Nanoscale dielectric microscopy of non-planar samples by lift-mode electrostatic force microscopy

Lift-mode electrostatic force microscopy (EFM) is one of the most convenient imaging modes to study the local dielectric properties of non-planar samples. Here we present the quantitative analysis of this imaging mode. We introduce a method to quantify and subtract the topographic crosstalk from the lift-mode EFM images, and a 3D numerical approach that allows for extracting the local dielectric constant with nanoscale spatial resolution free from topographic artifacts. We demonstrate this procedure by measuring the dielectric properties of micropatterned SiO2 pillars and of single bacteria cells, thus illustrating the wide applicability of our approach from materials science to biology

Internal hydration properties of single bacterial endospores probed by electrostatic force microscopy

We show that the internal hydration properties of single Bacillus cereus endospores in air under different relative humidity (RH) conditions can be determined through the measurement of its electric permittivity by means of quantitative electrostatic force microscopy (EFM). We show that an increase in the RH from 0% to 80% induces a large increase in the equivalent homogeneous relative electric permittivity of the bacterial endospores, from ∼4 up to ∼17, accompanied only by a small increase in the endospore height, of just a few nanometers. These results correlate the increase of the moisture content of the endospore with the corresponding increase of environmental RH. Three-dimensional finite element numerical calculations, which include the internal structure of the endospores, indicate that the moisture is mainly accumulated in the external layers of the endospore, hence preserving the core of the endospore at low hydration levels. This mechanism is different from what we observe for vegetative bacterial cells of the same species, in which the cell wall at high humid atmospheric conditions is not able to preserve the cytoplasmic region at low hydration levels. These results show the potential of quantitative EFM under environmental humidity control to study the hygroscopic properties of small-scale biological (and nonbiological) entities and to determine its internal hydration state. A better understanding of nanohygroscopic properties can be of relevance in the study of essential biological processes and in the design of bionanotechnological application

Regulation of ribonucleotide synthesis by the Pseudomonas aeruginosa two-component system AlgR in response to oxidative stress

Ribonucleotide reductases (RNR) catalyze the last step of deoxyribonucleotide synthesis, and are therefore essential to DNA-based life. Three forms of RNR exist: classes I, II, and III. While eukaryotic cells use only class Ia RNR, bacteria can harbor any combination of classes, granting them adaptability. The opportunistic pathogen Pseudomonas aeruginosa surprisingly encodes all three classes, allowing it to thrive in diferent environments. Here we study an aspect of the complex RNR regulation whose molecular mechanism has never been elucidated, the well-described induction through oxidative stress, and link it to the AlgZR two-component system, the primary regulator of the mucoid phenotype. Through bioinformatics, we identify AlgR binding locations in RNR promoters, which we characterize functionally through EMSA and physically through AFM imaging. Gene reporter assays in diferent growth models are used to study the AlgZR-mediated control on the RNR network under various environmental conditions and physiological states. Thereby, we show that the two-component system AlgZR, which is crucial for bacterial conversion to the mucoid phenotype associated with chronic disease, controls the RNR network and directs how the DNA synthesis pathway is modulated in mucoid and non-mucoid bioflms, allowing it to respond to oxidative stress

Hygroscopic properties of single bacterial cells and endospores studied by Electrostatic Force Microscopy

[eng] The large abundance of bacterial growth niches provide a rich diversity of bacterial traits. These are usually characterized using traditional microbiology research tools, and newer characterization techniques (which focus on addressing physical and physicochemical properties). Most of these techniques are performed at the level of colonies, where millions of cells are analysed and hinder the heterogeneity of single cells. The Atomic Force Microscope (AFM) is emerging as a promising nanotechnology tool for single bacterial cell studies (Nanomicrobiology), since it is capable of characterizing the structure and simultaneously obtaining other physical properties of interest under physiological conditions. To sustain harsh conditions, some bacterial species have the ability to produce endospores. This environmental resistance has been mainly attributed to the way endospores control its water content. A heterogeneous distribution of the water content plays a key role in the resistance. Despite the large existing literature in hydration properties of bacterial endospores, the hydration capabilities of endospores still present some open questions. In this work of thesis the hygroscopic properties of single bacterial cells and endospores are studied under different environmental conditions. To achieve these results, we have made use of the Electrostatic Force Microscopy (EFM), an adaptation of the AFM which can report changes in the dielectric properties of individual bacterial samples. Firstly of all, biocompatible gelatine was used to weakly attach bacterial cells, and the dynamic jumping mode was used to drastically reduce the shear forces provoked on bacterial samples during conventional AFM imaging. This methodology allowed us to observe in situ bacterial cell division at the single cell and nanoscale resolution. Due to the large morphology of bacterial samples, lift mode EFM had to be used. This electrical imaging mode hinders the intrinsic contribution of the sample under study due to topographical crosstalk contribution. A method was proposed to remove topographical crosstalk contribution, which revealed electrical homogeneity of inorganic calibration samples and of dried single bacterial cells. The use of a subsurface sample revealed the capabilities of the EFM as a tool for subsurface characterization. Such ability revealed the potential of the EFM to detect water distribution within the bacterial cell samples under study in this work of thesis. The electrical characterization of bacterial vegetative cells and bacterial endospores under a range of different relative humidity allowed us to study the difference in hygroscopic properties between the two samples. At low relative humidity, 40% RH, the bacterial endospores hardly hydrate in comparison to the bacterial vegetative cells. At high relative humidity, 80% RH, the bacterial vegetative cells drastically hydrate in comparison to the bacterial endospores. In the latter case, it has been demonstrated that the external layers accommodate most of the moisture absorbed, leaving the core at low hydration levels. In the case of the vegetative cells, the cell wall is not able to accommodate such high levels of moisture, forcing the cytoplasmic region to become highly hydrated. This discrepancy in the hydration behaviour seems key for the persistence of the core region as the driest region of the bacterial endospores in atmospheric conditions. Finally, electrical measurements performed under liquid conditions revealed the high hydration state of the living bacterial cells in contraposition to bacterial endospores. This lower hydration of the endospores under liquid conditions could be attributable to the difference in structure. All together, these results obtained in this work of thesis have shown the lower hydration properties of single bacterial endospores in contraposition to its vegetative cell in all environmental conditions, from dry conditions up to liquid environments.[cat] El microscopi de forces atòmiques (AFM) s'està convertint en una eina prometedora per a la caracterització de bacteris individuals, ja que presenten un ampli ventall de característiques. (Nanomicrobiologia). En particular, alguns bacteris són capaç de produir endòspores que resisteixen condicions ambientals extremes. S'ha observat que aquesta resistència està lligada al contingut de l'aigua, i en particular en la capacitat de mantenir el nucli sec. L'objectiu d'aquest treball de tesis és l'estudi de les seves propietats higroscòpiques en diferents condicions ambientals. En primer lloc es van obtenir imatges de bacteris individuals dividint-se amb resolució nanomètrica. L'ús de gelatina i un mètode d'imatge poc agressiu (dynamic jumping mode) va permetre imitar condicions naturals. A causa de la gran morfologia de les mostres bacterianes, es va utilitzar un mètode d'imatge elèctrica que emmascarava la contribució intrínseca de la mostra. La quantificació del sistema va permetre revelar homogeneïtat elèctrica de cèl·lules bacterianes individuals seques. L'ús d'una mostra subsuperficial va revelar el potencial del EFM per detectar la distribució de l'aigua dins de les cèl·lules bacterianes. La caracterització elèctrica de les cèl·lules vegetatives bacterianes i les endòspores bacterianes va revelar una major hidratació de les cèl·lules vegetatives bacterianes en contraposició a les endòspores bacterianes. A elevada humitat relativa, les cèl·lules vegetatives s'hidraten dràsticament i causen la hidratació de la regió citoplasmàtica, mentre que les endòspores tenen la capacitat de deixar el nucli en nivells baixos d'hidratació. Aquesta discrepància en el comportament d'hidratació sembla clau per a la persistència de la latència de les endòspores en condicions atmosfèriques. Finalment, mesures elèctriques realitzades en líquid van revelar un estat d'alta hidratació de les cèl·lules bacterianes vives en contraposició a les endòspores bacterianes. Aquesta hidratació inferior de les endòspores en condicions de líquid podria ser atribuïble a la diferència en l'estructura. Tot junt, aquests resultats obtinguts en aquest treball de tesi han demostrat una menor propietat d'hidratació en les endòspores bacterianes en contraposició a la seva cèl·lula vegetativa en totes les condicions ambients, des de condicions seques fins a líquides

Long-Lasting and Responsive DNA/Enzyme-Based Programs in Serum-Supplemented Extracellular Media

International audienceDNA molecular programs are emerging as promising pharmaceutical approaches due to their versatility for biomolecular sensing and actuation. However, the implementation of DNA programs has been mainly limited to serum-deprived in vitro assays due to the fast deterioration of the DNA reaction networks by the nucleases present in the serum. Here, we show that DNA/enzyme programs are functional in serum for 24 h but are later disrupted by nucleases that give rise to parasitic amplification. To overcome this, we implement three-letter code networks that suppress autocatalytic parasites while still conserving the functionality of DNA/enzyme programs for at least 3 days in the presence of 10% serum. In addition, we define a new buffer that further increases the biocompatibility and conserves responsiveness to changes in molecular composition across time. Finally, we demonstrate how serum-supplemented extracellular DNA molecular programs remain responsive to molecular inputs in the presence of living cells, having responses 6-fold faster than the cellular division rate, and are sustainable for at least three cellular divisions. This demonstrates the possibility of implementing in situ biomolecular characterization tools for serum-demanding in vitro models. We foresee that the coupling of chemical reactivity to our DNA programs by aptamers or oligonucleotide conjugations will allow the implementation of extracellular synthetic biology tools, which will offer new biomolecular pharmaceutical approaches and the emergence of complex and autonomous in vitro models

rEXPAR: An Isothermal Amplification Scheme That Is Robust to Autocatalytic Parasites

International audienceIn the absence of DNA, a solution containing the four deoxynucleotidetriphosphates (dNTPs), a DNA polymerase, and a nicking enzyme generates a self-replicating mixture of DNA species called parasite. Parasites are problematic in template-based isothermal amplification schemes such as EXPAR as well as in related molecular programming approaches, such as the PEN DNA toolbox. Here we show that using a nicking enzyme with only three letters (C, G, T) in the top strand of its recognition site, such as Nb.BssSI, allows us to change the sequence design of EXPAR templates in a way that prevents the formation of parasites when dATP is removed from the solution. This method allows us to make the EXPAR reaction robust to parasite contamination, a common feature in the laboratory, while keeping it compatible with PEN programs, which we demonstrate by engineering a parasite-proof bistable reaction network

Nanoscale dielectric microscopy of non-planar samples by lift-mode electrostatic force microscopy

Lift-mode electrostatic force microscopy (EFM) is one of the most convenient imaging modes to study the local dielectric properties of non-planar samples. Here we present the quantitative analysis of this imaging mode. We introduce a method to quantify and subtract the topographic crosstalk from the lift-mode EFM images, and a 3D numerical approach that allows for extracting the local dielectric constant with nanoscale spatial resolution free from topographic artifacts. We demonstrate this procedure by measuring the dielectric properties of micropatterned SiO2 pillars and of single bacteria cells, thus illustrating the wide applicability of our approach from materials science to biology

Nanoscale dielectric microscopy of non-planar samples by lift-mode electrostatic force microscopy

Lift-mode electrostatic force microscopy (EFM) is one of the most convenient imaging modes to study the local dielectric properties of non-planar samples. Here we present the quantitative analysis of this imaging mode. We introduce a method to quantify and subtract the topographic crosstalk from the lift-mode EFM images, and a 3D numerical approach that allows for extracting the local dielectric constant with nanoscale spatial resolution free from topographic artifacts. We demonstrate this procedure by measuring the dielectric properties of micropatterned SiO2 pillars and of single bacteria cells, thus illustrating the wide applicability of our approach from materials science to biology