Study On The Identification Of The Sulfur Dioxygenase From Acidithiobacillus Ferrooxidans And Its Role In Sulfur Oxidation

Acidithiobacillus ferrooxidans is a typical chemolithoautotrophic bacterium, which derives its energy from ferrous iron （Fe2+）, elemental sulfur （S0） and reduced inorganic sulfur compounds （RISCs） for CO2fixation and growth. It is a kind of dominant strain widely used in the bioleaching industry and also a model strain for research on ferrous iron and sulfur metabolism of the bioleaching bacteria.In the biomining process, besides ferrous iron oxidizing capacity, high efficient sulfur-oxidizing ability is essential for degrading and decomposing the sulfur layer that accumulated on the surface of the sulfide ores. However, due to multiple states of sulfur （-2to+6）, the metabolic mechanism is very complicated compared to iron oxidation which just possesses two states （+2and+3）. Currently, research on the mechanism of sulfur oxidation is largely based on biological informatics prediction and transcriptome studies, and the mechanism of elemtal sulfur oxidation which is the key point of the entire sulfur metabolism system is still not clear. Sulfur dioxgenase （SDO） is the key enzyme responsible for sulfur oxidation and also the hotspot of research worldwidely, but its encoding gene has not found in At. ferrooxidans. In this work, we firstly found that AFE₀269is the gene that encodes SDO by homologous sequence alignment and enzyme activity detection, and relevant research concerning SDO was carried out. First, we cloned sdo gene from At. ferrooxidans, and constructed a heterologous expression vector pET-22b（+）-sdo, which was introduced into E. coli, and the SDO was expressed and purified. Then the properties of the purified SDO were studied, which showed that:（1） The optimum temperature of SDO was around35℃, and its activity maintained at4℃-45℃, but the activity was not detected above50℃.（2） The optimum pH was around7.6with0.8mM GSH, and the activity lost when pH<6.（3） Most divalent metal ions showed strong inhibition effects against the enzyme except Mg2+, which possessed a moderate inhibition effect. DTT could improve SDO activity slightly, but the sulfhydryl modifiers of NEM strongly inhibited the activity of SDO.We further examined the expression of several key genes related to sulfur metabolism with Fe2+and S0as energy sources, and found that the sdo gene was upregulated4times, and doxD-1gene was upregulated4.5times with Fe2+as energy source compared that with S0as energy source. So the sdo gene was speculated to be constitutively expressed and there should be some important linkages between sulfur and ferrous oxidation in At. ferrooxidans.We constructed a sdo overexpressed strain At. ferrooxidans （pJRD215-psdo）, and examined the expression of the sulfur-oxidizing related genes of the engineered strain. It showed that sdo gene was upregulated80times compared to control strain At. ferrooxidans （pJRD215）, indicating that the sdo gene was highly transcripted under its own promoter in At. ferrooxidans. Moreover. doxD-1encoding thiosulfate:quinone reductase was also upregulated400times, which indicated that there should be a close link between the sdo and doxD-1, and both of them should play an important role in elemental sulfur oxidation. However, we found that the sdo overexpressed strain possessed a slight advantage in growth and sulfur oxiation against the control strain based on the growth curve and sulfur oxidizing activity determination.In order to specify the function of SDO in At. ferrooxidans, we decided to knock out the AFE₀269（sdo） gene. The suicide vector pKlT-Ksdo was successfully constructed and then conjugated into At. ferrooxidans, and single recombinants were obtained under kanamycin stress. Then, plasmid pJRD215-I-SceI was introduced into the single recombinant to induce the second recombination event. Now, the sdo gene knockout mutant was successfully obtained by preliminary test, though mixed with some single recombinants. So, the purification of the mutant is under way and the pure sdo knocout mutant will no doubt be obtained soon. Research on the characterization of the mutant would be carried out immediately.The research on sulfur dioxygenase of At. ferrooxidans provided us with the information of the SDO enzyme and also experimental data for perfecting the sulfur oxidation model of At. ferrooxidans, which had theoretical value. Moreover, it provided reference for constructing engineering bioleaching strain with high capacity of sulfur oxidation.