Norspermidine is not a self-produced trigger for biofilm disassembly.

Formation of Bacillus subtilis biofilms, consisting of cells encapsulated within an extracellular matrix of exopolysaccharide and protein, requires the polyamine spermidine. A recent study reported that (1) related polyamine norspermidine is synthesized by B. subtilis using the equivalent of the Vibrio cholerae biosynthetic pathway, (2) exogenous norspermidine at 25 µM prevents B. subtilis biofilm formation, (3) endogenous norspermidine is present in biofilms at 50-80 µM, and (4) norspermidine prevents biofilm formation by condensing biofilm exopolysaccharide. In contrast, we find that, at concentrations up to 200 µM, exogenous norspermidine promotes biofilm formation. We find that norspermidine is absent in wild-type B. subtilis biofilms at all stages, and higher concentrations of exogenous norspermidine eventually inhibit planktonic growth and biofilm formation in an exopolysaccharide-independent manner. Moreover, orthologs of the V. cholerae norspermidine biosynthetic pathway are absent from B. subtilis, confirming that norspermidine is not physiologically relevant to biofilm function in this species.

Lateral interactions govern self-assembly of the bacterial biofilm matrix protein BslA.

The soil bacterium Bacillus subtilis 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 B. subtilis 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 bslA 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.

Reciprocal sharing of extracellular proteases and extracellular matrix molecules facilitates Bacillus subtilis biofilm formation.

Extracellular proteases are a class of public good that support growth of Bacillus subtilis when nutrients are in a polymeric form. Bacillus subtilis biofilm matrix molecules are another class of public good that are needed for biofilm formation and are prone to exploitation. In this study, we investigated the role of extracellular proteases in B. subtilis biofilm formation and explored interactions between different public good producer strains across various conditions. We confirmed that extracellular proteases support biofilm formation even when glutamic acid provides a freely available nitrogen source. Removal of AprE from the NCIB 3610 secretome adversely affects colony biofilm architecture, while sole induction of WprA activity into an otherwise extracellular protease-free strain is sufficient to promote wrinkle development within the colony biofilm. We found that changing the nutrient source used to support growth affected B. subtilis biofilm structure, hydrophobicity and architecture. We propose that the different phenotypes observed may be due to increased protease dependency for growth when a polymorphic protein presents the sole nitrogen source. We however cannot exclude that the phenotypic changes are due to alternative matrix molecules being made. Co-culture of biofilm matrix and extracellular protease mutants can rescue biofilm structure, yet reliance on extracellular proteases for growth influences population coexistence dynamics. Our findings highlight the intricate interplay between these two classes of public goods, providing insights into microbial social dynamics during biofilm formation across different ecological niches.

Bacillus subtilis extracellular protease production incurs a context-dependent cost.

Microbes encounter a wide range of polymeric nutrient sources in various environmental settings, which require processing to facilitate growth. Bacillus subtilis, a bacterium found in the rhizosphere and broader soil environment, is highly adaptable and resilient due to its ability to utilise diverse sources of carbon and nitrogen. Here, we explore the role of extracellular proteases in supporting growth and assess the cost associated with their production. We provide evidence of the essentiality of extracellular proteases when B. subtilis is provided with an abundant, but polymeric nutrient source and demonstrate the extracellular proteases as a shared public good that can operate over a distance. We show that B. subtilis is subjected to a public good dilemma, specifically in the context of growth sustained by the digestion of a polymeric food source. Furthermore, using mathematical simulations, we uncover that this selectively enforced dilemma is driven by the relative cost of producing the public good. Collectively, our findings reveal how bacteria can survive in environments that vary in terms of immediate nutrient accessibility and the consequent impact on the population composition. These findings enhance our fundamental understanding of how bacteria respond to diverse environments, which has importance to contexts ranging from survival in the soil to infection and pathogenesis scenarios.

Bacillus subtilis Matrix Protein TasA is Interfacially Active, but BslA Dominates Interfacial Film Properties.

Microbial growth often occurs within multicellular communities called biofilms, where cells are enveloped by a protective extracellular matrix. Bacillus subtilis serves as a model organism for biofilm research and produces two crucial secreted proteins, BslA and TasA, vital for biofilm matrix formation. BslA exhibits surface-active properties, spontaneously self-assembling at hydrophobic/hydrophilic interfaces to form an elastic protein film, which renders B. subtilis biofilm surfaces water-repellent. TasA is traditionally considered a fiber-forming protein with multiple matrix-related functions. In our current study, we investigate whether TasA also possesses interfacial properties and whether it has any impact on BslA's ability to form an interfacial protein film. Our research demonstrates that TasA indeed exhibits interfacial activity, partitioning to hydrophobic/hydrophilic interfaces, stabilizing emulsions, and forming an interfacial protein film. Interestingly, TasA undergoes interface-induced restructuring similar to BslA, showing an increase in ß-strand secondary structure. Unlike BslA, TasA rapidly reaches the interface and forms nonelastic films that rapidly relax under pressure. Through mixed protein pendant drop experiments, we assess the influence of TasA on BslA film formation, revealing that TasA and other surface-active molecules can compete for interface space, potentially preventing BslA from forming a stable elastic film. This raises a critical question: how does BslA self-assemble to form the hydrophobic "raincoat" observed in biofilms in the presence of other potentially surface-active species? We propose a model wherein surface-active molecules, including TasA, initially compete with BslA for interface space. However, under lateral compression or pressure, BslA retains its position, expelling other molecules into the bulk. This resilience at the interface may result from structural rearrangements and lateral interactions between BslA subunits. This combined mechanism likely explains BslA's role in forming a stable film integral to B. subtilis biofilm hydrophobicity.

Plant-environment microscopy tracks interactions of Bacillus subtilis with plant roots across the entire rhizosphere.

Our understanding of plant-microbe interactions in soil is limited by the difficulty of observing processes at the microscopic scale throughout plants' large volume of influence. Here, we present the development of three-dimensional live microscopy for resolving plant-microbe interactions across the environment of an entire seedling growing in a transparent soil in tailor-made mesocosms, maintaining physical conditions for the culture of both plants and microorganisms. A tailor-made, dual-illumination light sheet system acquired photons scattered from the plant while fluorescence emissions were simultaneously captured from transparent soil particles and labeled microorganisms, allowing the generation of quantitative data on samples ∼3,600 mm3 in size, with as good as 5 µm resolution at a rate of up to one scan every 30 min. The system tracked the movement of Bacillus subtilis populations in the rhizosphere of lettuce plants in real time, revealing previously unseen patterns of activity. Motile bacteria favored small pore spaces over the surface of soil particles, colonizing the root in a pulsatile manner. Migrations appeared to be directed toward the root cap, the point of "first contact," before the subsequent colonization of mature epidermis cells. Our findings show that microscopes dedicated to live environmental studies present an invaluable tool to understand plant-microbe interactions.

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.

Microbial Primer: An introduction to biofilms - what they are, why they form and their impact on built and natural environments.

Biofilms are complex communities of microbes that are bound by an extracellular macromolecular matrix produced by the residents. Biofilms are the predominant form of microbial life in the natural environment and although they are the leading cause of chronic infections, they are equally deeply connected to our ability to bioremediate waste and toxic materials. Here we highlight the emergent properties of biofilm communities and explore notable biofilms before concluding by providing examples of their major impact on our health and both natural and built environments.

Pulcherrimin formation controls growth arrest of the Bacillus subtilis biofilm.

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.

The Intertwined Roles of Specialized Metabolites within the Bacillus subtilis Biofilm.

Bacteria produce specialized metabolites with a range of functions. In this issue of the Journal of Bacteriology, Schoenborn et al. study the production and role of secondary metabolites during biofilm development and sporulation in Bacillus subtilis (A. A. Schoenborn, S. M. Yannarell, E. D. Wallace, H. Clapper, et al., J Bacteriol 203:e00337-21, 2021, https://doi.org/https://doi.org/10.1128/JB.00337-21). Most metabolites studied are produced during differentiation, and six are required for the development of biofilms and/or spores. The authors propose a model for the timing of production and role in differentiation exerted by each secondary metabolite.

Biofilm Dispersal for Spore Release in Bacillus subtilis.

The dispersal of bacterial cells from a matured biofilm can be mediated either by active or passive mechanisms. In this issue of the Journal of Bacteriology, Nishikawa and Kobayashi demonstrate that the presence of calcium influences the dispersal of spores from the pellicle biofilm of Bacillus subtilis (M. Nishikawa and K. Kobayashi, J Bacteriol 203:e00114-21, 2021, https://doi.org/10.1128/JB.00114-21). The authors propose that temporal heterogeneity in matrix production and chelation of calcium by dipicolinic acid in spores weakens the biofilm matrix and causes passive dispersal.

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.

Biofilm hydrophobicity in environmental isolates of Bacillus subtilis.

Biofilms are communities of bacteria that are attached to a surface and surrounded by an extracellular matrix. The extracellular matrix protects the community from stressors in the environment, making biofilms robust. The Gram-positive soil bacterium Bacillus subtilis, particularly the isolate NCIB 3610, is widely used as a model for studying biofilm formation. B. subtilis NCIB 3610 forms colony biofilms that are architecturally complex and highly hydrophobic. The hydrophobicity is linked, in part, to the localisation of the protein BslA at the surface of the biofilm, which provides the community with increased resistance to biocides. As most of our knowledge about B. subtilis biofilm formation comes from one isolate, it is unclear if biofilm hydrophobicity is a widely distributed feature of the species. To address this knowledge gap, we collated a library of B. subtilis soil isolates and acquired their whole genome sequences. We used our novel isolates to examine biofilm hydrophobicity and found that, although BslA is encoded and produced by all isolates in our collection, hydrophobicity is not a universal feature of B. subtilis colony biofilms. To test whether the matrix exopolymer poly γ-glutamic acid could be masking hydrophobicity in our hydrophilic isolates, we constructed deletion mutants and found, contrary to our hypothesis, that the presence of poly γ-glutamic acid was not the reason for the observed hydrophilicity. This study highlights the natural variation in the properties of biofilms formed by different isolates and the importance of using a more diverse range of isolates as representatives of a species.

The Diverse Structures and Functions of Surfactant Proteins.

Surface tension at liquid-air interfaces is a major barrier that needs to be surmounted by a wide range of organisms; surfactant and interfacially active proteins have evolved for this purpose. Although these proteins are essential for a variety of biological processes, our understanding of how they elicit their function has been limited. However, with the recent determination of high-resolution 3D structures of several examples, we have gained insight into the distinct shapes and mechanisms that have evolved to confer interfacial activity. It is now a matter of harnessing this information, and these systems, for biotechnological purposes.

Genomic Differences between Listeria monocytogenes EGDe Isolates Reveal Crucial Roles for SigB and Wall Rhamnosylation in Biofilm Formation.

Listeria monocytogenes is a Gram-positive firmicute that causes foodborne infections, in part due to its ability to use multiple strategies, including biofilm formation, to survive adverse growth conditions. As a potential way to screen for genes required for biofilm formation, we harnessed the ability of bacteria to accumulate mutations in the genome over time, diverging the properties of seemingly identical strains. By sequencing the genomes of four laboratory reference strains of the commonly used L. monocytogenes EGDe, we showed that each isolate contains single nucleotide polymorphisms (SNPs) compared with the reference genome. We discovered that two SNPs, contained in two independent genes within one of the isolates, impacted biofilm formation. Using bacterial genetics and phenotypic assays, we confirmed that rsbU and rmlA influence biofilm formation. RsbU is the upstream regulator of the alternative sigma factor SigB, and mutation of either rsbU or sigB increased biofilm formation. In contrast, deletion of rmlA, which encodes the first enzyme for TDP-l-rhamnose biosynthesis, resulted in a reduction in the amount of biofilm formed. Further analysis of biofilm formation in a strain that still produces TDP-l-rhamnose but which cannot decorate the wall teichoic acid with rhamnose (rmlT mutant) showed that it is the decorated wall teichoic acid that is required for adhesion of the cells to surfaces. Together, these data uncover novel routes by which biofilm formation by L. monocytogenes can be impacted.IMPORTANCE Biofilms are an important mode of growth in many settings. Here, we looked at small differences in the genomes of the bacterium Listeria monocytogenes isolate EGDe and used them to find out how biofilms form. This important fundamental information may help new treatments to be developed and also highlights the fact that isolates of the same identity often diverge.

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.

Social behaviours by Bacillus subtilis: quorum sensing, kin discrimination and beyond.

Here, we review the multiple mechanisms that the Gram-positive bacterium Bacillus subtilis uses to allow it to communicate between cells and establish community structures. The modes of action that are used are highly varied and include routes that sense pheromone levels during quorum sensing and control gene regulation, the intimate coupling of cells via nanotubes to share cytoplasmic contents, and long-range electrical signalling to couple metabolic processes both within and between biofilms. We explore the ability of B. subtilis to detect 'kin' (and 'cheater cells') by looking at the mechanisms used to potentially ensure beneficial sharing (or limit exploitation) of extracellular 'public goods'. Finally, reflecting on the array of methods that a single bacterium has at its disposal to ensure maximal benefit for its progeny, we highlight that a large future challenge will be integrating how these systems interact in mixed-species communities.

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.

Spermidine promotes Bacillus subtilis biofilm formation by activating expression of the matrix regulator slrR.

Ubiquitous polyamine spermidine is not required for normal planktonic growth of Bacillus subtilis but is essential for robust biofilm formation. However, the structural features of spermidine required for B. subtilis biofilm formation are unknown and so are the molecular mechanisms of spermidine-stimulated biofilm development. We report here that in a spermidine-deficient B. subtilis mutant, the structural analogue norspermidine, but not homospermidine, restored biofilm formation. Intracellular biosynthesis of another spermidine analogue, aminopropylcadaverine, from exogenously supplied homoagmatine also restored biofilm formation. The differential ability of C-methylated spermidine analogues to functionally replace spermidine in biofilm formation indicated that the aminopropyl moiety of spermidine is more sensitive to C-methylation, which it is essential for biofilm formation, but that the length and symmetry of the molecule is not critical. Transcriptomic analysis of a spermidine-depleted B. subtilis speD mutant uncovered a nitrogen-, methionine-, and S-adenosylmethionine-sufficiency response, resulting in repression of gene expression related to purine catabolism, methionine and S-adenosylmethionine biosynthesis and methionine salvage, and signs of altered membrane status. Consistent with the spermidine requirement in biofilm formation, single-cell analysis of this mutant indicated reduced expression of the operons for production of the exopolysaccharide and TasA protein biofilm matrix components and SinR antagonist slrR Deletion of sinR or ectopic expression of slrR in the spermidine-deficient ΔspeD background restored biofilm formation, indicating that spermidine is required for expression of the biofilm regulator slrR Our results indicate that spermidine functions in biofilm development by activating transcription of the biofilm matrix exopolysaccharide and TasA operons through the regulator slrR.

Interfacial self-assembly of a bacterial hydrophobin.

The majority of bacteria in the natural environment live within the confines of a biofilm. The Gram-positive bacterium Bacillus subtilis forms biofilms that exhibit a characteristic wrinkled morphology and a highly hydrophobic surface. A critical component in generating these properties is the protein BslA, which forms a coat across the surface of the sessile community. We recently reported the structure of BslA, and noted the presence of a large surface-exposed hydrophobic patch. Such surface patches are also observed in the class of surface-active proteins known as hydrophobins, and are thought to mediate their interfacial activity. However, although functionally related to the hydrophobins, BslA shares no sequence nor structural similarity, and here we show that the mechanism of action is also distinct. Specifically, our results suggest that the amino acids making up the large, surface-exposed hydrophobic cap in the crystal structure are shielded in aqueous solution by adopting a random coil conformation, enabling the protein to be soluble and monomeric. At an interface, these cap residues refold, inserting the hydrophobic side chains into the air or oil phase and forming a three-stranded ß-sheet. This form then self-assembles into a well-ordered 2D rectangular lattice that stabilizes the interface. By replacing a hydrophobic leucine in the center of the cap with a positively charged lysine, we changed the energetics of adsorption and disrupted the formation of the 2D lattice. This limited structural metamorphosis represents a previously unidentified environmentally responsive mechanism for interfacial stabilization by proteins.