The putative role of the epipeptide EpeX in Bacillus subtilis intra-species competition.

Bacteria engage in competitive interactions with neighbours that can either be of the same or different species. Multiple mechanisms are deployed to ensure the desired outcome and one tactic commonly implemented is the production of specialised metabolites. The Gram-positive bacterium Bacillus subtilis uses specialized metabolites as part of its intra-species competition determinants to differentiate between kin and non-kin isolates. It is, however, unknown if the collection of specialized metabolites defines competitive fitness when the two isolates start as a close, interwoven community that grows into a densely packed colony biofilm. Moreover, the identity of specialized metabolites that have an active role in defining the outcome of an intra-species interaction has not been revealed. Here, we determine the competition outcomes that manifest when 21 environmental isolates of B. subtilis are individually co-incubated with the model isolate NCIB 3610 in a colony biofilm. We correlated these data with the suite of specialized metabolite biosynthesis clusters encoded by each isolate. We found that the epeXEPAB gene cluster was primarily present in isolates with a strong competitive phenotype. This cluster is responsible for producing the epipeptide EpeX. We demonstrated that EpeX is a competition determinant of B. subtilis in an otherwise isogenic context for NCBI 3610. However, when we competed the NCIB 3610 EpeX-deficient strain against our suite of environmental isolates we found that the impact of EpeX in competition is isolate-specific, as only one of the 21 isolates showed increased survival when EpeX was lacking. Taken together, we have shown that EpeX is a competition determinant used by B. subtilis that impacts intra-species interactions but only in an isolate-specific manner.

Density and temperature controlled fluid extraction in a bacterial biofilm is determined by poly-γ-glutamic acid production.

A hallmark of microbial biofilms is the self-production of an extracellular molecular matrix that encases the resident cells. The matrix provides protection from the environment, while spatial heterogeneity of gene expression influences the structural morphology and colony spreading dynamics. Bacillus subtilis is a model bacterial system used to uncover the regulatory pathways and key building blocks required for biofilm growth and development. In this work, we report on the emergence of a highly active population of bacteria during the early stages of biofilm formation, facilitated by the extraction of fluid from the underlying agar substrate. We trace the origin of this fluid extraction to the production of poly-γ-glutamic acid (PGA). The flagella-dependent activity develops behind a moving front of fluid that propagates from the boundary of the biofilm towards the interior. The extent of fluid proliferation is controlled by the presence of extracellular polysaccharides (EPS). We also find that PGA production is positively correlated with higher temperatures, resulting in high-temperature mature biofilm morphologies that are distinct from the rugose colony biofilm architecture typically associated with B. subtilis. Although previous reports have suggested that PGA production does not play a major role in biofilm morphology in the undomesticated isolate NCIB 3610, our results suggest that this strain produces distinct biofilm matrices in response to environmental conditions.

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.

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.

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.

Bifunctionality of a biofilm matrix protein controlled by redox state.

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.