<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

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

Characterization and anti-biofilm activity of extracellular polymeric substances produced by the marine biofilm-forming bacterium <i>Pseudoalteromonas ulvae</i> strain TC14

<p>This study investigated soluble (Sol-EPS), loosely bound (LB-EPS), and tightly bound extracellular polymeric substances (TB-EPS) harvested from biofilm and planktonic cultures of the marine bacterium <i>Pseudoalteromonas ulvae</i> TC14. The aim of the characterization (colorimetric methods, FTIR, GC-MS, NMR, HPGPC, and AFM analyses) was to identify new anti-biofilm compounds; activity was assessed using the BioFilm Ring Test®. A step-wise separation of EPS was designed, based on differences in water-solubility and acidity. An acidic fraction was isolated from TB-EPS, which strongly inhibited biofilm formation by marine bacterial strains in a concentration-dependent manner. The main constituents of this fraction were characterized as two glucan-like polysaccharides. An active poly(glutamyl-glutamate) fraction was also recovered from TB-EPS. The distribution of these key EPS components in Sol-EPS, LB-EPS, and TB-EPS was distinct and differed quantitatively in biofilm <i>vs</i> planktonic cultures. The anti-biofilm potential of the fractions emphasizes the putative antifouling role of EPS in the environment.</p

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