Biofilm formation is a process in which microbial cells aggregate to form collectives that are embedded in a self-produced extracellular matrix. Bacillus subtilis is a Gram-positive bacterium that is ...used to dissect the mechanisms controlling matrix production and the subsequent transition from a motile planktonic cell state to a sessile biofilm state. The collective nature of life in a biofilm allows emergent properties to manifest, and B. subtilis biofilms are linked with novel industrial uses as well as probiotic and biocontrol processes. In this Review, we outline the molecular details of the biofilm matrix and the regulatory pathways and external factors that control its production. We explore the beneficial outcomes associated with biofilms. Finally, we highlight major advances in our understanding of concepts of microbial evolution and community behaviour that have resulted from studies of the innate heterogeneity of biofilms.
Recent discoveries have implicated the gut microbiome in the progression and severity of Parkinson’s disease; however, how gut bacteria affect such neurodegenerative disorders remains unclear. Here, ...we report that the Bacillus subtilis probiotic strain PXN21 inhibits α-synuclein aggregation and clears preformed aggregates in an established Caenorhabditis elegans model of synucleinopathy. This protection is seen in young and aging animals and is partly mediated by DAF-16. Multiple B. subtilis strains trigger the protective effect via both spores and vegetative cells, partly due to a biofilm formation in the gut of the worms and the release of bacterial metabolites. We identify several host metabolic pathways differentially regulated in response to probiotic exposure, including sphingolipid metabolism. We further demonstrate functional roles of the sphingolipid metabolism genes lagr-1, asm-3, and sptl-3 in the anti-aggregation effect. Our findings provide a basis for exploring the disease-modifying potential of B. subtilis as a dietary supplement.
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•B. subtilis PXN21 inhibits and reverses α-syn aggregation in a C. elegans model•Spores and vegetative cells protect through different mechanisms•The probiotic inhibits α-syn aggregation by changing the host sphingolipid metabolism•Biofilm formation in the gut and bacterial metabolites reduce α-syn aggregation
How the gut microbiome affects Parkinson’s disease remains unclear. Goya et al. show that the probiotic B. subtilis strain PXN21 inhibits and clears α-synuclein aggregation in a C. elegans model. The bacterium acts via metabolites and biofilm formation to activate protective pathways in the host, including DAF-16/FOXO and sphingolipid metabolism.
Biofilms are communities of microbial cells that are encapsulated within a self-produced polymeric matrix. The matrix is critical to the success of biofilms in diverse habitats; however, many details ...of the composition, structure, and function remain enigmatic. Biofilms formed by the Gram-positive bacterium Bacillus subtilis depend on the production of the secreted film-forming protein BslA. Here, we show that a gradient of electron acceptor availability through the depth of the biofilm gives rise to two distinct functional roles for BslA and that these roles can be genetically separated through targeted amino acid substitutions. We establish that monomeric BslA is necessary and sufficient to give rise to complex biofilm architecture, whereas dimerization of BslA is required to render the community hydrophobic. Dimerization of BslA, mediated by disulfide bond formation, depends on two conserved cysteine residues located in the C-terminal region. Our findings demonstrate that bacteria have evolved multiple uses for limited elements in the matrix, allowing for alternative responses in a complex, changing environment.
BslA is a protein secreted by Bacillus subtilis which forms a hydrophobic film that coats the biofilm surface and renders it water-repellent. We have characterised three orthologues of BslA from ...Bacillus amyloliquefaciens, Bacillus licheniformis and Bacillus pumilus as well as a paralogue from B. subtilis called YweA. We find that the three orthologous proteins can substitute for BslA in B. subtilis and confer a degree of protection, whereas YweA cannot. The degree to which the proteins functionally substitute for native BslA correlates with their in vitro biophysical properties. Our results demonstrate the use of naturally-evolved variants to provide a framework for teasing out the molecular basis of interfacial self-assembly.
Highlights • Biofilms are structured bacterial communities encased in an extracellular matrix. • The biofilm formed by Bacillus subtilis is non-wetting. • The matrix component BslA confers ...hydrophobicity to the community. • BslA has a highly hydrophobic cap region appended to an immunoglobulin scaffold. • The BslA cap region alters its structure in an environmentally responsive manner.
Biofilm formation by Bacillus subtilis is a communal process that culminates in the formation of architecturally complex multicellular communities. Here we reveal that the transition of the biofilm ...into a nonexpanding phase constitutes a distinct step in the process of biofilm development. Using genetic analysis we show that B. subtilis strains lacking the ability to synthesize pulcherriminic acid form biofilms that sustain the expansion phase, thereby linking pulcherriminic acid to growth arrest. However, production of pulcherriminic acid is not sufficient to block expansion of the biofilm. It needs to be secreted into the extracellular environment where it chelates Fe3+ from the growth medium in a nonenzymatic reaction. Utilizing mathematical modeling and a series of experimental methodologies we show that when the level of freely available iron in the environment drops below a critical threshold, expansion of the biofilm stops. Bioinformatics analysis allows us to identify the genes required for pulcherriminic acid synthesis in other Firmicutes but the patchwork presence both within and across closely related species suggests loss of these genes through multiple independent recombination events. The seemingly counterintuitive self-restriction of growth led us to explore if there were any benefits associated with pulcherriminic acid production. We identified that pulcherriminic acid producers can prevent invasion by neighboring communities through the generation of an “iron-free” zone, thereby addressing the paradox of pulcherriminic acid production by B. subtilis.
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.
In the Bacillus subtilis biofilm matrix, TapA undergoes proteolytic cleavage. TapA secretion is aided by the action of a signal peptidease called SipW. An unknown protease results in the removal of the C‐terminus and the serine exoprotease Vpr cleaves the N‐terminus. Although proteolytic processing was not essential for TapA activity, it was found that a minimal functional form (yellow) was sufficient to facilitate rugose biofilm architecture.
The soil bacterium
is a model organism to investigate the formation of biofilms, the predominant form of microbial life. The secreted protein BslA self-assembles at the surface of the biofilm to give ...the
biofilm its characteristic hydrophobicity. To understand the mechanism of BslA self-assembly at interfaces, here we built a molecular model based on the previous BslA crystal structure and the crystal structure of the BslA paralogue YweA that we determined. Our analysis revealed two conserved protein-protein interaction interfaces supporting BslA self-assembly into an infinite 2-dimensional lattice that fits previously determined transmission microscopy images. Molecular dynamics simulations and in vitro protein assays further support our model of BslA elastic film formation, while mutagenesis experiments highlight the importance of the identified interactions for biofilm structure. Based on this knowledge, YweA was engineered to form more stable elastic films and rescue biofilm structure in
deficient strains. These findings shed light on protein film assembly and will inform the development of BslA technologies which range from surface coatings to emulsions in fast-moving consumer goods.
Membrane-anchored lipoproteins have a broad range of functions and play key roles in several cellular processes in Gram-positive bacteria. BA0330 and BA0331 are the only lipoproteins among the 11 ...known or putative polysaccharide deacetylases of Bacillus anthracis. We found that both lipoproteins exhibit unique characteristics. BA0330 and BA0331 interact with peptidoglycan, and BA0330 is important for the adaptation of the bacterium to grow in the presence of a high concentration of salt, whereas BA0331 contributes to the maintenance of a uniform cell shape. They appear not to alter the peptidoglycan structure and do not contribute to lysozyme resistance. The high resolution x-ray structure of BA0330 revealed a C-terminal domain with the typical fold of a carbohydrate esterase 4 and an N-terminal domain unique for this family, composed of a two-layered (4 + 3) β-sandwich with structural similarity to fibronectin type 3 domains. Our data suggest that BA0330 and BA0331 have a structural role in stabilizing the cell wall of B. anthracis.
Background: BA0330 and BA0331 are the only two lipoproteins among 11 known or putative polysaccharide deacetylases from Bacillus anthracis.
Results: Both proteins lack deacetylase activity and are important for cell shape maintenance (BA0331) or high salt stress adaptation of the bacterium (BA0330).
Conclusion: BA0330 and BA0331 stabilize the cell wall of B. anthracis.
Significance: Understanding the mechanisms by which lipoproteins maintain cell wall integrity.
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