High-resolution deep-etch EM micrographs of curliated E. coli and purification of curli fibers. (A and B) Representative freeze-fractured micrographs that have been rotary shadowed with platinum. The inset in (A) shows curli fibers. (C) MC4110 was absorbed onto glass and deep-etched without being fractured before rotary shadowing with platinum. (D) Coomassie stain SDS-PAGE of curli samples isolated from E. coli strain MC4100. Lanes 1 and 2 contain 40 μg of S6 wild-type curli without and with FA treatment, respectively. Lane 3 contains 20 μg of FA-treated GP curli. Molecular size markers (in kilodaltons) are indicated on the left. (E) Negative-stain EM micrographs of MC4100 grown on YESCA plates at 26°C for 48 hours. (F) Negative-stain EM micrograph of purified wild-type S6 curli. Bars: (A), 400 nm; (B) and (C), 60 nm; (E) and (F), 200 nm.

Curli biogenesis in the absence of CsgE and CsgF. (A) Negative-stain EM micrographs of MHR592 (csgF⁻) bacteria grown on YESCA plates at 26°C for 48 hours. (B) CsgA visualized by Western analysis with anti-CsgA (16) and bacteria grown at 26°C on YESCA plates for 48 hours. Circular plugs of 8 mm, including cells and underlying agar (to collect soluble, unpolymerized and secreted CsgA), were collected and resuspended in 200 μl of 1.5× SDS loading buffer either with or without prior FA treatment. The etracts loaded in each lane are as follows: lanes 1 and 2, MC4100 (wild type); lanes 3 and 4, LSR10 (csgA⁻); lanes 5 and 6, MHR480 (csgE⁻); lanes 7 and 8, MHR261 (csgB⁻); and lanes 9 and 10 MHR592 (csgF⁻). (C) Negative-stain EM micrographs of MHR480 (csgE⁻) bacteria grown on YESCA plates at 26°C for 48 hours. These fibers often looped into imperfect circles (see inset). (D) Interbacterial complementation and CR binding in csgE⁻ and csgF⁻ mutants. The CsgA⁺ donor strain MHR261 (I) and the CsgB⁺ recipient strain LSR10 (II) were streaked from the top of the plate to the bottom. The horizontal cross-streaks were made from left to right with the following strains: MC4100 (wild type) (I-1 and II-1), csgA⁻ (I-2), csgB⁻ (II-2), csgE⁻ (I-3 and II-3), csgEB⁻ (I-4 and II-4), csgF⁻ (I-5 and II-5), and csgFB⁻ (I-6 and II-6). Bars in (A) and (C), 200 nm.

Amyloid-like properties of curli. (A) The CD spectrum of wild-type S6 curli was measured with 300 μg of protein in 10 mM tris (pH 7.4) with a 0.02-cm cell in a JASCO J715 spectropolarimeter at 25°C. S6 and GP curli gave similar CD spectra. (B) A 10-μM solution of CR prepared in 10 mM tris (pH 7.4) and 100 mM NaCl was filtered through a 0.2-μm filter and mixed with 50 μl of buffer [10 mM tris (pH 7.4)] (◆), S6 curli (4 mg/ml stock) (○), or GP curli (4 mg/ml stock) (×) in a final volume of 1 ml. All spectra were normalized against the relevant non-CR–containing solutions. (C) Spectra representing the difference of CR with S6 curli and CR alone. (D) Fluorescence of 5 μM ThT alone (◇) or mixed with 40 μg of S6 curli (○) after excitation at 450 nm on an AlphaScan PTI fluorometer with a slit width of 4 nm. GP and S6 curli gave indistinguishable fluorescence results.

Purification and in vitro assembly of CsgA-his. (A) Western blot with anti-his (Covance, Richmond, California) to determine expression and cellular location of overexpressed CsgA-his. Log-phase cultures containing (lane 1) pMC3 (csgA-his) and pTrc99A (empty vector); (lane 2) pMC3 and pMC1 (csgG); or (lane 3) pMC3 and pMC5 (csgEFG) were induced with 0.5 mM IPTG for 1 hour; samples were removed and mixed with an equal amount of 2× SDS-PAGE dye and heated to 95°C before gel electrophoresis. CsgA-his expression with pTrc99A was detected only after overexposure of the blot. The cellular and supernatant fractions from cultures containing pMC3 and pMC5 were separated by centrifugation and loaded in lanes 4 and 5, respectively. (B) CsgA-his was purified from cleared LSR6/pMC3/pMC5 supernatants filtered through a 0.2-μm filter before loading on a disposable column packed with nickel NTA-agarose beads (Qiagen, Chatsworth, California). The column was washed with 10 column volumes of 10 mM tris (pH 7.4), 100 mM NaCl, and CsgA-his was eluted with 5 ml of wash buffer plus 100 mM imidazole and analyzed by Coomassie stain SDS-PAGE. Both the major band migrating at ~17.5 kD and the higher molecular weight, minor band (indicated by an asterisk) interacted with anti-CsgA (9). (C) The CD spectrum of wild-type S6 curli compared with that of 300 μg of soluble unpolymerized CsgA-his assayed immediately after purification. (D) High-resolution EM of pure CsgA-his preparations after a 1-week incubation at 4°C. Bar, 140 nm. (E) Absorbance of a 10 μM solution of CR with 100 μg of pure, unpolymerized CsgA-his (■) or pure polymerized CsgA-his (▲) after subtracting the absorbance of CR alone.