Nanomicrobiology (pdf)

Article PDF cannot be displayed. You can download it here:

https://link.springer.com/content/pdf/10.1007%2Fs11671-007-9077-1.pdf

Nanomicrobiology

Nanoscale Res Lett (2007) 2:365–372 DOI 10.1007/s11671-007-9077-1 NANO REVIEW Nanomicrobiology David Alsteens Æ Etienne Dague Æ Claire Verbelen Æ Guillaume Andre Æ Grégory Francius Æ Yves F. Dufrêne Received: 24 May 2007 / Accepted: 25 June 2007 / Published online: 19 July 2007 to the authors 2007 Abstract Recent advances in atomic force microscopy (AFM) are revolutionizing our views of microbial surfaces. While AFM imaging is very useful for visualizing the surface of hydrated cells and membranes on the nanoscale, force spectroscopy enables researchers to locally probe biomolecular forces and physical properties. These unique capabilities allow us to address a number of questions that were inaccessible before, such as how does the surface architecture of microbes change as they grow or interact with drugs, and what are the molecular forces driving their interaction with antibiotics and host cells? Here, we provide a flavor of recent achievements brought by AFM imaging and single molecule force spectroscopy in microbiology. Keywords AFM Cells Imaging Force spectroscopy Molecular recognition Single molecule Ultrastructure Introduction During the past 40 years, the importance of the microbial cell surface in biology, medicine, industry, and ecology has been increasingly recognized. Because they constitute the frontier between the cells and their environment, microbial cell walls play several key functions: supporting the internal turgor pressure of the cell, protecting the cytoplasm from the outer environment, imparting shape to the D. Alsteens E. Dague C. Verbelen G. Andre G. Francius Y. F. Dufrêne (&) Unité de Chimie des Interfaces, Université Catholique de Louvain, Croix du Sud 2/18, B-1348 Louvain-la-Neuve, Belgium e-mail: organism, acting as a molecular sieve, controlling molecular recognition and cell adhesion, and being the target of antibiotics. These functions have major consequences in biotechnology (wastewater treatment, bioremediation, and immobilized cells in reactors), industrial systems (biofouling and contamination) and medicine (interactions of pathogens with animal host tissues, accumulation on implants and prosthetic devices). This emphasizes the need to develop new techniques for probing the structure, properties and interactions of microbial surfaces. Traditionally, probing of the cell surface architecture relies on transmission (TEM) and scanning (SEM) electron microscopy techniques [1–5]. Although cryo-methods have allowed researchers to get more natural views of bacterial cell envelopes, these approaches are very demanding in terms of sample preparation and analysis and are only applied in a few laboratories worldwide. Valuable information on the composition, properties and interactions of cell surfaces can also be gained using electron microscopy approaches, biochemical analysis, biophysical techniques and surface analysis methods [4, 5]. These techniques usually involve cell manipulation prior to examination and often provide averaged information obtained on large ensembles of cells. However, recent advances in atomic force microscopy (AFM) are helping to overcome these problems by providing three-dimensional images of hydrated cells and membranes with nanometer resolution [6, 7], and enabling researchers to probe a variety of molecular forces and physical properties on cell surfaces, including the unfolding pathways of single membrane proteins [8], the elasticity of cell walls [9], the molecular forces responsible for cell–cell and cell–solid interactions [10], and the localization of specific molecular recognition sites [11]. The number of publications in which AFM is applied to microbiological samples has increased continuously 123 366 over the past years, indicating that a new field is born, i.e., nanomicrobiology. The general principle of AFM is to scan a sharp tip over the surface of a sample, while sensing the so-called nearfield physical interactions between the tip and the sample. This allows three-dimensional images to be generated directly in aqueous solution. The sample is mounted on a piezoelectric scanner which ensures three-dimensional positioning with high accuracy. While the tip (or sample) is being scanned in the (x, y) directions, the force interacting between tip and specimen is monitored with piconewton sensitivity. This force is measured by the deflection of a soft cantilever which is detected by a laser beam focused on the free end of the cantilever and reflected into a photodiode. A number of different AFM imaging modes are available, which differ mainly in the way the tip is moving over the sample. In the so-called contact mode, the AFM tip is raster scanned over the sample while the cantilever deflection, thus the force applied to the tip, is kept constant using feedback control. In dynamic or intermittent mode, an oscillating tip is scanned over the surface and the amplitude and phase of the cantilever are monitored near its resonance frequency. Because lateral forces during imaging are greatly reduced with dynamic modes, they are advantageous for imaging soft biological samples. In force spectroscopy, the cantilever deflection is recorded as a function of the vertical displacement of the piezoelectric scanner, i.e., as the sample is pushed toward the tip and retracted. This results in a cantilever deflection versus scanner displacement curve, which can be transformed into a force-distance curve using appropriate corrections. For most microbiological applications, accurate determination of the contact point (zero separation distance) between the AFM tip and the soft sample is rather delicate due to the complex contributions of surface forces and mechanical deformation [9]. Force-distance curves can be recorded either at single, well-defined locations of the (x, y) plane or at multiple locations to yield a so-called ‘force-volume image.’ In doing so, spatially resolved maps of physical properties (elasticity and adhesion) and molecular interactions can be produced (for a review on force spectroscopy methodology and applications, see [12]). Structural Imaging Membrane Proteins Two-dimensional crystals of membrane proteins, and more recently native membranes, have proven to be particularly well-suited for high-resolution AFM imaging and 123 Nanoscale Res Lett (2007) 2:365–372 manipulation [6]. Owing to continuous progress in instrumentation, sample preparation methods and recording conditions, structural information can now be routinely obtained on membrane proteins to a resolution of 0.5–1 nm and under physiological conditions, which makes AFM a complementary tool to X-ray and electron crystallography. Examples of such protein crystalline arrays that have been visualized with subnanometer resolution include Bacillus S-layers [13], the hexagonally packed intermediate (HPI) layer of Deinococcus radiodurans [14], purple membrane from the archeon Halobacterium [15] and porins cry (...truncated)