Two-dimensional polyacrylamide gel electrophoresis (2D PAGE) in combination with mass spectrometry is currently the most widely used technology for comparative bacterial proteomic analysis (Gygi et al. 2000). A set of A. ferrooxidans ATCC
19859 proteins differentially expressed when grown in metal sulfides, thiosulfate, elemental sulfur, and ferrous iron media were characterized by using 2D PAGE (Ramirez et al. 2004). N-terminal amino acid sequencing and tandem mass spec-trometry analysis of these proteins allowed the identification and localization of their corresponding genes in the available genomic sequence of A. ferrooxidans ATCC 23270. The genomic context around several of these genes suggests their involvement in the energy metabolism of A. ferrooxidans. Two groups of proteins could be distinguished. Proteins highly upregulated by growth on sulfur compounds but downregulated by growth on ferrous iron are particularly interesting, such as a
44-kDa outer-membrane protein, an exported 21-kDa putative thiosulfate sulfur transferase protein, a 33-kDa putative thiosulfate/sulfate binding protein, and a
45-kDa putative capsule polysaccharide export protein (WcbC). Polysaccharides may play a role in the adherence capability of bacteria to solid surfaces. In this regard, it is known that most leaching bacteria grow attached to the surface of the solid substrates such as elemental sulfur and metal sulfides.
On the other hand, A. ferrooxidans proteins that are downregulated when growing on sulfur but upregulated when growing on ferrous iron were also analyzed by 2D PAGE (Ramirez et al. 2004). These include rusticyanin, a cytochrome c552, a putative phosphate binding protein (PstS), the small and large subunits of ribulose-1, 5-bisphosphate carboxylase/oxygenase (Rubisco), and a 30-kDa putative CbbQ protein, amongst others. These results suggested a separation of the iron and sulfur utilization pathways. Rusticyanin in addition of being highly expressed on ferrous iron was also newly synthesized as determined by metabolic labeling, although at lower levels during growth on sulfur compounds and iron-free metal sulfides (Ramirez et al. 2004). These results were in agreement with those of Yarzabal et al. (2004). The capacity of A. ferrooxidans to oxidize thiosulfate and tetrathionate was found to be inhibited by the presence of ferrous iron (Das et al. 1993). However, during the growth of A. ferrooxidans on iron-containing metal sulfides, such as pyrite and chalcopyrite, we found elevated expression of proteins involved in both ferrous iron and sulfur compound utilization, indicating that the two energy-generating pathways are simultaneously induced depending on the type and the concentration of the available oxidizable substrates (Ramirez et al. 2004). In agreement with these results, it was previously suggested that A. ferrooxidans can simultaneously utilize both ferrous iron and elemental sulfur as energy sources (Espejo and Romero 1987).
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