Multiscale X-ray study of Bacillus subtilis biofilms reveals interlinked structural hierarchy and elemental heterogeneity.

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.

Bacillus subtilis biofilms characterized as hydrogels. Insights on water uptake and water binding in biofilms.

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 in 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.

Colloidal-like aggregation of a functional amyloid protein.

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.

Titanium complexes affect Bacillus subtilis biofilm 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.

Calcium Carbonate Formation in the Presence of Biopolymeric Additives.

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.