© to the authors 2007
Received: 24 May 2007
Accepted: 25 June 2007
Published: 19 July 2007
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.
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 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 , the elasticity of cell walls , the molecular forces responsible for cell–cell and cell–solid interactions , and the localization of specific molecular recognition sites . The number of publications in which AFM is applied to microbiological samples has increased continuously 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 near-field 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 . 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 ).
Two-dimensional crystals of membrane proteins, and more recently native membranes, have proven to be particularly well-suited for high-resolution AFM imaging and manipulation . 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 , the hexagonally packed intermediate (HPI) layer of Deinococcus radiodurans , purple membrane from the archeon Halobacterium  and porins crystals of Escherichia coli . Function-related conformational changes can be monitored on single proteins, as nicely demonstrated for porin OmpF in which voltage and pH-induced channel closure was observed . There is also clear evidence that the instrument is evolving from an imaging technique to a multifunctional tool, enabling the measurement of multiple properties of membrane proteins . In particular, the combined use of AFM imaging and single molecule force spectroscopy makes it possible to pull on single membrane proteins, thereby providing novel molecular insight into their unfolding pathways and assembly forces [18, 19].
Yet, an important bottleneck has limited the widespread use of the technique in membrane research, i.e., the need to firmly attach the specimens onto a solid support for analysis, meaning the very central concept of a native membrane separating two aqueous compartments is not preserved. One-way to circumvent this limitation is to combine AFM with patch-clamp techniques in the same experimental setup, thereby offering the exciting possibility to image non-supported membrane patches and to study currents through single ion channels . More recently, a two-chamber AFM set-up was developed, by adsorbing membrane patches on holey silicon surfaces with nanoscale hole diameters and periodicities , enabling investigators to probe the structure, elasticity and energy of interaction of membrane proteins separating two aqueous compartments, and over which membrane gradients can be established.
Atomic force microscopy has also revealed the native ultrastructure of non-crystalline native membranes of different photosynthetic bacteria, including Rhodopseudomonas viridis , Rhodospirillum photometricum , Rhodobacter sphaeroides , Rhodobacter blasticus , and Rhodopseudomonas palustris , providing new insight on the supramolecular organization of membrane protein complexes. In one such study , AFM was used to investigate how the composition and architecture of photosynthetic membranes of R. photometricum change in response to light. Despite large modifications in the membrane composition, the local environment of core complexes remained unaltered, whereas specialized paracrystalline light-harvesting antenna domains grew under low-light conditions. It was concluded that such structural adaptation ensures efficient photon capture under low-light conditions and prevents photodamage under high-light conditions.
Progress in applying AFM imaging to microbial cells have been slower due essentially to difficulties associated with sample preparation and imaging conditions. While isolated membranes are generally well-immobilized by simple adsorption on mica , stronger attachment is often needed for whole cells . This can be achieved by drying the sample or by bonding the cells covalently to the support. However, these treatments may cause significant rearrangement, denaturation or contamination of the cell walls, yielding information that may no longer be representative of the native state. More appropriate approaches include treating the support with a polycation to strengthen cell adhesion or immobilizing the cells mechanically in an agar gel or in a porous membrane. In the agar method, the gel is used as a soft, deformable immobilization matrix, thereby allowing direct visualization of growth processes . In the porous membrane method, spherical cells are trapped in a polymer membrane with a pore size comparable to the dimensions of the cell, allowing repeated imaging in buffer solutions without cell detachment or cell damage .
Despite these technical difficulties, AFM is being used increasingly to visualize ultrastructural details on hydrated cells. Saccharomyces cerevisiae , Phanerochaete chrysosporium , Aspergillus oryzae , Aspergillus nidulans , Pinnularia viridis , lactic acid bacteria , Bacillus spores , Staphylococcus aureus , and Mycobacterium bovis [39, 40] are just a few examples of microbial species that have been explored by AFM in recent years.
Along the same line, cell growth and division events in S. aureus were monitored using AFM combined with thin-section TEM . Nanoscale holes were seen around the septal annulus at the onset of division and attributed to cell wall structures possessing high-autolytic activity. After cell separation, concentric rings were observed on the surface of the new cell wall and suggested to reflect newly formed peptidoglycan.
Single Molecule Measurements
Molecular recognition between receptors and cognate ligands plays a central role in cellular behaviors. For instance, cell adhesion and aggregation is usually mediated through specific cell adhesion proteins such as lectins and adhesins. Using single molecule force spectroscopy, the molecular forces driving receptor–ligand interactions can be directly measured on live cells. Further, affinity imaging offers a means to localize specific molecules on cells, such as cell adhesion proteins and antibiotic binding sites [11, 44].
Molecular Recognition Forces
Molecular recognition studies with the AFM implies functionalizing the tips (and solid surfaces) with relevant biomolecules, using procedures that meet the following requirements [11, 44]: (a) the forces which immobilize the molecules should be stronger than the intermolecular force being studied; (b) the attached biomolecules should have enough mobility so that they can freely interact with complementary molecules; (c) the contribution of non-specific adhesion to the measured forces should be minimized; (d) attaching biomolecules at low surface density is recommended in order to ensure single molecule detection; (e) site-directed coupling may be desired to orientate all the interacting molecules in the same way.
Molecular recognition forces are measured by recording force curves between modified tips and sample and then assessing the unbinding force between complementary receptor and ligand molecules from the adhesion force observed upon retraction. The measured unbinding forces are typically in the 50–400 pN range, depending on the experimental conditions [45–56]. Control experiments should always be carried out to demonstrate the specificity of the measured unbinding forces, which is best achieved by block experiments in which the receptor sites are masked by adding free ligands or by exploiting genetic mutation. Using these force spectroscopy experiments, a variety of ligand–receptor forces have been measured at the single molecule level including those associated with avidin/streptavidin [45, 46], antibodies , DNA , lectins , cadherins , integrins , and selectins . The four later studies are particularly interesting in cellular biology since they concern the specific forces associated with cell adhesion proteins, thereby contributing to refine our understanding of cellular interactions.
This review shows that nanomicrobiology––the exploration of microbial cells on the nanoscale—is an exciting research field that has developed very rapidly in the past years. AFM imaging enables investigators to visualize, under physiological conditions, the surface structure of membranes and cell surfaces, with unprecedented resolution. Conformational changes of membrane proteins can be detected at subnanometer resolution in relation with function. Time-lapse imaging offers a means to follow dynamic events occurring at cell surfaces, such as cell growth and drug-induced alterations. Single molecule force spectroscopy allows researchers to measure the forces and dynamics of receptor–ligand interactions and to identify and localize specific sites on live cells. These nanoscale studies provide new avenues in many areas, particularly in biomedicine for investigating the mode of action of drugs, and for elucidating the molecular basis of host–pathogen interactions.
This work was supported by the National Foundation for Scientific Research (FNRS), the Foundation for Training in Industrial and Agricultural Research (FRIA), the Région wallonne, the Université Catholique de Louvain (Fonds Spéciaux de Recherche), and the Federal Office for Scientific, Technical and Cultural Affairs (Interuniversity Poles of Attraction Programme). Y. F. D. is a Research Associate of the FNRS.
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