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Fingerprinting Species and Strains of Bacilli Spores by Distinctive Coat Surface Morphology Rong Wang,*,† Soumya N. Krishnamurthy,† Jae-Sun Jeong,† Adam Driks,‡ Manav Mehta,† and Bruce A. Gingras§ Department of Biological, Chemical and Physical Sciences, Illinois Institute of Technology, Chicago, Illinois 60616, Department of Microbiology and Immunology, Loyola UniVersity Medical Center, Maywood, Illinois 60153, and IIT Research Institute, Chicago, Illinois 60616 ReceiVed June 15, 2007. In Final Form: July 18, 2007 In this work, we applied high-resolution atomic force microscopy (AFM) to identify and characterize similarities and differences in the spore surface morphology of strains from four species of Bacilli: B. anthracis, B. cereus, B. pumilis, and B. subtilis. Common features of the examined spores in the dry state included ridges that spanned the long axis of each spore, and nanometer-scale fine rodlets that covered the entire spore surface. However, important differences in these features between species permitted them to be distinguished by AFM. Specifically, each species possessed significant variation in ridge architecture, and the rodlet width in B. anthracis was significantly less than that of the other species. To characterize similarities and differences within a species, we examined three B. subtilis strains. The ridge patterns among the three strains were largely the same; however, we detected significant differences in the ridge dimensions. Taken together, these experiments provide important information about natural variation in spore surface morphology, define structural features that can serve as species- and strain-specific signatures, and give insight into the dynamics of spore coat flexibility and its role during spore dormancy and germination.
Introduction Spores are produced by a variety of Bacilli and Clostridia species in response to starvation. While these highly resilient dormant cell types can withstand extremes of heat, radiation, chemical assault, and time,1-3 they quickly convert to actively growing vegetative cells through germination4-6 upon the return of nutrients to the environment. These remarkable characteristics allow pathogenic spores, such as B. anthracis and C. botulinum, to serve as potent biological weapons.7,8 As spore germination is essential for the contamination of pathogenic spore-related diseases, and it is triggered by interactions between the spore surface and the environment, a comprehensive understanding of the surface of a spore holds the key to inhibit and eliminate the diseases. While spore surfaces have been extensively examined via conventional biological methods, microscopic imaging can provide additional information useful for both fundamental studies of individual spores and forensic investigation. The outer structures of a spore surface have been the focus of a significant amount of structural analysis. The most prominent surface structure common to all bacterial spores is the coat, a * To whom correspondence should be addressed. E-mail:
[email protected]. Tel: 312-567-3121. Fax: 312-567-3494. † Illinois Institute of Technology. ‡ Loyola University Medical Center. § IIT Research Institute. (1) Fairhead, H.; Setlow, B.; Waites, W. M.; Setlow, P. Appl. EnViron. Microbiol. 1994, 60, 2647-2649. (2) Setlow, P. In Bacterial Stress Responses; Storz, G., Hengge-Aronis, R., Eds.; American Society for Microbiology: Washington, D.C., 2000; pp 217230. (3) Takamatsu, H.; Watabe, K. Cell. Mol. Life Sci. 2002, 59, 434-444. (4) Moir, A.; Smith, D. A. Annu. ReV. Microbiol. 1990, 44, 531-553. Moir, A.; Corfe, B. M.; Behravan, J. Cell. Mol. Life Sci. 2002, 59, 403-409. (5) Nicholson, W. L.; Setlow, P. In Molecular Biology Methods for Bacillus; Hardwood, C. R., Cutting, S. M., Eds.; Wiley: Farmington, CT, 1990; pp 391450. (6) Paidhungat, M.; Setlow, P. In Bacillus subtilis and Its Closest RelatiVes: From Genes to Cells; Sonenshein, A. L., Hoch, J. A., Losick, R., Eds.; American Society for Microbiology: Washinton, D.C., 2002; pp 537-548. (7) Broussard, L. A. Mol. Diagn. 2001, 6, 323-333. (8) Robinson-Dunn, B. Arch. Pathol. Lab Med. 2002, 126, 291-294.
multilayered proteinaceous shell,9,10 which is critical to spore survival and germination.11-14 Thin-section electron microscopy (EM) reveals that the coat is comprised of distinct layers whose number and specific appearance varies with the species.15,16 This approach cannot reveal, however, the topographical structure of the coat surface. We have been engaged in extending the information derived from thin-section EM by imaging the spore surface using an atomic force microscope (AFM).17 This approach generates highresolution three-dimensional (3D) images without the need of any significant sample preparation. In previous work,17 we showed that B. subtilis, B. cereus, and B. anthracis spore surfaces possess ridges that, for the most part, traverse the long axis of the spore. Similar ridges have been observed by others using scanning electron microscopy (SEM), freeze-etch microscopy,18,19 and AFM.20 However, our work identified striking, previously undetected species-specific differences in ridge structures. Examination of a set of B. subtilis coat protein gene mutants indicated that the absence of a single coat protein species can alter the ridge structures tremendously.17,21,22 Therefore, the ridge (9) Driks, A. In Bacillus subtilis and Its Closest RelatiVes: From Genes to Cells; Sonenshein, A. L., Hoch, J. A., Losick, R., Eds.; American Society for Microbiology: Washington, D.C., 2002; pp 527-536. (10) Henriques, A. O.; Moran, C. P., Jr. Methods 2000, 20, 95-110. (11) Bagyan, I.; Setlow, P. J. Bacteriol. 2002, 184, 1219-1224. (12) Hullo, M. F.; Moszer, I.; Danchin, A.; Martin-Verstraete, I. J. Bacteriol. 2001, 183, 5426-5430. (13) Kuwana, R.; Kasahara, Y.; Fujibayashi, M.; Takamatsu, H.; Ogasawara, N.; Watabe, K. Microbiol. 2002, 148, 3971-3982. (14) Lai, E. M.; Phadke, N. D.; Kachman, M. T.; Giorno, R.; Vazquez, S.; Vazquez, J. A.; Maddock, J. R.; Driks, A. J. Bacteriol. 2003, 185, 1443-1454. (15) Driks, A. Microbiol. Mol. Biol. ReV. 1999, 63, 1-20. (16) Popham, D. L. Cell. Mol. Life Sci. 2002, 59, 426-433. (17) Chada, V. G.; Sanstad, E. A.; Wang, R.; Driks, A. J. Bacteriol. 2003, 185, 6255-6261. (18) Aronson, A. I.; Fitz-James, P. Bacteriol. ReV. 1976, 40, 360-402. (19) Holt, S. C.; Leadbetter, E. R. Bacteriol. ReV. 1969, 33, 346-378. Holt, S. C.; Gauthier, J. J.; Tipper, D. J. J. Bacteriol. 1975, 122, 1322-1338. (20) Plomp, M.; Leighton, T. J.; Wheeler, K. E.; Malkin, A. J. Biophys. J. 2005, 88, 603-608. (21) McPherson, D. C.; Kim, H.; Hahn, M.; Wang, R.; Grabowski, P.; Eichenberger, P.; Driks, A. J. Bacteriol. 2005, 187 (24), 8278-8290.
10.1021/la701788d CCC: $37.00 © 2007 American Chemical Society Published on Web 08/28/2007
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Table 1. Dimensions of Spores, Ridges, and Rodlets of Air-Dried Bacillus Spores Measured from AFM Images spore width (µm) meana B. anthracis B. cereus B. pumilis B. subtilis
PY79 3610 Natto
0.89 0.80 0.88 0.76 0.73 0.70
std deva 0.13 0.08 0.14 0.12 0.06 0.16
spore length (µm) mean 1.55 1.40 1.42 1.36 1.34 1.19
std dev 0.18 0.13 0.20 0.14 0.11 0.10
spore height (µm) mean 0.30 0.34 0.34 0.34 0.34 0.47
ridge width (nm)
ridge height (nm)
rodlet period (nm)
rodlet width (nm)
std dev
mean
std dev
mean
std dev
mean
std dev
mean
std dev
0.02 0.06 0.05 0.02 0.05 0.08
86b
18 14 18 16 27 15
11 14 26 33 25 14
9 13 15 13 8
6.9 8.0 7.3 8.0 7.9 7.6
0.5 0.4 0.6 0.5 0.8 0.4
2.7 4.2 4.2 4.4 5.0 4.7
0.5 0.7 0.5 0.6 0.5 0.6
107 93 114 168 99
a The mean and standard derivation (std dev) values were calculated based on more than 60 individual measurements on different spores and on separately prepared samples. b The ridge width of B. anthracis indicates the width of one of the paired ridges.
structures may reflect differences in protein organization between species as well as between strains within a species. Insight into a possible role for the ridges comes from our finding that, when the spore swells during germination (due to entry of water into the spore core),23 the ridges disappear. Taken together with other data,24 we proposed that the ridges permit the coat to accommodate changes in the spore core volume that are a normal part of spore formation and germination.17,23,25 While the ridges are the most striking feature of the coat at low resolution, classical freeze-etch analyses18,19 as well as more recent AFM studies20 have also identified fine features in the coat surface, referred to as rodlets, whose dimensions are consistent with the possibility that they represent a fundamental structural unit of the outer coat. In the current study, we carried out high-resolution AFM analysis of spores from a variety of species and strains within one species. The ridge and rodlet patterns and dimensions differed sufficiently between species and strains to permit species-specific and strain-specific identifications. We also observed that the rodlet width does not differ detectably between the wet and dry states; however, differences in rodlet periodicity are significant. Taken as a whole, our data indicate that the spore coat undergoes a significant physical change during spore dehydration and rehydration, whereas the spore dormancy is retained. This is ascribed to the stability of the building blocks of spore coats at high resolution (individual rodlets) as well as the flexibility and integrity of the coat architect. Materials and Methods Bacterial Strains and Sporulation. We examined B. anthracis (Sterne strain 34F2), B. cereus (strain 569), B. pumilis (wild strain ADL1391, isolated from rabbit intestine26), as well as the B. subtilis PY79 strains,27 the B. subtilis 3610 strain (from ATCC) and a commercial B. subtilis Natto strain (Takahashi Foods Corp., Kyoto, Japan). Sporulation was performed by nutrient exhaustion.17 In brief, we allowed cells to grow in a rich medium (Difco Sporulation Medium) until they had exhausted the available nutrient sufficiently to begin sporulation. This sporulation regimen mimics conditions in the environment and is suitable for a wide range of species. All strains of spores were prepared under the same conditions. AFM Imaging. AFM imaging was carried out using a Nanoscope IIIa (Digital Instruments, Santa Barbara, CA) equipped with a J-scanner. Most of the images were captured under ambient conditions. In this case, 50 µL of an aqueous spore suspension was spread on a piece of mica (1 cm2), which was then mounted onto a standard sample holder. After allowing the spores to settle on the substrate, dry, and reach equilibrium in an ambient environment (temperature: 25 °C, relative humidity: 60-70%), the sample was placed in an AFM chamber for imaging. Commercial single-crystal silicon cantilevers were used to acquire images in air tapping mode, which was performed at a resonant frequency of ∼250 kHz and a loading force of 200-800 pN. Images were also collected in pure water to examine the spores under wet conditions. To avoid detachment of spores from the substrate support in water, we modified the substrate with a monolayer of succinimidyl-3-(2-pyridyldithio)-
propionate (Molecular Probes, Eugene, OR) as previously reported by us28-31 and others.32,33 Images in aqueous media were collected in fluid tapping mode using a fluid cell and an oxide-sharpened Si3N4 tip at a thermal resonance frequency of 8-10 kHz. All images were collected with 512 pixels per line at a scan rate of 1 Hz or less. To prove that the surface features are intrinsic to the species and strains, AFM measurements were repeated on spores of each species and strain from at least three separate cultures under the fixed sporulation conditions. Results in Table 1 were summarized on the basis of all data.
Results B. anthracis Spore Surface Morphology. We first examined the B. anthracis spore coat of air-dried spores. B. anthracis spores possess an outermost layer that encases the entire spore (and, therefore, covers the coat), called the exosporium, that is present only on a subset of species (including B. cereus and B. thuringiensis, but not B. subtilis or B. pumilis). The exosporium surface is rather smooth, and can be readily distinguished from a spore coat surface, on which ridge structures are characteristic.17 Since our goal in this work is to image and compare structures on the coat surface of Bacilli spores, we examined only spores in which the exosporium was absent. Figure 1a shows a typical 3D height image. Dry B. anthracis spores are ellipsoid and, on average, are 1.55 µm long, 0.89 µm wide, and 0.30 µm high. Consistent with our previous observation, we found that the spore surface possesses ridges that run mostly along the long axis of the spore; each ridge is usually composed of two parallel strands of similar dimension and shape,17 as indicated by arrows in Figure 1a,b. When imaged at higher resolution, we observed rodlet structures both on the ridges and in the valleys between them. In most cases, rodlets appeared to be oriented along one direction, slightly deviated from the long axis of the ellipsoid spores (within 20°). In rare cases, we also observed rodlets oriented perpendicularly to the rest (Figure 1c). We ascribe them to rodlets in adjacent layers, (22) Kim, H.; Hahn, M.; Grabowski, P.; McPherson, D. C.; Otte, M. M.; Wang, R.; Ferguson, C. C.; Eichenberger, P.; Driks, A. Mol. Microbiol. 2006, 59 (2), 487-502. (23) Setlow, P. Curr. Opin. Microbiol. 2003, 6 (6), 550-556. (24) Westphal, A. J.; Price, P. B.; Leighton, T. J.; Wheeler, K. E. Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 3461-3466. (25) Driks, A. Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 3007-3009. (26) Rhee, K. J.; Sethupathi, P.; Driks, A.; Lanning, D. K.; Knight, K. L. J. Immunol. 2004, 172 (2), 1118-1124. (27) Youngman, P.; Perkins, J. B.; Losick, R. Plasmid 1984, 12, 1-9. (28) Reddy, C. V.; Malinowska, K.; Menhart, N.; Wang, R. Biochim. Biophys. Acta 2004, 1667, 15-25. (29) Tang, Q.; Zhang, Y.; Chen, L.; Yan, F.; Wang, R. Nanotechnology 2005, 16, 1062-1068. (30) Vengasandra, S.; Sethumadhavan, G.; Yan, F.; Wang, R. Langmuir 2003, 19, 10940-10946. (31) Yan, F.; Chen, L.; Tang, Q.; Wang, R. Bioconjugate Chem. 2004, 15, 1030-1036. (32) Dammer, U.; Hegner, M.; Anselmetti, D.; Wagner, P.; Dreier, M.; Huber, W.; Guntherodt, H. J. Biophys. J. 1996, 70, 2437-2441. (33) Harada, Y.; Kuroda, M.; Ishida, A. Langmuir 2000, 16, 708-715.
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Figure 1. Air-dried B. anthracis spores imaged in air. (a) 3D height image of a spore. (xy-axis: 500 nm/division; z-axis: 300 nm/division). The arrows point to the double ridge structure. (b) Amplitude image of the spores at a large scale (scale bar: 875 nm). (c) Fine rodlet structures at high resolution (scale bar: 162 nm).
consistent with the suggestions by Holt and Leadbetter who made similar observations on B. subtilis spores in a freeze-etch study.19 The average periodicity (peak-to-peak rodlet separation) is 6.9 ( 0.5 nm, and the average rodlet width is 2.7 ( 0.5 nm. When we examined B. anthracis spores in water, ridges were no longer evident (Figure 2a). These spores had average dimensions of 1.68 µm long, 1.03 µm wide and 1.00 µm high, making their volume approximately four times larger than that of a dry spore (Figure 2b). Overall, these size changes are similar to what we observed previously for B. subtilis spores.17 The degree of swelling is reproducible and is much higher than that reported by others.20,24 The difference between our result and that of others is most likely due to different conditions for spore swelling. In our case, spores were immersed in pure water overnight to reach hydration saturation, hence spore swelling was maximized. Like in the dry state, spores in water possessed rodlets that were largely similar in their pattern on the spore surface with an average width of 2.5 ( 0.6 nm (Figure 2c). However, the rodlet periodicity is 8.0 ( 0.6 nm, which is somewhat greater than that on dry spores. Spores in the wet state showed parallel rodlets uniformly covering the entire spore surface, whereas rodlets were usually observed in small patches in the dry state. This is not a surprise since the significant surface
Wang et al.
Figure 2. Wet B. anthracis spores imaged in pure water. (a) 3D height image of a spore (xy-axis: 500 nm/division; z-axis: 300 nm/division). (b) Amplitude image of the spores at a large scale (scale bar: 875 nm). (c) Fine rodlet structures at high resolution (scale bar: 200 nm).
morphological changes (e.g., ridge formation and coat contraction) lead to a greater degree of surface roughness in the dry state, making the acquisition of fine rodlet structures possible at only local areas. Spore Surface Morphology of Three Species of Bacilli. To identify potential differences at high resolution, we also imaged B. cereus, B. pumilis, and B. subtilis spores. Like B. anthracis spores, B. cereus spores are encased in an exosporium. As before, we imaged only those spores that were clearly free of exosporium. Spores from all three species were ellipsoid in shape. As shown in Figures 3 and 4, and also summarized in Table 1, the width, length, and height of the spores vary slightly. In general, B. anthracis, B. cereus, and B. pumilus spores are similar in size, whereas B. subtilis spores are relatively small (Table 1). A striking difference among the four species lies in the ridge pattern. B. subtilis and B. anthracis ridges differ in that the B. anthracis ridge appears to be composed of two parallel strands, whereas B. subtilis possesses single-strand ridges. B. cereus differs from both of those strains in that its ridges zigzag from pole to pole. In the case of B. pumilis, we discovered yet another ridge pattern: these ridges are parallel to each other, are in close
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specific variations in ridge dimensions, rodlet periodicities and widths were in the ranges of 7.6 ( 0.4 to 8.0 ( 0.7 nm and 4.4 ( 0.6 to 5.0 ( 0.5 nm, respectively, for all strains (Table 1).
Discussion
Figure 3. (a) A typical B. cereus spore showing the angularly bent ridges (scale bar: 368 nm). (b) Rodlet structures on the B. cereus spore at high resolution (scale bar: 188 nm). (c) A typical B. pumilis spore showing the high-density ridges (scale bar: 396 nm). (b) Rodlet structures on the B. pumilis spore at high resolution (scale bar: 200 nm). The air-dried spores were imaged in air.
apposition, extend along the long axis, and cover the entire spore surface at high density. A major goal of the present work is to characterize spore surfaces at high resolution. We found that, regardless of the species, very similar rodlet structures were readily and reproducibly observed (Figures 1c, 2c, and 3c,d). Rodlets on the ridges and in the valleys were oriented along a single direction, which usually deviated slightly from that of the ridge. B. cereus, B. pumilis, and B. subtilis rodlets have rodlet periods in the narrow range of 7.3 ( 0.6 nm to 8.0 ( 0.7 nm. In contrast, the B. anthracis rodlet period averaged 6.9 ( 0.5 nm, a difference of 16% between this species and the other three (Table 1). The widths of B. cereus, B. pumilis, and B. subtilis rodlets were between 4.2 ( 0.7 to 5.0 ( 0.5 nm, while those of B. anthracis were 2.7 ( 0.5 nm, which is about 35% less than those of the other three species. The measurements of rodlet periodicity and width may lack some precision because of tip convolution effects. However, because the reported values are from more than 100 measurements on different samples and with different tips, they clearly demonstrate that rodlet dimensions fall into two distinct classes, even with the consideration of uncertainty brought from data distribution. Spore Surface Morphology of Three Strains of B. subtilis. In revealing the differences in ridge patterns among the four species of Bacilli, we chose one popular strain for each species. This brings up the question of whether the ridge patterns are species-specific or strain-specific. To address this question and to further examine the strain-specific features, we examined three strains of B. subtilis: PY79 (a common laboratory strain), 3610, and a commercial Natto strain. All these strains possessed ridges that were largely similar in pattern to our previous analysis (Figure 4). Strikingly, ridge width varied significantly between strains (Table 1). Strain 3610 has the widest ridges (at 168 ( 27 nm on average), whereas Natto shows the narrowest (at 99 ( 15 nm on average). Although ridge height varies in a broad range, PY79 ridges are, in general, much deeper. In contrast to the strain-
We have used AFM to image spores of four species of Bacilli and three strains of one of those species. We draw three major conclusions from this work. First, we have shown that, for each species, there are significant differences in the ridge patterns of dry spores, permitting species-specific identification. In the wet state, the ridges disappeared on the spores. With the disappearance of ridges in the wet state, the packing of rodlets is slightly relieved as indicated by the increase in periodicity. Second, although spores of three B. subtilis strains showed similar ridge patterns, we were able to distinguish them by analysis of the ridge dimensions. Finally, we have shown that rodlet dimensions distinguish B. anthracis from the other three species. An important implication of this work is that high-resolution surface imaging can have significant utility in fingerprinting spore surface features, which can be used to distinguish species and strains. We observed that heights of ridges on the spores range from 11 to 33 nm, depending on the species. According to the thin-section EM analysis, the thickness of the outer coat and inner coat of a spore is typically on the order of several tens of nanometers.15 Thus ridge formation involves folding of both the outer coat and the inner coat. We speculate that the consequence of spore coat folding is to reduce the number of contacts between the spore coat and the spore interior. The folding extent may differ among the species, giving rise to the difference in the number and location of the contacts and hence distinct ridge patterns. It is striking that strains of a single species (B. subtilis) are so readily distinguished even though they share a common ridge pattern. The differences in ridge dimension among the three strains of B. subtilis may be regulated by the relative extent of spore coat contraction upon the dehydration. A future goal will be to identify the molecular determinants of these differences by studying mutants of nonconservative and conservative coat proteins. It is unlikely that the variations in ridge dimensions we have observed are the consequence of a variation in a single coat protein gene. As genomes of Bacillus strains become sequenced, we hope to identify collections of genes that collaborate in the appearance of specific coat surface features. As already discussed, spores appear to exist in contracted and expanded states, where the dry spore corresponds to the most contracted state.24,25,34 During germination, the coat is maximally expanded and is quickly shed. However, placing spores in water allows the coat to expand significantly without providing the opportunity for germination (which requires additional small molecules).20,24 The ability of the coat to accommodate significant physical changes in interior volume is striking, and does not obviously contribute to a known spore function or protective ability. The ability of the coat to fold and unfold is either important to spore survival or a side consequence of a highly conserved aspect of the design of the coat. An important aspect of our data is that, while the ridge morphologies of the spores of each of the four species studied were distinct, the rodlet widths and spacings were similar for three species, with those of B. anthracis differing. Rodlets appear to be the building blocks of the spore coat architect. Because of the rodlets’ dimensions, their parallel alignment, and their uniform coverage of the entire spore surface, a rodlet is most likely the line-up of several closely associated coat surface proteins. In the (34) Santo, L. Y.; Doi, R. H. J. Bacteriol. 1974, 120, 475-481.
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Figure 4. Images of three strains of B. subtilis spores at two different scales. (a,d) PY79 strain; (b,e) 3610 strain; (c,f) Natto strain. Scale bars: (a) 2.35 µm; (b) 2.50 µm; (c) 2.50 µm; (d) 146 nm; (e) 150 nm; (f) 125 nm. The air-dried spores were imaged in air.
case of B. subtilis, the major coat surface proteins appear to be, at a minimum, CotB, CotC, and CotG.22,35-37 It is interesting to note that, while these proteins are not especially well conserved among the Bacilli, the rodlet structure is, to a large degree. Therefore, regardless of whether orthologues of CotB, CotC, and CotG comprise the surfaces of spores of species other than B. subtilis, it is likely that interactions between coat surface protein assemblies in these species are conserved. The differences between the B. anthracis spore surface morphology and those of the other species we analyzed is not readily explained by comparisons of these organisms by taxonomic or phylogenic considerations. Possibly, a coat protein species unique to B. anthracis imparts its distinctive morphology. We also found that, while rodlet width does not change significantly when the coat transits between the folded (dry) and unfolded (wet) states, rodlet periodicity increases by 15% in the unfolded state. While the coat protein association and composition within a rodlet is responsible for the width, the periodicity is a measure of the interactions between neighboring rodlets. Thus the folded to unfolded state transition reflects the mechanical distortion of the spore coat in response to environmental humidity changes. The
differing interactions between rodlets are likely to be an important aspect of coat flexibility. The lack of change of the rodlet width indicates that the spore coat building block remains unchanged, consistent with the fact that, in the absence of nutrients, spore hydration does not lead to spore germination. Our data emphasize an important mystery: Why is there such diversity in spore coat morphology when the underlying function of the structure is likely to be conserved? We feel that insight to this question will require analysis of the spore surfaces of a larger group of species and strains. By characterizing the range of variation as well as the similarities among species and strains, we expect to better understand the fundamental basis of variation in coat surface morphology. Such extensive studies will also build up a comprehensive database and provide the foundation of using AFM surface analysis, an alternative approach to genotyping, to identify spore species and strains. In our preliminary research, we also found that spores treated with a vacuum or detergent remarkably changed in surface morphology while they maintained high dormancy. Thus AFM-based surface analysis will be also useful in forensic investigation for tracking the source of pathogenic spores.
(35) Isticato, R.; Cangiano, G.; Tran, H. T.; Ciabattini, A.; Medaglini, D.; Oggioni, M. R.; De Felice, M.; Pozzi, G.; Ricca, E. J. Bacteriol. 2001, 183 (21), 6294-6301. (36) Kim, J. H.; Lee, C. S.; Kim, B.G. Biochem. Biophys. Res. Commun. 2005, 331 (1), 210-214. (37) Mauriello, E. M.; Due Le, H.; Isticato, R.; Cangiano, G.; Hong, H. A.; De Felice, M.; Ricca, E.; Cutting, S. M. Vaccine 2004, 22 (9), 1177-1187.
Acknowledgment. This research was supported by a research grant provided by the IIT Research Institute. It was also partly supported by NSF (IBN-0103080). LA701788D
