Fig 2.

Effect of amino acid substitutions near the redox switch and within helix α4 of Spx on trxB-lacZ transcription. (A) The IPTG-inducible alleles encoding a proteolysis-resistant form of SpxDD or SpxDD with other amino acid substitutions was introduced into the amyE locus of the Spx null mutant strain bearing a trxB-lacZ fusion. Strains were grown in DS medium, and when the OD₆₀₀ was 0.4, 1 mM IPTG was added to induce the SpxDD proteins. Samples were taken at time intervals, and β-galactosidase activities were measured. The highest activities of Spx mutants were selected and used to calculate the ratio of mutant activity to SpxDD activity, where 1 represents the activity observed in the strain expressing the parent SpxDD. (B) Cells of each strain were harvested 1.5 h following IPTG addition and lysed with the bead-beating method to extract total cellular protein. Spx expression levels in these strains were determined by Western blotting (WB) using anti-Spx antibody.