Fig. 5. The GapA-associated copper pool is absent in the ΔgapA mutant strain. The S. aureus SH1000 wild type and the ΔgapA mutant strain were cultured in TM medium (0.5 L) with no added glucose but containing 50 μM CuSO4. Extracts were prepared and resolved under anaerobic conditions by AEC, and the fraction eluted by 400 mM NaCl from the wild type extract was found to contain most of the GAPDH activity. Aliquots (500 μL) of the 400 mM NaCl AEC fractions from each strain were further resolved by SEC on a Superdex 200 Increase column in 10 mM Tris, 150 mM NaCl, pH 7.5. SEC fractions were analysed for copper (blue circles: closed = WT, open = ΔgapA mutant) by ICP-MS and for GAPDH activity using an enzyme assay (black triangles) (A). Note that the SEC fractions from the ΔgapA mutant strain were devoid of GAPDH activity (data not shown). SDS-PAGE analysis of selected SEC fractions from both the wild type (B) and the ΔgapA mutant (C) confirmed that the protein distribution was essentially unchanged between the two chromatographs, with the exception of the presence of the ∼36 kDa GapA band (highlighted by a red asterisk) in the wild type fractions that was absent in the ΔgapA mutant samples (also indicated with a red asterisk).
