Molecular mechanism study of arsenate, uranyl oxide and chromate resistance and reduction by Desulfovibrio desulfuricans G20.

摘要

Desulfovibrio desulfuricans G20 is a laboratory constructed strain of the wild-type sulfate reducing bacterium G100A originally isolated from an oil well corrosion site. It is known to reduce As(VI), U(VI) and Cr(VI). Strain G20 can grow and reduce 20 mM As(V) to As(III) in lactate-sulfate media. Sequence analysis showed that the genome of strain G20 contains one copy of arsC and one arsRBCC operon. Two mutants in this strain with defects in arsenate resistance were generated by nitrosoguanidine mutagenesis. arsRBCC operons are intact in both mutants but each has one point mutation in the arsC region. Either arsC or arsRBCC can restore the arsenate resistance of the mutants and recombinant with arsRBCC operon can resist arsenate up to 70 mM. Furthermore, both arsC and arsRBCC can be expressed in Escherichia coli strain DH5alpha and increase arsenate resistance. Escherichia coli recombinants with arsRBCC operon can grow and reduce 50 mM arsenate in LB broth. Real time PCR has shown that arsC is constitutively expressed and its product will reduce As(V) in the cytoplasm. Also As(III) will activate the arsRBCC operon.;Strain G20 can grow in lactate sulfate medium with up to 4 mM uranyl acetate. In order to determine which genes are required for growth at high uranium concentrations, 5760 Tn10 transposon mutants were screened for U(VI) resistance defects. 24 mutants lost U(VI) resistance when grown in the lactate sulfate medium with 2 mM uranyl acetate. From these isolates, it was determined that 22 genes were disrupted by the transposon insertion. One tranposon is located in a non-coding area and another transposon is located in a non-coding region within one operon. Ten mutants were completely inhibited by 2 mM uranyl acetate in lactate sulfate medium. Of the disrupted genes, three are involved in DNA repair, one is involved in rRNA methylation, three are involved in regulation of expression and one is involved in RNA polymerase renaturation. This latter mutant was previously identified as a sediment non-survivor. Fourteen mutants were partially inhibited by 2 mM U(VI). The disrupted genes include: one involved in DNA repair; one involved in regulation of expression; two involved in membrane transporter; four encode hypothetical proteins and five are involved in other functions. Only one mutant of these showed a total loss of the ability to reduce U(VI) to U(IV) in the washed cell test. This last mutant was disrupted in a cAMP-binding protein gene. The realtime PCR data suggest that this gene controls its own operon's expression. The operon contains thioredoxin, thioredoxin reductase and one oxidoreductase gene. The whole thioredoxin operon was cloned into Escherichia coli strain JM109 and the transformant showed increased U(VI) resistance and the ability to reduce U(VI) to U(IV). The oxidoreductase protein was purified and has shown to possess U(VI) reduction activity in vitro with addition of thioredoxin, thioredoxin reductase and NADPH.