Microscopy and modelling investigations on the morphology of the biofilm exopolysaccharide produced by Burkholderia multivorans strain C1576.

Bacteria form very often biofilms where they embed in a self-synthesized matrix exhibiting a gel-like appearance. Matrices offer several advantages, including defence against external threats and the easiness of intercellular communication. In infections, biofilm formation enhances bacteria resistance against antimicrobials, causing serious clinical problems for patients' treatments. Biofilm matrices are composed of proteins, extracellular DNA, and polysaccharides, the latter being the major responsible for matrix architecture. The repeating unit of the biofilm polysaccharide synthesized by Burkholderia multivorans strain C1576 contains two mannoses and two sequentially linked rhamnoses, one of them 50 % methylated on C-3. Rhamnose, a 6-deoxysugar, has lower polarity than other common monosaccharides and its methylation further reduces polarity. This suggests a possible role of this polysaccharide in the biofilm matrix; in fact, computer modelling and atomic force microscopy studies evidenced intra- and inter-molecular non-polar interactions both within polysaccharides and with aliphatic molecules. In this paper, the polysaccharide three-dimensional morphology was investigated using atomic force microscopy in both solid and solution states. Independent evidence of the polymer conformation was obtained by transmission electron microscopy which confirmed the formation of globular compact structures. Finally, data from computer dynamic simulations were used to model the three-dimensional structure.

Potential of mean force conformational energy maps for disaccharide linkages of the Burkholderia multivorans exopolysaccharide C1576 in aqueous solution.

Potential of Mean Force Ramachandran energy maps in aqueous solution have been prepared for all of the glycosidic linkages found in the C1576 exopolysaccharide from the biofilms of the bacterial species Burkholderia multivorans, a member of the Burkholderia cepacian complex that was isolated from a cystic fibrosis patient. C1576 is a rhamnomannan with a tetrasaccharide repeat unit. In general, for the four linkage types in this polymer, hydration did not produce dramatic changes in the Ramachandran energy surfaces, with the 3-methyl-α-d-rhamnopyranose-(1→3)-α-d-rhamnopyranose case exhibiting the greatest hydration change, with the global minimum energy conformation shifting by more than 80° in ψ. However, hydration did reduce the rigidity of all the linkages, increasing the overall flexibility of this polysaccharide.

The biofilm of Burkholderia cenocepacia H111 contains an exopolysaccharide composed of l-rhamnose and l-mannose: Structural characterization and molecular modelling.

Burkholderia cenocepacia belongs to the Burkholderia Cepacia Complex, a group of 22 closely related species both of clinical and environmental origin, infecting cystic fibrosis patients. B. cenocepacia accounts for the majority of the clinical isolates, comprising the most virulent and transmissible strains. The capacity to form biofilms is among the many virulence determinants of B. cenocepacia, a characteristic that confers enhanced tolerance to some antibiotics, desiccation, oxidizing agents, and host defenses. Exopolysaccharides are a major component of biofilm matrices, particularly providing mechanical stability to biofilms. Recently, a water-insoluble exopolysaccharide produced by B. cenocepacia H111 in biofilm was characterized. In the present study, a water-soluble exopolysaccharide was extracted from B. cenocepacia H111 biofilm, and its structure was determined by GLC-MS, NMR and ESI-MS. The repeating unit is a linear rhamno-tetrasaccharide with 50% replacement of a 3-α-L-Rha with a α-3-L-Man. [2)-α-L-Rhap-(1→3)-α-L-[Rhap or Manp]-(1→3)-α-L-Rhap-(1→2)-α-L-Rhap-(1→]n Molecular modelling was used to obtain information about local structural motifs which could give information about the polysaccharide conformation.

Burkholderia cenocepacia H111 Produces a Water-Insoluble Exopolysaccharide in Biofilm: Structural Determination and Molecular Modelling.

Biofilms are a multicellular way of life, where bacterial cells are close together and embedded in a hydrated macromolecular matrix which offers a number of advantages to the cells. Extracellular polysaccharides play an important role in matrix setup and maintenance. A water-insoluble polysaccharide was isolated and purified from the biofilm produced by Burkholderia cenocepacia strain H111, a cystic fibrosis pathogen. Its composition and glycosidic linkages were determined using Gas-Liquid Chromatography-Mass Spectrometry (GLC-MS) on appropriate carbohydrate derivatives while its complete structure was unraveled by 1D and 2D NMR spectroscopy in deuterated sodium hydroxide (NaOD) aqueous solutions. All the collected data demonstrated the following repeating unit for the water-insoluble B. cenocepacia biofilm polysaccharide: [3)-α-d-Galp-(1→3)-α-d-Glcp-(1→3)-α-d-Galp-(1→3)-α-d-Manp-(1→]n Molecular modelling was used, coupled with NMR Nuclear Overhauser Effect (NOE) data, to obtain information about local structural motifs which could give hints about the polysaccharide insolubility. Both modelling and NMR data pointed at restricted dynamics of local conformations which were ascribed to the presence of inter-residue hydrogen bonds and to steric restrictions. In addition, the good correlation between NOE data and calculated interatomic distances by molecular dynamics simulations validated potential energy functions used for calculations.

Ramachandran conformational energy maps for disaccharide linkages found in Burkholderia multivorans biofilm polysaccharides.

Ramachandran conformational energy maps have been prepared for all of the glycosidic linkages found in the C1576 exopolysaccharide that constitutes the biofilms of the bacterial species Burkholderia multivorans, a member of the Burkholderia cepacian complex that was isolated from a cystic fibrosis patient. This polysaccharide is a rhamnomannan with a tetrasaccharide repeat unit containing two mannose residues and two rhamnose residues, -[3-α-d-Man-(1→2)-α-d-Man-(1→2)-α-d-Rha-(1→3)-α-d-Rha-(1→]n-, where approximately 50% of the rhamnoses are randomly methylated on their O3 hydroxyl groups, further increasing the overall hydrophobicity of the chains. Because of the methylation, the tetrasaccharide repeat unit actually contains six possible linkages. The conformational energy maps are fully adiabatic relaxed maps in which the energy for each (ϕ,ψ) grid point on the map represents the lowest possible energy for the molecule in that conformation, considering all the combinations of the other degrees of freedom, such as hydroxyl orientations. Molecular dynamics simulations were used to verify that these maps indeed describe the conformational dynamics of these linkages. All six linkages were found to be quite restricted in possible ϕ angles, but to exhibit several possible low-energy ψ angles, suggesting that these chains could be quite flexible.

Protein permeation through an electrically tunable membrane.

Protein filtration is important in many fields of science and technology such as medicine, biology, chemistry, and engineering. Recently, protein separation and filtering with nanoporous membranes has attracted interest due to the possibility of fast separation and high throughput volume. This, however, requires understanding of the protein's dynamics inside and in the vicinity of the nanopore. In this work, we utilize a Brownian dynamics approach to study the motion of the model protein insulin in the membrane-electrolyte electrostatic potential. We compare the results of the atomic model of the protein with the results of a coarse-grained and a single-bead model, and find that the coarse-grained representation of protein strikes the best balance between the accuracy of the results and the computational effort required. Contrary to common belief, we find that to adequately describe the protein, a single-bead model cannot be utilized without a significant effort to tabulate the simulation parameters. Similar to results for nanoparticle dynamics, our findings also indicate that the electric field and the electro-osmotic flow due to the applied membrane and electrolyte biases affect the capture and translocation of the biomolecule by either attracting or repelling it to or from the nanopore. Our computational model can also be applied to other types of proteins and separation conditions.

Nanopore gating with an anchored polymer in a switching electrolyte bias.

In this work, we theoretically study the interaction between a solid state membrane equipped with a nanopore and a tethered, negatively charged polymer chain subjected to a time-dependent applied electrolyte bias. In order to describe the movement of the chain in the biomolecule-membrane system immersed in an electrolyte solution, Brownian dynamics is used. We show that we can control the polymer's equilibrium position with various applied electrolyte biases: for a sufficiently positive bias, the chain extends inside the pore, and the removal of the bias causes the polymer to leave the pore. Corresponding to a driven process, we find that the time it takes for a biomolecular chain to enter and extend into a nanopore in a positive bias almost increases linearly with chain length while the amount of time it takes for a polymer chain to escape the nanopore is mainly governed by diffusion.

Charged particle separation by an electrically tunable nanoporous membrane.

We study the applicability of an electrically tunable nanoporous semiconductor membrane for the separation of nanoparticles by charge. We show that this type of membrane can overcome one of the major shortcomings of nanoporous membrane applications for particle separation: the compromise between membrane selectivity and permeability. The computational model that we have developed describes the electrostatic potential distribution within the system and tracks the movement of the filtered particle using Brownian dynamics while taking into consideration effects from dielectrophoresis, fluid flow, and electric potentials. We found that for our specific pore geometry, the dielectrophoresis plays a negligible role in the particle dynamics. By comparing the results for charged and uncharged particles, we show that for the optimal combination of applied electrolyte and membrane biases the same membrane can effectively separate same-sized particles based on charge with a difference of up to 3 times in membrane permeability.

DNA translocation through a nanopore in a single-layered doped semiconductor membrane.

Recently, we developed a computational model that allowed us to study the influence a semiconductor membrane has on a DNA molecule translocating through a nanopore in this membrane. Our model incorporated both the self-consistent Poisson-Nernst-Planck simulations for the electric potential of a solid state membrane immersed in an electrolyte solution together with the Brownian dynamics of the biomolecule. In this paper, we study how the applied electrolyte bias, the semiconductor membrane bias, and the semiconductor material type (n-Si or p-Si) affect the translocation dynamics of a single-stranded DNA moving through a nanopore in a single-layered semiconductor membrane. We show that the type of semiconductor material used for the membrane has a prominent effect on the biomolecule's translocation time, with DNA exhibiting much longer translocation times through the p-type membrane than through the n type at the same electrolyte and membrane potentials, while the extension of the biomolecule remains practically unchanged. In addition, we find the optimal combination for the membrane-electrolyte system's parameters to achieve the longest translocation time and largest DNA extension. With our single-layered electrically tunable membranes, the DNA translocation time can be manipulated to have an order of magnitude increase.