Amyloid fibers provide structural integrity to Bacillus subtilis biofilms

Abstract

Bacillus subtilis forms biofilms whose constituent cells are held together by an extracellular matrix. Previous studies have shown that the protein TasA and an exopolysaccharide are the main components of the matrix. Given the importance of TasA in biofilm formation, we characterized the physicochemical properties of this protein. We report that purified TasA forms fibers of variable length and 10-15 nm in width. Biochemical analyses, in combination with the use of specific dyes and microscopic analyses, indicate that TasA forms amyloid fibers. Consistent with this hypothesis, TasA fibers required harsh treatments (e.g., formic acid) to be depolymerized. When added to a culture of a tasA mutant, purified TasA restored wild-type biofilm morphology, indicating that the purified protein retained biological activity. We propose that TasA forms amyloid fibers that bind cells together in the biofilm.

Fig. 1.

 The presence of fibers in pellicles is related to the presence of TasA. Pellicle formation in MSgg at 30 °C as observed under a stereomicroscope at a magnification of 32× of (A) wild type, (B) eps mutant, and (C) tasA mutant. (D–F) Electron micrographs of negatively stained, immunogold labeled samples from pellicles after 24 h of incubation. (D) Wild type, (E) eps mutant, and (F) tasA mutant. (Scale bars, 500 nm.) The rectangles shown in D, E, and F represent the areas shown at higher magnification in G, H, and I, respectively.

Fig. 2.

 Dye binding properties of colonies and cell extracts. (A) Colonies of B. subtilis wild-type and matrix mutants grown for 72 h on Congo Red indicator plates. (B) Absorbance of a 10 μM solution of Congo Red (CR) with 1 M NaCl extracts of wild-type and matrix mutants. (C) Immunogold-labeled electron micrographs of TasA fibers from 1 M NaCl extracts of wild-type B. subtilis. (Scale bar, 500 nm.)

Fig. 3.

 Amyloid properties of purified TasA. Negative staining electron micrographs of (A) freshly prepared TasA polymerized in long fibers of large MW and (B) depolymerization of fibrils into smaller oligomers with broad distribution of lower MW. Insets show chromatography traces of the samples run over a sizing column and standards of known molecular weights (dotted lines). (C) Differential spectra of the Congo Red absorbance after addition of polymerized (■) or unpolymerized TasA (▲). (D) Fluorescence of 20 μM Thioflavin solution alone or mixed with polymerized (■) or unpolymerized TasA (▲) after excitation at 450 nm. (Scale bars, 100 nm.)

Fig. 4.

 Repolymerization of TasA in vitro. Electron micrographs of TasA immediately after 1-min (A) or 5-min (B) treatment with formic acid. Note that small fibers are seen in A but not in B. (Scale bars, 200 nm.) (C) Kinetics of TasA repolymerization in vitro. Purified TasA treated for 1 min (◆) or 5 min (▪) with 10% formic acid. After evaporation of formic acid, the repolymerization was followed by the Thioflavin T assay.

Fig. 5.

 TasA particles and aggregates react with A11 antibody. (A) Detection of transient intermediates during polymerization of TasA. Samples were blotted onto nitrocellulose membranes and probed with anti-TasA (1:20,000) or A11 (1:10,000) antibodies. Samples were removed during the time course for TEM analysis. (B) No structures were observed immediately after treatment with formic acid. (C) After 3 h, when some A11 reactivity was observed in the dot-blot, small homogenous particles (5–12 nm wide) were observed. Detail of one particle (12 nm) is shown in the Inset at higher magnification. (D) After 5 h, larger amorphous aggregates were observed that coincided with the strongest reactivity with A11. (E) At 24 h, when A11 reactivity had dropped, fibers morphologically similar to those in untreated samples were observed. (Scale bars, 100 nm; Inset, 20 nm.)

Fig. 6.

Addition of purified TasA restores pellicle formation to a tasA mutant. Cells were grown in MOLP broth and incubated for 48 h without agitation at 30 °C: (A) wild-type, (B) tasA mutant, and (C) tasA mutant after addition of purified (40 μg) polymerized TasA. Immunogold-labeled electron micrographs of TasA fibers between cells of wild-type (D and G) and tasA mutant after addition of purified TasA (F and I) and absence of fibers in a tasA mutant alone (E and H). (Scale bars, 500 nm.) The rectangles shown in D, E, and F, represent the areas shown at higher magnification in G, H, and I, respectively.