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Research Article

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

Diego O. Serra, Anja M. Richter, Gisela Klauck, Franziska Mika, Regine Hengge
Roberto Kolter, Editor
Diego O. Serra
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Anja M. Richter
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Gisela Klauck
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Franziska Mika
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Regine Hengge
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Roberto Kolter
Harvard Medical School
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DOI: 10.1128/mBio.00103-13
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  • FIG 1 
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    FIG 1 

    Regulation of flagella, CsgD, and curli and the role of c-di-GMP in macrocolony morphology. (A) The regulatory cascade that drives flagellum synthesis and operation (FlhDC/σ70, σFliA, and its anti-sigma factor, FlgM; left side of the figure) (25) and the transcription factor cascade that drives biosynthesis of curli fibers (σS, MlrA, CsgD; right side of the figure) inhibit each other by two mechanisms: (i) FliZ, which is under positive FlhDC control, downregulates a subset of σS-dependent genes, including ydaM, mlrA, and csgD (20, 34), and (ii) two separate c-di-GMP control modules, each consisting of a diguanylate cyclase(s) (light red ovals) and a phosphodiesterase (blue hexagons), downregulate motility and activate csgD transcription (YegE/YedQ and YhjH), or activate csgD only (YdaM and YciR) (20, 30). A third c-di-GMP control module (YaiC and YoaD) controls the activity of the cellulose synthase BcsA. C-di-GMP-binding effectors are shown as purple pentagons. (B and C) Macrocolonies of strain W3110 and the indicated mutant derivatives were grown for 5 days on CR-containing salt-free LB plates.

  • FIG 2 
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    FIG 2 

    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.

  • FIG 3 
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    FIG 3 

    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.

  • FIG 4 
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    FIG 4 

    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.

  • FIG 5 
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    FIG 5 

    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.

  • FIG 6 
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    FIG 6 

    Expression of the key biofilm regulator CsgD in a developing macrocolony biofilm. (A) Cellular levels of csgD mRNA in developing W3110 macrocolonies determined by Northern blot analysis. Macrocolonies were harvested after growth for 1, 2, 3, 4, and 7 days as indicated. The arrow indicates the csgD mRNA, which is the main product of the csgDEFG operon. (B) Expression pattern of csgD::gfp in different zones of a representative cross section of a 2-day-old WT macrocolony. Combined, both images visualize the whole vertical section of the macrocolony at the central region (zone III). Macrocolonies of the W3110 derivative harboring a single-copy csgD::gfp reporter fusion were grown on salt-free LB plates, cryoembedded, cross-sectioned, and visualized by fluorescence microscopy.

Supplemental Material

  • Figures
  • Additional Files
  • Figure S1

    Morphology of mature W3110 macrocolonies in the presence or absence of CR or TS. (A) Macrocolony of strain W3110 developing over time on a CR-containing salt-free LB plate. (B) Top-view images of 7-day-old macrocolonies of W3110 and its ΔcsgB mutant derivative grown at 28°C on salt-free LB plates without or with supplementation of CR or TS. Download Figure S1, JPG file, 2.9 MB.

    Copyright © 2013 Serra et al.

    This is an open-access article distributed under the terms of the Creative Commons Attribution-Noncommercial-ShareAlike 3.0 Unported license, which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original author and source are credited.

  • Figure S2

    Surface of mature macrocolonies of W3110 and its ΔcsgD and ΔbcsA mutant derivatives. (A) Top-view SEM images (at ×6,000 and ×50,000 magnification) of the surface of 7-day-old macrocolonies of W3110 (upper panels) and the ΔcsgD mutant (middle panels) are shown. Note that the surface of the ΔcsgD mutant macrocolony is essentially identical to that of the ΔcsgB mutant macrocolony (Fig. 4I), whereas top-view SEM images (at ×6,000 and ×50,000 magnification) of the surface of a 7-day-old ΔbcsA mutant macrocolony (lower panels) are essentially undistinguishable from those of the W3110 macrocolonies (upper panels). (B) High-resolution top-view SEM images (×50,000 and ×100,000 magnification) showing empty curli baskets that preserve their form after the release of the hosted bacteria during preparation for SEM. Download Figure S2, JPG file, 2.9 MB.

    Copyright © 2013 Serra et al.

    This is an open-access article distributed under the terms of the Creative Commons Attribution-Noncommercial-ShareAlike 3.0 Unported license, which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original author and source are credited.

  • Figure S3

    Surface and bottom of mature macrocolonies of W3110 and its ΔfliC and ΔmotA mutant derivatives. High-resolution SEM images (at ×50,000 and ×100,000 magnification) show details of size, morphology, and spatial arrangement of bacterial cells and flagella at the bottom of a 7-day-old W3110 macrocolony (upper- and middle-left panels). The high-resolution SEM image (at ×100,000 magnification) shows details of the coiled state of individual flagellar filaments at the bottom of a 7-day-old ΔmotA mutant macrocolony (lower-left panel). The right panels show high-resolution SEM images (at ×50,000 magnification) of the surface of 7-day-old macrocolonies of W3110 and the indicated mutants. Arrows indicate flagella, which can be distinguished from shorter curli fibers by their length and larger diameter. Download Figure S3, JPG file, 2.8 MB.

    Copyright © 2013 Serra et al.

    This is an open-access article distributed under the terms of the Creative Commons Attribution-Noncommercial-ShareAlike 3.0 Unported license, which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original author and source are credited.

  • Figure S4

    Physiological stratification inside the macrocolony biofilm. (A) The low-magnification (×180) side-view SEM image shows a cross section of the ring-forming area of a W3110 macrocolony. (B and E) Side-view SEM images at ×12,000 and ×50,000 magnification show small, ovoid bacteria encased by the dense curli network in the upper layers of the W3110 macrocolony. Images were taken within the zone boxed in green in panel A. (C and F) Side-view SEM images (also at ×12,000 and ×50,000 magnification) show a transition zone composed of patches and strips of curli-encased bacteria and flagellated curli-free bacteria. Images were taken within the zone boxed in red in panel A. (D and G) Side-view SEM images (at ×12,000 and ×50,000 magnification) show elongated, rod-shaped bacteria tethered to each other by flagella. Images were taken within the zone boxed in blue in panel A. Download Figure S4, JPG file, 2.9 MB.

    Copyright © 2013 Serra et al.

    This is an open-access article distributed under the terms of the Creative Commons Attribution-Noncommercial-ShareAlike 3.0 Unported license, which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original author and source are credited.

  • Figure S5

    Internal regions close to the surface and the bottom of mature ΔcsgB and ΔfliC mutant macrocolonies. Side-view SEM images (at ×12,000 magnification) of a 7-day-old ΔcsgB mutant macrocolony show curli-free, small, ovoid bacteria in the upper layers (upper-left panel) and flagellated, rod-shaped bacteria in the bottom layers of the macrocolony (upper-right panel). Side-view SEM images (at ×12,000 magnification) of a 7-day-old ΔfliC macrocolony show small, ovoid bacteria encased by the curli network in the upper layers (lower-left panel) and nonflagellated, rod-shaped bacteria in the bottom layers of the macrocolony (lower-right panel). Download Figure S5, JPG file, 2.7 MB.

    Copyright © 2013 Serra et al.

    This is an open-access article distributed under the terms of the Creative Commons Attribution-Noncommercial-ShareAlike 3.0 Unported license, which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original author and source are credited.

  • Table S1

    Oligonucleotide primers used in this study. Table S1, DOC file, 0.1 MB.

    Copyright © 2013 Serra et al.

    This is an open-access article distributed under the terms of the Creative Commons Attribution-Noncommercial-ShareAlike 3.0 Unported license, which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original author and source are credited.

Additional Files

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    Supplementary Data

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    • Figure sf01, JPG - Figure sf01, JPG
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    • Figure sf03, JPG - Figure sf03, JPG
    • Figure sf04, JPG - Figure sf04, JPG
    • Figure sf05, JPG - Figure sf05, JPG
    • Table st1, DOC - Table st1, DOC
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Microanatomy at Cellular Resolution and Spatial Order of Physiological Differentiation in a Bacterial Biofilm
Diego O. Serra, Anja M. Richter, Gisela Klauck, Franziska Mika, Regine Hengge
mBio Mar 2013, 4 (2) e00103-13; DOI: 10.1128/mBio.00103-13

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Microanatomy at Cellular Resolution and Spatial Order of Physiological Differentiation in a Bacterial Biofilm
Diego O. Serra, Anja M. Richter, Gisela Klauck, Franziska Mika, Regine Hengge
mBio Mar 2013, 4 (2) e00103-13; DOI: 10.1128/mBio.00103-13
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