Figure 7.

Effects of alanine exchanges of residues with potential catalytic function. A, effect of the indicated single-alanine exchanges on the formation of fluorescent pyoverdine and control of the presence of the respective PvdO variants in the periplasm by SDS-PAGE/Western blotting. B, subcellular fractionation of all analyzed PvdO variants. Cytoplasmic (C), membrane (M), and periplasmic (P) fractions were analyzed for the presence of PvdO by SDS-PAGE/Western blotting. Note than in case of stable PvdO variants, the protein is exclusively detectable in the periplasm, as expected for Sec transport. C, Coomassie-stained PvdO preparations that had been used for metal detections. D, sequence logo of the mutated regions (see “Experimental procedures” for details). Exchanged residues in PvdO_A506 are colored red, and distinct regions are highlighted in yellow and blue. The residue numbers on top of the sequence logo indicate the residue position in PvdO_A506. E, homology model of the PvdO_A506 structure based on the known structure of PvdO from P. aeruginosa (PDB code 5HHA). Exchanged residues are highlighted in red. Left, overview of the structure; right, close-up view. The model was visualized using Chimera (36). Masses of marker proteins (in kDa) are indicated on the left side of blots.