This grant proposal addresses open questions. First, when C. metallidurans was incubated with 50 µM Au(III) tetrachloride, the cells accumulated Au after 1 min in the cytoplasm. Accumulation continued and after 6 h, all of the Au was converted into an Au(I)-S species. "Hot spots indicating formation of Au nanoparticles in the periplasm occurred from 72 h on, in parallel with transformation of the Au(I)-S species into metallic Au(0) and an Au(I)-C species, which could be Au(I)-CH3 or Au(I)-CN. Consequently, CopA not only oxidizes Au(I) back to prevent its re-import into the cytoplasm, it also frees Au(I) from a nearly covalent Au(I)-S bond to facilitate formation of the nanoparticles. But how can a cytoplasmic Au(I)-S species be transported back to the periplasm? Second, silver may also be a component of auriferous soil, with a mean content of 50 ng/g soil, comparable to that of gold at 80 ng/g. Silver induced up-regulation of cup and gig components for a copper efflux pump and products of gold induced genes without known function. A cupC/AR copF mutant of strain CH34 displayed decreased silver resistance. How does Ag(I) interfere with copper and gold resistance? Third, the CopC cytoplasmic metal chaperone binds Au(I). How does CopC contribute to the picture? Does it inhibit Au(I)-mediated inhibition of copper transport by CupA or is it responsible for this process? Last, C. metallidurans is not the only bacterium associated with gold grains. How does the bacterium interact with other bacteria in the golden environment?
Consequently, five topics will be addressed:
- Connection between Au(I), Ag(I), Cu(I) export from the cytoplasm and glutathione.
- Active excretion of gold nanoparticles by complemented gshA mutants, which cannot synthesize glutatione.
- Interaction between copper-exporting PIB1-type ATPases, Au(I), Ag(I), CupC and glutathione.
- Interaction between periplasmic Cu(I) oxidases and glutathione.
- Comparison of gold resistance and mobilization of C. metallidurans with other gold-associated bacteria.
Topic 1 will be the major goal of the work, topics 2 and 3 minor goals, topics 4 and 5 back-up and test-bed experiments that may be further pursued in a second period.