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

Abstract

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.

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.