Microanatomy at Cellular Resolution and Spatial Order of Physiological Differentiation in a Bacterial Biofilm

Spatial distribution of curli in different biofilm zones visualized by thioflavin S fluorescence. Seven-day-old E. coli macrocolony biofilms grown on salt-free LB medium supplemented with TS were cryoembedded and sectioned perpendicular to the plane of the macrocolony at a thickness of 5 µm. Thin sections were visualized by fluorescence microscopy. Fluorescence images were false-colored green for TS. (A and B) Merged bright-field and fluorescence images at low magnification of representative cross sections of macrocolonies of W3110 and its ΔcsgB derivative, respectively. Images visualize the whole vertical section of the macrocolonies at the central region. Bright-field images appear in a gray background to better visualize the location of the fluorescence. (C and D) Fluorescence images of a representative 5-µm-thick section showing upper and inside-middle parts of the W3110 macrocolony biofilm, respectively. The upper-right insets show enlarged views of the respective boxed areas.

Small, ovoid cells and curli fiber network at the surface of the macrocolony biofilm. Seven-day-old E. coli macrocolonies were prepared for SEM examination as described in Materials and Methods. SEM imaging was performed in high vacuum mode. (A) Low-magnification (×80) perspective-view SEM image of a representative sector of a W3110 macrocolony displaying wrinkles and ring patterns. (B and C) Top-view SEM images at ×100 and ×400 magnification showing the wrinkles of the W3110 macrocolony. (D and E) Further-magnified top views (×3,000 and ×12,000) of the surface of the wrinkles. (F and G) High-resolution top-view SEM images (×50,000 and ×100,000) showing in fine detail small, ovoid bacteria encased by a dense network of curli fibers at the surface of a W3110 macrocolony. (H) Top-view SEM image at ×6,000 magnification of the surface of a macrocolony of W3110 ΔcsgD. (I) High-resolution top-view SEM image (×50,000 magnification) showing curli-free, small, ovoid bacteria arranged in a plane at the surface of the ΔcsgB macrocolony.

Growing cells and flagella are found at the bottom and the outer rim of the W3110 macrocolony biofilm. (A and D) SEM images at ×6,000 and ×50,000 magnification reveal details of the mesh of entangled filaments formed by bacteria at the bottom of the macrocolony. (B and E) SEM images (also at ×6,000 and ×50,000 magnification) show long, rod-shaped bacteria completely devoid of the filaments at the bottom of a ΔfliC mutant macrocolony, indicating that the filaments are flagella. (C and F) SEM images of a ΔmotA mutant macrocolony at ×6,000 and ×50,000 magnification show a more relaxed state of the mesh of flagella, which allows coiling of individual flagellar filaments at the bottom of the macrocolony. (G and J) Top-view SEM images (at ×3,000 and ×6,000 magnification) display the outer-edge area of a W3110 macrocolony. (H and I) High-resolution top-view SEM images (×24,000 and ×50,000) of the outer-edge area show rod-shaped W3110 cells tied together by flagella. (K) The top-view SEM image (at ×3,000 magnification) shows the outer-edge area of a ΔfliC mutant macrocolony. The image reveals the almost complete loss of the growth zone at the edge, which occurred during the preparation procedure for SEM. (L) Magnified top-view SEM image (×12,000) of the edge of a ΔfliC mutant macrocolony. The image shows a remaining sector of the growth zone composed by nonflagellated, rod-shaped cells.

Morphology and horizontal and vertical distributions of flagella and curli in different surface zones of a 2-day-old W3110 macrocolony biofilm. (A) The top-view image shows a 2-day-old macrocolony grown on salt-free LB medium supplemented with CR. (B and E) Top-view SEM images (at ×12,000 and ×50,000 magnification) of the macrocolony surface were taken at zone I as indicated in panel A. Red arrows indicate flagella. (C and F) Top-view SEM images (at ×12,000 and ×50,000 magnification) of the macrocolony surface taken at zone II as indicated in panel A. Blue arrows indicate patches and strips of curli-encased bacteria. (D and G) Top-view SEM images (at ×12,000 and ×50,000 magnification) of the macrocolony surface were taken at zone III as indicated in panel A. (H to J) Merged bright-field and fluorescence images of representative 5-µm-thick cryosections at zones I, II, and III of a 2-day-old W3110 macrocolony grown on salt-free LB supplemented with TS. (K) Side-view SEM image at ×3,000 magnification showing a cross section of a 2-day-old W3110 macrocolony at zone II. (L) Side-view SEM image at ×12,000 magnification of the upper layers of the 2-day-old macrocolony at zone II. Blue arrows indicate curli formation. (M) Side-view SEM images at ×12,000 magnification of the upper layers of the 2-day-old macrocolony at zone III.