Z-form extracellular DNA is a structural component of the bacterial biofilm matrix.

Biofilms are community architectures adopted by bacteria inclusive of a self-formed extracellular matrix that protects resident bacteria from diverse environmental stresses and, in many species, incorporates extracellular DNA (eDNA) and DNABII proteins for structural integrity throughout biofilm development. Here, we present evidence that this eDNA-based architecture relies on the rare Z-form. Z-form DNA accumulates as biofilms mature and, through stabilization by the DNABII proteins, confers structural integrity to the biofilm matrix. Indeed, substances known to drive B-DNA into Z-DNA promoted biofilm formation whereas those that drive Z-DNA into B-DNA disrupted extant biofilms. Importantly, we demonstrated that the universal bacterial DNABII family of proteins stabilizes both bacterial- and host-eDNA in the Z-form in situ. A model is proposed that incorporates the role of Z-DNA in biofilm pathogenesis, innate immune response, and immune evasion.

Extracellular DNA Promotes Efficient Extracellular Electron Transfer by Pyocyanin in Pseudomonas aeruginosa Biofilms.

Redox cycling of extracellular electron shuttles can enable the metabolic activity of subpopulations within multicellular bacterial biofilms that lack direct access to electron acceptors or donors. How these shuttles catalyze extracellular electron transfer (EET) within biofilms without being lost to the environment has been a long-standing question. Here, we show that phenazines mediate efficient EET through interactions with extracellular DNA (eDNA) in Pseudomonas aeruginosa biofilms. Retention of pyocyanin (PYO) and phenazine carboxamide in the biofilm matrix is facilitated by eDNA binding. In vitro, different phenazines can exchange electrons in the presence or absence of DNA and can participate directly in redox reactions through DNA. In vivo, biofilm eDNA can also support rapid electron transfer between redox active intercalators. Together, these results establish that PYO:eDNA interactions support an efficient redox cycle with rapid EET that is faster than the rate of PYO loss from the biofilm.

Regulation of Biofilm Exopolysaccharide Biosynthesis and Degradation in Pseudomonas aeruginosa.

Microbial communities enmeshed in a matrix of macromolecules, termed as biofilms, are the natural setting of bacteria. Exopolysaccharide is a critical matrix component of biofilms. Here, we focus on biofilm matrix exopolysaccharides in Pseudomonas aeruginosa. This opportunistic pathogen can adapt to a wide range of environments and can form biofilms or aggregates in a variety of surfaces or environments, such as the lungs of people with cystic fibrosis, catheters, wounds, and contact lenses. The ability to synthesize multiple exopolysaccharides is one of the advantages that facilitate bacterial survival in different environments. P. aeruginosa can produce several exopolysaccharides, including alginate, Psl, Pel, and lipopolysaccharide. In this review, we highlight the roles of each exopolysaccharide in P. aeruginosa biofilm development and how bacteria coordinate the biosynthesis of multiple exopolysaccharides and bacterial motility. In addition, we present advances in antibiofilm strategies targeting matrix exopolysaccharides, with a focus on glycoside hydrolases.

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.

Combined pH ratiometry and fluorescence lectin-binding analysis (pH-FLBA) for microscopy-based analyses of biofilm pH and matrix carbohydrates.

Bacterial biofilms have a complex and heterogeneous three-dimensional architecture that is characterized by chemically and structurally distinct microenvironments. Confocal microscopy-based pH ratiometry and fluorescence lectin-binding analysis (FLBA) are well-established methods to characterize pH developments and the carbohydrate matrix architecture of biofilms at the microscale. Here, we developed a combined analysis, pH-FLBA, to concomitantly map biofilm pH and the distribution of matrix carbohydrates in bacterial biofilms while preserving the biofilm microarchitecture. As a proof of principle, the relationship between pH and the presence of galactose- and fucose-containing matrix components was investigated in dental biofilms grown with and without sucrose. The pH response to a sucrose challenge was monitored in different areas at the biofilm base using the ratiometric pH-sensitive dye C-SNARF-4. Thereafter, the fucose- and galactose-specific fluorescently labeled lectins Aleuria aurantia lectin (AAL) and Morus nigra agglutinin G (MNA-G) were used to visualize carbohydrate matrix components in the same biofilm areas and their immediate surroundings. Sucrose during growth significantly decreased biofilm pH (P < 0.05) and increased the amounts of both MNA-G- and AAL-targeted matrix carbohydrates (P < 0.05). Moreover, it modulated the biofilm composition towards a less diverse community dominated by streptococci, as determined by 16S rRNA gene sequencing. Altogether, these results suggest that the production of galactose- and fucose-containing matrix carbohydrates is related to streptococcal metabolism and, thereby, pH profiles in dental biofilms. In conclusion, pH-FLBA using lectins with different carbohydrate specificities is a useful method to investigate the association between biofilm pH and the complex carbohydrate architecture of bacterial biofilms.IMPORTANCEBiofilm pH is a key regulating factor in several biological and biochemical processes in environmental, industrial, and medical biofilms. At the microscale, microbial biofilms are characterized by steep pH gradients and an extracellular matrix rich in carbohydrate components with diffusion-modifying properties that contribute to bacterial acid-base metabolism. Here, we propose a combined analysis of pH ratiometry and fluorescence lectin-binding analysis, pH-FLBA, to concomitantly investigate the matrix architecture and pH developments in microbial biofilms, using complex saliva-derived biofilms as an example. Spatiotemporal changes in biofilm pH are monitored non-invasively over time by pH ratiometry, while FLBA with lectins of different carbohydrate specificities allows mapping the distribution of multiple relevant matrix components in the same biofilm areas. As the biofilm structure is preserved, pH-FLBA can be used to investigate the in situ relationship between the biofilm matrix architecture and biofilm pH in complex multispecies biofilms.

Cutibacterium acnes biofilm formation is influenced by bone microenvironment, implant surfaces and bacterial internalization.

BACKGROUND: The bacterial persistence, responsible for therapeutic failures, can arise from the biofilm formation, which possesses a high tolerance to antibiotics. This threat often occurs when a bone and joint infection is diagnosed after a prosthesis implantation. Understanding the biofilm mechanism is pivotal to enhance prosthesis joint infection (PJI) treatment and prevention. However, little is known on the characteristics of Cutibacterium acnes biofilm formation, whereas this species is frequently involved in prosthesis infections. METHODS: In this study, we compared the biofilm formation of C. acnes PJI-related strains and non-PJI-related strains on plastic support and textured titanium alloy by (i) counting adherent and viable bacteria, (ii) confocal scanning electronic microscopy observations after biofilm matrix labeling and (iii) RT-qPCR experiments. RESULTS: We highlighted material- and strain-dependent modifications of C. acnes biofilm. Non-PJI-related strains formed aggregates on both types of support but with different matrix compositions. While the proportion of polysaccharides signal was higher on plastic, the proportions of polysaccharides and proteins signals were more similar on titanium. The changes in biofilm composition for PJI-related strains was less noticeable. For all tested strains, biofilm formation-related genes were more expressed in biofilm formed on plastic that one formed on titanium. Moreover, the impact of C. acnes internalization in osteoblasts prior to biofilm development was also investigated. After internalization, one of the non-PJI-related strains biofilm characteristics were affected: (i) a lower quantity of adhered bacteria (80.3-fold decrease), (ii) an increase of polysaccharides signal in biofilm and (iii) an activation of biofilm gene expressions on textured titanium disk. CONCLUSION: Taken together, these results evidenced the versatility of C. acnes biofilm, depending on the support used, the bone environment and the strain.

Challenges and opportunities in elucidating the structures of biofilm exopolysaccharides: A case study of the Pseudomonas aeruginosa exopolysaccharide called Pel.

Biofilm formation protects bacteria from antibiotic treatment and host immune responses, making biofilm infections difficult to treat. Within biofilms, bacterial cells are entangled in a self-produced extracellular matrix that typically includes exopolysaccharides. Molecular-level descriptions of biofilm matrix components, especially exopolysaccharides, have been challenging to attain due to their complex nature and lack of solubility and crystallinity. Solid-state nuclear magnetic resonance (NMR) has emerged as a key tool to determine the structure of biofilm matrix exopolysaccharides without degradative sample preparation. In this review, we discuss challenges of studying biofilm matrix exopolysaccharides and opportunities to develop solid-state NMR approaches to study these generally intractable materials. We specifically highlight investigations of the exopolysaccharide called Pel made by the opportunistic pathogen, Pseudomonas aeruginosa. We provide a roadmap for determining exopolysaccharide structure and discuss future opportunities to study such systems using solid-state NMR. The strategies discussed for elucidating biofilm exopolysaccharide structure should be broadly applicable to studying the structures of other glycans.

OprF Impacts Pseudomonas aeruginosa Biofilm Matrix eDNA Levels in a Nutrient-Dependent Manner.

The biofilm matrix is composed of exopolysaccharides, eDNA, membrane vesicles, and proteins. While proteomic analyses have identified numerous matrix proteins, their functions in the biofilm remain understudied compared to the other biofilm components. In the Pseudomonas aeruginosa biofilm, several studies have identified OprF as an abundant matrix protein and, more specifically, as a component of biofilm membrane vesicles. OprF is a major outer membrane porin of P. aeruginosa cells. However, current data describing the effects of OprF in the P. aeruginosa biofilm are limited. Here, we identify a nutrient-dependent effect of OprF in static biofilms, whereby ΔoprF cells form significantly less biofilm than wild type when grown in media containing glucose or low sodium chloride concentrations. Interestingly, this biofilm defect occurs during late static biofilm formation and is not dependent on the production of PQS, which is responsible for outer membrane vesicle production. Furthermore, while biofilms lacking OprF contain approximately 60% less total biomass than those of wild type, the number of cells in these two biofilms is equivalent. We demonstrate that P. aeruginosa ΔoprF biofilms with reduced biofilm biomass contain less eDNA than wild-type biofilms. These results suggest that the nutrient-dependent effect of OprF is involved in the maintenance of P. aeruginosa biofilms by retaining eDNA in the matrix. IMPORTANCE Many pathogens form biofilms, which are bacterial communities encased in an extracellular matrix that protects them against antibacterial treatments. The roles of several matrix components of the opportunistic pathogen Pseudomonas aeruginosa have been characterized. However, the effects of P. aeruginosa matrix proteins remain understudied and are untapped potential targets for antibiofilm treatments. Here, we describe a conditional effect of the abundant matrix protein OprF on late-stage P. aeruginosa biofilms. A ΔoprF strain formed significantly less biofilm in low sodium chloride or with glucose. Interestingly, the defective ΔoprF biofilms did not exhibit fewer resident cells but contained significantly less extracellular DNA (eDNA) than wild type. These results suggest that OprF is involved in matrix eDNA retention in biofilms.

Manganese Acts as an Environmental Inhibitor of Pseudomonas aeruginosa Biofilm Development by Inducing Dispersion and Modulating c-di-GMP and Exopolysaccharide Production via RbdA.

The opportunistic human pathogen Pseudomonas aeruginosa causes chronic infections that involve multicellular aggregates called biofilms. Biofilm formation is modulated by the host environment and the presence of cues and/or signals, likely affecting the pool of the bacterial second messenger cyclic diguanylate monophosphate (c-di-GMP). The manganese ion Mn2+ is a divalent metal cation that is essential for pathogenic bacterial survival and replication during the infection in a host organism. In this study, we investigated how Mn2+ alters P. aeruginosa biofilm formation via the regulation of c-di-GMP levels. Exposure to Mn2+ was found to temporally enhance attachment but impair subsequent biofilm development, apparent by reduced biofilm biomass accumulation and lack of microcolony formation due to the induction of dispersion. Moreover, exposure to Mn2+ coincided with reduced production of the exopolysaccharides Psl and Pel, decreased transcriptional abundance of pel and psl, and decreased levels of c-di-GMP. To determine whether the effect of Mn2+ was linked to the activation of phosphodiesterases (PDEs), we screened several PDE mutants for Mn2+-dependent phenotypes (attachment and polysaccharide production) as well as PDE activity. The screen revealed that the PDE RbdA is activated by Mn2+ and is responsible for Mn2+-dependent attachment, inhibition of Psl production, and dispersion. Taken together, our findings suggest Mn2+ is an environmental inhibitor of P. aeruginosa biofilm development that acts through the PDE RbdA to modulate c-di-GMP levels, thereby impeding polysaccharide production and biofilm formation but enhancing dispersion. IMPORTANCE While diverse environmental conditions such as the availability of metal ions have been shown to affect biofilm development, little is known about the mechanism. Here, we demonstrate that Mn2+ affects Pseudomonas aeruginosa biofilm development by stimulating phosphodiesterase RbdA activity to reduce the signaling molecule c-di-GMP levels, thereby hindering polysaccharide production and biofilm formation but enhancing dispersion. Our findings demonstrate that Mn2+ acts as an environmental inhibitor of P. aeruginosa biofilms, further suggesting manganese to be a promising new antibiofilm factor.

Combined Application of Antisense Oligomers to Control Transcription Factors of Candida albicans Biofilm Formation.

Antisense oligomers (ASOs) have been little exploited to control determinants of Candida albicans virulence. Biofilm formation is an important virulence factor of C. albicans, that is regulated by a complex network of transcription factors (such as EFG1, BRG1 and ROB1). Thus, the main goal of this work was to project ASOs, based on the 2'-OMethyl chemical modification, to target BRG1 and ROB1 mRNA and to validate its application either alone or in combination with the EFG1 mRNA target, to reduce C. albicans biofilm formation. The ability of ASOs to control gene expression was evaluate by qRT-PCR. The effect on biofilm formation was determined by the total biomass quantification, and simultaneously the carbohydrates and proteins reduction on extracellular matrix. It was verified that all the oligomers were able to reduce the levels of gene expression and the ability of C. albicans to form biofilms. Furthermore, the combined application of the cocktail of ASOs enhances the inhibition of C. albicans biofilm formation, minimizing biofilm thickness by reducing the quantity of matrix content (protein and carbohydrate). So, our work confirms that ASOs are useful tools for research and therapeutic development on the control of Candida species biofilm formation.

Fusion of a bacterial cadherin-like domain and green fluorescent protein as a specific probe to study biofilm matrix formation in Rhizobium spp.

Rhizobium adhering proteins or 'Raps' are secreted proteins identified in a very restricted group of rhizobial strains, specifically those belonging to R. leguminosarum and R. etli. The distinctive feature of members of the Rap family is the presence of one or two cadherin-like domains or CHDLs that are also present in numerous extracellular bacterial and archaeal proteins and were proposed to confer carbohydrate binding ability. We have previously made an in-depth characterization of RapA2, a calcium-binding lectin, composed by two CHDLs, involved in biofilm matrix remodelling in R. leguminosarum bv. viciae 3841. In this study, CHDLs derived from RapA2 were analysed in detail, finding significant structural and functional differences despite their considerable sequence similarity. Only the carboxy-terminal CHDL retained properties similar to those displayed by RapA2. Our findings were used to obtain a novel fluorescent probe to study biofilm matrix development by confocal laser scanning microscopy, and also to shed some light on the role of the ubiquitous CHDL domains in bacterial secreted proteins.

Osmotic stress relief antibiotic tolerance of 1,8-cineole in biofilm persister cells of Escherichia coli O157:H7 and expression of toxin-antitoxin system genes.

The control of E. coli activity from forming biofilm and persister cells is an essential factor in both the health and food industries. The efficacy of antimicrobial treatment is often limited due to their low penetrability as biofilm formation protect cells within from physical or chemical threats. Among other factors, osmotic stress has shown to have a high capacity to enhance the antimicrobial activities against various pathogens. Thus, this study aimed to test the hypothesis that the antimicrobial activity of cineole (CN) could be enhanced under osmotic stress to inhibit biofilm and persister cells. Time-kill analysis revealed that CN under NaCl-induced osmotic stress (CN-S) had better inhibitory effect on E. coli biofilm. 5% CN-S altered the integrity, hydration, motilities and exopolysaccharide production of E. coli cells. Also, the outer membrane permeability, surface roughness and hydrophobicity which determine initial cell adhesion, aggregation and colony assembly were significantly perturbed. Furthermore, the expression levels of virulence genes stx1, stx2, eae, flhD, and the TA system antitoxin genes mazE, hipB were downregulated. When applied to cucumber, the rate of increase in internalized bacterial cells significantly reduced after storage at 4 °C for 48 h. Thus, the results suggested that the application of osmotic stress could minimize the working concentration of antimicrobials in real food systems, which could be helpful in counteracting the growing concern of microbial resistance.

Seaweed Extracts: A Promising Source of Antibiofilm Agents with Distinct Mechanisms of Action against Pseudomonas aeruginosa.

The organization of bacteria in biofilms is one of the adaptive resistance mechanisms providing increased protection against conventional treatments. Thus, the search for new antibiofilm agents for medical purposes, especially of natural origin, is currently the object of much attention. The objective of the study presented here was to explore the potential of extracts derived from three seaweeds: the green Ulva lactuca, the brown Stypocaulon scoparium, and the red Pterocladiella capillacea, in terms of their antibiofilm activity against P. aeruginosa. After preparation of extracts by successive maceration in various solvents, their antibiofilm activity was evaluated on biofilm formation and on mature biofilms. Their inhibition and eradication abilities were determined using two complementary methods: crystal violet staining and quantification of adherent bacteria. The effect of active extracts on biofilm morphology was also investigated by epifluorescence microscopy. Results revealed a promising antibiofilm activity of two extracts (cyclohexane and ethyl acetate) derived from the green alga by exhibiting a distinct mechanism of action, which was supported by microscopic analyses. The ethyl acetate extract was further explored for its interaction with tobramycin and colistin. Interestingly, this extract showed a promising synergistic effect with tobramycin. First analyses of the chemical composition of extracts by GC-MS allowed for the identification of several molecules. Their implication in the interesting antibiofilm activity is discussed. These findings suggest the ability of the green alga U. lactuca to offer a promising source of bioactive candidates that could have both a preventive and a curative effect in the treatment of biofilms.

Calcium Prevents Biofilm Dispersion in Bacillus subtilis.

Biofilm dispersion is the final stage of biofilm development, during which biofilm cells actively escape from biofilms in response to deteriorating conditions within the biofilm. Biofilm dispersion allows cells to spread to new locations and form new biofilms in better locations. However, dispersal mechanisms have been elucidated only in a limited number of bacteria. Here, we investigated biofilm dispersion in Bacillus subtilis. Biofilm dispersion was clearly observed when B. subtilis was grown under static conditions in modified LB medium containing glycerol and manganese. Biofilm dispersion was synergistically caused by two mechanisms: decreased expression of the epsA operon encoding exopolysaccharide synthetases and the induction of sporulation. Indeed, constitutive expression of the epsA operon in the sporulation-defective ΔsigK mutant prevented biofilm dispersion. The addition of calcium to the medium prevented biofilm dispersion without significantly affecting the expression of the epsA operon and sporulation genes. In synthetic medium, eliminating calcium did not prevent the expression of biofilm matrix genes and, thereby, biofilm formation, but it attenuated biofilm architecture. These results indicate that calcium structurally stabilizes biofilms and causes resistance to biofilm dispersion mechanisms. Sporulation-dependent biofilm dispersion required the spoVF operon, encoding dipicolinic acid (DPA) synthase. During sporulation, an enormous amount of DPA is synthesized and stored in spores as a chelate with calcium. We speculate that, during sporulation, calcium bound to biofilm matrix components may be transported to spores as a calcium-DPA complex, which weakens biofilm structure and leads to biofilm dispersion. IMPORTANCE Bacteria growing as biofilms are notoriously difficult to eradicate and sometimes pose serious threats to public health. Bacteria escape from biofilms by degrading them when biofilm conditions deteriorate. This process, called biofilm dispersion, has been studied as a promising strategy for safely controlling biofilms. However, the regulation and mechanism of biofilm dispersion has been elucidated only in a limited number of bacteria. Here, we identified two biofilm dispersion mechanisms in the Gram-positive, spore-forming bacterium Bacillus subtilis. The addition of calcium to the medium stabilized biofilms and caused resistance to dispersal mechanisms. Our findings provide new insights into biofilm dispersion and biofilm control.

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.

Approaches to Targeting Bacterial Biofilms in Cystic Fibrosis Airways.

The treatment of lung infection in the context of cystic fibrosis (CF) is limited by a biofilm mode of growth of pathogenic organisms. When compared to planktonically grown bacteria, bacterial biofilms can survive extremely high levels of antimicrobials. Within the lung, bacterial biofilms are aggregates of microorganisms suspended in a matrix of self-secreted proteins within the sputum. These structures offer both physical protection from antibiotics as well as a heterogeneous population of metabolically and phenotypically distinct bacteria. The bacteria themselves and the components of the extracellular matrix, in addition to the signaling pathways that direct their behaviour, are all potential targets for therapeutic intervention discussed in this review. This review touches on the successes and failures of current anti-biofilm strategies, before looking at emerging therapies and the mechanisms by which it is hoped they will overcome current limitations.

Extracellular DNA (eDNA). A Major Ubiquitous Element of the Bacterial Biofilm Architecture.

After the first ancient studies on microbial slime (the name by which the biofilm matrix was initially indicated), multitudes of studies on the morphology, composition and physiology of biofilms have arisen. The emergence of the role that biofilms play in the pathogenesis of recalcitrant and persistent clinical infections, such as periprosthetic orthopedic infections, has reinforced scientific interest. Extracellular DNA (eDNA) is a recently uncovered component that is proving to be almost omnipresent in the extracellular polymeric substance (EPS) of biofilm. This macromolecule is eliciting unprecedented consideration for the critical impact on the pathogenesis of chronic clinical infections. After a systematic review of the literature, an updated description of eDNA in biofilms is presented, with a special focus on the latest findings regarding its fundamental structural role and the contribution it makes to the complex architecture of bacterial biofilms through interactions with a variety of other molecular components of the biofilm matrix.

Various biofilm matrices of the emerging pathogen Staphylococcus lugdunensis: exopolysaccharides, proteins, eDNA and their correlation with biofilm mass.

Staphylococcus lugdunensis is an emerging high-virulent pathogen causative of hospital-acquired infections. Biofilm formation is a complex pathogenic process that leads to well-established bacterial communities. There is a paucity of data on the composition of the biofilm matrix among S. lugdunensis strains. Here, twenty-two S. lugdunensis clinical isolates, mainly from orthopaedic infections but also from other clinical sources, were sub-grouped by ribotyping and dendrogram analysis. Biofilms were analysed by fluorimetric methods based on FITC-Wheat Germ Agglutinin, SYPRO Ruby and TOTO-1 dyes to detect exopolysaccharides, proteins and extracellular DNA (eDNA), respectively. Biofilm morphology was investigated under confocal laser scanning microscopy (CLSM). Isolates displayed intriguing diversities in biofilm mass and matrix composition. The content of exopolysaccharides was found to be to be strongly associated with the biofilm mass (R2 = 0.882), while the content of proteins turned out to be weakly (R2 = 0.465) and that of eDNA very weakly associated (R2 = 0.202) to the biofilm mass.

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.

A pyrene-inhibitor fluorescent probe with large Stokes shift for the staining of Aß1-42, α-synuclein, and amylin amyloid fibrils as well as amyloid-containing Staphylococcus aureus biofilms.

Amyloid fibrils formed by a variety of peptides are biological markers of different human diseases, such as Alzheimer's disease, Parkinson's disease, and type II diabetes, and are structural constituents of bacterial biofilms. Novel fluorescent probes offering improved sensitivity or specificity toward that diversity of amyloid fibrils or providing alternative spectral windows are needed to improve the detection or the identification of amyloid structures. One potential source for such new probes is offered by molecules known to interact with fibrils, such as the inhibitors of amyloid aggregation found in drug discovery projects. Here we show the feasibility of the approach by designing, synthesizing, and testing several pyrene-based fluorescent derivatives of a previously discovered inhibitor of the aggregation of the Aß1-42 peptide. All the derivatives tested retain the interaction with the amyloid architecture and allow its staining. The most soluble derivative, N-acetyl-2-(2-methyl-4-oxo-5,6,7,8-tetrahydro-4H-benzo[4,5]thieno[2,3-d][1,3]oxazin-7-yl)-N-(pyren-1-ylmethyl)acetamide (compound 1D), stains similarly well amyloid fibrils formed by Aß1-42, α-synuclein, or amylin, provides a sensitivity only slightly lower than that of thioflavin T, displays a large Stokes shift, allows efficient excitation in the UV spectral region, and is not cytotoxic. Compound 1D can also stain amyloid fibrils formed by staphylococcal peptides present in biofilm matrices and can be used to distinguish, by direct staining, Staphylococcus aureus biofilms containing amyloid-forming phenol-soluble modulins from those lacking them. Graphical abstract ᅟ.