Figure 2.

Compound 12-resistant mutations using two different selections. A, nucleotide changes found in dsbB after selection of LptD₄₂₁₃ growth on aerobic minimal media in the presence of 10 μm compound 12 (top). Nucleotide changes found in dsbB after selection on anaerobic minimal media in the presence of 2 μm compound 12 (bottom). The mutations studied in this work are indicated with an orange circle. B, location of the mutations (orange) in the structure of the DsbA-DsbB complex. DsbA is shown in green, DsbB in cyan, Cys-44 in red, and ubiquinone-1 (UQ1) in purple. PyMOL was used to visualize the structure (2ZUP) of the crystallized complex when Cys-30 of DsbA is forming a disulfide bond with Cys-104 of DsbB and Cys-41 and Cys-44 of DsbB are disulfide bonded. C, α-DsbB immunoblot analysis of strains carrying dsbB at λ att site under the control of trc₂₀₄ promoter (CL591 to CL596 strains). Cells were grown for 4 h in M63 minimal medium with 0.2% glucose and 1 mm IPTG to induce expression of DsbB. Cells were TCA-precipitated, and protein pellets were resuspended in 100 mm Tris, 1% SDS buffer. β-Mercaptoethanol was used to reduce the proteins. 10 μg of total protein samples were loaded in 12% acrylamide gel. α-RpoA was used as a loading control. The relative amount was calculated using arbitrary levels given by Image Lab 5.2 software.