Biomineralization describes the process of mineral precipitation from soluble precursors by living organisms. It is sometimes associated with single bacterial cells, for example, the formation of magnetosomes by magnetotactic bacteria, as well as with groups of bacterial cells that form biofilms and precipitate calcium carbonate (CaCO₃). Recently, there has been growing evidence connecting isolated bacteria and bacterial biofilms with calcium oxalate (CaOx) formation in kidney stones. Therefore, in this study, we examined the effect of a principal exopolysaccharide bacterial biofilm component on the crystallization of CaOx. We observed that the exopolysaccharide, identified as levan, induced the formation of both octahedral CaOx dihydrate (COD, Weddellite) and pancake-like CaOx monohydrate crystals (COM, Whewellite) in a concentration-dependent manner. A combined analysis of the CaOx crystals that formed in the presence of levan, using scanning electron microscopy, Raman spectroscopy, and X-ray diffraction, indicated that levan affects both the nucleation and the growth of CaOx and that its interaction with CaOx is stereospecific. Given the emerging relation between bacterial biofilms and kidney stones, which are prevalent within approximately 12% of the worldwide population, it is important to decipher the effect of biofilm extracellular polymers on the formation of CaOx crystals as it may assist in the development of future treatments to interfere with kidney stone formation.

Biofilms are surface or interface-associated communities of bacterial cells, embedded in a self-secreted extracellular matrix (ECM). Cells in biofilms are 100–1000 times more resistant to antibiotic treatment relative to planktonic cells due to various reasons, including the ECM acting as a diffusion barrier to antibiotic molecules, the presence of persister cells that divide slowly and are less susceptible to cell-wall targeting drugs, and the activation of efflux pumps in response to antibiotic stress. In this study we tested the effect of two titanium(IV) complexes that have been previously reported as potent and non-toxic anticancer chemotherapeutic agents on Bacillus subtilis cells in culture and in biofilm forming conditions. The Ti(IV) complexes tested, a hexacoordinate diaminobis(phenolato)-bis(alkoxo) complex (phenolaTi) and a bis(isopropoxo) complex of a diaminobis(phenolato) “salan”-type ligand (salanTi), did not affect the growth rate of cells in shaken cultures, however they did affect biofilm formation. Surprisingly, while phenolaTi inhibited biofilm formation, the presence of salanTi induced the formation of more mechanically robust biofilms. Optical microscopy images of biofilm samples in the absence and presence of Ti(IV) complexes suggest that Ti(IV) complexes affect cell–cell and/or cell–matrix adhesion, and that these are interfered with phenolaTi and enhanced by salanTi. Our results highlight the possible effect of Ti(IV) complexes on bacterial biofilms, which is gaining interest in light of the emerging relations between bacteria and cancerous tumors.

Many bacteria in nature exist in multicellular communities termed biofilms, where cells are embedded in an extracellular matrix that provides rigidity to the biofilm and protects cells from chemical and mechanical stresses. In the Gram-positive model bacterium Bacillus subtilis, TasA is the major protein component of the biofilm matrix, where it has been reported to form functional amyloid fibres contributing to biofilm structure and stability. Here, we present electron cryomicroscopy structures of TasA fibres, which show that, rather than forming amyloid fibrils, TasA monomers assemble into fibres through donor-strand exchange, with each subunit donating a β-strand to complete the fold of the next subunit along the fibre. Combining electron cryotomography, atomic force microscopy, and mutational studies, we show how TasA fibres congregate in three dimensions to form abundant fibre bundles that are essential for B. subtilis biofilm formation. Our study explains the previously observed biochemical properties of TasA and shows how a bacterial extracellular globular protein can assemble from monomers into β-sheet-rich fibres, and how such fibres assemble into bundles in biofilms.

Hydrogels are 3-dimensional networks composed of synthetic or natural polymers that absorb and retain water. They are formed when polymeric chains are cross-linked by covalent or non-covalent bonds. Hydrogels formed by peptides and proteins are unique due to their bioactivity and biocompatibility. Particularly interesting peptide- or protein-based hydrogels are metallogels, hydrogels that form by proteins and peptides that interact with metal ions. Metallogels are potential candidates for medical applications because metal ions are essential for living organisms, for example in respiration, proper growth and development of tissues, red blood cell formation, and immune system stimulation. In this review, we describe general principles of hydrogel formation and we demonstrate them using specific metallogels examples. We focus on physically cross-linked hydrogels and briefly review their formation mechanism, characterization, mechanical properties, and applications.

Antibiotic resistance of bacteria in the biofilm has become a major problem for the treatment of chronic infections. Nanotechnology offers a new approach to fighting against biofilm-associated infections. Here, we report on the synthesis of silver nanoparticles (AgNPs) with epigallocatechin gallate (EGCG) ligand, which is known to present antibacterial activity. We further describe the interaction of EGCG-AgNPs with lysozyme, an antibacterial enzyme abundant in fluids like serum, saliva and tears, and the formation of EGCG-AgNP-lysozyme bioconjugates. Our results demonstrate the formation of lysozyme protein corona as shown by UV-Vis, dynamic light scattering and SDS-PAGE analyses. Using circular dichroism spectroscopy we found that lysozyme preserves its secondary structure at low concentration of EGCG-AgNPs, whereas higher concentrations induce protein structural changes. Furthermore, we have shown in bacterial growth and biofilm formation assays that EGCG presents no antibacterial activity on planktonic Bacillus subtilis cells when bound to AgNPs. It was found that EGCG-AgNPs (>40 nM) inhibit B. subtilis in biofilm forming conditions and the inhibition effect is reduced by lysozyme corona formation on EGCG-AgNPs. Our results demonstrate that EGCG-AgNPs could be used as antibiofilm agents because they showed lower cytotoxicity and significant inhibitory effect on pellicle formation.

Biofilms are multicellular microbial communities that encase themselves in an extracellular matrix (ECM) of secreted biopolymers and attach to surfaces and interfaces. Bacterial biofilms are detrimental in hospital and industrial settings, but they can be beneficial, for example, in agricultural as well as in food technology contexts. An essential property of biofilms that grants them with increased survival relative to planktonic cells is phenotypic heterogeneity, the division of the biofilm population into functionally distinct subgroups of cells. Phenotypic heterogeneity in biofilms can be traced to the cellular level; however, the molecular structures and elemental distribution across whole biofilms, as well as possible linkages between them, remain unexplored. Mapping X-ray diffraction across intact biofilms in time and space, we revealed the dominant structural features in Bacillus subtilis biofilms, stemming from matrix components, spores, and water. By simultaneously following the X-ray fluorescence signal of biofilms and isolated matrix components, we discovered that the ECM preferentially binds calcium ions over other metal ions, specifically, zinc, manganese, and iron. These ions, remaining free to flow below macroscopic wrinkles that act as water channels, eventually accumulate and may possibly lead to sporulation. The possible link between ECM properties, regulation of metal ion distribution, and sporulation across whole, intact biofilms unravels the importance of molecular-level heterogeneity in shaping biofilm physiology and development.

Biofilms are surface-associated soft microbial communities, which may be either detrimental or beneficial to their hosting environment. They develop from single cells into mature colonies, that are composed of cells and sometimes (in Firmicutes phylum) spores, held together by an extracellular matrix (ECM) of secreted biomolecular components. Biofilm development is a dynamic process, during which cells organize into subgroups, creating functionally distinct regions in space. Specific examples of functional-spatial division in Bacillus subtilis biofilms include matrix and spore formation as well as water channels that form beneath wrinkles. An interesting question arising is whether the division of labor in biofilms is also reflected in the molecular-level order across whole biofilms. Using combined X-ray diffraction (XRD)/X-ray fluorescence (XRF), we studied the molecular order in intact biofilms across multiple length scales. We discovered that biofilms display a distinct spatio-temporal XRD signature that depends on highly ordered structures in spores and on cross β sheet structures in matrix components. Spore signal is found especially enhanced with water molecules and metal-ions signals along macroscopic wrinkles, known to act as water channels. Demonstrating in situ the link between molecular structures, metal ions distribution and division of labor across whole biofilms in time and space, this study provides new pivotal insight to the understanding biofilm development.

Functional amyloid proteins often appear as fibers in extracellular matrices of microbial soft colonies. In contrast to disease-related amyloid structures, they serve a functional goal that benefits the organism that secretes them, which is the reason for the title “functional”. Biofilms are a specific example of a microbial community in which functional amyloid fibers play a role. Functional amyloid proteins contribute to the mechanical stability of biofilms and mediate the adhesion of the cells to themselves as well as to surfaces. Recently, it has been shown that functional amyloid proteins also play a regulatory role in biofilm development. TasA is the major proteinaceous fibrilar component of the extracellular matrix of biofilms made of the soil bacterium and Gram-positive Bacillus subtilis. We have previously shown, as later corroborated by others, that in acidic solutions, TasA forms compact aggregates that are composed of tangled fibers. Here, we show that in a neutral pH and above a certain TasA concentration, the fibers of TasA are elongated and straight and that they bundle up in highly concentrated salt solutions. TasA fibers resemble the canonic amyloid morphology; however, these fibers also bear an interesting nm-scale periodicity along the fiber axis. At the molecular level, TasA fibers contain a twisted β-sheet structure, as indicated by circular dichroism measurements. Our study shows that the morphology of TasA fibers depends on the environmental conditions. Different fibrilar morphologies may be related with different functional roles in biofilms, ranging from granting biofilms with a mechanical support to acting as antibiotic agents.

Functional amyloid proteins are self-secreted by microbial cells that aggregate into extracellular networks and provide microbial colonies with mechanical stability and resistance to antibiotic treatment. In order to understand the formation mechanism of functional amyloid networks, their aggregation has been studied in vitro under different physical conditions, such as temperature, salt concentration, and pH. Typical aggregates' morphologies include fibers or plaques, the latter resembling amyloid aggregates in neurodegenerated brains. Here, we studied the pH-reduction-induced aggregation of TasA, an extracellular functional amyloid appearing as fibers in biofilms of the soil bacterium, Bacillus subtilis. We used turbidity and zeta potential measurements, electron microscopy, atomic force microscopy, and static light scattering measurements, to characterize the aggregates of TasA and to compare them with colloidal aggregates. We further studied the aggregation of TasA in the presence of negatively charged nanoparticles and showed that nanoparticles co-aggregated with TasA, and that the co-aggregation was hindered sterically. Based on these studies, we concluded that, similarly to colloidal aggregation, TasA aggregation occurs due to surface potential modulations and that the aggregation is followed by a rearrangement process. Shedding light on the aggregation mechanism of TasA, our results can be used for the design of TasA aggregation inhibitors and promoters.

Biofilms are aggregates of cells that form on surfaces or at the air-water interface. Cells in a biofilm are encased in a self-secreted extracellular matrix (ECM) that provides them with mechanical stability and protects them from antibiotic treatment. From a soft matter perspective, biofilms are regarded as colloidal hydrogels, with the cells playing the role of colloids and the ECM compared with a cross-linked hydrogel. Here, we examined whole biofilms of the soil bacterium Bacillus subtilis utilizing methods that are commonly used to characterize hydrogels in order to evaluate the uptake of water and the water properties in the biofilms. Specifically, we studied wild-type as well ECM mutants, lacking the protein TasA and the exopolysaccharide (EPS). We characterized the morphology and mesh size of biofilms using electron microscopy, studied the state of water in the biofilms using differential scanning calorimetry, and finally, we tested the biofilms' swelling properties. Our study revealed that Bacillus subtilis biofilms resemble cross-linked hydrogels is their morphology and swelling properties. Strikingly, we discovered that all the water in biofilms was bound water and there was no free water in the biofilms. Water binding was mostly related with the presence of solutes and much less so with the major ECM components, the protein TasA and the polysaccharide EPS. This study sheds light on water uptake and water binding in biofilms and it is therefore important for the understanding of solute transport and enzymatic function inside biofilms.

The term amyloid defines a group of proteins that aggregate into plaques or fibers. Amyloid fibers gained their fame mostly due to their relation with neurodegenerative diseases in humans. However, secreted by lower organisms, such as bacteria and fungi, amyloid fibers play a functional role: for example, when they serve as cement in the extracellular matrix of biofilms. Originating either in humans or in microorganisms, the sequence of amyloid proteins is decorated with hexapeptides with high propensity to form fibers, known as steric zippers. We have found that steric zippers form globular structures on route to making fibers and exhibit a characteristic force–distance (F-D) fingerprint when pulled with an atomic force microscope (AFM) tip. Particularly, the F-D pulling curves showed force plateau steps, suggesting that the globular structures were composed of chains that were unwound like a yarn ball. Force plateau analysis showed that the F-D characteristic parameters were sequence sensitive, representing differences in the packing of the hexapeptides within the globules. These unprecedented findings show that steric zippers exhibit a characteristic nanomechanical signature in solution in addition to previously observed characteristic crystallographic structure. Getting to the fundamental interactions that govern the unzipping of full-length amyloid fibers may initiate the development of antiamyloid methods that target the physical in addition to the structural properties of steric zippers.

Biomineralization is the formation of minerals in the presence of organic molecules, often related with functional and/or structural roles in living organisms. It is a complex process and therefore a simple, in vitro, system is required to understand the effect of isolated molecules on the biomineralization process. In many cases, biomineralization is directed by biopolymers in the extracellular matrix. In order to evaluate the effect of isolated biopolymers on the morphology and structure of calcite in vitro, we have used the vapor diffusion method for the precipitation of calcium carbonate, scanning electron microscopy and micro Raman for the characterization, and ultraviolet-visible (UV/Vis) absorbance for measuring the quantity of a biopolymer in the crystals. In this method, we expose the isolated biopolymers, dissolved in a calcium chloride solution, to gaseous ammonia and carbon dioxide that originate from the decomposition of solid ammonium carbonate. Under the conditions where the solubility product of calcium carbonate is reached, calcium carbonate precipitates and crystals are formed. Calcium carbonate has different polymorphs that differ in their thermodynamic stability: amorphous calcium carbonate, vaterite, aragonite, and calcite. In the absence of biopolymers, under clean conditions, calcium carbonate is mostly present in the calcite form, which is the most thermodynamically stable polymorph of calcium carbonate. This method examines the effect of the biopolymeric additives on the morphology and structure of calcium carbonate crystals. Here, we demonstrate the protocol through the study of an extracellular bacterial protein, TapA, on the formation of calcium carbonate crystals. Specifically, we focus on the experimental set up, and characterization methods, such as optical and electron microscopy as well as Raman spectroscopy.

Biofilms are aggregates of microbial cells that form on surfaces and at interfaces and are encased in an extracellular matrix. In biofilms made by the soil bacterium Bacillus subtilis, the protein TapA mediates the assembly of the functional amyloid protein TasA into extracellular fibers and it also anchors these fibers to the cell surface. Here we used circular dichroism and NMR to show that, unlike the structured TasA, TapA is disordered. In addition, TapA is composed of two weakly interacting domains, a disordered C‐terminal domain and a more structured N‐terminal domain. These two domains also exhibited different structural changes in response to changes in external conditions, such as increased temperatures and the presence of lipid vesicles. While the two TapA domains weakly interacted in solution, their cooperative interaction with lipid vesicles prevented the vesicles' disruption, that was otherwise observed in the presence of the C‐terminal domain alone. Our findings therefore suggest that the two‐domain composition of TapA is important for its interaction with a single or with multiple partners in the extracellular matrix in biofilms.

Biomineralization is a mineral precipitation process occurring in the presence of organic molecules and used by various organisms to serve a structural and/or a functional role. Many biomineralization processes occur in the presence of extracellular matrices that are composed of proteins and polysaccharides. Recently, there is growing evidence that bacterial biofilms induce CaCO₃ mineralization and that this process may be related with their extracellular matrix (ECM). In this study we explore, in vitro, the effect of two bacterial ECM proteins, TasA and TapA, and an exopolysaccharide, EPS, on calcium carbonate crystallization. We have found that all the three biopolymers induce the formation of complex CaCO₃ structures. The crystals formed in the presence of the EPS are very diverse in morphology and they are either calcite or vaterite in structure. However, more uniformly sized calcite crystals are formed in the presence of the proteins; these crystals are composed of single crystalline domains that assemble together into spherulites (in the presence of TapA) or dumbbell-like shapes (in the presence of TasA). Our results suggest the EPS affects the nucleation of calcium carbonate when it induces the formation of vaterite crystals and that unlike EPS, the proteins stabilize preformed calcite nuclei and induce their aggregation into complex calcite structures. Biomineralization processes induced by bacterial ECM macromolecules make biofilms more robust and difficult to remove when they form, for example, on pipes and filters in water desalination systems or on ship hulls. Understanding the formation conditions and mechanism of formation of calcium carbonate in the presence of bacterial biopolymers may lead to the design of suitable mineralization inhibitors.

Biofilms are aggregates of cells that form surface-associated communities. The cells in biofilms are interconnected with an extracellular matrix, a network that is made mostly of polysaccharides, proteins, and sometimes nucleic acids. Some extracellular matrix proteins form fibers, termed functional amyloid or amyloid-like, to differentiate their constructive function from disease-related amyloid fibers. Recent functional amyloid assembly studies have neglected their interaction with membranes, despite their native formation in a cellular environment. Here, we use TasA, a major matrix protein in biofilms of the soil bacterium Bacillus subtilis, as a model functional amyloid protein and ask whether the bacterial functional amyloid interacts with membranes. Using biochemical, spectroscopic, and microscopic tools, we show that TasA interacts distinctively with bacterial model membranes and that this interaction mutually influences the morphology and structure of the protein and the membranes. At the protein level, fibers of similar structure and morphology are formed in the absence of membranes and in the presence of eukaryotic model membranes. However, in the presence of bacterial model membranes, TasA forms disordered aggregates with a different β sheet signature. At the membrane level, fluorescence microscopy and anisotropy measurements indicate that bacterial membranes deform more considerably than eukaryotic membranes upon interaction with TasA. Our findings suggest that TasA penetrates bacterial more than eukaryotic model membranes and that this leads to membrane disruption and to reshaping the TasA fiber formation pathway. Considering the important role of TasA in providing integrity to biofilms, our study may direct the design of antibiofilm drugs to the protein–membrane interface.

Controlling the interaction of drug delivery systems (DDS) with tissues is critical for the success of therapies. Specifically in cancer, due to the high density of the tumors, tissue penetration of DDS is critical and may be challenging. In previous work we have shown that Solidified Polymer Micelles (SPMs) rapidly internalize into cells and tissues. Using AFM analysis, in the present work we measured differences in rigidity of SPM compared with Wet Polymer Micelles (WPM). We further examined whether the semi-solid form of hydrated SPMs has an effect on the interaction with tumor cells both in mono-layer systems and in multi-layer clusters of cells as spheroids. For that we have performed detailed characterization of SPM compared to WPM, including examinations of particle size, stability, drug release kinetics and cell transcytosis, in melanoma A-375 cells. Cell uptake measurements were done using fluorescent signal analysis, FACS and microscopy imaging, showing enhanced abilities of SPMs to penetrate cells and tissues. A simple physical model is presented that well agrees with the experiments and provides insight about the role of particle rigidity in the engulfment mechanism. We conclude that particle rigidity enhances cellular uptake and tissue penetration and that SPMs have a promising potential as an effective and highly permeable DDS. Our findings can be important in future rational design of DDS for particle adjustment to specific tissues and pathologies.

Biofilms are surface-associated groups of microbial cells that are embedded in an extracellular matrix (ECM). The ECM is a network of biopolymers, mainly polysaccharides, proteins, and nucleic acids. ECM proteins serve a variety of structural roles and often form amyloid-like fibers. Despite the extensive study of the formation of amyloid fibers from their constituent subunits in humans, much less is known about the assembly of bacterial functional amyloid-like precursors into fibers. Using dynamic light scattering, atomic force microscopy, circular dichroism, and infrared spectroscopy, we show that our unique purification method of a Bacillus subtilis major matrix protein component results in stable oligomers that retain their native α-helical structure. The stability of these oligomers enabled us to control the external conditions that triggered their aggregation. In particular, we show that stretched fibers are formed on a hydrophobic surface, whereas plaque-like aggregates are formed in solution under acidic pH conditions. TasA is also shown to change conformation upon aggregation and gain some β-sheet structure. Our studies of the aggregation of a bacterial matrix protein from its subunits shed new light on assembly processes of the ECM within bacterial biofilms.

Bacteria often live in the form of surface-associated communities of cells termed biofilms. Within biofilms, there is a division of labor in which genetically identical cells differentiate to serve distinct functions. This cellular differentiation results from a response to extracellular signals that occur due to changes in the local environment of a cell or in response to signaling molecules that the cells themselves produce. In this review, we discuss differentiation in biofilms, focusing on the molecular mechanisms that regulate differentiation in the bacterium Bacillus subtilis. In this organism, there is a subpopulation of cells within a biofilm that produces a signal, while a different subpopulation of cells responds to it. Studying what signals cells use to communicate with each other within a biofilm will allow for better design of strategies to prevent and disrupt biofilms.