New copper resistance determinants in the extremophile acidithiobacillus ferrooxidans: a quantitative proteomic analysis.

Acidithiobacillus ferrooxidans is an extremophilic bacterium used in biomining processes to recover metals. The presence in A. ferrooxidans ATCC 23270 of canonical copper resistance determinants does not entirely explain the extremely high copper concentrations this microorganism is able to stand, suggesting the existence of other efficient copper resistance mechanisms. New possible copper resistance determinants were searched by using 2D-PAGE, real time PCR (qRT-PCR) and quantitative proteomics with isotope-coded protein labeling (ICPL). A total of 594 proteins were identified of which 120 had altered levels in cells grown in the presence of copper. Of this group of proteins, 76 were up-regulated and 44 down-regulated. The up-regulation of RND-type Cus systems and different RND-type efflux pumps was observed in response to copper, suggesting that these proteins may be involved in copper resistance. An overexpression of most of the genes involved in histidine synthesis and several of those annotated as encoding for cysteine production was observed in the presence of copper, suggesting a possible direct role for these metal-binding amino acids in detoxification. Furthermore, the up-regulation of putative periplasmic disulfide isomerases was also seen in the presence of copper, suggesting that they restore copper-damaged disulfide bonds to allow cell survival. Finally, the down-regulation of the major outer membrane porin and some ionic transporters was seen in A. ferrooxidans grown in the presence of copper, indicating a general decrease in the influx of the metal and other cations into the cell. Thus, A. ferrooxidans most likely uses additional copper resistance strategies in which cell envelope proteins are key components. This knowledge will not only help to understand the mechanism of copper resistance in this extreme acidophile but may help also to select the best fit members of the biomining community to attain more efficient industrial metal leaching processes.

Response to copper of Acidithiobacillus ferrooxidans ATCC 23270 grown in elemental sulfur.

The response of Acidithiobacillus ferrooxidans ATCC 23270 to copper was analyzed in sulfur-grown cells by using quantitative proteomics. Forty-seven proteins showed altered levels in cells grown in the presence of 50 mM copper sulfate. Of these proteins, 24 were up-regulated and 23 down-regulated. As seen before in ferrous iron-grown cells, there was a notorious up-regulation of RND-type Cus systems and different RND-type efflux pumps, indicating that these proteins are very important in copper resistance. Copper also triggered the down-regulation of the major outer membrane porin of A. ferrooxidans in sulfur-grown bacteria, suggesting they respond to the metal by decreasing the influx of cations into the cell. On the contrary, copper in sulfur-grown cells caused an overexpression of putative TadA and TadB proteins known to be essential for biofilm formation in bacteria. Surprisingly, sulfur-grown microorganisms showed increased levels of proteins related with energy generation (rus and petII operons) in the presence of copper. Although rus operon is overexpressed mainly in cells grown in ferrous iron, the up-regulation of rusticyanin in sulfur indicates a possible role for this protein in copper resistance as well. Finally, copper response in A. ferrooxidans appears to be influenced by the substrate being oxidized by the microorganism.