Keith M. Bromley

BslA is a self-assembling bacterial hydrophobin that coats the Bacillus subtilis biofilm

Biofilms represent the predominant mode of microbial growth in the natural environment. Bacillus subtilis is a ubiquitous Gram-positive soil bacterium that functions as an effective plant growth-promoting agent. The biofilm matrix is composed of an exopolysaccharide and an amyloid fiber-forming protein, TasA, and assembles with the aid of a small secreted protein, BslA. Here we show that natively synthesized and secreted BslA forms surface layers around the biofilm. Biophysical analysis demonstrates that BslA can self-assemble at interfaces, forming an elastic film. Molecular function is revealed from analysis of the crystal structure of BslA, which consists of an Ig-type fold with the addition of an unusual, extremely hydrophobic “cap” region. A combination of in vivo biofilm formation and in vitro biophysical analysis demonstrates that the central hydrophobic residues of the cap are essential to allow a hydrophobic, nonwetting biofilm to form as they control the surface activity of the BslA protein. The hydrophobic cap exhibits physiochemical properties remarkably similar to the hydrophobic surface found in fungal hydrophobins; thus, BslA is a structurally defined bacterial hydrophobin. We suggest that biofilms formed by other species of bacteria may have evolved similar mechanisms to provide protection to the resident bacterial community.

An alternate route to phosphorylating DegU of Bacillus subtilis using acetyl phosphate

BackgroundTwo-component signal transduction pathways allow bacteria to sense and respond to the environment. Typically such pathways comprise a sensor histidine kinase and a response regulator. Phosphorylation of the response regulator commonly results in its activation, allowing the protein to bind to target promoter elements to regulate transcription. Several mechanisms are used to prevent inappropriate phosphorylation of the response regulator, thereby ensuring a specific response. In Bacillus subtilis, the DegS-DegU two-component system controls transcription of target genes in a manner dependent on the level of the phosphorylated response regulator, DegU. Previous work has tentatively indicated that DegU, and DegU H12L, a DegU variant which displays enhanced stability of the phosphoryl moiety, can be phosphorylated in the absence of the kinase, DegS.ResultsThe data presented here reveal that DegU H12L requires aspartic acid 56 (D56), the identified DegU phosphorylation site, for its activity. By indirectly measuring the level of DegU ~ P in the cell by assessment of several well recognised DegU regulated processes it was shown that DegU H12L retains its activity in the absence of DegS, and that mutation of D56 produced an inactive protein. Further experiments designed to raise the level of acetyl phosphate within the cell suggest that DegU can be phosphorylated by acetyl phosphate in the absence of degS. Additionally, the phenotypic and biochemical experiments presented indicate that DegU H12L can reliably mimic high levels of phosphorylated DegU.ConclusionsThe ability of acetyl phosphate to modify DegU, and indeed DegU H12L, reveal an additional layer of regulation for DegU phosphorylation that will be relevant when the level of DegS is low or in the absence of degS. Given the number of processes that DegU can activate or inhibit, extensive regulation at a number of levels is required to ensure that the system is not inappropriately stimulated. DegS has both kinase and phosphatase activity and our findings demonstrate that the phosphatase activity of DegS is essential to control the level of DegU phosphate. Overall we contribute to our understanding of how the intricate signalling pathway DegS-DegU is regulated in B. subtilis.

Formation of functional, non-amyloidogenic fibres by recombinant Bacillus subtilis TasA

Bacterial biofilms are communities of microbial cells encased within a self-produced polymeric matrix. In the Bacillus subtilis biofilm matrix the extracellular fibres of TasA are essential. Here a recombinant expression system allows interrogation of TasA, revealing that monomeric and fibre forms of TasA have identical secondary structure, suggesting that fibrous TasA is a linear assembly of globular units. Recombinant TasA fibres form spontaneously, and share the biological activity of TasA fibres extracted from B. subtilis, whereas a TasA variant restricted to a monomeric form is inactive and subjected to extracellular proteolysis. The biophysical properties of both native and recombinant TasA fibres indicate that they are not functional amyloid-like fibres. A gel formed by TasA fibres can recover after physical shear force, suggesting that the biofilm matrix is not static and that these properties may enable B. subtilis to remodel its local environment in response to external cues. Using recombinant fibres formed by TasA orthologues we uncover species variability in the ability of heterologous fibres to cross-complement the B. subtilis tasA deletion. These findings are indicative of specificity in the biophysical requirements of the TasA fibres across different species and/or reflect the precise molecular interactions needed for biofilm matrix assembly. Contributions Conceived and designed the experiments: CE, EE, RG, CEM, RJM, MS, NSW; Performed the experiments: KB, LC, CE, EE, PKF, RG, CEM, RJM, MS, TS; Contributed new analytical tools: CE, EE, RG, TS; Analysed the data: CE, EE, CEM, RJM, MS, LCS, NSW; Wrote the paper: EE, RJM, CEM, MS, NSW.

A phenomenological description of BslA assemblies across multiple length scales

Intrinsically interfacially active proteins have garnered considerable interest recently owing to their potential use in a range of materials applications. Notably, the fungal hydrophobins are known to form robust and well-organized surface layers with high mechanical strength. Recently, it was shown that the bacterial biofilm protein BslA also forms highly elastic surface layers at interfaces. Here we describe several self-assembled structures formed by BslA, both at interfaces and in bulk solution, over a range of length scales spanning from nanometres to millimetres. First, we observe transiently stable and highly elongated air bubbles formed in agitated BslA samples. We study their behaviour in a range of solution conditions and hypothesize that their dissipation is a consequence of the slow adsorption kinetics of BslA to an air–water interface. Second, we describe elongated tubules formed by BslA interfacial films when shear stresses are applied in both a Langmuir trough and a rheometer. These structures bear a striking resemblance, although much larger in scale, to the elongated air bubbles formed during agitation. Taken together, this knowledge will better inform the conditions and applications of how BslA can be used in the stabilization of multi-phase materials. This article is part of the themed issue ‘Soft interfacial materials: from fundamentals to formulation’.

Bifunctionality of a biofilm matrix protein controlled by redox state

Significance The biofilm matrix is a critical target in the hunt for novel strategies to destabilize or stabilize biofilms. Knowledge of the processes controlling matrix assembly is therefore an essential prerequisite to exploitation. Here, we highlight that the complexity of the biofilm matrix is even higher than anticipated, with one matrix component making two independent functional contributions to the community. The influence the protein exerts is dependent on the local environmental properties, providing another dimension to consider during analysis. These findings add to the evidence that bacteria can evolve multifunctional uses for the extracellular matrix components. 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.

Formation of functional, non-amyloidogenic fibres by recombinant Bacillus subtilis TasA: Functional recombinant non-amyloidogenic TasA fibres

Bacterial biofilms are communities of microbial cells encased within a self‐produced polymeric matrix. In the Bacillus subtilis biofilm matrix, the extracellular fibres of TasA are essential. Here, a recombinant expression system allows interrogation of TasA, revealing that monomeric and fibre forms of TasA have identical secondary structure, suggesting that fibrous TasA is a linear assembly of globular units. Recombinant TasA fibres form spontaneously, and share the biological activity of TasA fibres extracted from B. subtilis, whereas a TasA variant restricted to a monomeric form is inactive and subjected to extracellular proteolysis. The biophysical properties of both native and recombinant TasA fibres indicate that they are not functional amyloid‐like fibres. A gel formed by TasA fibres can recover after physical shear force, suggesting that the biofilm matrix is not static and that these properties may enable B. subtilis to remodel its local environment in response to external cues. Using recombinant fibres formed by TasA orthologues we uncover species variability in the ability of heterologous fibres to cross‐complement the B. subtilis tasA deletion. These findings are indicative of specificity in the biophysical requirements of the TasA fibres across different species and/or reflect the precise molecular interactions needed for biofilm matrix assembly.

BslA-stabilized emulsion droplets with designed microstructure

Emulsions are a central component of many modern formulations in food, pharmaceuticals, agrichemicals and personal care products. The droplets in these formulations are limited to being spherical as a consequence of the interfacial tension between the dispersed phase and continuous phase. The ability to control emulsion droplet morphology and stabilize non-spherical droplets would enable the modification of emulsion properties such as stability, substrate binding, delivery rate and rheology. One way of controlling droplet microstructure is to apply an elastic film around the droplet to prevent it from relaxing into a sphere. We have previously shown that BslA, an interfacial protein produced by the bacterial genus Bacillus, forms an elastic film when exposed to an oil- or air–water interface. Here, we highlight BslAs ability to stabilize anisotropic emulsion droplets. First, we show that BslA is capable of arresting dynamic emulsification processes leading to emulsions with variable morphologies depending on the conditions and emulsification technique applied. We then show that frozen emulsion droplets can be manipulated to induce partial coalescence. The structure of the partially coalesced droplets is retained after melting, but only when there is sufficient free BslA in the continuous phase. That the fidelity of replication can be tuned by adjusting the amount of free BslA in solution suggests that freezing BslA-stabilized droplets disrupts the BslA film. Finally, we use BslAs ability to preserve emulsion droplet structural integrity throughout the melting process to design emulsion droplets with a chosen shape and size.

Natural variations in the biofilm-associated protein BslA from the genus Bacillus

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.

Evolutionary variations in the biofilm-associated protein BslA from the genus Bacillus

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.