Figure 2

Suppression of the lethal phenotype of surA skp cells by multicopy ppiD. (A) Schematic representation of PpiD and its variants used in this study, with amino acid residues numbered as in the full-length PpiD polypeptide. Diagonally striped box: transmembrane segment; white box: N-terminal region; Gray box: parvulin domain, with alanine substitutions indicated by black bars. PpiDΔTM was preceded by the SurA signal peptide so that it would be secreted into the periplasm (see Methods). (B) Growth of the SurA-depletion strain P _Llac-O1 -surA Δskp (SB44452) carrying pASK75 (empty vector), pSurA, pSkp, and pPpiD, respectively. Cells were grown overnight in the presence of IPTG, after dilution spotted on LB plates ± IPTG, and incubated at 37°C for 16-24 h. (C) Growth of the strains P _Llac-O1 -surA (SB44454) and P _Llac-O1 -surA Δskp (SB44452) at 37°C in liquid LB with (solid lines) and without (dotted lines) IPTG, resulting in the indicated "genotypes" wild-type (WT), surA, skp, and surA skp. The asterisk marks the point of sub-culturing (see Methods). Within the framed interval samples were taken for further analysis. Note that the Δskp surA strain containing pASK75 or pPpiDΔTM resumed growth after ~360-minute cultivation without IPTG. Western blotting revealed that at this point the cells also resumed production of SurA (see additional file 3). In contrast, SurA levels remained low in Δskp surA pPpiD cells during the entire course of growth, indicating that increased PpiD levels compensate for the simultaneous lack of SurA and Skp. (D) PpiD proteins in P _Llac-O1 -surA Δskp cells after 240-minute growth in LB without IPTG. Extracts from 4 × 10⁷ cells were loaded in each lane and analyzed by western blotting. Lane 8 shows lane 6 after prolonged development of the blot to visualize the protein. Cytoplasmic Hsc66 served as a loading control. Data for one representative experiment are shown.