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In Vivo Atomic Force Microscopy of Surface Proteins on Deinococcus radiodurans T. E. Lister and P. J. Pinhero* Idaho National Engineering and Environmental Laboratory, PO Box 1625, Idaho Falls, Idaho 83415-2218 Received October 16, 2000. In Final Form: February 12, 2001 We present in vivo images of the outer cell wall of the bacteria Deinococcus radiodurans using noncontact atomic force microscopy (AFM). High-resolution imaging shows a hexagonal-packed lattice. This lattice is consistent with previous observations of a hexagonally packed intermediate layer from extracted cell wall material. The ordered hexagonal structures were primarily observed at the interfaces between adjoining bacteria, while rougher, “orange-peel-like” structures were observed in other regions. It is possible that the bacteria were in a dormant state during imaging as the desiccation tolerance of D. radiodurans is well documented. The only previous observations of the HPI layer of D. radiodurans have been performed on chemically extracted S-layers applied to flat substrates. Our report is the first in vivo observation of these structures in D. radiodurans without invasive sample preparation. Additionally, this is the first known report of in vivo observation of bacterial S-layers by AFM.
Introduction The outer surfaces of bacteria, surface (S-) layer, possess unique structures that may initially determine its response to variations in environmental conditions. The roles fulfilled by the S-layer may include various functions such as providing cell protection, determining both cell adhesion and cell recognition, acting as structural scaffolds for enzymes, and providing virulence factors.1 It is known from electron microscopy (EM) that S-layer subunits may possess various symmetries. Linear (p1, p2), tetragonal (p4), and hexagonal symmetries (p3, p6) have been observed.2 Due to the highly periodic nature of S-layers, a variety of applications have been proposed. Most of these depend on the in vitro self-assembly capabilities of isolated S-layer subunits on the surfaces of solid substrates (e.g., silicon wafers, polymer composites, and metals), Langmuir-lipid films, and liposomes. EM traditionally has been the tool of choice for viewing submicrometer structural details of S-layers. Nevertheless, due to the limited electron scattering efficiencies of the lighter elements (hydrogen, carbon, nitrogen, and oxygen) and the susceptibility of biomacromolecules to damage induced by a beam of high-energy electrons, biomolecules must often be processed prior to visualization with EM. Processing eventually extracts water and thus contributes to denaturing of the biological structures which ultimately means that most of the details elucidating information about function is lost. Scanning probe microscopy (SPM) is a well-suited alternative technique for observing submicrometer detail of S-layers. SPM is based upon the interaction of a finely constructed submicrometer probe with a small area of a specimen surface. A supreme advantage of SPM over EM is that it is nonintrusive and, therefore, should yield a more natural, in vivo, representation of the true native structure of an organism. The S-layer of Deinococcus radiodurans has p6 (i.e., hexagonal) symmetry. It is therefore commonly referred to as the hexagonally packed intermediate (HPI) layer. A lattice constant of 18 nm was determined with contact (1) Firtel, M.; Beveridge, T. J. Micron 1995, 26, 347-62. (2) Sara, M.; Sleytr, U. B. J. Bacteriol. 2000, 182, 859-868.
mode atomic force microscopy (AFM).3-5 It should be noted that the HPI layer observed in these studies is extracted from the host organism with sodium dodecyl sulfate and immobilized on glass substrates by photo-cross-linking. Most of the published studies focus on the inner surface of the HPI layer, despite the fact that the outer surface is much more important in determining interactions with the surrounding medium. Due to preimage processing, the images are no more representative of the true in situ HPI structure than are the surfaces observed with EM techniques. Here we show the first published AFM images of the HPI layer of D. radiodurans acquired in vivo. This is a first step in tailoring imaging techniques toward observing bacteria in their native state, unperturbed by sample preparation. Information about the native outer surface of D. radiodurans may shed light on its uniqueness with respect to radiation tolerance and desiccation resistance. The radiation tolerance of D. radiodurans has been well documented by our research group6 as well as others.7 Though the level of radiation tolerance displayed by D. radiodurans may be wholly a product of genetic factors, our group observed a loose correlation between γ-radiation tolerance and cell wall structure in the cited previous study. The issue of desiccation resistance obviously is strongly related to variations in HPI structure. As bacteria begin to dehydrate, their surfaces contract and fold as a result of the reduction in volume. We have observed in other studies8 that adjacent bacteria can self-assemble as a cooperative response to dehydration processes. Examination of individual bacteria and changes in their as(3) Engel, A.; Baumeister, W.; Saxton, W. O. Proc. Natl. Acad. Sci. 1982, 79, 4050-4. (4) Baumeister, W.; Karrenberg, F.; Rachel, R.; Engel, A.; Heggeler, B. t.; Saxton, W. O. Eur. J. Biochem. 1982, 21, 535-44. (5) Baumeister, W.; Barth, M.; Hegerl, R.; Guckenberger, R.; Hahn, M.; Saxton, W. O. J. Mol. Biol. 1986, 187, 241-250. (6) Bruhn, D. F.; Breckenridge, C. R.; Tsang, M. N.; Watkins, C. S.; Windes, W. E.; Roberto, F. F.; Wright, R. N.; Pinhero, P. J.; Brey, R. F. Irradiation of Microbes from Spent Nuclear Fuel Storage Pool Environments; Global ’99: International Conference on Future Nuclear Systems, Jackson Hole, WY, 1999. (7) Daly, M. J.; Minton, K. W. Science 1995, 270, 1318. (8) Breckenridge, C. R.; Lister, T. E.; Pinhero, P. J. 2000.
10.1021/la001448g CCC: $20.00 © 2001 American Chemical Society Published on Web 04/05/2001
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sociated S-layer structure may illuminate how biofilms respond to drying conditions. Experimental Procedures Deinococcus radiodurans bacteria were cultured from pure ATCC standard strains. The bacteria were grown on agarsolidified growth medium and removed from the plates by swabbing with a plastic wand and applying them to a 304 stainless steel disks. The stainless steel disks were 12.7 mm in diameter and 1.59 mm thick and were finely polished to 0.05 µm using diamond grit. The specimens were cleaned by sonicating sequentially in acetone, then methanol, and finally deionized (DI) water. A drop of DI water was used to spread the bacteria evenly into an area approximately 5 mm in diameter. The sample was allowed to air-dry in a covered Petri dish. After drying, the bacteria form a smooth continuous film across the applied area. Imaging was performed on samples dried for at least 2 h. After the initial drying process, no change in the imaging characteristics of the bacteria was observed over a several day period. The DI water was obtained from a Barnstead four-stage filtration system that yielded a resistivity of 17.9 MΩ‚cm. Acetone and methanol were HPLC grade (Aldrich). AFM measurements were performed using a Molecular Imaging PicoScan system (Molecular Imaging Inc., Tempe, AZ) operating in the magnetic AC (MAC) noncontact mode. Specially coated AFM tips (type II MAC tips, Molecular Imaging, Inc.) were employed. These tips are coated with a magnetically conductive material and possess a lower spring constant range (1.2-5.5 N/m) than common tapping mode models due to the lower amplitude vibrations encountered with employing MAC mode. The advantage of this configuration is the lower contact forces compared to other noncontact or intermittent contact configurations. The tips were composed of monolithic doped Si with radii rated better than 10 nm. All images were taken using an AFM “M” piezo scanner having a maximum scan range of 30 µm2 and a maximum z-range of 6 µm. The imaging was performed in an acoustically isolated cabinet outfitted with a conventional vibration isolation (elastic cord suspended block) stage upon which the AFM resides during scanning. Images are displayed in two modes, height mode which is the movement of the z-piezo as it tracks the surface using feedback to maintain constant oscillation amplitude and deflection mode is the direct measure of amplitude change due to changes in topography.
Results and Discussion This work is a component of a larger research area examining the effects of drying processes upon biofilms formed on the surfaces of materials used in the handling and storage of spent nuclear fuel. One of the fundamental issues that needed to be addressed at the outset was to determine the morphologies of individual bacteria within natural environments. Most published information is based upon characterization of bacteria in environments perturbed by stains, reduced pressure, or cryogenics. D. radiodurans was of particular interest in this study, as mentioned above, due to its known radiation tolerance and desiccation resistance. An AFM micrograph of D. radiodurans is shown Figure 1A-B. In this image four bacteria are forming a tetrad, a common structural conformation present in this particular bacteria.9 From the measured height profile, the bacteria are protruding approximately 500 nm above the level background. It is difficult to determine a real height as the bacteria likely extend below the surrounding material. This surrounding background material is likely not the substrate, but a mixture of extracellular polymeric substances (EPS), culturing media, and salt precipitates. Centrifuging the bacteria prior to application seems to eliminate most of this material, but without any noticeable improvement in imaging quality for the bacteria. Our data suggest that (9) Battista, J. R. Annu. Rev. Microbiol. 1997, 51, 203-24.
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the bacteria are partially desiccated, since the measurements show they are roughly half as high as they are wide (∼1 µm). In addition to determining the general shape and size, finer detail of the bacteria cell wall was observed (Figure 1A). This finer detail appears in the form of a folded structure resembling an orange peel texture. Within the orange peel region are linear structures protruding approximately 25 nm from the surroundings. At higher magnification further detail in the linear structures was observed, as demonstrated in Figure 1B-C. This image was acquired directly on the top of a single bacterium, resolving 20-50 nm structures. At significantly higher magnifications, hexagonal lattices were observed near the interfaces between adjoining bacteria (Figure 2).As mentioned in the Introduction, D. radiodurans possess a hexagonally packed intermediate (HPI) protomer structure on their outer surface. For D. radiodurans, each hexagonal-shaped protomer unit consists of six proteins having a combined molecular mass of 655 kDa.10 Figure 2A is the intersection of three bacteria, a triad, showing two somewhat different hexagonal domains. The domain to the lower left has units with hexagonal shape and a central pore clearly visible. This domain has characteristics (unit diameter with pore) most commonly observed in our study. The more unusual domain located on the right side of the intersection has a somewhat larger unit size and a less distinct pore although some units resemble those in the lower left. There appears to be continuity between the orientation of the domains on what is likely two different bacteria going from lower left to upper right. A similar relationship is observed in Figure 2B where the image was acquired near a tetrad interface. The center of the intersection in Figure 2B (cross hair in image) is near the center of the image with each of the four bacteria (numbered in the image) extending into the corners of the image. The two bacteria on the left appear to have HPI structures where the rows of hexamers are oriented in a similar fashion, as do the bacteria on the right. However, the HPI rows rotate at the vertical interface of the bacteria indicating a misorientation between the bacteria on the left versus right. The angle of misorientation was measured to be approximately 30°. It is possible that the HPI layers are oriented due to a previous cell division (reproduction) event. The highest resolution images of the HPI lattice are shown in Figure 3. These images clearly demonstrate the hexagonal symmetry characteristic of the HPI layer. They appear close-packed with the central pore having a measured depth of about 0.5 nm. Previous imaging of the HPI layer extracted and assembled onto mica surfaces has resolved six individual proteins composing each unit.5 We were not able to achieve this level of resolution. Possible reasons for the lower resolution in this study are (1) noncontact AFM does not yield the level of resolution afforded by contact mode AFM because of the shorter interaction times between the probe tip and the surface, or (2) the outer carbohydrate structure remains intact in our studies, where in previous studies the layer might have been stripped by the extraction process or by using contact mode AFM. The in vivo images of the HPI layer show other interesting features when compared to layers formed by extraction/self-assembly. Figure 3A (and other images in the paper) shows irregular packing of the units, with the rows deviating from linearity and height being quite varied along a row. Another interesting feature shown in Figure (10) Muller, D. J.; Baumeister, W.; Engel, A. Proc. Natl. Acad. Sci. 1999, 96, 13170-13174.
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Figure 1. AFM micrographs of D. radiodurans showing both height (left) and deflection (right) images: (A + B) 3.3 µm2 image of a tetrad; (C + D) 850 nm2 image taken directly on top of a bacterium. The height mode scale is indicated in the bar below image.
Figure 2. AFM micrographs (deflection mode) showing the HPI layer (A) at the intersection of three bacteria (580 nm2) and (B) at the center of a tetrad (680 nm2), where the numbers indicate the four bacteria and the cross shows approximate intersection.
3B is a crevice defect in the HPI lattice that may have been induced by dehydration stress. The deviations of the HPI structures compared to those imaged using extracted proteins are not likely to be instrumental in nature.
Potential errors in these measurements can result from slow scan axis drift or piezo hysteresis. This was confirmed by taking measurements using both directions of the slow scan (vertical) axis or by using a larger scan area. In
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Figure 3. Two high-resolution AFM micrographs (height mode) of the HPI layer near the intersection of bacteria. Scan areas are 200 nm2 (A) and 102 nm2 (B).
Figure 4. An AFM micrograph (deflection mode) showing a mixed structure of HPI and folding structures.
another case, as shown in Figure 4, we have observed the HPI layer mixed with something close to the folding structure suggesting that perhaps the defects we are seeing are due to a drying effect where the integrity of the cell wall is compromised by loss of internal volume. As we saw a great variety of interunit distances in the HPI lattices, statistical measurements of the HPI layer were performed. The packing distances tabulated from 42 micrographs (acquired from different locations on three separate specimens) measured along all three row directions give an average distance of 21.21 ( 2.52 nm. The previous value for HPI spacing from extracted proteins self-assembled onto mica was 18.0 nm.3,4 Electron microscopy of freeze-etched/thin-sectioned samples has been performed and communicated,11,12 but the details were not published to our knowledge. Our measurements are somewhat larger than the values from the extracted (11) Emde, B.; Heibe, E.; Karrenberg, F.; Baumeister, W. Structural investigations on the cell wall of Micrococcus radiodurans; Second International Congress on Cell Biology, Berlin, 1980. (12) Emde, B.; Wehrli, E.; Baumeister, W. The topography of the cell wall of Micrococcus radiodurans; 7th European Congress on Electron Microscopy, The Hague, 1980.
samples. However, the extreme values of unit spacing (measured over at least four units) observed over all micrographs was 16.39 and 28.40 nm (not including the folded structures), which encompasses the values for the extracted protein. The range and high standard deviation also point to the large variation we see in the spacing of the HPI layer, values which lie beyond the measurement uncertainty for a calibrated scanning probe instrument. Thus we are confident in assessing most of the observed variability to the sample. Although we have seen the HPI layer near the interfaces of adjoining bacteria, it was not often observed at the tops of bacteria. However, in isolated cases, as shown in Figure 5, we were able to image a periodic array on the top part of a bacterium. The reason we see the HPI structure mostly at the edges of bacteria likely has to do with the dehydration process. Our measurements show the surface roughness is concentrated toward the center of the bacteria. The centers of the bacteria are composed of a folded structure that has some periodicity in the chains. Large patches of HPI are likely not seen due to the roughness in this area. We expect that the volume loss at the center of dehydrated bacteria would be largest. We are currently attempting to explain this observation with further imaging using different preparation and imaging conditions. Although not definitive proof, it appears that the regular structures observed near the interfaces of the bacteria are due to the HPI (S-layer) layer. Our observations parallel those reported using transmission electron microscopy (TEM) and AFM of self-assembled proteins.10,13,14 This is a significant finding if indeed it is the S-layer, as this demonstrates the potential for imaging outer cell wall structures in a natural state. We have reason to believe that the cells were in a living yet dormant state due to the numerous reports of D. radiodurans surviving dry conditions.9 Some preliminary attempts were made at imaging the bacteria under solution; however the bacteria appeared to float off the surface, even if the drying and solution addition were done sequentially. To prevent this, some methods of attachment should be attempted such as surface modification, e.g., surface roughening, to facilitate (13) Muller, D. J.; Baumeister, W.; Engel, A. J. Bacteriol. 1996, 178, 3025-3030. (14) Moller, C.; Allen, M.; Elings, V.; Engel, A.; Muller, D. J. Biophys. J. 1999, 77, 1150-1158.
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Figure 5. An AFM micrograph (625 nm2) of a bacterium with HPI layer extending across the surface. Height mode (A) and deflection mode (B) images are shown.
attachment. Imaging the S-layer in solution on the bacteria surface would be a significant step beyond the progress described in this paper. One important question our images raise is why there is a variance between structures imaged on the top of the bacteria where the corrugated fold structure is seen versus images obtained at the outer edges where the HPI structure is observed. One possible explanation is that the bacteria are flattened by desiccation and that the folded structure is due to the outer cell wall buckling to form the rougher orange-peel texture. While it is tempting to think that the linear chains may be the HPI layer turned on its side, many of the observed folds exceed the approximately 4-9 nm height measured previously by a variety of techniques.5,15 However it is possible that we are not achieving high resolution due to the rough corrugation of the cell wall or other factors. In that case one would not expect to find a regular structure, but a random arrangement with significant z-corrugation. Indeed the structures that we observe in the center portions of the bacteria are of similar dimensions to the S-layer, and the linear chains possess unidirectional order. We also observe significant disorder in the HPI structures compared to those near perfect lattices observed by AFM and TEM. We ascribe this distortion to the collapsing of the inner structure and distortion of the cell wall due to these strains. (15) Wang, Z. H.; Hartmann, T.; Baumeister, W.; Guckenberger, R. Proc. Natl. Acad. Sci.1990, 87 (23), 9343-7.
Summary High-resolution images of the outer cell surface of Deinococcus radiodurans have been obtained in vivo using tapping mode AFM. A variety of structures were observed varying in size from 10 to 50 nm, including ordered domains that appear to be the surface protein layer (Sor HPI-layer). At the highest achievable resolution, the lattice is a close-packed configuration of hexagon-shaped subunits containing a pore at their center. Extensive measurements indicated that the structures are not isotropic. While the HPI layer was primarily found at the edges of adjoining bacteria, the tops of the organisms registered a more disordered structure that is believed to be a result of loss of internal volume due to partial desiccation. Attempts to reconcile this observation by imaging in solution have been unsuccessful due to problems in attaching the cells to our substrates. Future studies will concentrate on this issue. Acknowledgment. The authors acknowledge the helpful reviews and suggestions of R. N. Wright and F. S. Colwell. Additionally, we wish to acknowledge our financial support from the U.S. Department of Energy under Contract DE-AC-07-99ID13727. LA001448G
