Production of Extracellular Glycogen by Pseudomonas fluorescens: Spectroscopic Evidence and Conformational Analysis by Biomolecular Recognition

Glycogen is mainly found as the principal storage form of glucose in cells. Many bacteria are able to synthesize large amounts of glycogen under unfavorable life conditions. By combining infrared spectroscopy, single molecule force spectroscopy (SMFS) and immuno-staining technique, we evidenced that planktonic P. fluorescens (Pf) cells are also able to produce glycogen as an extracellular polymeric substance. For this purpose, Pf suspensions were examined at 3 and 21 h of growth in nutritive medium (LB, 0.5 g/L). The conformation of the extracellular glycogen, revealed through its infrared spectral signature, has been investigated by SMFS measurements using Freely Jointed Chain model. The analysis of force versus distance curves showed over growth time that the increase of glycogen production was accompanied by an increase in glycogen contour lengths and ramifications. These results demonstrated that the production of extracellular bacterial glycogen can occur even if the cells are not subjected to unfavorable life conditions

Diffusion of Fluorescently Labeled Bacteriocin from Edible Nanomaterials and Embedded Nano-Bioactive Coatings

Application of nano-biotechnology to improve the controlled release of drugs or functional agents is widely anticipated to transform the biomedical, pharmaceutical, and food safety trends. The purpose of the current study was to assess and compare the release rates of fluorescently labeled antimicrobial peptide nisin (lantibiotic/biopreservative) from liposomal nanocarriers. The elevated temperature, high electrostatic attraction between anionic bilayers and cationic nisin, larger size, and higher encapsulation efficiency resulted in rapid and elevated release through pore formation. However, acidic pH and optimal ethanol concentration in food simulating liquid (FSL) improved the stability and retention capacity of loaded drug. Thus, controlling various factors had provided partition coefficient <i>K</i> values from 0.23 to 8.78 indicating variation in nisin affinity toward encapsulating macromolecule or FSL. Interaction between nisin and nanoscale bilayer systems by atomic force (AFM) and transmission electron microscopy demonstrated membrane activity of nisin from adsorption and aggregation to pore formation. Novel nanoactive films with preloaded nanoliposomes embedded in biodegradable polymer revealed improved morphological, topographic, and roughness parameters studied by confocal microscopy and AFM. Pre-encapsulated nanoactive biopolymer demonstrated excellent retention capacity as drug carriers by decreasing the partition coefficient value from 1.8 to 0.66 (∼30%) due to improved stability of nanoliposomes embedded in biopolymer network

<i>In Situ</i> Analysis of Bacterial Extracellular Polymeric Substances from a <i>Pseudomonas fluorescens</i> Biofilm by Combined Vibrational and Single Molecule Force Spectroscopies

Extracellular polymeric substances (EPS) play an important role in biofilm cohesion and adhesion to surfaces. EPS of a <i>P. fluorescens</i> biofilm were characterized through their vibrational spectra (infrared and Raman) and their conformational properties using single molecule force spectroscopy with specific probes for glucose, galactose, and <i>N</i>-acetyl glucosamine-rich EPS. Vibrational spectra evidenced the overproduction of glycogen and other carbohydrates in the biofilm. The conformational analysis was performed from both the freely jointed chain (FJC) and worm like chain (WLC) models. The results of the FJC fittings showed highly ramified and/or folded structures for all the detected EPS with molecular elongations up to 1000–2500 nm, and typical Kuhn lengths of glycogen macromolecules. The characteristics of galactose-rich EPS have been found to be significantly different from those of glucose- and <i>N</i>-acetyl glucosamine-rich EPS. On the basis of the theoretical fittings with the WLC model, our results suggested that carbohydrates may be associated with peptide domains

Origin of the Differential Nanoscale Reactivity of Biologically and Chemically Formed Green Rust Crystals Investigated by Chemical Force Spectroscopy

Iron-containing nanoparticles, such as green rusts, can be formed by either chemical (c-GR) or biological (b-GR) pathways. It is known that c-GRs display very high reactivity toward organic and inorganic contaminants and thus have great potential for the remediation of contaminated environments, whereas b-GRs are very weakly reactive. This reactivity difference is usually attributed to much higher surface/volume ratio of c-GR compared to b-GR. Using atomic and chemical force microscopy to probe the reactivity at the nanoscale of both types of nanoparticles, we are able to show that the primary reason for the low reactivity of b-GR is not the low surface/volume ratio but the passivation of the surface due to the presence of biological exopolymers (EPS). This conclusion should hold true for many biological nanoparticles and allows us to explain their often observed low, yet unexplained, reactivity

Thermo-Regulated Adhesion of the Streptococcus thermophilus <i>Δrgg0182</i> Strain

The physicochemical determinants governing the temperature-dependent adhesion of Streptococcus thermophilus to abiotic surfaces are identified under physiological condition for cells either lacking or not the Rgg₀₁₈₂ transcriptional regulator involved in their thermal adaptation. For that purpose, the wild type LMG18311 strain and <i>Δrgg₀₁₈₂</i> mutant were imaged using highly resolved atomic force microscopy (AFM) at various cell growth temperatures (42 to 55 °C). The corresponding hydrophobic/hydrophilic balance of the cells was quantitatively addressed via the measurement by chemical force microcopy of their adhesion to a reference hydrophobic surface. Analysis of force–separation distance curves further allowed us to discriminate cell surfaces according to the presence or absence of biopolymers. These results were interpreted in relation to the measured adhesion of the <i>Δrgg</i>_<i>0182</i> mutant onto the hydrophobic wall of microwells in the temperature range from 46 to 52 °C. It is evidenced that the viscoelastic <i>Δrgg</i>_<i>0182</i> cell envelop behaves as a thermo-responsive film whose hydrophobicity increases with increasing temperature, thereby favoring cell attachment to hydrophobic surfaces. Regardless cell growth temperature, wild-type cells do not attach to hydrophobic surfaces and the presence of the Rgg₀₁₈₂ transcriptional regulator is associated with the synthesis of hydrophilic cell surface biopolymers. Throughout, the impact of electrostatics on bioadhesion is ruled out upon examination of electrohydrodynamic cell properties at 50 °C

Auxiliary Biomembranes as a Directional Delivery System To Control Biological Events in Cell-Laden Tissue-Engineering Scaffolds

Delivery of growth factors is an indispensable part of tissue engineering. Here, we describe a detachable membrane-based release system composed of extracellular matrix components that can be attached to hydrogels to achieve directional release of bioactive molecules. This way, the release of cytokines/growth factors can be started at a desired point of tissue maturation or directly in vivo. As a model, we develop thin films of an interpenetrating network of double-cross-linked gelatin and hyaluronic acid derivatives. The use of the auxiliary release system with vascular endothelial growth factor results in extensive sprouting by encapsulated vascular endothelial cells. The presence of the release system with interleukin-4 results in clustering of encapsulated macrophages with a significant decrease in M1 macrophages (proinflammatory). This system can be used in conjunction with three-dimensional structures as an auxiliary system to control artificial tissue maturation and growth

Design of Flexible Free Standing Plasma Polymer-Based Films As Hosts for Enzyme Immobilization

This article deals with the elaboration and characterization of an original and easy way to produce templates able to host dyes and enzymes. The template films are made of plasma methacrylic acid (MAA) and ethylene glycol dimethacrylate (EGDMA) copolymers and allow for the design of a free-standing matrix able to host active enzymes. Plasma copolymers based on 80/20% molar ratio MAA and EGDMA were prepared to obtain stable chemical plasma films containing carboxyl groups. The amount of such functional groups was quantified by chemical derivatization, Fourier transform infrared spectroscopy and nuclear magnetic resonance spectroscopies. The analysis reveals the possibility to produce stable coatings with accessible functional groups to control the affinity of the film for dyes such as toluidine blue and enzymes such as alkaline phosphatase. We showed that this kind of plasma film is able to entrap enzymes whose catalytic activity is still preserved. Enzymatic activity was studied by measuring the production of paranitrophenol from the hydrolysis of paranitrophenyl phosphate (PNP). This distribution of the enzyme across the film thickness was investigated by means of laser confocal scanning microscopy and time of flight secondary ion mass spectrometry. Some desorption of enzyme was observed during and after the first enzymatic assay, but stable activity was obtained thereafter. The mechanism by which the negatively charged enzyme is entrapped in the plasma polymer matrix, also carrying negatively charged groups, is briefly discussed

Biomimetic Cryptic Site Surfaces for Reversible Chemo- and Cyto-Mechanoresponsive Substrates

Chemo-mechanotransduction, the way by which mechanical forces are transformed into chemical signals, plays a fundamental role in many biological processes. The first step of mechanotransduction often relies on exposure, under stretching, of cryptic sites buried in adhesion proteins. Likewise, here we report the first example of synthetic surfaces allowing for specific and fully reversible adhesion of proteins or cells promoted by mechanical action. Silicone sheets are first plasma treated and then functionalized by grafting sequentially under stretching poly(ethylene glycol) (PEG) chains and biotin or arginine-glycine-aspartic acid (RGD) peptides. At unstretched position, these ligands are not accessible for their receptors. Under a mechanical deformation, the surface becomes specifically interactive to streptavidin, biotin antibodies, or adherent for cells, the interactions both for proteins and cells being fully reversible by stretching/unstretching, revealing a reversible exposure process of the ligands. By varying the degree of stretching, the amount of interacting proteins can be varied continuously