Figure 3.

Effects of CsgE on the CsgG-dependent secretion of PapD2 and A22-PapD2.

(A) A schematic diagram of the PapD2 and A22-PapD2 constructs used in the secretion assays. PapD2 consists of the 21 amino acid sec-dependent signal sequence of PapD (ss^D) fused to domain 2 of PapD with a C-terminal 6-his tag; domain 2 is the C-terminal 93 amino acids of PapD encompassing the second domain of this 2-domain chaperone. A22-PapD2 consists of the PapD signal sequence (ss^D), the N-terminal 22 amino acids of mature CsgA (A22), domain 2 of PapD and a C-terminal 6-his tag. The carat indicates the sec cleavage site.

(B-C) CsgG-dependent protein secretion assay: cells were grown in LB shaking cultures at 37°C to OD₆₀₀~0.8, protein expression induced for 1 hr, then whole cell and supernatant (or concentrated supernatant) fractions were subjected to Western blot analysis using PapD-specific antisera. < corresponds to the predicted size of ss^D-A22-PapD2 (i.e., prior to sec signal cleavage); * indicates a non-specific background band.

(B) The bacterial strain LSR11 (MC4100Δcsg) expressing PapD2 (lanes 1-2; pAN87) or A22-PapD2 (lanes 3-4; pAN88) and an empty vector (lanes 1 and 3; pTrc99A) or CsgG (lanes 2 and 4; pMC1).

(C) The bacterial strain LSR35 (MC4100ΔcsgEF::kan^R) expressing CsgG from the chromosome contained the following plasmids: PapD2 (lanes 5-7; pAN93) or A22-PapD2 (lanes 8-10; pAN94); and, empty vector (lanes 5 and 8; pBAD33), CsgE (lanes 6 and 9; pLR42) or CsgF (lanes 7 and 10; pLR75).

(D) Whole cell samples of the strains in panel B and C were subjected to Western blot analysis using CsgG-specific antiseria. Lane numbers correlate to those of panels B and C.