Figure 7

Confirmation of membrane topology of MsrR. (A) Schematic drawing of MsrR showing the predicted secondary structure (red barrel, α-helix; blue arrow, β-sheet) and transmembrane helix (TM). Black arrows indicate the positions where MsrR was fused to PhoA yielding fusion constructs number 1 (Δ295, deletion of 295 aa from the C-terminus), 2 (Δ246), 3 (Δ147), 4 (Δ80), and 5 (full length). (B) PhoA activity of fusion constructs after induction with arabinose as well as of uninduced control samples. The average and standard deviations of the results obtained using three different clones of each fusion construct are shown. The dashed line indicates a threshold of 120 Miller Units, which equals twice the highest background activity measured. (C) Detection of PhoA fusion constructs by Western blot hybridization using α-PhoA antibodies. For all fusion constructs, bands corresponding to the expected size were detected (marked by an asterisk): 52.6 kDa (1), 58.0 kDa (2), 69.3 kDa (3), 76.9 kDa (4), and 85.5 kDa (5). In the lanes containing constructs number 3–5, several smaller bands were present, which most probably represent degradation products.