Fig. 2.

Distribution of active TMAO reductase in cytoplasmic and periplasmic fractions of cells expressing tatC suppressors. Cell cultures (15 ml) were grown for 22-24 h anaerobically in Cohen and Rickenberg medium²⁴ as previously described³⁶ with 0.4% glucose and 0.4% TMAO (Sigma-Aldrich). Cells were normalized based on OD₆₀₀ to a total of OD₆₀₀ 5 and then fractionated by the cold osmotic shock procedure.²⁵ Briefly, cells were resuspended in fractionation buffer (30 mM Tris-HCl, pH 8.0, 20% (w/v) sucrose, 1 mM EDTA, pH 8.0), incubated at room temperature for 10 min, and then after centrifugation were resuspended in ice-cold MgSO₄ (5 mM). Spheroplasts were sonicated. Of the obtained fractions, 12.5% were loaded, and proteins were separated under native condition on a 7.5% Tris gel at pH 6.5. Gels were incubated with 20 ml of 100 mM sodium phosphate buffer (pH 7); 1 mM methyl viologen was added and reduced by an excess of a 1% dithionite solution in 0.1 mM NaOH. Gels were incubated under mild shaking until stained blue; 10 ml of a 2 M TMAO in the same phosphate buffer was added to visualize TorA activity as can be seen in the transparent lanes. MC4100 and B1lk0 (ΔtatC) containing pBR322-tatC were used as positive controls. GB22KK-C cells contain the gene encoding TorA(R11K,R12K) in the chromosome and the deletion of dmsABC∷kan.¹⁷