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Fig. S1. PlpD tertiary structure prediction. The N-terminal part of PlpD (residues 25–235) can fold according the potato Part17 protein structure (Rydel et al., 2003). In this putative PlpD structure, the four homology blocks (shown in blue, purple, green and yellow respectively) are located in a pocket, where the side-chains of the putative active-site residues Ser60 and Asp207 (shown in red) are facing each other. This catalytic region is presumably wrapped in between two layers of α-helices.

Fig. S2. PlpDV5/His6 is produced as a full-length protein in E. coli. The recombinant C-terminal V5/His6 tagged PlpD was produced in E. coli BL21(DE3) from the pRS4 plasmid. After SDS-PAGE separation (10%), the total cellular proteins were immunodetected with an anti-PlpD (left panel) and anti-V5 tag (right panel) antisera. A specific product with an apparent size of ∼90 kDa that could represent the full-length form off PlpDV5His6 was specifically found upon IPTG induction (2 h at the indicated concentration). In E. coli the passenger domain is thus not cleaved. The molecular markers are indicated in kDa on the left, a non-specific background protein with an asterisk.

Fig. S3. PlpDV5/His6 is targeted to the inner membrane (IM) of E.coli. Bacteria producing PlpDV5/His6 were submitted to fractionation. After sonication of cell lysate, half of PlpD was recovered in inclusion bodies (data not shown). The envelope (Env) and soluble fractions (C/P) were obtained by ultracentrifugation, and the IM proteins were then solubilized with SLS (2%). The TolA protein and the OmpA porin were used respectively as inner and outer membrane (OM) markers of E. coli. The recombinant PlpD was detected with anti-V5 tag antiserum in the same fraction as TolA, indicating that it was localized in the inner membrane of E. coli.

Fig. S4. The PlpD passenger domain processing. We examined the possible role in the process of two P. aeruginosa outer membrane proteins, the OprD protein (PA0958), a porin with protease activity (Yoshihara et al., 1996), and SprS (PA3535), an annotated AT with a serine-protease passenger domain (Ma et al., 2003; Hardie et al., 2009). Pseudomonas aeruginosa PA14 and isogenic oprD and oprS (PA3535) mutant strains were grown in LB to early stationary phase. Cells and TCA-precipitated proteins of supernatant were separated by SDS-PAGE for immunoblotting with anti-PlpD-specific antiserum. The bacterial culture equivalent of 0.5 units of OD600 for cells (C) and of 5 units of OD600 for supernatants (SN) were loaded on a 10% acrylamide gel. PlpDC-ter is indicated with a black arrow, PlpDN-ter with a white arrow. The fate of the PA14 PlpD orthologue is similar to what was observed in PA01 albeit the secreted domain was detected at a much lower level in the PA14 strain. However, in the oprD and oprS transposon-insertion mutant strains, the ∼47 kDa fragment of PlpD was immunodetected in the cell fraction showing that these two proteases are not required for PlpD maturation.

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