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* This work was supported, in whole or in part, by National Institutes of Health Grants GM58213 (to R. K.) and GM18546 (to R. L.). This work was also supported by grants from the BASF (Badische Anilin- und Soda-Fabrik) Advanced Research Initiative at Harvard University (to R. L. and R. K.). 1 Present address: Departamento de Microbiología, Facultad de Ciencias, Instituto de Hortofruticultura Subtropical y Mediterránea La Mayora, IHSM-UMA-CSIC, Universidad de Málaga, Málaga, Spain.
Biofilms are surface-associated groups of microbial cells that are embedded in an extracellular matrix (ECM). The ECM is a network of biopolymers, mainly polysaccharides, proteins, and nucleic acids. ECM proteins serve a variety of structural roles and often form amyloid-like fibers. Despite the extensive study of the formation of amyloid fibers from their constituent subunits in humans, much less is known about the assembly of bacterial functional amyloid-like precursors into fibers. Using dynamic light scattering, atomic force microscopy, circular dichroism, and infrared spectroscopy, we show that our unique purification method of a Bacillus subtilis major matrix protein component results in stable oligomers that retain their native α-helical structure. The stability of these oligomers enabled us to control the external conditions that triggered their aggregation. In particular, we show that stretched fibers are formed on a hydrophobic surface, whereas plaque-like aggregates are formed in solution under acidic pH conditions. TasA is also shown to change conformation upon aggregation and gain some β-sheet structure. Our studies of the aggregation of a bacterial matrix protein from its subunits shed new light on assembly processes of the ECM within bacterial biofilms.
Background: TasA is an extracellular matrix protein that makes amyloid-like fibers in Bacillus subtilis biofilms.
Results: An isolated TasA matrix precursor self-assembled in vitro into fibers on hydrophobic surfaces and in acidic solutions.
Conclusion: TasA is purified as stable, structured oligomers that aggregate in response to simple physical external cues.
Significance: TasA aggregation principles can be used to design new anti-biofilm drugs and surfaces.
Biofilms are surface-associated communities of microbial cells. They originate from individual cells that, upon encountering a suitable environment, form multicellular communities that are encased in self-produced extracellular matrix (ECM).
ECM proteins in bacteria serve a variety of structural roles and often form fibrous appendages. These include adhesive fimbriae, type IV pili, and flagella (which are also used for motility), and proteins that form amyloid-like proteins such as curli (
). Curli fibers are composed of a major and minor subunit, CsgA and CsgB, respectively. Their assembly involves four accessory proteins, CsgC, CsgE, CsgF, and CsgG. CsgG forms a pore in the outer membrane through which the curli proteins are secreted. CsgE acts as a chaperone for CsgA and helps prevent CsgA polymerization in vitro, whereas CsgF is required for CsgB surface exposure and aggregation of CsgA (
). These proteins are encoded in an operon with a third gene, sipW, which encodes the signal peptidase responsible for processing and secretion of TasA and TapA. There are no other accessory proteins analogous to the Csg system, possibly because in B. subtilis there is no outer membrane that must be crossed prior to fiber assembly. Both TasA and TapA are present in purified biofilm matrix protein preparations that have been extracted from the cell surface (
). When these matrix protein preparations were analyzed on an electron microscopy (EM) grid, fibers were observed. In addition, fibers that labeled with anti-TasA antibody connected cells within a biofilm (
Here, using dynamic light scattering (DLS) and atomic force microscopy (AFM) we show that B. subtilis matrix protein preparations are not purified in the form of fibers. Rather, these preparations contain oligomers that do not spontaneously aggregate in solution. In contrast to the purification methods described for most amyloid precursors, we did not purify the protein from inclusion bodies, and therefore our preparations resulted in a nondenatured, α-helical, and stable protein. Because these oligomers did not aggregate spontaneously in solution, we were able to determine which external cues gave rise to protein aggregation into fibrous structures where the constituent protein gained in β-sheet content. In particular, we show that hydrophobic (but not hydrophilic) surfaces, as well as acidic solution conditions, trigger the oligomers to form fibers.
) under shaking conditions. By choosing to work with cells that lack their eps genes, we ensured the lack of the exopolysaccharide component in our preparations. At the same time, the cells produced more TasA than the wild-type strain because they were also mutated in their sinR gene, which encodes a repressor of tasA transcription (
). The supernatant was then collected after an additional centrifugation (10,000 × g, 15 min) and filtered through a 0.4-μm PolyEtherSulfone (PES) bottle-top filter. To precipitate less soluble proteins we added ammonium sulfate (ground into a fine powder) to 30% (w/v) saturation. The supernatant was collected after centrifugation (20,000 × g, 10 min), concentrated with Amicon centrifugal filter tubes and passed through a HiLoad 26/60 Superdex S200 sizing column that was preequilibrated with a 50 mm NaCl, 20 mm Tris solution at pH 8. TasA eluted in the void volume and concentrated with Amicon centrifugal filter tubes. We have shown previously that following this protein purification protocol, TasA is purified with some TapA (
). We nevertheless refer to the purified protein as TasA because the TapA fraction was below the detection limits of the Edman degradation analysis.
Atomic Force Microscopy
We show the height-field AFM images of TasA adsorbed on the different surfaces (see sample preparation below). We used an Asylum SPM with AC mode for high resolution visualization of the protein on the surfaces. Probes were etched silicon (NSC18, 75 kHz, 3.5 N/m) from MikroMasch. We used the freeware Gwyddiion to analyze AFM images unless otherwise specified.
Sample Preparation for AFM
Preparation of Hydrophobic Surfaces
Silicon wafers (University Wafers, 3″, p-type, test grade) were cut to 5 × 5-mm pieces and cleaned by boiling in toluene and ethanol. The surfaces were further cleaned with an oxygen plasma for 10 min and immersed immediately in a 0.4 mm trichloro(octyl)silane (OTS) (97%; Sigma-Aldrich) solution in toluene for 40 min. After rinsing with toluene and subsequently immersing for 10–30 min in toluene the surfaces came out dry and were further purged in a stream of nitrogen.
We used freshly cleaved mica (Grade I; S&J Trading Inc., New York). The surface of freshly cleaved mica is clean, atomically smooth, and hydrophilic.
TasA Adsorption on Surfaces
A 3–5-μl drop of TasA (5 μm in 50 mm NaCl, 20 mm Tris, pH 8) was placed on the relevant (hydrophobic or hydrophilic) surface for a period of 2 h. To prevent water evaporation, we kept the surfaces in a humid, sealed, container throughout the adsorption process. The surfaces were then immersed in aqueous solutions: phosphate-buffered saline (PBS) and deionized water, under shaking conditions for a period of 10 and 20 min, respectively. The surfaces were taken out of the deionized water container and allowed to dry in ambient air.
Dynamic Light Scattering
DLS measurements were performed with an ALV system, equipped with an ALV/SP-125 goniometer and an AVL-5000 correlator excited with a Coherent Verdi 2W laser (532 nm) running at 0.2–0.4 watts. Measurements were performed by focusing a vertically polarized light onto the sample and collecting the scattered light with a detector at 90 °C. We used the CUMULANTS method (
) to analyze the normalized intensity correlation function and extract an average decay rate, Γav. A translational diffusion coefficient Deff was then calculated using the equation
where q is a wave vector, q = (4π n/λ) sin (θ/2), n is the refractive index of the medium, λ is the laser wavelength, and θ is the scattering angle. Finally, we used the Stokes-Einstein relation to calculate the average hydrodynamic radius,
where KB is the Boltzmann constant, T is the absolute temperature, η is the viscosity of the medium at a given temperature, and Deff is the particle effective diffusion constant. Scattering of a TasA (5 μm in 50 mm NaCl, 20 mm Tris, pH 8) solution was collected for 30–60 s at 30–60-s intervals with a count rate of (20–150) × 103 counts/s. The temperature was kept constant at 25 °C.
In the DLS experiments where we probed the growth of the protein with time after adjusting the solution pH to 2.5 (Fig. 6A) the protein concentration was 2 μm. To adjust the pH to 2.5 (Fig. 6A) we used formic acid, but similar results were obtained when we used hydrochloric acid. Aggregation is a concentration-dependent process and starting with a higher concentration resulted with larger aggregates that settle faster. At higher protein concentrations, lower pH, and times longer than 4 h we could not probe the growth of the aggregates due to their faster settling. To observe the aggregates that were formed after 3 h from the point when we adjusted the pH in solution, we placed a 3–5-μl droplet of the protein solution that we used for DLS on both hydrophobic or hydrophilic surfaces (described above). After 15–30 min the surface was rinsed with PBS and then water, for 10 and 20 min, respectively, and allowed to air dry prior to examining by AFM.
Circular Dichroism (CD) Measurements
CD was measured with a JASCO apparatus both in 10-mm and 1-mm optical path cuvettes. Using the 10-mm cuvette enabled us to stir (at 270 rpm) the solution during the aggregation process so that signal was not lost due to settling of the aggregates. In balancing between the need to get a reasonable CD signal and the need to work in a concentration comparable with that used for DLS, we measured the CD spectra of 0.5–1 μm and 3–4 μm protein with cuvettes of 10- and 1-mm optical lengths, respectively. In addition, because Tris buffer gives a large background signal with CD measurements, we dialyzed the protein against 10 mm potassium phosphate buffer, pH 7.4, for these measurements. CD was measured at pH 7.4 and 3, with the latter obtained by adding concentrated formic acid. Because formic acid absorbs the light substantially in the UV range, we were limited to working at pH values higher than 3. Similar results were obtained when using hydrochloric acid and pH adjustment to 3 or 2.5.
The spectrum of SDS-treated TasA was performed in 10 mm sodium phosphate because the potassium salt of SDS is water-insoluble. Guanidine HCl (ultrapure, MP Biomedicals 99% min) was used as a denaturing agent.
Thioflavin T Fluorescence Measurements
TasA was mixed with a thioflavin T (ThT) stock solution (that was prepared in 50 mm NaCl, 20 mm Tris, pH 8) to a final concentration of 2–4 μm protein, 25 mm ThT. The final volume was 200 μl, and measurements were performed in 96-well plates with a clear bottom (Costar). We used a BMG LABTECH microplate reader equipped with fixed excitation/emission filter of 430/480 nm, respectively. Data were collected after adjusting the pH to 2.5 or at pH 8 (the latter served as the control experiment) every 2–5 min. The plate was shaking with an orbital mode between measurements. In the reported experiments we did not block the plates before use, but similar results were obtained with plates that were blocked with 2.5 mg/ml bovine serum albumin (BSA) prior to the addition of the protein solutions.
Fourier Transform Infrared (FTIR) Spectroscopy
We used a Bruker Tensor 27 FTIR spectrometer, equipped with a Germanium Attenuated Total Reflection module and a liquid N2-cooled MCT detector to measure the spectra of the protein monolayers on the surfaces. As a hydrophobic surface we used the OTS-hydrophobized silicon/silicon oxide surface, and the model hydrophilic surface was either freshly cleaved mica or pieces of a silicon wafer that was cleaned with oxygen plasma prior to protein adsorption. For each sample, 1000 interferograms were accumulated at a spectral resolution of 4 cm−1. The spectra of the protein-free surfaces were recorded under conditions identical to those of the protein/surface samples and subtracted from the protein spectra.
The spectra of the protein in aqueous solutions were recorded with a Bruker Alpha spectrometer in Absorbance mode. We used 0.3–0.5 mm protein solutions in phosphate buffer (pH 7.4), and hydrochloric acid was used to adjust the pH to 2.5. For each sample, 512 interferograms were accumulated at a spectral resolution of 4 cm−1. The spectra of the buffer were recorded under conditions identical to those of the protein samples and subtracted from the protein spectra. All spectra were scaled independently to a full scale on the ordinate axis. The first and second derivatives of the amide I band were calculated and used to identify maximum and the additional spectral components of the band, respectively.
Transmission Electron Microscopy
To prepare the samples we used carbon-coated grids that were placed on top of a 3–5-μl drop of TasA in 50 mm NaCl, 20 mm Tris, pH 8 (we used 2.5 μm because the fibers formed from a 5-μm solution formed a dense mesh of fibers, and single fibers could not be resolved from the images) for 1–5 min. The grid was floated briefly on a drop of water, and the excess liquid was blotted off on a filter paper (Whatman no. 1) and negatively stained with uranyl acetate (1–2% aqueous solution). The samples were dried and examined in a JEOL 1200EX transmission electron microscope at an accelerating voltage of 80 kV. Images were taken with an AMT 2k CCD camera.
Contact Angle Measurements
We used a ramé-hart goniometer to measure the contact angle of water on mica and on a silicon/silicon oxide surface modified with a monolayer of trichloro(octyl)silane.
AFM images were passed through a threshold filter to filter the background (noise). This resulted in black and white images such as those in Fig. 5, A and B. The surface coverage of the protein was then calculated from such images where the protein appeared as white pixels on a black background. To separate the contribution of fibers and oligomers in the total surface coverage (such as in Fig. 5, D and E) we applied a Matlab particle analysis algorithm that sorts the particles in the images based on surface area. We chose an area corresponding to r = 30 nm as a cutoff below which the particles were considered to be oligomers. Particles with larger area were considered to be fibers.
We purified a matrix protein, TasA, that forms amyloid-like fibers in biofilms (
). Using DLS and AFM we showed that the purified protein appeared as soluble oligomers. These oligomers remained stable in solutions of pH larger than the protein isoelectric point (the calculated isoelectric point of TasA lacking its signal peptide is 5). Under these conditions TasA is negatively charged, and this might explain the stability of the oligomers in solution.
Previously, when we added such protein preparations to the growth medium of mutants lacking TasA they were biologically active in that they complemented the lack of TasA: biofilms that were thin and fragile in the absence of TasA became robust and similar to those made by wild-type cells after the addition of TasA (
). The complementation ability of the purified oligomers indicated that they were precursor to the formation of amyloid-like fibers. In contrast to commonly used purification protocols of recombinant proteins from inclusion bodies, our purification protocol of the matrix protein from the surface of B. subtilis cells yielded an amyloid-like precursor that preserved its native α-helical structure.
Recently, α-synuclein, a human amyloid precursor that is related to Parkinson disease, was isolated under nondenaturing conditions in the presence of phospholipid small unilamellar vesicles where it appeared as α-helically structured tetramers (
). This is the only report to date of a human amyloid precursor that was isolated in a native form. We show here that a bacterial amyloid-like precursor, TasA, can also be purified in a natively folded state and that in similarity with the human example, it is α-helical as well. For comparison, the extensively studied bacterial amyloid precursor CsgA, which is the major subunit of the functional amyloid curli, is commonly purified by overexpression in E. coli as CsgA-His under denaturing conditions (
). Our purification method for TasA therefore offers an alternative that will enable the study of the aggregation of functional amyloids from their natively folded precursors and will therefore represent their condition in vivo.
We further showed that exposure of such oligomers to a hydrophobic surface triggered their aggregation into stretched fibers whereas exposure to acidic solution conditions induced their aggregation into fibrous aggregates. The AFM images of these aggregates showed that they were composed of lined-up oligomers. Formation of similar fibers by the islet amyloid polypeptide, related to type 2 diabetes, has been recently reported (
). The AFM images of a single aggregate being composed of lined-up oligomers revealed a novel perspective on amyloid plaques that have been previously analyzed at lower resolution. Whereas TasA is composed of interspersed hydrophobic/hydrophilic segments, the fact that the fibrous aggregates adsorb better to a hydrophobic surface suggests that they are hydrophobic themselves. Similarly to other amyloid and amyloid-like precursors, we have shown that the aggregates of TasA on hydrophobic surfaces and in an acidic solution changed structure and gained β-sheet characteristics upon aggregation.
What are the in vivo conditions that may be responsible for a local hydrophobic and/or acidic environment within biofilms? We speculate that the assembly of TasA into fibers or fibrous networks over time and space during biofilm formation is triggered by external conditions, similar to those demonstrated here. This idea is supported by the conversion of (what we now identify as) TasA oligomers into amyloid-like fibers in biofilms made by tasA mutant cells (
), might provide the physical triggers required for the aggregation of TasA in vivo.
We thank David Weitz and Susan Lindquist for illuminating discussions, Tom Kodger for help with dynamic light scattering measurements, Martin Mwangi for help with contact angle measurements, Maria Ericsson for help with electron microscopy, the Harvard Center for Nanoscale Systems (CNS) for use of their SEM and AFM imaging facilities (CNS is a member of the National Nanotechnology Infrastructure Network), and the Harvard Microchemistry and Proteomics Analysis Facility for MS and Edman Analysis of TasA.