Fig. 2: Expression of Esp_N using the curli-dependent amyloid generator (C-DAG) system.
From: The biofilm-associated surface protein Esp of Enterococcus faecalis forms amyloid-like fibers
a Representative scheme of C-DAG. Potential amyloid domains are expressed under the control of the inducible PBAD promoter, which allows the expression of the epitope fused to the first 42 residues of the signal sequence of CsgA. CsgG is expressed under the control of an IPTG-inducible promoter. Outer membrane (OM), cytoplasmic membrane (CM), periplasmic space (PS). b Variation in colony color phenotype of E. coli cells exporting the N-terminal domain of Esp (Esp_N), a C-repeat (Esp_C), and AB-repeats (Esp_AB) on agar supplemented with CR. E. coli expressing Bap_B and Bap_A from S. aureus was used as positive and negative controls respectively. c CR solubilized using ethanol–acetone from E. coli cells expressing Esp_N, Esp_C, Esp_AB, Bap_B, and Bap_A. d Quantification of CR. Absorbance was measured at 500 nm. Bars represent the standard deviations of the results of six independent experiments (n = 6). Statistically significant differences were determined using Mann–Whitney test *p < 0.05, **p < 0.01. Transmission electron micrographs of negatively stained fiber-like structures formed by E. coli cells that express Esp_N e, f and Bap_B h. E. coli cells that express Bap_A i, Esp_C j and Esp_AB k did not show the presence of fibers as shown by transmission electron microscopy. Immunogold labeled of fiber-like structures of samples from E. coli cells that express Esp_N using anti-His antibodies g. Scale bar of panels e, j, k represents 500 nm. Scale bar of panels f, h, i represents 200 nm. Scale bar of panel g represents 100 nm.
