Surface Structure and Nanomechanical Properties of Shewanella putrefaciens Bacteria at Two pH values (4 and 10) Determined by Atomic Force Microscopy (original) (raw)
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PLoS ONE, 2011
The physicochemical properties and dynamics of bacterial envelope, play a major role in bacterial activity. In this study, the morphological, nanomechanical and electrohydrodynamic properties of Escherichia coli K-12 mutant cells were thoroughly investigated as a function of bulk medium ionic strength using atomic force microscopy (AFM) and electrokinetics (electrophoresis). Bacteria were differing according to genetic alterations controlling the production of different surface appendages (short and rigid Ag43 adhesins, longer and more flexible type 1 fimbriae and F pilus). From the analysis of the spatially resolved force curves, it is shown that cells elasticity and turgor pressure are not only depending on bulk salt concentration but also on the presence/absence and nature of surface appendage. In 1 mM KNO 3 , cells without appendages or cells surrounded by Ag43 exhibit large Young moduli and turgor pressures (,700-900 kPa and ,100-300 kPa respectively). Under similar ionic strength condition, a dramatic ,50% to ,70% decrease of these nanomechanical parameters was evidenced for cells with appendages. Qualitatively, such dependence of nanomechanical behavior on surface organization remains when increasing medium salt content to 100 mM, even though, quantitatively, differences are marked to a much smaller extent. Additionally, for a given surface appendage, the magnitude of the nanomechanical parameters decreases significantly when increasing bulk salt concentration. This effect is ascribed to a bacterial exoosmotic water loss resulting in a combined contraction of bacterial cytoplasm together with an electrostatically-driven shrinkage of the surface appendages. The former process is demonstrated upon AFM analysis, while the latter, inaccessible upon AFM imaging, is inferred from electrophoretic data interpreted according to advanced soft particle electrokinetic theory. Altogether, AFM and electrokinetic results clearly demonstrate the intimate relationship between structure/flexibility and charge of bacterial envelope and propensity of bacterium and surface appendages to contract under hypertonic conditions.
Journal of Molecular Recognition, 2012
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.
Spatially resolved force spectroscopy of bacterial surfaces using force-volume imaging
Colloids and Surfaces B: Biointerfaces, 2008
Force spectroscopy using the atomic force microscope (AFM) is a powerful technique for measuring physical properties and interaction forces at microbial cell surfaces. Typically for such a study, the point at which a force measurement will be made is located by first imaging the cell using AFM in contact mode. In this study, we image the bacterial cell Shewanella putrefaciens for subsequent force measurements using AFM in force-volume mode and compare this to contact-mode images. It is known that contact-mode imaging does not accurately locate the apical surface and periphery of the cell since, in contact mode, a component of the applied load laterally deforms the cell during the raster scan. Here, we illustrate that contact-mode imaging does not accurately locate the apical surface and periphery of the cell since, in contact mode, a component of the applied load laterally deforms the cell during the raster scan. This is an artifact due to the deformability and high degree of curvature of bacterial cells. We further show that force-volume mode imaging avoids the artifacts associated with contact-mode imaging due to surface deformation since it involves the measurement of a grid of individual force profiles. The topographic image is subsequently reconstructed from the zero-force height (the contact distance between the AFM tip and the surface) at each point on the cell surface. We also show how force-volume measurements yield applied load versus indentation data from which mechanical properties of the cell such as Young's modulus, cell turgor pressure and elastic and plastic energies can be extracted.
Microbial Surfaces Investigated Using Atomic Force Microscopy
Biotechnology Progress, 2004
This paper is dedicated to atomic force microscopy (AFM) as a progressive tool for imaging bacterial surfaces and probing their properties. The description of the technique is complemented by the explanation of the method's artifacts typical, in particular, for the imaging of bacterial cells. Sample preparation techniques are summarized in a separate section. Special attention is paid to the differences in imaging of Gram-positive and Gram-negative bacteria. Probing of mechanical properties, including elastic modulus, fragility, and adhesion of the cell walls is emphasized. The advantages of AFM in the studies of real-time cellular dynamical processes are illustrated by the experiment with the germination of spores.
Atomic Force Microscopy Measurement of Heterogeneity in Bacterial Surface Hydrophobicity
Langmuir, 2008
The structure and physicochemical properties of microbial surfaces at the molecular level determine their adhesion to surfaces and interfaces. Here, we report the use of atomic force microscopy (AFM) to explore the morphology of soft, living cells in aqueous buffer, to map bacterial surface heterogeneities, and to directly correlate the results in the AFM force-distance curves to the macroscopic properties of the microbial surfaces. The surfaces of two bacterial species, Acinetobacter Venetianus RAG-1 and Rhodococcus erythropolis 20S-E1-c, showing different macroscopic surface hydrophobicity were probed with chemically functionalized AFM tips, terminating in hydrophobic and hydrophilic groups. All force measurements were obtained in contact mode and made on a location of the bacterium selected from the alternating current mode image. AFM imaging revealed morphological details of the microbial-surface ultrastructures with about 20 nm resolution. The heterogeneous surface morphology was directly correlated with differences in adhesion forces as revealed by retraction force curves and also with the presence of external structures, either pili or capsules, as confirmed by transmission electron microscopy. The AFM force curves for both bacterial species showed differences in the interactions of extracellular structures with hydrophilic and hydrophobic tips. A. Venetianus RAG-1 showed an irregular pattern with multiple adhesion peaks suggesting the presence of biopolymers with different lengths on its surface. R. erythropolis 20S-E1-c exhibited long-range attraction forces and single rupture events suggesting a more hydrophobic and smoother surface. The adhesion force measurements indicated a patchy surface distribution of interaction forces for both bacterial species, with the highest forces grouped at one pole of the cell for R. erythropolis 20S-E1-c and a random distribution of adhesion forces in the case of A. Venetianus RAG-1. The magnitude of the adhesion forces was proportional to the three-phase contact angle between hexadecane and water on the bacterial surfaces.
Nanoscale Mapping of the Elasticity of Microbial Cells by Atomic Force Microscopy
Langmuir, 2003
Single microbial cells can show important local variations of elasticity due to the complex, anisotropic composition of their walls. An example of this is the yeast during cell division, where chitin is known to accumulate in the localized region of the cell wall involved in budding. We used atomic force microscopy (AFM) to measure quantitatively the local mechanical properties of hydrated yeast cells. Topographic images and spatially resolved force maps revealed significant lateral variations of elasticity across the cell surface, the bud scar region being significantly stiffer than the surrounding cell wall. To get quantitative information on sample elasticity, force curves were converted into force vs indentation curves. The curves were then fitted with the Hertz model, yielding Young's modulus values of 6.1 ( 2.4 and 0.6 ( 0.4 MPa for the bud scar and surrounding cell surface, respectively. These data lead us to conclude that in yeast, the bud scar is 10 times stiffer than the surrounding cell wall, a finding which is consistent with the accumulation of chitin in the bud scar region. This is the first report in which spatially resolved AFM force curves are used to distinguish regions of different elasticity at the surface of single microbial cells in relation with function (i.e., cell division). In future research, this approach will provide fundamental insights into the spatial distribution of physical properties at heterogeneous microbial cell surfaces.