillus ferrooxidans was incubated in 9K base medium under aeration. The pH of the medium was controlled at 2.0 and the temperature at 30℃.The viable count was continually measured. We found that thegrowth curves of Thiobacillus ferrooxidans followed the same lag, logarithmic, stationary and aging phase as seen in other bacteria. The logarithmic phases were from 10 to 32 hours for adding Fe2+as energy resource, 20-60 hrs at PO4 " starvation additionally adding Fe as energy resource and 4¹2 days for adding elemental sulphur as energy resource. The typical doubling times for Thiobacillus ferrooxidans under such conditions were about 1.59hrs, 3.48 hrs and 11.91 hrs respectively (measured by viable count).We researched the effect of L-methionine and L-cysteine on metal sulphide's bioleaching by Thiobacillus ferrooxidans. By adding suitable amount of the amino acid L-cysteine to acidic solution in contact with sphalerite or pyrite, the activity of Thiobacillus Ferrooxidans is largely enhanced. But, at comparable high concentration of L-cysteine, a deleterious effect in bacterial activity was found, which could be due to the toxic effect of this amino acid at higher concentrations to microbes. The adding of L-met would be greatly inhibition to the bioleaching no matter how much it had applied, which indicate L-met is harmful for bioleaching. The results of were coincidence with previous reports. The quite different effects on bioleaching between L-cysteine and L-met lies in that L-cysteine has a SH group which is helpful in metal sulfide bioleaching by Thiobacillus ferrooxidans.We also studied the effect of Cu2+> Cd2+ on metal sulphide'sbioleaching by Thiobacillus ferrooxidans. Results indicated that these two kinds of metal ions could inhibit the activity of Thiobacillus ferrooxidans undomesticated at different degrees. On the contrary, the leaching activity of Thiobacillus ferrooxidans domesticated by corresponding metal ion, would not be affected even when certain amount of metal ions (Cu or Cd ) was added. All these research work has given clues and foundation for latter further comparative proteomics research of Thiobacillus ferrooxidans cultivated under different condition.To explore the proteomics' experiments for Thiobacillus ferrooxidans, we firstly optimized 2-dimential electrophoresis, such as focusing time at 8000 voltages, the pH range of prefab dry strips, the amount of protein loaded et ah Results indicated that the separation results would be excellent when adopting pH4-7 prefab dry strips, 5 hrs' focusing at 8000 voltages, more than 400ug protein loaded, and there would be about 540 protein spots. We also compared the methods of gel-staining, and found that the "blue Silver" colloidal Coomassie Blue staining would get good separation effect when the protein loaded was more than 900fxg, and the superority for Coomassie Blue staining was that it had excellent compatibility with latter protein-identification by mass spectrum when compared with silver staining.In this thesis, we studied the proteome response of Thiobacillus ferrooxidans to phosphate starvation. The proteins expressed ofThiobacillus ferrooxidans were separated by high resolution two-dimensional gel electrophoresis with immobilized pH gradients in the first dimension (IPG-DALT) combining pH3-10 with pH4-7, and in the 12.5% SDS-PAGE for the second dimensional separation. The separated proteins were identified by using matrix-assited laser desorption/time-of-flight mass spectrometry (MALDI-TOF). We got clear and good reproducibility of two dimensional electrophoresis gel images for phosphate-starvation cultivated Thiobacillus ferrooxidans and its comparison. The resulted Images were quantitatively analyzed using PDQuest 2D analysis software. Image analysis indicated that about 540+15 protein spots were visualized by silver staining each gel. t was also indicated that there existed more than 70 differential-expressed protein spots, among which the amount of 21 proteins expressed was increased at the cultivation condition of phosphate starvation, while 50 protein decreased. 10 Differential-expressed protein spots were excised and subjected to in-gel digestion with trypsin. The resulted peptides were measured by MALDI-TOF, and 6 spots were identified through Mascot searching in genome database. Among the proteins identified, there included NADH Dehydrogenase I chain D, Recombination protein recA, RNA helicase, AP2 domain-containing transcription factor et al. Due to the lackage of the technology of 2-DE, which is unable to separate low molecular proteins especially those of low than lOkDa, we adopted threetypes of SELDI protein chips to study on the low molecular part of Thiobacillus ferrooxidans' proteome response to phosphate starvation. Results indicated that there were 13 distinct differential-expressed proteins, the molecular weights of which are between 6360.18 15659.5 IDa.We also studied the comparative proteomics of Thiobacillus ferrooxidans grown under different energy resources (Fe2+ and sulphur). The proteins expressed of Thiobacillus ferrooxidans were separated by high resolution two-dimensional gel electrophoresis with immobilized pH gradients in the first dimension (IPG-DALT) combining pH3-10 with pH4-7, and in 12.5% SDS-PAGE for the second dimension. The separated proteins were identified by using matrix-assited laser desorption/time-of-light mass spectrometry (MALDI-TOF) and electrospray ionization mass spectrometry (ESI-MS/MS). The resulted Images were quantitatively analyzed using PDQuest 2D analysis software. Image analysis indicated that about 540+15 protein spots were visualized by silver staining in a gel. It was also indicated that there existed almost 80 differential-expressed protein spots, among which the amount of 45 proteins expressed was increased at the cultivation condition of phosphate absence, while 36 protein decreased. 30 Differential-expressed protein spots were excised and subjected to in-gel digestion with trypsin. The resulted peptides were measured byMALDI-TOF/MS, and 20 spots were identified through Mascot search in genome database. Among the proteins identified, there are PcaR protein, Acyl-coA Dehydrogenase, ATP synthase Beta chain, RNA polymerase beta prime subunit, NAP/NADP transhydrogenase alpha subunit Subunit, Enoyl-CoA hydratase/carnithine racemase, Ribose-5-phosphate Isomerase, ATP synthase alpha chain, short chain dehydrogenase, Polyribonucleotide nucletidyltransferase, Cytochrome C, cytochrome oxidase II, ATPase components of ABC transporters with duplicated ATPase domains, long-chain-fatty-acid-CoA ligase (fadD-4), et ah We also adopted three types of SELDI protein chips to study the low molecular part of thiobacillus ferrooxidans' proteome, and there are 35 differential expressed proteins between the range of 5642.43-20887.64 Da.