Figure 1A. Congo Red depletion assay. Fn 12230 (black), the fadA-deletion mutant US1 (∆fadA) (orange) and the spontaneous lam mutant (blue) (top panel) were grown to different OD600. E. coli MC4100, ∆curli, and ∆csgA (bottom panel) were grown on TSA-blood agar plate at 26oC for 48hr. The bacteria were suspended in PBS to OD600 of 0.5, 1.0, 1.5 and 2.0, followed by incubation in 10 μg/ml Congo Red (CR) for 10 min. After centrifugation, the supernatants were measured at OD500. The results shown are the average of five independent experiments performed in duplicate. The error bars indicate SD. *p<0.05, ** p<0.01, ***p<0.001 (compared to wild type, t-test). B. Kinetics of FadA production in Fn. Fn 12230 and US1 were inoculated to OD600 = 0.1. 10 OD600 units of bacteria were harvested by centrifugation at 12-hr intervals. Bacteria were lysed with 2 mg/ml lysozyme, followed by incubation in 1% sarkosyl at 4° C for 20 minutes, which became the 'total' sample. Lysates were then centrifuged at 100,000 g for 20 minutes and the supernatants and pellets were collected. An aliquot of 5 μl is loaded onto each lane, followed by immunoblot using anti-FadA monoclonal antibody (mAb) 7H7 at 1:4,000 dilution to detect FadA protein in total, supernatant, and pellet fractions. Ponceau staining of lysozyme is shown as a loading control. Pre-FadA and mFadA are pointed by arrows. Note pre-FadA migrates faster than mFadA on SDS-PAGE as previously reported (Han et al., 2005). C. Analysis of detergent-resistant FadA polymers in wild-type Fn and its mutants by semi-denaturing detergent agarose gel electrophoresis (SDD-AGE) and Western blot analysis using mAb 7H7. Fn12230 (wt), fadA-deletion mutant US1 (∆fadA) and spontaneous mutant lam (lam) were grown to log (OD600 0.3-0.4) or stationary phase (OD600 >0.8), E. coli MC4100, ∆curli, and ∆csgA were grown to stationary phase. The bacteria were harvested by centrifugation. Following sequential incubation in lysis buffer containing 2 mg/ml lysozyme and 1% sarkosyl, the insoluble pellets were obtained by centrifugation. An aliquot of 100 μg of each pellet was loaded onto 1.7% agarose gel followed by overnight electrophoresis in 0.5xTAE and 0.1% SDS. Following transfer to nitrocellulose membrane, Western blot was performed using mAb 7H7 at 1:4,000 dilution to detect the FadA proteins or anti-CsgA antibody at 1:15,000 dilution. The heterogeneously large detergent resistant FadA polymers were detected in wild-type Fn in the stationary phase, defective in US1 and lam, and absent in all in log phase. D. Immunohistochemical analysis of FadA in log and stationary phases. Fn12230 (Fn), fadA-deletion mutant US1 (∆fadA) and spontaneous mutant lam in log and stationary phase were fixed before being placed onto glass slides and stained by mAb 7H7 at 1:800 dilution, followed by incubation of HRP-conjugated anti-mouse IgG and developed by DAB. The large FadA aggregates are detected specifically in wild-type Fn 12230 in the stationary but not in log phase. FadA was detected on lam, but without secreted aggregates. No FadA was detected in US1. The imagines were taken using 40X objective. Scale bar equals 20 μm. E. Double immunofluorescent staining of Fn12230, fadA-deletion mutant US1 (∆fadA) and spontaneous mutant lam in log and stationary phases using mAb 7H7 at 1:800 dilution and anti-human β-amyloid polyclonal antibody A11 at 1:500 dilution, followed by incubation with Alexa Fluor 680-conjugated donkey anti-rabbit and Alexa Fluor 555-conjugated goat anti-mouse, both at 1:1,000 dilution. Co-staining of FadA and A11 was observed in Fn in stationary phase, not in log phase, or in US1 or lam. The imagines were taken under 60X objective. Scale bar equals 5 μm. F. Scanning electron microscopy of Fn 12230, US1 (∆fadA) and lam in log and stationary phase at 10,000x magnification. Note the fibrous structure coating Fn in stationary phase, pointed by the clear arrows, but not in log phase. Scale bar equals 1 μm.