M. A. Cotta

The role of conditioning film formation and surface chemical changes on Xylella fastidiosa adhesion and biofilm evolution

Biofilms are complex microbial communities with important biological functions including enhanced resistance against external factors like antimicrobial agents. The formation of a biofilm is known to be strongly dependent on substrate properties including hydrophobicity/hydrophilicity, structure, and roughness. The adsorption of (macro)molecules on the substrate, also known as conditioning film, changes the physicochemical properties of the surface and affects the bacterial adhesion. In this study, we investigate the physicochemical changes caused by Periwinkle wilt (PW) culture medium conditioning film formation on different surfaces (glass and silicon) and their effect on X. fastidiosa biofilm formation. Contact angle measurements have shown that the film formation decreases the surface hydrophilicity degree of both glass and silicon after few hours. Atomic force microscopy (AFM) images show the glass surface roughness is drastically reduced with conditioning film formation. First-layer X. fastidiosa biofilm on glass was observed in the AFM liquid cell after a period of time similar to that determined for the hydrophilicity changes. In addition, attenuation total reflection-Fourier transform infrared (ATR-FTIR) spectroscopy supports the AFM observation, since the PW absorption spectra increases with time showing a stronger contribution from the phosphate groups. Although hydrophobic and rough surfaces are commonly considered to increase bacteria cell attachment, our results suggest that these properties are not as important as the surface functional groups resulting from PW conditioning film formation for X. fastidiosa adhesion and biofilm development.

Role of group V exchange on the shape and size of InAs/InP self-assembled nanostructures

We have studied the influence of Group V overpressure on the final shape and size of InAs nanostructures grown on (001) InP substrates. The mechanisms leading to postgrowth modifications in the InAs nanostructures are discussed. The simultaneous action of Group V overpressure and stress field—produced by the InAs nanostructures—can induce strong material transport. The direction of this material net current depends on the type of Group V element used for the overpressure flux. In situ reflection high-energy electron diffraction, atomic force microscopy, and transmission electron microscopy measurements were used to characterize the transitions in morphology. Our results show that morphological studies considering the grown surface that do not take into account postgrowth processes can be misleading to understand the growth mechanisms governing the self-assembling process.

Spatiotemporal distribution of different extracellular polymeric substances and filamentation mediate Xylella fastidiosa adhesion and biofilm formation

Microorganism pathogenicity strongly relies on the generation of multicellular assemblies, called biofilms. Understanding their organization can unveil vulnerabilities leading to potential treatments; spatially and temporally-resolved comprehensive experimental characterization can provide new details of biofilm formation, and possibly new targets for disease control. Here, biofilm formation of economically important phytopathogen Xylella fastidiosa was analyzed at single-cell resolution using nanometer-resolution spectro-microscopy techniques, addressing the role of different types of extracellular polymeric substances (EPS) at each stage of the entire bacterial life cycle. Single cell adhesion is caused by unspecific electrostatic interactions through proteins at the cell polar region, where EPS accumulation is required for more firmly-attached, irreversibly adhered cells. Subsequently, bacteria form clusters, which are embedded in secreted loosely-bound EPS, and bridged by up to ten-fold elongated cells that form the biofilm framework. During biofilm maturation, soluble EPS forms a filamentous matrix that facilitates cell adhesion and provides mechanical support, while the biofilm keeps anchored by few cells. This floating architecture maximizes nutrient distribution while allowing detachment upon larger shear stresses; it thus complies with biological requirements of the bacteria life cycle. Using new approaches, our findings provide insights regarding different aspects of the adhesion process of X. fastidiosa and biofilm formation.

Synthetic melanin thin films: Structural and electrical properties

Scanning probe microscopy was used to investigate the structural and electrical organization at the nanoscopic level of hydrated melanin thin films synthesized by oxidizing L-3-(3,4-dihydroxyphenyl)-alanine (L-dopa) in dimethyl sulfoxide. Atomic force microscopy (AFM) provided the morphologies of the L-dopa melanin films. Electrostatic force microscopy and conductive-AFM were used to spatially resolve the electrical properties of the material. Using a simple parallel plate capacitor model a method to measure the charge distribution on the sample was developed. The correlations between topography, electric charge, and current images of the sample demonstrated that the hydration process produces a restructuring of melanin observed not only through topographic variations, but also through the creation of areas with different electrical properties.

Faceting evolution during self-assembling of InAs/InP quantum wires

The self-assembling of InAs quantum wires on (001) InP substrates during chemical beam epitaxy has been studied. The samples were characterized by reflection high-energy electron diffraction (RHEED), atomic force microscopy, and high-resolution transmission electron microscopy (HRTEM). By monitoring the RHEED chevron structures along the [110] direction, we studied the facets formation during the initial states of InAs growth. The facets angles measured by HRTEM are in perfect agreement with the angles between chevron streaks. A time dependence of the chevron streaks angles is reported and correlated to the wire formation. These results can be interpreted using nonequilibrium models existing in literature.

Synthetic melanin films : Assembling mechanisms, scaling behavior, and structural properties

In this work we report on the surface characterization of melanin thin films prepared using both water-based and organic solvent-based melanin syntheses. Atomic force microscopy (AFM) analysis of these films suggests that the organic solvent synthesis provides relatively planar basic melanin structures; these basic structures generate surface steps with height in the range of 2–3nm and small tendency to form larger aggregates. The scaling properties obtained from the AFM data were used to infer the assembling mechanisms of these thin films which depend on the solvent used for melanin synthesis. The behavior observed in organic solvent-based melanin suggests a diffusion-limited aggregation process. Thus films with good adhesion to the substrate and smoother morphologies than water-prepared melanin films are obtained. Electronic structure calculations using a conductorlike screening model were also performed in order to elucidate the microscopic processes of thin film formation. Our results suggest that the...

Surface physicochemical properties at the micro and nano length scales: role on bacterial adhesion and Xylella fastidiosa biofilm development.

The phytopathogen Xylella fastidiosa grows as a biofilm causing vascular occlusion and consequently nutrient and water stress in different plant hosts by adhesion on xylem vessel surfaces composed of cellulose, hemicellulose, pectin and proteins. Understanding the factors which influence bacterial adhesion and biofilm development is a key issue in identifying mechanisms for preventing biofilm formation in infected plants. In this study, we show that X. fastidiosa biofilm development and architecture correlate well with physicochemical surface properties after interaction with the culture medium. Different biotic and abiotic substrates such as silicon (Si) and derivatized cellulose films were studied. Both biofilms and substrates were characterized at the micro- and nanoscale, which corresponds to the actual bacterial cell and membrane/ protein length scales, respectively. Our experimental results clearly indicate that the presence of surfaces with different chemical composition affect X. fastidiosa behavior from the point of view of gene expression and adhesion functionality. Bacterial adhesion is facilitated on more hydrophilic surfaces with higher surface potentials; XadA1 adhesin reveals different strengths of interaction on these surfaces. Nonetheless, despite different architectural biofilm geometries and rates of development, the colonization process occurs on all investigated surfaces. Our results univocally support the hypothesis that different adhesion mechanisms are active along the biofilm life cycle representing an adaptation mechanism for variations on the specific xylem vessel composition, which the bacterium encounters within the infected plant.

Optical emission of InAs nanowires

Wurtzite InAs nanowire samples grown by chemical beam epitaxy have been analyzed by photoluminescence spectroscopy. The nanowires exhibit two main optical emission bands at low temperatures. They are attributed to the recombination of carriers in quantum well structures, formed by zincblende-wurtzite alternating layers, and to the donor-acceptor pair. The blue-shift observed in the former emission band when the excitation power is increased is in good agreement with the type-II band alignment between the wurtzite and zincblende sections predicted by previous theoretical works. When increasing the temperature and the excitation power successively, an additional band attributed to the band-to-band recombination from wurtzite InAs appears. We estimated a lower bound for the wurtzite band gap energy of approximately 0.46 eV at low temperature.

Spontaneous periodic diameter oscillations in InP nanowires: the role of interface instabilities.

We have observed that thin InP nanowires generated by vapor-liquid-solid growth display spontaneous periodic diameter oscillations when large group III supersaturations are used. Diameter variations are associated with a large number of stacking faults and crystallographic phase changes(wurtzite/zinc-blende); also the axial distance between oscillations depends on the indium precursor flow used during the run. We attribute the morphology changes to a substantial deformation of the triple phase line (vapor-liquid-solid) at the catalyst nanoparticle edge originated from multistep nucleation during growth. The deformation alters the mechanical force balance acting on the nanoparticle during growth in such a way that the particle displaces from the nanowire top and wets the nanowire sidewall. Subsequently, as catalytic growth occurs at the sidewall, the associated increase in diameter will eventually push the NP back to its original wire-top position until the onset of a new instability at the triple phase line.

Development of a recombinant fusion protein based on the dynein light chain LC8 for non-viral gene delivery

The low efficiency of gene transfer is a recurrent problem in DNA vaccine development and gene therapy studies using non-viral vectors such as plasmid DNA (pDNA). This is mainly due to the fact that during their traffic to the target cells nuclei, plasmid vectors must overcome a series of physical, enzymatic and diffusional barriers. The main objective of this work is the development of recombinant proteins specifically designed for pDNA delivery, which take advantage of molecular motors like dynein, for the transport of cargos from the periphery to the centrosome of mammalian cells. A DNA binding sequence was fused to the N-terminus of the recombinant human dynein light chain LC8. Expression studies indicated that the fusion protein was correctly expressed in soluble form using E. coli BL21(DE3) strain. As expected, gel permeation assays found the purified protein mainly present as dimers, the functional oligomeric state of LC8. Gel retardation assays and atomic force microscopy proved the ability of the fusion protein to interact and condense pDNA. Zeta potential measurements indicated that LC8 with DNA binding domain (LD4) has an enhanced capacity to interact and condense pDNA, generating positively charged complexes. Transfection of cultured HeLa cells confirmed the ability of the LD4 to facilitate pDNA uptake and indicate the involvement of the retrograde transport in the intracellular trafficking of pDNA:LD4 complexes. Finally, cytotoxicity studies demonstrated a very low toxicity of the fusion protein vector, indicating the potential for in vivo applications. The study presented here is part of an effort to develop new modular shuttle proteins able to take advantage of strategies used by viruses to infect mammalian cells, aiming to provide new tools for gene therapy and DNA vaccination studies.