| The genus Acidithiobacillus is one of the important leaching microorganisms. Many of fluorite ores produce high concentration of fluorine ion in acidic leaching environment, which influence the metabolism and growth of A.ferrooxidans, and inhibit its efficiency in the process of leaching ore. Therefore, higher fluoride resistant strains can effectively improve the leaching efficiency. To study the molecular mechanism of its anti-fluoride will help us to select the excellent strains. According to the Acidithiobacillus ferrooxidans genomes have been reported, through bioinformatics analysis found that most of the A.ferrooxidans also contains fluoride resistance related gene crc B gene exists in many bacteria, it has been demonstrated that some bacteria in the fluoride resistant properties of crc B gene encoding fluorine ion transporters, but crc B genes from A.ferrooxidans have the same effect needs to be further verified. We obtained two strains of different A.ferrooxidans strains through separation, an anti fluoride, a non fluoride resistance, were measured and their anti fluoride and the results showed that fluoride resistant bacteria in fluoride concentration in growth medium 20mg/L rate significantly weaker than fluoride resistant bacteria, anti bacteria in fluorine ion concentration is not the medium 60mg/L growth, while anti fluoride bacteria can still grow, but the growth rate was lower, The poor resistance of fluoride is tentatively called A.ferrooxidans BKF, which has a strong fluoride resistance to the tentative was named A.ferrooxidans KF.Based on A.ferrooxidans ATCC 23270 complete genome sequence, specific primers were designed respectively cloning the two strains of gene crc B. The full-length crc B gene sequences of A.ferrooxidans KF and A.ferrooxidans BKF were Obtained by PCR cloning. The sequence alignment and homology modeling. Crc B physicochemical properties of and 3D structure model was determined. The results showed that three amino acid differences exist in A.ferrooxidans KF and A.ferrooxidans BKF Crc B proteins.The 43,44 amino acid differences lead to protein three-dimensional structure of a certain degree of change, these amino acid mutations may lead to anti fluoride related functions of the Crc B. The changes of transcription level crc B gene of A.ferrooxidans KF in the fluoride stress on the analysis by Real-time Quantitative PCR, the results showed that A.ferrooxidans KF of the crc B gene in fluoride conditions(Fluorine ion concentration 20mg/L) transcriptional level increased 18 times than no fluoride conditions, initially proved that crc B is closely related to A.ferrooxidans gene and anti fluoride process. Because the crc B gene of A.ferrooxidans bacteria in the encoding is an antiporter, to construct recombinant plasmid p ET30a-crc B, transformed into expression host Escherichia.coli(BL21(DE3)), the recombinant sequence was proved to be correct after fluoride conditions induced by water load the plasmid of E.coli as the control group, each group had three parallel two hours interval measured OD values, results showed that the recombinant plasmid p ET30a-crc B containing recombinant bacteria in culture medium containing fluorine and growth speed of Ming Significantly faster than the control group, which showed that the single gene crc B can play a certain role in anti fluoride.This study using biological information to analyze the differences of the amino acid sequences of fluoride resistant strain and fluoride resistant strains Crc B. By simulating the three-dimensional structure was preliminary analysis of the mutation point difference. At the transcription level of fluoride resistant strains in fluorine ion on the level of transcription crc B gene differences. In E.coli of the heterologous expression and analysis of the gene crc B can play a certain resistance to fluorine. In the future, two strains will be through resequencing and transcriptome analysis. The analysis and the differences of the fluorine resistance related gene at the transcriptional level, will explain molecular mechanism of the anti fluoride. |
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