The protective layer of biofilm: a repellent function for a new class of amphiphilic proteins

Bacteria can survive harsh conditions when growing in complex communities of cells known as biofilms. The matrix of the biofilm presents a scaffold where cells are attached to each other and to the surface. The biofilm matrix is also a protective barrier that confers tolerance against various antimicrobial agents. In this issue of Molecular Microbiology, Kobayashi and Iwano (2012) show that the liquid permeability of Bacillus subtilis biofilms is determined by a small secreted protein, i.e. BslA (formerly called YuaB). BslA is important for the proper development of biofilms, but unlike exopolysaccharide and TasA, is not directly involved in cell cluster formation, and is synthesized following the production of exopolysaccharide and amyloid fibres. The amphiphilic BslA protein forms a polymer in vitro and localizes in vivo to the surface of the biofilm. The microstructures of the biofilm wrinkles are reduced in the bslA mutant strain and the liquid repellency of the biofilm surface is diminished. Exogenously added BslA(42-181) protein complements the bslA mutation and restores not only water repellency, but also the formation of aerial structures. This study demonstrates that amphiphilic proteins have an important role in liquid repellency of biofilms and it suggests that these polymers contribute to antimicrobial resistance.     

A major protein component of the Bacillus subtilis biofilm matrix

Microbes construct structurally complex multicellular communities (biofilms) through production of an extracellular matrix. Here we present evidence from scanning electron microscopy showing that a wild strain of the Gram positive bacterium Bacillus subtilis builds such a matrix. Genetic, biochemical and cytological evidence indicates that the matrix is composed predominantly of a protein component, TasA, and an exopolysaccharide component. The absence of TasA or the exopolysaccharide resulted in a residual matrix, while the absence of both components led to complete failure to form complex multicellular communities. Extracellular complementation experiments revealed that a functional matrix can be assembled even when TasA and the exopolysaccharide are produced by different cells, reinforcing the view that the components contribute to matrix formation in an extracellular manner. Having defined the major components of the biofilm matrix and the control of their synthesis by the global regulator SinR, we present a working model for how B. subtilis switches between nomadic and sedentary lifestyles.     

Functional Analysis of the Accessory Protein TapA in Bacillus subtilis Amyloid Fiber Assembly

Bacillus subtilis biofilm formation relies on the assembly of a fibrous scaffold formed by the protein TasA. TasA polymerizes into highly stable fibers with biochemical and morphological features of functional amyloids. Previously, we showed that assembly of TasA fibers requires the auxiliary protein TapA. In this study, we investigated the roles of TapA sequences from the C-terminal and N-terminal ends and TapA cysteine residues in its ability to promote the assembly of TasA amyloid-like fibers. We found that the cysteine residues are not essential for the formation of TasA fibers, as their replacement by alanine residues resulted in only minor defects in biofilm formation. Mutating sequences in the C-terminal half had no effect on biofilm formation. However, we identified a sequence of 8 amino acids in the N terminus that is key for TasA fiber formation. Strains expressing TapA lacking these 8 residues were completely defective in biofilm formation. In addition, this TapA mutant protein exhibited a dominant negative effect on TasA fiber formation. Even in the presence of wild-type TapA, the mutant protein inhibited fiber assembly in vitro and delayed biofilm formation in vivo. We propose that this 8-residue sequence is crucial for the formation of amyloid-like fibers on the cell surface, perhaps by mediating the interaction between TapA or TapA and TasA molecules.     

Tapping into the biofilm: insights into assembly and disassembly of a novel amyloid fibre in Bacillus subtilis

The Bacillus subtilis biofilm matrix harbours a functionally important amyloid fibre network composed of the protein TasA. Previous studies showed that the protein TapA (formerly YqxM) plays roles in TasA fibre formation and disassembly. In this issue, Romero et al., 2011 show that TapA is a component of the fibre and links it to the peptidoglycan. Therefore, TapA directs TasA fibre formation, links it to the cell wall and, most likely, participates in fibre dispersal. These results provide important insights into the control of biofilm formation in B. subtilis and, potentially, the regulation of amyloid fibre formation in diverse species.     

Bistability and biofilm formation in Bacillus subtilis

Biofilms of Bacillus subtilis consist of long chains of cells that are held together in bundles by an extracellular matrix of exopolysaccharide and the protein TasA. The exopolysaccharide is produced by enzymes encoded by the epsA-O operon and the gene encoding TasA is located in the yqxM-sipW-tasA operon. Both operons are under the control of the repressor SinR. Derepression is mediated by the antirepressor SinI, which binds to SinR with a 1:1 stoichiometry. Paradoxically, in medium promoting derepression of the matrix operons, the overall concentration of SinR in the culture greatly exceeded that of SinI. We show that under biofilm-promoting conditions sinI, which is under the control of the response regulator Spo0A, was expressed only in a small subpopulation of cells, whereas sinR was expressed in almost all cells. Activation of Spo0A is known to be subject to a bistable switch, and we infer that SinI reaches levels sufficient to trigger matrix production only in the subpopulation of cells in which Spo0A is active. Additionally, evidence suggests that sinI is expressed at intermediate, but not low or high, levels of Spo0A activity, which may explain why certain nutritional conditions are more effective in promoting biofilm formation than others.     

Extracellular DNA facilitates the formation of functional amyloids in Staphylococcus aureus biofilms

Persistent staphylococcal infections often involve surface‐associated communities called biofilms. Staphylococcus aureus biofilm development is mediated by the co‐ordinated production of the biofilm matrix, which can be composed of polysaccharides, extracellular DNA (eDNA) and proteins including amyloid fibers. The nature of the interactions between matrix components, and how these interactions contribute to the formation of matrix, remain unclear. Here we show that the presence of eDNA in S. aureus biofilms promotes the formation of amyloid fibers. Conditions or mutants that do not generate eDNA result in lack of amyloids during biofilm growth despite the amyloidogeneic subunits, phenol soluble modulin peptides, being produced. In vitro studies revealed that the presence of DNA promotes amyloid formation by PSM peptides. Thus, this work exposes a previously unacknowledged interaction between biofilm matrix components that furthers our understanding of functional amyloid formation and S. aureus biofilm biology.     

An accessory protein required for anchoring and assembly of amyloid fibres in B. subtilis biofilms

Cells within Bacillus subtilis biofilms are held in place by an extracellular matrix that contains cell-anchored amyloid fibres, composed of the amyloidogenic protein TasA. As biofilms age they disassemble because the cells release the amyloid fibres. This release appears to be the consequence of incorporation of D-tyrosine, D-leucine, D-tryptophan and D-methionine into the cell wall. Here, we characterize the in vivo roles of an accessory protein TapA (TasA anchoring/assembly protein; previously YqxM) that serves both to anchor the fibres to the cell wall and to assemble TasA into fibres. TapA is found in discrete foci in the cell envelope and these foci disappear when cells are treated with a mixture of D-amino acids. Purified cell wall sacculi retain a functional form of this anchoring protein such that purified fibres can be anchored to the sacculi in vitro. In addition, we show that TapA is essential for the proper assembly of the fibres. Its absence results in a dramatic reduction in TasA levels and what little TasA is left produces only thin fibres that are not anchored to the cell.     

The majority of the matrix protein TapA is dispensable for Bacillus subtilis colony biofilm architecture

Biofilm formation is a co-operative behaviour, where microbial cells become embedded in an extracellular matrix. This biomolecular matrix helps manifest the beneficial or detrimental outcome mediated by the collective of cells. Bacillus subtilis is an important bacterium for understanding the principles of biofilm formation. The protein components of the B. subtilis matrix include the secreted proteins BslA, which forms a hydrophobic coat over the biofilm, and TasA, which forms protease-resistant fibres needed for structuring. TapA is a secreted protein also needed for biofilm formation and helps in vivo TasA-fibre formation but is dispensable for in vitro TasA-fibre assembly. We show that TapA is subjected to proteolytic cleavage in the colony biofilm and that only the first 57 amino acids of the 253-amino acid protein are required for colony biofilm architecture. Through the construction of a strain which lacks all eight extracellular proteases, we show that proteolytic cleavage by these enzymes is not a prerequisite for TapA function. It remains unknown why TapA is synthesised at 253 amino acids when the first 57 are sufficient for colony biofilm structuring; the findings do not exclude the core conserved region of TapA having a second role beyond structuring the B. subtilis colony biofilm.     

Isolation,Characterization, and Aggregation of a Structured Bacterial Matrix Precursor

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.     

The FapF Amyloid Secretion Transporter Possesses an Atypical Asymmetric Coiled Coil

Gram-negative bacteria possess specialized biogenesis machineries that facilitate the export of amyloid subunits, the fibers of which are key components of their biofilm matrix. The secretion of bacterial functional amyloid requires a specialized outer-membrane protein channel through which unfolded amyloid substrates are translocated. We previously reported the crystal structure of the membrane-spanning domain of the amyloid subunit transporter FapF from Pseudomonas. However, the structure of the periplasmic domain, which is essential for amyloid transport, is yet to be determined. Here, we present the crystal structure of the N-terminal periplasmic domain at 1.8-Å resolution. This domain forms a novel asymmetric trimeric coiled coil that possesses a single buried tyrosine residue as well as an extensive hydrogen-bonding network within a glutamine layer. This new structural insight allows us to understand this newly described functional amyloid secretion system in greater detail.     

Inhibitory effects of D-amino acids on Staphylococcus aureus biofilm development

Biofilms are communities of cells held together by a self-produced extracellular matrix typically consisting of protein, exopolysaccharide, and often DNA. A natural signal for biofilm disassembly in Bacillus subtilis is certain D-amino acids, which are incorporated into the peptidoglycan and trigger the release of the protein component of the matrix. D-amino acids also prevent biofilm formation by the related Gram-positive bacterium Staphylococcus aureus. Here we employed fluorescence microscopy and confocal laser scanning microscopy to investigate how D-amino acids prevent biofilm formation by S. aureus. We report that biofilm formation takes place in two stages, initial attachment to surfaces, resulting in small foci, and the subsequent growth of the foci into large aggregates. D-amino acids did not prevent the initial surface attachment of cells but blocked the subsequent growth of the foci into larger assemblies of cells. Using protein- and polysaccharide-specific stains, we have shown that D-amino acids inhibited the accumulation of the protein component of the matrix but had little effect on exopolysaccharide production and localization within the biofilm. We conclude that D-amino acids act in an analogous manner to prevent biofilm development in B. subtilis and S. aureus. Finally, to investigate the potential utility of D-amino acids in preventing device-related infections, we have shown that surfaces impregnated with D-amino acids were effective in preventing biofilm growth.     

Dimethyl sulfoxide and ethanol elicit increased amyloid biogenesis and amyloid-integrated biofilm formation in Escherichia coli

Escherichia coli directs the assembly of functional amyloid fibers termed "curli" that mediate adhesion and biofilm formation. We discovered that E. coli exhibits a tunable and selective increase in curli protein expression and fiber assembly in response to moderate concentrations of dimethyl sulfoxide (DMSO) and ethanol. Furthermore, the molecular alterations resulted in dramatic functional phenotypes associated with community behavior, including (i) cellular agglutination in broth, (ii) altered colony morphology, and (iii) increased biofilm formation. Solid-state nuclear magnetic resonance (NMR) spectra of intact pellicles formed in the presence of [(13)C(2)]DMSO confirmed that DMSO was not being transformed and utilized directly for metabolism. Collectively, the chemically induced phenotypes emphasize the plasticity of E. coli's response to environmental stimuli to enhance amyloid production and amyloid-integrated biofilm formation. The data also support our developing model of the extracellular matrix as an organized assembly of polymeric components, including amyloid fibers, in which composition relates to bacterial physiology and community function.