Force volume and stiffness tomography investigation on the dynamics of stiff material under bacterial membranes

Laboratory of Physics of Living Matter, EPFL, Lausanne, Switzerland.
Journal of Molecular Recognition (Impact Factor: 2.15). 05/2012; 25(5):278-84. DOI: 10.1002/jmr.2171
Source: PubMed


The determination of the characteristics of micro-organisms in clinical specimens is essential for the rapid diagnosis and treatment of infections. A thorough investigation of the nanoscale properties of bacteria can prove to be a fundamental tool. Indeed, in the latest years, the importance of high resolution analysis of the properties of microbial cell surfaces has been increasingly recognized. Among the techniques available to observe at high resolution specific properties of microscopic samples, the Atomic Force Microscope (AFM) is the most widely used instrument capable to perform morphological and mechanical characterizations of living biological systems. Indeed, AFM can routinely study single cells in physiological conditions and can determine their mechanical properties with a nanometric resolution. Such analyses, coupled with high resolution investigation of their morphological properties, are increasingly used to characterize the state of single cells. In this work, we exploit the capabilities and peculiarities of AFM to analyze the mechanical properties of Escherichia coli in order to evidence with a high spatial resolution the mechanical properties of its structure. In particular, we will show that the bacterial membrane is not mechanically uniform, but contains stiffer areas. The force volume investigations presented in this work evidence for the first time the presence and dynamics of such structures. Such information is also coupled with a novel stiffness tomography technique, suggesting the presence of stiffer structures present underneath the membrane layer that could be associated with bacterial nucleoids.

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Available from: Giovanni Longo, Aug 05, 2014
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    • "In previous studies, the localized accumulation or compartmentalization of DNA, nucleoids, and proteins such as stressinduced DNA-binding protein Dps, have been associated with increase in the stiffness of bacterial cell membrane [25] [26]. Our results show that DC treatment using carbon electrodes resulted in statistically significant increase in the stiffness of PAO1 cells. "

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    • "So that the bacterial membrane and its higher stiffness would appear only at a restricted spots and thus generate an important roughness upon the bacterium we did not observe; (ii) the Nostoc is moving as a caterpillar. The Nostoc would retract from the sample from place to place, leading to a disruption in mechanical contact with the glass slide and consequently causing a vanishing of stiffness; (iii) a mechanical wave with an alternation of high and low stiffness zones running through the filamentous bacterium during its gliding; (iv) the presence of mobile hard structures, as nucleoids, present underneath the membrane layer [44]. However as we did not measure noticeable variations of height along the nodules the two first hypotheses might be ruled out. "
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    ABSTRACT: We present a study about AFM imaging of living, moving or self-immobilized, bacteria in their genuine physiological liquid medium. No external immobilization protocol, neither chemical nor mechanical, was needed. For the first time, the native gliding movements of Gram-negative Nostoc cyanobacteria upon the surface, at speeds up to 900m/h, were studied by AFM. This was possible thanks to an improved combination of a gentle sample preparation process and an AFM procedure based on fast and complete force-distance curves made at every pixel, drastically reducing lateral forces. No limitation in spatial resolution or imaging rate was detected. Gram-positive and non-motile Rhodococcus wratislaviensis bacteria were studied as well. From the approach curves, Young modulus and turgor pressure were measured for both strains at different gliding speeds and are ranging from 20±3 to 105±5MPa and 40±5 to 310±30kPa depending on the bacterium and the gliding speed. For Nostoc, spatially limited zones with higher values of stiffness were observed. The related spatial period is much higher than the mean length of Nostoc nodules. This was explained by an inhomogeneous mechanical activation of nodules in the cyanobacterium. We also observed the presence of a soft extra cellular matrix (ECM) around the Nostoc bacterium. Both strains left a track of polymeric slime with variable thicknesses. For Rhodococcus, it is equal to few hundreds of nanometers, likely to promote its adhesion to the sample. While gliding, the Nostoc secretes a slime layer the thickness of which is in the nanometer range and increases with the gliding speed. This result reinforces the hypothesis of a propulsion mechanism based, for Nostoc cyanobacteria, on ejection of slime. These results open a large window on new studies of both dynamical phenomena of practical and fundamental interests such as the formation of biofilms and dynamic properties of bacteria in real physiological conditions.
    PLoS ONE 03/2013; 8(4). DOI:10.1371/journal.pone.0061663 · 3.23 Impact Factor
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    • "Any images of this microorganism are therefore impossible to record. Recent works by Longo et al. (2012) present nanoindentation images of E. coli cells. This is also a way to image this bacterial species, however, the lateral resolution shown by the authors is of 32 px 2 , which is very low and does not allow a detailed observation of the bacterial cell wall. "
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    ABSTRACT: Since the last 10 years, AFM has become a powerful tool to study biological samples. However, the classical modes offered (imaging or tapping mode) often damage sample that are too soft or loosely immobilized. If imaging and mechanical properties are required, it requests long recording time as two different experiments must be conducted independently. In this study we compare the new QI™ mode against contact imaging mode and force volume mode, and we point out its benefit in the new challenges in biology on six different models: Escherichia coli, Candida albicans, Aspergillus fumigatus, Chinese hamster ovary cells and their isolated nuclei, and human colorectal tumor cells.
    Micron 02/2013; 48. DOI:10.1016/j.micron.2013.02.003 · 1.99 Impact Factor
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