Impact of Growth Conditions on Pseudomonas fluorescens Morphology Characterized by Atomic Force Microscopy.

This work is dedicated to the characterization by Atomic Force Microscopy (AFM) of Pseudomonas fluorescens, bacteria having high potential in biotechnology. They were first studied first in optimal conditions in terms of culture medium and temperature. AFM revealed a more-or-less elongated morphology with typical dimensions in the micrometer range, and an organization of the outer membrane characterized by the presence of long and randomly distributed ripples, which are likely related to the organization of lipopolysaccharides (LPS). The outer membrane also presents invaginations, some of them showing a reorganization of ripples, which could be the first sign of a bacterial stress response. In a second step, bacteria grown under unfavorable conditions were characterized. The choice of the medium appeared to be more critical in the case of the second generation of cells, the less adapted medium inducing not only changes in the membrane organization but also larger damages in bacteria. An increased growth temperature affected both the usual "swollen" morphology and the organization of the outer membrane. Here also, LPS likely contribute to membrane remodelling, which makes them potential markers to track cell state changes.

Characterization of cadmium accumulation and phytoextraction in three species of the genus Atriplex (canescens, halimus and nummularia) in the presence or absence of salt.

This study aims to establish for the first time a comparison between the resistance to cadmium (Cd) stress of three halophyte species, Atriplex canescens, Atriplex halimus and Atriplex nummularia in addition to their already known tolerance for salt and drought. Plants were exposed to CdCl2 (20 and 50 µM) in the presence or in the absence of salt (50 mM NaCl) for one and two months. The amount of accumulated Cd was determined in the roots and leaves as well as the amount excreted on the surface of the leaves. Physiological parameters such as chlorophyll content and stress biomarkers, including malondialdehyde and enzymatic activities, were then analyzed. The results show that these plants are able to neutralize the excess of reactive oxygen species resulting from treatments by activating the antioxidant defense mechanisms in order to restore the homeostasis of cells. All three species are also able to accumulate high amounts of Cd in the leaves (several hundred mg of Cd/kg of dry leaves) and this phenomenon is amplified in the presence of salt. All together our results allow to consider the three Atriplex species as hyperaccumulators in the presence/absence of salt and as good candidates in a strategy of Cd phytoextraction in the presence of low concentrations of the pollutant. Nevertheless, both A. canescens and A. nummularia species seem to have a higher capacity to hyper-accumulate Cd when the concentration of Cd reaches higher level of contamination.

Hydrogenated and fluorinated surfactants derived from Tris(hydroxymethyl)-acrylamidomethane allow the purification of a highly active yeast F1-F0 ATP-synthase with an enhanced stability.

Loss of stability and integrity of large membrane protein complexes as well as their aggregation in a non-lipidic environment are the major bottlenecks to their structural studies. We have tested C(12)H(25)-S-poly-Tris-(hydroxymethyl)acrylamidomethane (H(12)-TAC) among many other detergents for extracting the yeast F(1)F(0) ATP-synthase. H(12)-TAC was found to be a very efficient detergent for removing the enzyme from mitochondrial membranes without altering its sensitivity towards specific ATP-synthase inhibitors. This extracted enzyme was then solubilized by either dodecyl maltoside (DDM), H(12)-TAC or fluorinated surfactants such as C(2)H(5)-C(6)F(12)-C(2)H(4)-S-poly-Tris-(hydroxymethyl)acrylamidomethane (H(2)F(6)-TAC) or C(6)F(13)-C(2)H(4)-S-poly-Tris-(hydroxymethyl)acrylamidomethane (F(6)-TAC), two surfactants exhibiting a comparable polar head to H(12)-TAC but bearing a fluorinated hydrophobic tail. Preparations from enzymes purified in the presence of H(12)-TAC were found to be more adapted for AFM imaging than ATP-synthase purified with DDM. Keeping H(12)-TAC during the Ni-NTA IMAC purification step or replacing it by DDM at low concentrations did not however allow preserving enzyme activity, while fluorinated surfactants H(2)F(6)-TAC and F(6)-TAC were found to enhance enzyme stability and integrity as indicated by sensitivity towards inhibitors. ATPase specific activity was higher with F(6)-TAC than with H(2)F(6)-TAC. When enzymes were mixed with egg phosphatidylcholine, ATP-synthases purified in the presence of H(2)F(6)-TAC or F(6)-TAC were more stable upon time than the DDM purified enzyme. Furthermore, in the presence of lipids, an activation of ATP-synthases was observed that was transitory for enzymes purified with DDM, but lasted for weeks for ATP-synthases isolated in the presence of molecules with Tris polyalcoholic moieties. Relipidated enzymes prepared with fluorinated surfactants remained highly sensitive towards inhibitors, even after 6 weeks.

Detrimental impact of silica nanoparticles on the nanomechanical properties of Escherichia coli, studied by AFM.

Despite great innovative and technological promises, nanoparticles (NPs) can ultimately exert an antibacterial activity by affecting the cell envelope integrity. This envelope, by conferring the cell its rigidity and protection, is intimately related to the mechanical behavior of the bacterial surface. Depending on their size, surface chemistry, shape, NPs can induce damages to the cell morphology and structure among others, and are therefore expected to alter the overall mechanical properties of bacteria. Although Atomic Force Microscopy (AFM) stands as a powerful tool to study biological systems, with high resolution and in near physiological environment, it has rarely been applied to investigate at the same time both morphological and mechanical degradations of bacteria upon NPs treatment. Consequently, this study aims at quantifying the impact of the silica NPs (SiO2-NPs) on the mechanical properties of E. coli cells after their exposure, and relating it to their toxic activity under a critical diameter. Cell elasticity was calculated by fitting the force curves with the Hertz model, and was correlated with the morphological study. SiO2-NPs of 100 nm diameter did not trigger any significant change in the Young modulus of E. coli, in agreement with the bacterial intact morphology and membrane structure. On the opposite, the 4 nm diameter SiO2-NPs did induce a significant decrease in E. coli Young modulus, mainly associated with the disorganization of lipopolysaccharides in the outer membrane and the permeation of the underlying peptidoglycan layer. The subsequent toxic behavior of these NPs is finally confirmed by the presence of membrane residues, due to cell lysis, exhibiting typical adhesion features.

Probing the threshold of membrane damage and cytotoxicity effects induced by silica nanoparticles in Escherichia coli bacteria.

The engineering of nanomaterials, because of their specific properties, is increasingly being developed for commercial purposes over the past decades, to enhance diagnosis, cosmetics properties as well as sensing efficiency. However, the understanding of their fate and thus their interactions at the cellular level with bio-organisms remains elusive. Here, we investigate the size- and charge-dependence of the damages induced by silica nanoparticles (SiO2-NPs) on Gram-negative Escherichia coli bacteria. We show and quantify the existence of a NPs size threshold discriminating toxic and inert SiO2-NPs with a critical particle diameter (Φc) in the range 50nm-80nm. This particular threshold is identified at both the micrometer scale via viability tests through Colony Forming Units (CFU) counting, and the nanometer scale via atomic force microscopy (AFM). At this nanometer scale, AFM emphasizes the interaction between the cell membrane and SiO2-NPs from both topographic and mechanical points of view. For SiO2-NPs with Φ>Φc no change in E. coli morphology nor its outer membrane (OM) organization is observed unless the NPs are positively charged in which case reorganization and disruption of the OM are detected. Conversely, when Φ<Φc, E. coli exhibit unusual spherical shapes, partial collapse, even lysis, and OM reorganization.

Morphological and nanostructural surface changes in Escherichia coli over time, monitored by atomic force microscopy.

The present study aims at evaluating intrinsic changes in Escherichia coli (E. coli) surface over time, by Atomic Force Microscopy (AFM). For that purpose, bacteria were immobilized on mica or on mica previously functionalized by the deposition of a polyelectrolyte multilayer cushion. AFM images reveal that E. coli population goes through different stages. Firstly, after a week, the number of healthy bacteria decreases resulting in a release of cellular components which likely become, in turn, a nutrition source for increasing the healthy population after around two weeks. Finally, after one month, most of the bacteria is dead. Our study shows a transition of a healthy rod-shaped bacterium to a dead collapsed one. Most importantly, along with the morphological evolution of bacteria, are the structure changes and the mechanical properties of their outer membrane, emphasized by AFM phase images with very high resolution. Indeed, the surface of healthy bacteria is characterized by a phase separation pattern, thereafter mentioned as "ripples". Bacterial ageing goes along with the loss of this organized structure, turning into circular areas with irregular boundaries. These changes are likely caused by a re-organization, due to external stress, of mainly lipopolysaccharides (LPS) present in the outer membrane of E. coli.

Influence of oxidized lipids on palmitoyl-oleoyl-phosphatidylcholine organization, contribution of Langmuir monolayers and Langmuir-Blodgett films.

In this work, we studied the interaction of two oxidized lipids, PoxnoPC and PazePC, with POPC phospholipid. Mean molecular areas obtained from (π-A) isotherms of mixed PoxnoPC-POPC and PazePC-POPC monolayers revealed different behaviors of these two oxidized lipids: the presence of PoxnoPC in the monolayers induces their expansion, mean molecular areas being higher than those expected in the case of ideal mixtures. PazePC-POPC behave on the whole ideally. This difference can be explained by a different conformation of oxidized lipids. Moreover the carboxylic function of PazePC is protonated under our experimental conditions, as shown by (π-A) isotherms of PazePC at different pH values. Both oxidized lipids induce also an increase of the monolayer elasticity, PoxnoPC being slightly more efficient than PazePC. These monolayers were transferred from the air-water interface onto mica supports for a study by AFM. AFM images are on the whole homogenous, suggesting the presence of only one lipid phase in both cases. However, in the case of PazePC-POPC monolayers, AFM images show also the presence of areas thicker of 7nm to 10nm than the surrounding lipid phase, probably due to the local formation of multilayer systems induced by compression.

Oscillatory dissipation of a simple confined liquid.

We present a sensitive measurement of the dissipation and the effective viscosity of a simple confined liquid (octamethylcyclotetrasiloxane) using an atomic force microscope. The experimental data show that the damping and the effective viscosity increase and present oscillations as the gap between the cantilever tip and the surface is diminished. To our knowledge, the damping and the viscosity modulation are reported here with such good accuracy for the first time. Such an experimental result is different from what has been reported earlier where only a continuous increase of the damping and the viscosity are observed.