Native-PAGE Enzyme Activity Assay

Precast SDS-PAGE gels were run at 30 mA per gel for 1 h on a BioRad Ready Strip IPG/Protean II system. Gels were stained with ReadyBlue Protein Gel Stain (Sigma-Aldrich). Enzyme activity staining was performed with Native-PAGE gels and cytoplasmic cell fractions under anoxic conditions (52 (link)). P. fastidiosa phosphite-oxidizing activity was stained by immersing the gel strips in 25 mM HEPES buffer, pH 8.0, containing 3 mM MgCl₂, and D. phosphitoxidans phosphite-oxidizing activity in 25 mM MOPS buffer, pH 7.2, containing 3 mM MgCl₂ and 17 mM NaCl, each with addition of 2 mM AMP, 2 mM NAD⁺, 10 mM sodium phosphite, and 0.4 mM iodonitrotetrazolium chloride (INT) as the final electron acceptor in order to indirectly detect NADH formation. Protein bands excised from stained Native-PAGE gels and SDS-PAGE gels were identified by peptide mass fingerprinting at the Proteomics Facility of the University of Konstanz.