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.</jats:p

The Role of Functional Amyloids in Multicellular Growth and Development of Gram-Positive Bacteria

Amyloid fibrils play pivotal roles in all domains of life. In bacteria, these fibrillar structures are often part of an extracellular matrix that surrounds the producing organism and thereby provides protection to harsh environmental conditions. Here, we discuss the role of amyloid fibrils in the two distant Gram-positive bacteria, Streptomyces coelicolor and Bacillus subtilis. We describe how amyloid fibrils contribute to a multitude of developmental processes in each of these systems, including multicellular growth and community development. Despite this variety of tasks, we know surprisingly little about how their assembly is organized to fulfill all these roles

<i>Bacillus subtilis</i> 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 β-strand secondary structure. Unlike BslA, TasA rapidly reaches the interface and forms non-elastic 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

Biofilm Matrixome: Extracellular Components in Structured Microbial Communities

Biofilms consist of microbial communities embedded in a 3D extracellular matrix. The matrix is composed of a complex array of extracellular polymeric substances (EPS) that contribute to the unique attributes of biofilm lifestyle and virulence. This ensemble of chemically and functionally diverse biomolecules is termed the 'matrixome'. The composition and mechanisms of EPS matrix formation, and its role in biofilm biology, function, and microenvironment are being revealed. This perspective article highlights recent advances about the multifaceted role of the 'matrixome' in the development, physical-chemical properties, and virulence of biofilms. We emphasize that targeting biofilm-specific conditions such as the matrixome could lead to precise and effective antibiofilm approaches. We also discuss the limited knowledge in the context of polymicrobial biofilms, and the need for more in-depth analyses of the EPS matrix in mixed communities that are associated with many human infectious diseases. Keywords: extracellular matrix; extracellular polymeric substances (EPSs); microenvironments; polymicrobial biofilm; spatial organization; virulence

Constituent of extracellular polymeric substances (EPS) produced by a range of soil bacteria and fungi

Extracellular polymeric substances (EPS) produced by soil bacteria and fungi are crucial for microbial growth and provide many functions for the soil and its microbes. EPS composition may depend on microbial community composition and the soil physical and chemical environment, nevertheless, not much is known about the EPS constituents’ specific roles nor how they interact to alter biofilm’s functions. We hypothesized that EPS production would be enhanced with the presence of a surface and with a more labile carbon source. Also, that even though carbohydrates and proteins are the main constituents of EPS, we could still find quantifiable amounts of mannosamine and galactosamine (two amino sugars previously shown to be part of microbial biofilms). Ten soil bacterial and ten soil fungal species were cultured with glycerol or starch and with or without a quartz matrix. After a 4-day cultivation, EPS were extracted, and seven constituents were determined: carbohydrates, DNA, proteins, muramic acid, mannosamine, galactosamine, and glucosamine. We found EPS composition was strongly modified by microbial type, whereas differences in EPS production were driven mostly by environmental conditions. The EPS-carbohydrate/protein ratio was higher for cultures grown in starch media than in glycerol and increased in the presence of quartz. EPS-carbohydrate concentration reflected environmental changes of substrate quality and surface presence. Contrastingly, changes in the other EPS constituent composition are likely due to intrinsic microbial characteristics. Our findings open the pathway to study microbial biofilms in more complex environments (such as soils) and shed light to the importance of extracellular structures to microbial processes

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.</p

Biofilm inspired fabrication of functional bacterial cellulose through ex-situ and in-situ approaches

Bacterial cellulose (BC) has been explored for use in a range of applications including tissue engineering and textiles. BC can be produced from waste streams, but sustainable approaches are needed for functionalisation. To this end, BslA, a B. subtilis biofilm protein was produced recombinantly with and without a cellulose binding module (CBM) and the cell free extract was used to treat BC either ex-situ, through drip coating or in-situ, by incorporating during fermentation. The results showed that ex-situ modified BC increased the hydrophobicity and water contact angle reached 120°. In-situ experiments led to a BC film morphological change and mechanical testing demonstrated that addition of BslA with CBM resulted in a stronger, more elastic material. This study presents a nature inspired approach to functionalise BC using a biofilm hydrophobin, and we demonstrate that recombinant proteins could be effective and sustainable molecules for functionalisation of BC materials

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.</p

Bacillus subtilis biofilm formation and social interactions

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.</p

Impact of cell signals and pyocyanin on mixed-biofilms of Pseudomonas aeruginosa PaO1 and Escherichia coli K-12

Biofilms occur when microorganisms attach to surfaces, replicate and secrete dense polymers to the extent where the organisms become physically encapsulated. As biofilms often arise where there is a high level of diversity of microbial species, there is a high-likelihood that biofilms will contain more than one species of microorganism. These mixed-species biofilms have been shown to be advantageous to either one or multiple species within these biofilms. Pseudomonas aeruginosa and Escherichia coli are two bacteria of interest that are commonly involved in infections of humans and are known biofilm-producers that have generated dual-species biofilms in vitro. In recent years it has been shown that the formation of mixed-species biofilms of these two bacterial species is beneficial to either one or both bacteria in terms of adherence to surfaces, formation of the biofilm matrix, viable cell concentrations within mixed-species biofilms and resistance to external threats in the form of synthetic antimicrobial agents. The interactions between these two organisms in large part had been attributed to secretion of quorum-sensing signals and others molecules produced as a by-product of metabolic processes. This study investigated how the cell-signalling molecules, indole produced by E. coli and n-acyl homoserine lactones, n-butyryl homoserine lactone (C4-HSL) and n-3-oxo-dodecanoyl (3-oxo-C12-HSL) produced by P. aeruginosa would affect interactions between these organisms within mixed-species biofilms. Previous work had suggested that cell signally molecules would result in cross species effects. The work conducted within this study had found that n-acyl homoserine lactones had no discernible effect on E. coli biofilm formation and that indole did not have a strong influence over biofilm and production of the metabolic by-product and virulence factor pyocyanin, produced by P. aeruginosa PaO1. Pyocyanin however produced within mixed-species biofilms was shown to reduce viable wild-type E. coli K-12 to similar concentrations to that of an indole-deficient strain. The results within this thesis also found that reducing the incubation temperature increased biofilm formation of E. coli. For instance, viable cell retention increased from 2.20x 105 cfu at 37ºC to 8.73x107 cfu at 25ºC within 72 hours whereas total biofilm production increased from an absorbance reading of 0.22 at 37ºC to an absorbance reading of 0.98 at 30ºC within 24 hours. Reductions in pyocyanin in P. aeruginosa PaO1 cultures also coincided with reductions in temperature thus reducing the temperature from 37ºC to 25ºC reduced pyocyanin concentrations of P. aeruginosa PaO1 cultures, grown for 72 hours from 149.34 μM to 23.10 μM. A result of reducing the incubation temperature of mixed-species biofilm cultures, other than noticeable reductions in concentrations of pyocyanin, was an increase in E. coli cell recovery. For instance, within 72 hours E. coli viable cell recovery increased from 1.9x102 cfu to 2.0 x105 cfu when the temperature was reduced from 37ºC to 25ºC. This indicated that E. coli within mixed-species biofilms alongside P. aeruginosa in an enclosed system was more likely to survive at lower temperatures than higher temperatures ergo are more likely to survive within mixed-species biofilms outside rather than inside of the human body with a core temperature of approximately 37ºC. This highlights a potential issue where the bacteria contained within these biofilms that have been shown in some instances to be more resistant to antimicrobials than single-species biofilms, have a higher likelihood of remaining viable for an extended period of time outside of the host reservoir. Increasing the duration of viability of E. coli and P. aeruginosa in mixed-species biofilms in areas that are prone to containing biofilms and are commonly associated with the presence of human beings can potentially increase the chances of contracting these infectious agents