Fig. 3. Metal distribution in S. aureus cytosolic extracts. Cytosolic extracts were prepared from S. aureus SH1000 cells, cultured in TSB medium either (A) without or (B) with addition of 500 μM CuSO4. Extracts were resolved by anion exchange chromatography (1 mL HiTrap Q HP column) in 20 mM Hepes, pH 8.0, and eluted with stepwise increases in NaCl concentration. Resulting fractions were subjected to a second dimension of separation by HPLC size exclusion chromatography (SW3000 column) in 5 mM Hepes, 50 mM NaCl, pH 7.5. Elemental composition of the resulting fractions were determined by ICP-MS, shown by the height of the peaks on the z-axis. Accumulation of copper in one major pool was observed in cells cultured in excess copper, whereas the accumulation of zinc was decreased in high molecular weight pools. Manganese accumulation was unchanged. All lysate preparation and chromatography steps were performed under anaerobic conditions.
