Liraz Chai

A self-produced trigger for biofilm disassembly that targets exopolysaccharide.

SUMMARY Biofilms are structured communities of bacteria that are held together by an extracellular matrix consisting of protein and exopolysaccharide. Biofilms often have a limited lifespan, disassembling as nutrients become exhausted and waste products accumulate. D-amino acids were previously identified as a selfproduced factor that mediates biofilm disassembly by causing the release of the protein component of the matrix in Bacillus subtilis. Here we report that B. subtilis produces an additional biofilmdisassembly factor, norspermidine. Dynamic light scattering and scanning electron microscopy experiments indicated that norspermidine interacts directly and specifically with exopolysaccharide. D-amino acids and norspermidine acted together to break down existing biofilms and mutants blocked in the production of both factors formed long-lived biofilms. Norspermidine, but not closely related polyamines, prevented biofilm formation by B. subtilis, Escherichia coli ,a ndStaphylococcus aureus.

Dynamic metabolic exchange governs a marine algal-bacterial interaction

Emiliania huxleyi is a model coccolithophore micro-alga that generates vast blooms in the ocean. Bacteria are not considered among the major factors influencing coccolithophore physiology. Here we show through a laboratory model system that the bacterium Phaeobacter inhibens, a well-studied member of the Roseobacter group, intimately interacts with E. huxleyi. While attached to the algal cell, bacteria initially promote algal growth but ultimately kill their algal host. Both algal growth enhancement and algal death are driven by the bacterially-produced phytohormone indole-3-acetic acid. Bacterial production of indole-3-acetic acid and attachment to algae are significantly increased by tryptophan, which is exuded from the algal cell. Algal death triggered by bacteria involves activation of pathways unique to oxidative stress response and programmed cell death. Our observations suggest that bacteria greatly influence the physiology and metabolism of E. huxleyi. Coccolithophore-bacteria interactions should be further studied in the environment to determine whether they impact micro-algal population dynamics on a global scale. DOI: http://dx.doi.org/10.7554/eLife.17473.001

Osmotic pressure can regulate matrix gene expression in Bacillus subtilis

Many bacteria organize themselves into structurally complex communities known as biofilms in which the cells are held together by an extracellular matrix. In general, the amount of extracellular matrix is related to the robustness of the biofilm. Yet, the specific signals that regulate the synthesis of matrix remain poorly understood. Here we show that the matrix itself can be a cue that regulates the expression of the genes involved in matrix synthesis in Bacillus subtilis. The presence of the exopolysaccharide component of the matrix causes an increase in osmotic pressure that leads to an inhibition of matrix gene expression. We further show that non‐specific changes in osmotic pressure also inhibit matrix gene expression and do so by activating the histidine kinase KinD. KinD, in turn, directs the phosphorylation of the master regulatory protein Spo0A, which at high levels represses matrix gene expression. Sensing a physical cue such as osmotic pressure, in addition to chemical cues, could be a strategy to non‐specifically co‐ordinate the behaviour of cells in communities composed of many different species.

Isolation, Characterization, and Aggregation of a Structured Bacterial Matrix Precursor

Background: TasA is an extracellular matrix protein that makes amyloid-like fibers in Bacillus subtilis biofilms. Results: An isolated TasA matrix precursor self-assembled in vitro into fibers on hydrophobic surfaces and in acidic solutions. Conclusion: TasA is purified as stable, structured oligomers that aggregate in response to simple physical external cues. Significance: TasA aggregation principles can be used to design new anti-biofilm drugs and surfaces. 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.

Interactions between molecularly smooth gold and mica surfaces across aqueous solutions.

Using a surface force balance, we measured the forces between an ultrasmooth (0.2 nm rms roughness) template-stripped gold surface and a molecularly smooth mica surface. Comparison of these forces in both low salt (conductivity water, equivalent to 10(-6)-10(-5) M 1:1 salt) and high salt (10 mM KClO4) regimes enabled us to examine the properties of water layers confined between a metal and a dielectric to films of a few nanometers or less in thickness. We find that the long-range forces between gold and mica are similar to those between two mica surfaces, indicating a net effective negative charge density on the gold similar to that on the mica. Differences were more pronounced at small separations, manifested by the larger jump-in distance in pure water and the weaker hydration repulsion in high salt between a gold and a mica surface compared with two mica surfaces. However, despite these short-ranged differences, replacing one mica surface with gold does not measurably alter the viscosity of nanoconfined water layers, either as free molecules or as bound hydration layers, relative to their confinement by two mica sheets.

Rigidity of polymer micelles affects interactions with tumor cells.

ABSTRACT 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.

Bacterial Model Membranes Reshape Fibrillation of a Functional Amyloid Protein

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.