Better explicating the strengths and shortcomings of these models will help refine projections and improve transparency in the years ahead.
Image credit: Witsawat.S.
Edited by Scott J. Hultgren, Washington University School of Medicine, St. Louis, MO, and approved July 8, 2013 (received for review April 11, 2013)
Article Figures & SI
Figures
- Fig. 1.
In situ analysis of BslA localization in the complex colony biofilm. Confocal scanning laser microscopy images of cross-sections through complex colonies formed by either (A and C) wild-type cells (3610, sacA::Phy-spank-gfp; NRS1473) or (B and D) the bslA mutant strain (3610, bslA::cat, sacA::Phy-spank-gfp; NRS3812). The smaller images show the region highlighted by the white box at higher magnification. Fluorescence from the GFP within the cells is shown in green in the large panels and in the merged images and the fluorescence associated with DyLight594, representing immuno-labeled BslA staining, is shown in red. (Scale bar, 50 µm.)
- Fig. 2.
In situ analysis of BslA localization in the floating biofilm. Confocal scanning laser microscopy images of xy sections through a typical pellicle of wild-type strain NRS1473 (3610, sacA::Phy-spank-gfp) after immunofluorescence staining. (A) −0.2 μm, (B) 0 μm, and (C) 0.4 μm into the height of the pellicle. Fluorescence from the GFP within the cells is false-colored green and fluorescence associated with DyLight594, representing immuno-labeled BslA staining, is false-colored red in the merged image. (Scale bar, 5 μm.)
- Fig. 3.
Analysis of bslA expression by cells forming complex colonies. (A and B) Flow cytometry analysis of expression of (A) bslA (NRS2289; 3610, sacA::PbslA-gfp) and (B) tapA-sipW-tasA (NRS2394; 3610, sacA::PtapA-gfp) in cells isolated from complex colonies after 18 h of growth at 37 °C. The gray-shaded zone represents the fluorescence observed for wild-type NCIB3610 containing no gfp. (C and D) Single-cell microscopy of cells isolated from complex colonies carrying either the (C) PbslA-gfp (NRS2289) or (D) PtapA-gfp (NRS2394) transcriptional reporters. (Scale bars, 10 µm.)
- Fig. 4.
In vitro analysis of BslA self-assembly into an elastic protein film. Pendant droplet analysis of purified BslA42–181 protein shows elastic film formation at the protein–oil interface. (A) A 40-µL droplet of BslA42–181 (0.2 mg/mL in 25 mM phosphate buffer, pH 7) was expelled into glyceryl trioctanoate, and following 20 min of equilibration compressed by retraction of 5 µL. Wrinkles formed in the neck of the drop, indicative of film formation. (B) Film relaxation after droplet compression, as measured by loss of surface wrinkles (expressed as the reduction in normalized grayscale values). Wild-type BslA42–181 shown by black circles; also shown are BslA42–181 containing the amino acid substitutions L76K (red circles), L77K (green triangles), and L79K (yellow triangles).
- Fig. 5.
Structural analysis of BslA. (A and B) Topological representation of (A) BslA and (B) the structurally similar β2-microglobulin (34) constructed using TopDraw (44). Yellow and green β-strands represent conservation with the canonical Ig fold and the hydrophobic cap, respectively. Blue β-strands and the red α−helix represent secondary structure not part of the classical Ig fold. (C and D) Ribbon representation of the structure of (C) BslA and (D) β2-microglobulin using the same color scheme as in A and B. The surface-exposed leucine, isoleucine, and valines are represented as sticks with magenta carbon atoms. (E and F) The hydrophobic regions of (E) BslA and (F) HFBII (35). The hydrophobic region is shown in green, and surface-exposed leucine, isoleucine, and valines are annotated.
- Fig. 6.
In vivo biofilm analysis of the hydrophobic cap of BslA. (A) Complex colony morphologies of strains containing leucine/isoleucine-to-lysine mutations in the β-sheets CAP2 and CAP3 alongside wild-type (NCIB3610), bslA− (NRS2097), and bslA+ (NRS2299) controls. (B) Pellicle morphology of the CAP2 and CAP3 mutants shown in A. (C) Sessile water-drop analysis of colony hydrophobicity of CAP2 and CAP3 mutants. Colonies were grown as for morphology analysis and 5-µL water drops placed on top. (D) Complex colony morphologies of strains containing mutations in the central CAP1 β-sheet. (E) Pellicle morphology of the CAP1 mutants shown in D. (F) Water-droplet analysis of colony hydrophobicity of CAP1 mutants. Table S3 gives strain details.
- Fig. 7.
Model of BslA film formation and relaxation after compression. In the equilibrium state, BslA will form a film at the water–oil interface, with both lateral protein–protein interactions between BslA monomers and interactions between the hydrophobic cap (shown in magenta) and the oil–water interface. After compression (by removal of some of the water) the monomers are moved closer together, creating the visible wrinkles. For the wild-type proteins the surface activity of the hydrophobic cap prevents monomers from being released from the BslA film, causing long-lasting wrinkles. However, in the proteins containing mutations in the CAP1 β-sheet the surface activity is lowered enough to allow release of some of the BslA monomers in the film, allowing the film to return to an equilibrium state and the relaxation of the wrinkles.
Data supplements
Supporting Information
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Self-assembling hydrophobin coats biofilm
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