Investigation of bacterial spore structure by high resolution solid-state nuclear magnetic resonance spectroscopy and transmission electron microscopy

International Journal of Food Microbiology 63 (2001) 35–50 www.elsevier.nl / locate / ijfoodmicro

Investigation of bacterial spore structure by high resolution solid-state nuclear magnetic resonance spectroscopy and transmission electron microscopy Renata G.K. Leuschner*, Peter J. Lillford Unilever Research Colworth, Sharnbrook, Bedford MK44 1 LQ , UK Received 17 January 2000; received in revised form 9 June 2000; accepted 27 July 2000

Abstract High resolution solid-state nuclear magnetic resonance spectroscopy (NMR) in combination with transmission electron microscopy (TEM) of spores of Bacillus cereus, an outer coatless mutant B. subtilis 322, an inner coatless mutant B. subtilis 325 and of germinated spores of B. subtilis CMCC 604 were carried out. Structural differences in the coats, mainly protein of spores were reflected by NMR spectra which indicated also differences in molecular mobility of carbohydrates which was partially attributed to the cortex. Dipicolinic acid (DPA) of spores of B. cereus displayed a high degree of solid state order and may be crystalline. Heat activation was studied on spores of B. subtilis 357 lux 1 and revealed a structural change when analysed by TEM but this was not associated with increases in molecular mobility since no effects were measured by NMR.  2001 Elsevier Science B.V. All rights reserved. Keywords: Nuclear magnetic resonance; Transmission electron microscopy; Spores; Bacillus subtilis; Bacillus cereus; Heat activation

1. Introduction Solid state NMR is a spectroscopic technique that can be used to identify and quantify structural features both in bulk materials and at surfaces and interfaces (Eckert, 1996). The application of nuclear magnetic resonance spectroscopy (NMR) provide non-invasive methods *Corresponding author. Present address: Central Science Laboratory, Sand Hutton, York YO41 1LZ, UK. Tel.: 1 44-1904-462668; fax: 1 44-1904-462-111. E-mail address: [email protected] (R.G.K. Leuschner).

to study in vivo molecular and supramolecular properties of spores. Bacterial spores have a compartmentalised structure. The central protoplast is encased by a wide cortex that consists mainly of peptidoglycan and is further surrounded by a proteinaceous coat (Gould, 1999). High-resolution solid-state NMR reveals an insight into distinct areas of spores which contain protein, polysaccharides, phosphorus or dipicolinic acid. The main advantage of NMR is its unprecedented selectivity (SchmidtRohr and Spiess, 1994). The main components of coats are proteins which comprise 50–80% of the total protein in mature

0168-1605 / 01 / $ – see front matter  2001 Elsevier Science B.V. All rights reserved. PII: S0168-1605( 00 )00396-2

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spores (Zheng et al., 1988). The structure and chemical composition of spore coats has been extensively studied (Vinter, 1959; Warth et al., 1963; Kadota et al., 1965; Aronson and Fitz-James, 1968; Gould et al., 1970; Hiragi, 1972). Spore coats of B. subtilis are arranged in three morphological layers, an outer layer, an inner layer and an amorphous layer between the inner coat and the cortex (Seyler et al., 1997). The outer electron dense coat and an inner lamellar coat can be easily distinguished by transmission electron microscopy (TEM). Spore coat biogenesis in B. subtilis has been reviewed by Ricca et al. (1997). Mutants have been isolated and characterised which lack the inner or outer coat (Zheng et al., 1988; Driks et al., 1994). Coats of B. cereus differ in their structure and chemical composition from those of B. subtilis. B. cereus does not contain a thick electron dense outer coat as B. subtilis. It has an outer coat and an under coat. The outer coat can be further subdivided into a cross-patched and an underlying pitted layer. (Ohye and Murrell, 1973; Aronson and Fitz-James, 1976; Stelma et al., 1978). A core specific component dipicolinic acid (DPA) was reported to be mainly located in the spore core. Kozuka et al. (1985) investigated the localisation of DPA by immunoelectron microscopy, Lenz and Gilvarg (1973) measured the beta-attenuation of intact spores grown on tritium-labeled DPA and Matano et al. (1993) provided evidence that DPA was bound to macromolecules in spores such as DNA. Despite their extreme dormancy, spores retain an alert sensory mechanism which is able to respond to specific germinants within minutes and triggers a series of sequentially interrelated degradative events known as germination (Moir et al., 1994). Degradation during germination has been described to occur initially in the cortex but also in the coat area (Atrih et al., 1998, 1999). Phosphorus is located to more than 90% in the core of dormant spores of B. megaterium and B. cereus. The coat was described to contain phosphorus whereas the cortex displayed the lowest phosphorus concentrations (Johnstone et al., 1980; Stewart et al., 1980; Nishihara et al., 1980). High phosphorus concentrations were found in the outer coat of B. coagulans (Stewart et al., 1981). NMR provides the opportunity to investigate noninvasively in vivo molecular mobility in structural spore compartments without destroying or invading the spore structure. The literature offers a broad

range of applications for NMR on complex systems. The investigation of 31 P and 13 C molecular mobility are routinely applied for complex systems such as food (Gidley, 1992). Proteins, polysaccharides and carbohydrates were shown to display characteristic spectra (Gidley, 1992; Kalichevsky et al., 1992). Cell walls of B. subtilis were studied by 13 C and 15 N magic angle spinning NMR (Forrest et al., 1991). The spore characteristic component DPA has been identified in spores of B. coagulans, B. subtilis, B. megaterium by Lundin and Sacks (1988). NMR spectra of crystalline and amorphous DPA were compared with the corresponding spectra in B. subtilis spores (Ablett et al., 1999). Phosphorus components in dormant spores of B. cereus and the dependence of the chemical shift of phosphoric acid from the pH have been examined by Shibata et al. (1984) using 31 P NMR. NMR was complemented in this study with conventional transmission electron microscopy (TEM) because it provides sample characteristics related to the bulk properties whereas microscopy allows studies of individual particles. The purpose of this study was to relate both techniques to further an understanding of spore structure in the dormant, heat activated and resuscitated state.

2. Material and methods

2.1. Bacterial strains Bacillus subtilis CMCC 604 and B. cereus T was obtained from the Colworth Microbiology Culture Collection. Outer and inner coatless mutants of B. subtilis strain 322 and 325 were kindly provided by Prof. R. Losick, Havard University, Cambridge, USA and were described by Zheng et al. (1988) and Driks et al. (1994). Bacillus subtilis PSB 357 lux 1 was provided by Dr. P.J. Hill and Prof. G.S.A.B. Stewart, Nottingham University, UK. The properties and application of spores of this strain as indicators for inimical processes has been described by (Hill et al., 1994).

2.2. Preparation of spore suspensions Fresh overnight cultures of Bacillus strains were prepared by agitation at 308C in Heart Infusion broth (Difco Laboratories, Detroit, USA). Aliquots of 1 ml

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were plated on Heart Infusion agar plates. The agar plates were incubated at 308C until preparations had 99% phase-bright spores as examined by phasecontrast light microscopy (Ortholux II Leitz, Leica Microsystems Ltd., Milton Keynes, UK). Medium used for cultivation and sporulation of B. subtilis PBS 357 lux 1 was supplemented with 150 mg / ml erythromycin (Sigma Chemical, Poole, Dorset, UK). Spores were washed from agar plates with 10 ml aliquots of cold, sterile distilled water. The combined suspensions were centrifuged at 4000 3 g (Rotor: JLA-10.500, Beckman, California, USA) for 20 min at 48C. The spore pellet was resuspended in distilled water and centrifuged at 4000 3 g for 30 min at 48C. This washing procedure was repeated at least 4–5 times. Final purity and homogeneity of spore crops were investigated as described previously (Leuschner et al., 1999a). The wet spore pellet was divided into aliquots and stored at 2 208C. In this study spores originating from the same spore crop were used.

2.3. Preparation of germinated spores B. subtilis CMCC 604 displayed a germination rate of . 90% (Leuschner and Lillford, 1999b). Spores of B. subtilis CMCC 604 were incubated in L-alanine buffer at 378C for 1 h, centrifugated at 4000 3 g for 30 min at 48C and washed twice with distilled water.

2.4. NMR spectroscopy A Bruker MSL-300 (for phosphorus analysis) or a DSX 300 (for carbon analysis) (Bruker, Karlsruhe, Germany) spectrometer system equipped with a wide-bore super conducting magnet system and a multinuclear, double bearing 7 mm CP-MAS VT probe (Bruker) and associated with high-power pulse amplifiers provided high resolution solid spectra of natural abundance 13 C and 31 P at a frequency of 75.48 and 121.497 MHz, respectively. Cross polarization magic angle spinning spectra (CPMAS) were acquired using total suppression of side bands (TOSS) (Dixon et al., 1982) for 13 C which effectively suppressed spinning sidebands. Typical spectrometer conditions were proton excitation p / 2 pulse length 4.5 / 6 ms, a contact time of 1 ms and the resulting spectra were acquired under high power

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decoupling. SPMAS spectra were obtained using a single p / 2 excitation pulse of 4.7–4.8 ms for carbon or 5.5 ms for phosphorus with a spectrum being acquired under high power proton decoupling conditions. Measurements were carried out using phase cycling of pulses in multipulse sequences (depth) in the manner of Bendall and Gordon (1983) to reduce the residual signal which comes from the probe. All spore spectra were obtained at 48C in a rotor (Bruker) with an outside diameter of 7 mm, a length of 1.5 cm and a volume of 360 ml. An acceptable signal-to-noise ratio was usually obtained for 13 C after 20 000 transients. Data were acquired by DISMEL (MSL) or XWINNMR (DSX) software package (Bruker) for Fourier transformation. Exponential apodization, giving a line broadening of 20 Hz for 13 C, was normally used. NMR spectra were referenced for 31 P with respect to the CPMAS di-sodium hydrogen orthophosphate (FSA Laboratory Supply, Loughborough, UK) at 208C which resonance maximum was set to 0 ppm. It has a chemical shift of 2 3.5 (Jones and Katritzky, 1960) relative to orthophosphoric acid which is often used as a reference. After each experiment the appearance of spores by phase-contrast light microscopy and by TEM was recorded to evaluate possible effects on their structure during NMR analysis.

2.5. Electron microscopy Spores were preserved in cacodylate buffer (0.1 m) (TAAB Laboratories, Aldermaston, UK) containing OsO 4 (1% w / v). For transmission electron microscopical (TEM) examination preserved spores were embedded in 2% agar, cut into small pieces and were transferred into 1% uranyl acetate (TAAB) for 3 h. They were dehydrated in 70%, 90% and twice in absolute ethanol for 1 h each. The samples were transferred into 100% acetone (two changes) for 30 min, and was stored overnight in a mixture of 1:1 acetone and resin (TAAB). The sample was soaked for 24 h in 100% resin, transferred into fresh resin and finally polymerised at 608C for 2 days. Sections were cut at 100 nm thickness using an Ultracut E (Reichert-Jung, Leica) and a diamond knife (Diatome, TAAB). Sections were collected onto 200 mesh copper grids and stained in lead citrate before examination in a TEM [JEOL 1200 EX mk 2 TEM (Welwyn Garden City, UK)].

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2.6. Heat activation and inactivation

2.8. Analysis of recorded data

Heat activation was performed in a temperature controlled water bath at 608C for 1 s, 10 min, 30 min and 1 h. Two eppencorf tubes with approximately 20 ml of a thick spore slurry were heated whereby one underwent the fixing procedure after heating in hot OsO 4 while the second was returned to 208C for 3 h and fixed in cold OsO 4 . For NMR analysis heating was carried out in the NMR rotor directly in the machine. Two experiments were conducted. In one the sample was heated to 708C and hold for 5 min and spectra were acquired subsequently. In the second experiment a NMR spectra were acquired while a sample was kept at 708C. Heat inactivation of spores was performed in a commercial autoclave (M.D.H., Andover, Hampshire, UK) at 1218C and 1348C for 15 and 35 min, respectively.

Phase contrast images were captured and saved using an image analysis software (Qwin, Leica Quantimet 570C, Leica Microsystems Ltd.). The total magnification on the analysis monitor was 6000 3 . The diameters of spores on the screen were 6 mm. Quantitative measurements were performed manually. The measurement of absolute greylevels of spores after various heat treatments was carried out by determining the grey intensity in the centre of approximately 300 spores. The software calculated the mean and statistical parameters such as standard deviation and standard error of greylevels within a spore population.

3. Results

2.7. Phase contrast light microscopy

3.1. Cross polarization ( CPMAS) and single pulse ( SPMAS) magic angle spinning 13 C nuclear magnetic resonance ( NMR) spectroscopy

The quantification of grey intensities in spores under phase contrast optics was performed according to Leuschner and Lillford (1999b). Preparation of a slide for phase contrast light microscopy was carried out as follows: A drop (4 ml) of a spore suspension was transferred onto a glass microscope slide. The drop was covered with a 22 3 40 mm cover slip and sealed with nail varnish to avoid dehydration. The slide was imaged under phase-contrast optics of a light microscope (Ortholux II Leitz). Groups of single spores which were attached to the underside of the cover slip were chosen for analysis. An important criteria was that all spores were in a well defined focal plane and immobilised under the cover slip surface. An objective magnification of 60 3 was chosen (Nikon 3 60, Plan Apo 1.40 na, Kingston, Surrey, UK). The microscope lamp was maintained at constant level throughout. As an internal standard, latex spheres of a diameter of 1.14 mm (Malvern Instruments Ltd., Worcestershire, UK) were mixed into heat inactivated suspensions of spores and served as constant controls for phase-brightness. This was particularly important in calibrating spore intensities during subsequent image analysis.

A CPMAS and SPMAS spectra of B. cereus T are shown in Fig. 1a and b. The CPMAS spectrum displayed a sharp peak of high intensity at 150 ppm and further immobilised carbon components at 170– 180, 90–50 and 50–10 ppm. Considerable amount of mobility was observed in the SPMAS spectrum complementary to immobilised components with exception of the peak at 150 ppm. DPA was only present in the CPMAS spectrum. Resolved peak maxima were identified at 181, 175, 75, 69, 60, 55, 51, 39, 33, 30, 27, 22 and 17 ppm in the SPMAS spectrum. NMR spectra of an outer coatless mutant B. subtilis 322 and an inner coatless mutant B. subtilis 325 are presented in Figs. 2a, b and 3a, b, respectively. These spectra are very similar and the CPMAS spectra and revealed a lower intensity of cross polarizable components between 50 and 10 ppm than was observed in the CPMAS spectrum of B. cereus. Similarly, the SPMAS spectra of the coat mutants resembled each other and resolved peaks were detected at 175, 98, 75, 73, 72, 69, 66, 65, 64, 60, 55, 30 and 22 ppm. 31 P NMR SPMAS and CPMAS spectra of germinated spores of B. subtilis are shown in Fig. 4. In the

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Fig. 1. (a) Nuclear magnetic resonance 13 C Cross polarization magic angle spinning (CPMAS) spectrum of dormant Bacillus cereus spores. (b) Nuclear magnetic resonance 13 C Single pulse magic angle spinning (SPMAS) spectrum of dormant Bacillus cereus spores.

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Fig. 2. (a) Nuclear magnetic resonance 13 C Cross polarization magic angle spinning (CPMAS) spectrum of outer coatless mutant Bacillus subtilis 322 spores. (b) Nuclear magnetic resonance 13 C Single pulse magic angle spinning (SPMAS) spectrum of outer coatless mutant Bacillus subtilis 322 spores.

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Fig. 3. (a) Nuclear magnetic resonance 13 C Cross polarisation magic angle spinning (CPMAS) spectrum of inner coatless mutant Bacillus subtilis 325 spores. (b) Nuclear magnetic resonance 13 C Single pulse magic angle spinning (SPMAS) spectrum of inner coatless mutant Bacillus subtilis 325 spores.

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60, 55, 39, 30, 28, 23 and 19. This finding corresponded to low signal intensities of cross polarisable components in the 13 C CPMAS spectrum around 80–100 ppm and 150 ppm. Germination led to high molecular mobility throughout the SPMAS spectrum.

3.2. Transmission electron microscopy ( TEM) Transmission electron micrographs of thin sections of B. cereus, B. subtilis 322, B. subtilis 325 and germinated spores of B. subtilis CMCC 604 are displayed in Figs. 6–9. The outer coatless mutant (B. subtilis 322) has a loosely attached inner coat which is missing in the inner coatless mutant (B. subtilis 325). The outer coat of B. subtilis 325 appeared to be less electron dense than observed for B. subtilis 357 lux 1 . Germinated spores contained an outer and inner coat but a cortex was not observed in the majority of germinated spore sections. The conditions during NMR analysis displayed no effects on the spore structure when analysed by TEM or on their viability according to plate count determination.

3.3. Heat treatment studies

Fig. 4. 31 P Single pulse magic angle spinning (SPMAS) NMR spectrum of germinated spores of B. subtilis 604 (top) and corresponding cross polarisation magic angle spinning spectrum (CPMAS) bottom.

CPMAS spectrum a low intensity was observed indicating low amounts of immobilised phosphorus components in the sample. The SPMAS spectrum indicated a high phosphorus mobility and in agreement with the CPMAS spectrum a flat broad underlying peak ranging from 1 to 2 9 ppm. Three resolved peaks were present in the germinated sample with chemical shifts at 2 2.5 (1), 2 5.9 (2) and 2 7.5 (3) ppm. The half height width of the tallest peak at 2 5.9 ppm was 0.5 ppm. 13 C NMR SPMAS and CPMAS spectra of germinated spores are shown in Fig. 5a and b. High molecular mobility was observed in germinated spore preparations in the 13 C SPMAS spectrum. Narrow and resolved resonances were primarily observed in the region of 45–100 ppm. Resolved peaks were identified at chemical shifts of 175, 98, 79, 75, 73, 70, 66, 65, 64,

Spores of B. subtilis 357 were heated in a water bath at 608C for up to an hour and no decrease in their phase-brightness was detected. Electron micrographs of heat activated spores of B. subtilis 357 lux 1 are shown in Figs. 10 and 11. An effect on the internal structure (core and cortex) can clearly be seen which was dependent on the heating period. We investigated a possible effect of hot OsO 4 during fixing on dormant spores and found that heating of spores for 1 s revealed no visible effects. After 3 h at room temperature the observed activated state had reverted and spores retained their dormant appearance (Fig. 12). NMR studies revealed no effects on molecular mobility of carbon and phosphorus depending on heat activation with both experimental conditions as described under Section 2.6. When spores were heat inactivated by heating at 1218C for 15 min and at 1348C for 35 min they turned phase grey and their brightness decreased to 64% and 53%, respectively. The greylevel of these spores did not change any further. Transmission electron micrographs of heat inactivated spores showed a non-reversible effect (Fig. 13). TEM displayed no distinct differences between the two

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Fig. 5. (a) Nuclear magnetic resonance 13 C Cross polarisation magic angle spinning (CPMAS) spectrum of germinated spores of B. subtilis 604. (b) Nuclear magnetic resonance 13 C Single pulse magic angle spinning (SPMAS) spectrum of germinated spores of B. subtilis 604.

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Fig. 6. Transmission electron micrograph of Bacillus cereus spores. Magnification 3 200 000.

Fig. 7. Transmission electron micrograph of Bacillus subtilis 322 spores. Magnification 3 200 000.

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Fig. 8. Transmission electron micrograph of Bacillus subtilis 325 spores. Magnification 3 100 000.

Fig. 9. Transmission electron micrograph of a germinated spore of Bacillus subtilis 604. Magnification 3 200000.

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Fig. 10. Transmission electron micrograph of heat activated (608C / 10 min) spores of Bacillus subtilis 357 lux 1 . Magnification 3 200 000.

Fig. 11. Transmission electron micrograph of heat activated (608C / 1 h) spores of Bacillus subtilis 357 lux 1 . Magnification 3 200 000.

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Fig. 12. Transmission electron micrograph reverted spores of Bacillus subtilis 357 lux 1 after storage for 3 h at room temperature following heat activation (608C / 1 h). Magnification 3 200 000.

heat inactivation conditions which related to the differences in phase brightness except that the harsher treatment appeared to display the structural damage more explicitly.

4. Discussion Our results revealed that NMR has a potential to detect structural characteristics in defined compartments in particles of micrometer size such as spores. NMR studies were described to reveal a noninvasive insight into the central part of spores (Lundin and Sacks, 1988; Ablett et al., 1999) where a DPA signal was identified at 150 ppm. The authors investigated spores of B. subtilis. We observed a sharper more intense DPA signal for spores of B. cereus than has been observed in B. subtilis (Leuschner and Lillford, 2000). A reason for the peak intensity was that the 13 C CPMAS NMR spectrum of B. cereus was less dominated by carbonyl resonances ( | 175 ppm) and resonances between 10 and 50 ppm than that of B. subtilis which resulted in more equal

signals intensity or that more DPA is in the solid state in B. cereus spores. It appears that spores of B. cereus contain DPA in a high degree of solid state order which may therefore be crystalline. Ablett et al. (1999) investigated the 13 C CPMAS NMR spectra of pure crystalline and amorphous DPA. The DPA signal we observed in the 13 C CPMAS spectrum of B. cereus displayed sharp characteristics of crystalline DPA. No DPA signals were observed in both coat mutant spore preparations of B. subtilis 322 and 325. An explanation might be that the mutant spores released their DPA and that it was subsequently discarded during washing with distilled water. Both coat mutants of B. subtilis had lower signal intensities between 10 and 50 ppm an area where protein components resonate than did spores with normal coat properties such as B. cereus and B. subtilis. B. cereus had less protein signals than did B. subtilis 357 lux 1 (Leuschner and Lillford, 2000) because of its less electron dense coat. Resonances of the coat mutants in the area where carbohydrates resonate between 50 and 100 ppm were compatible with D-2-acetamido-2-deoxy-D-galactopyranose (b-

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Fig. 13. Transmission electron micrograph of heat inactivated (1218C / 15 min) spores Bacillus subtilis 357 lux. Magnification 3 200 000.

GalNAc) which has carbon resonances at: 96.6 ppm (C-1), 55 ppm (C-2), 72.2 ppm (C-3), 69 ppm (C-4), 75.1 ppm (C-5) and 69 ppm (C-6) (Bock et al., 1984). These resonances were described for bacterial cell wall polysaccharides (Reddy et al., 1994). B. cereus did not display such a mobile SPMAS spectrum in the carbohydrate area as the coat mutant spores and therefore could not be clearly attributed to distinct components. The SPMAS spectrum of B. cereus was more resolved than that of B. subtilis 357 lux 1 (Leuschner and Lillford, 2000) which could be attributed to different components or less molecular mobility in B. subtilis spores. NMR resonance spectra of germinated spores differed significantly from those of dormant B. subtilis spores (Leuschner and Lillford, 2000). The resonance peak of DPA at a chemical shift of 150 ppm (Lundin and Sacks, 1988) was not present in these experiments in the 13 C CPMAS spectrum of germinated spores. This is consistent with a mobilisation and excretion of this substance during germination (Moir et al., 1994). DPA was not observed in the 13 C SPMAS spectrum because the germinated spores were washed repetitively with distilled water

prior to NMR analyses whereby released DPA was discarded. The resonances at 75 ppm and 105 ppm can be assigned to polysaccharide and carbohydrates (Gidley, 1992; Kalichevsky et al., 1992). Similar chemical shifts were reported for cell walls of B. subilis cells (Forrest et al., 1991). Peaks in the SPMAS spectrum of germinated spores between 50 and 100 ppm would be compatible with a-2-acetamido-2-deoxy-D-galactopyranose (Bock et al., 1984) and a-D-Galactopyranose (Reddy et al., 1994). TEM examinations of germinated spores revealed a disappearance of the cortex and this was in accordance with low signal intensities in this specific area in the 13 C CPMAS spectra. Since 50–80% of the total protein of mature spores was made up by coat proteins (Jenkinson et al., 1981) strong protein related signals in the 13 C CPMAS and SPMAS NMR spectra of dormant and germinated spores were observed which resembled the NMR spectra of albumin (Gidley, 1992). Coat degradation during germination was evident in the ultrastructural analysis in this study and in accordance with reports in the literature (Hashimoto and Conti, 1971), but the material was still present in the

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spores presented to NMR analysis and revealed a large degree of solid-like order. 31 P phosphorus spectra from spores shown by Shibata et al. (1984) displayed different resonance peaks than our spectra. The authors assigned peaks at 0 and 1.67 to internal ortho-phosphate. Their spectra had a very high noise to signal ratio and they report difficulties in acquiring spectra without the addition of EDTA. We acquired reasonable NMR spectra without an addition of EDTA which could be a result of washing germinated spores with distilled water. 31 P SPMAS spectra which we acquired showed immobilised and mobile phosphorus in accordance with Rasmussen et al. (1997). A significant decrease of immobilised phosphorus on germination relative to dormant spores (Leuschner and Lillford, 2000) indicated the reorganisation of the core to, e.g. cytoplasm of vegetative cells which was also evident in TEM micrographs of germinated spores. The results obtained by high-resolution solid state NMR reflect well structural changes which are taking place during germination. Since we did not observe any effects of high speed spinning or electric fields on spore viability and internal structure it was a genuinely non-invasive measurement technique to obtain general characteristics on a molecular level of a spore sample which could afterwards be used for further analysis. Heat activation resulted in structural changes which were seen by TEM but they were not associated with detectable increases in molecular mobility which was an unexpected observation. Transmission electron micrographs of heat activated spores displayed a structural change which became more explicit with longer heating times. This structural change was reversible. The observed effect could be related to expansion of the spore which was not correlated with molecular mobility increases because we observed no changes in molecular mobility of heat activated spores during NMR analysis. Observations by Beaman et al. (1988) provided evidence that heat activation affects permeability of spores. The authors measured decreases in spore density with increased heating times at heat activation temperatures. Our study shows that a combination of NMR and TEM resulted in a valuable insight of dormant bacterial spore structure and dynamical changes caused by germination. Both techniques revealed

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characteristic information on properties of individual spores and of the whole sample bulk at the same time. Internal structure seen by TEM was reflected in corresponding NMR spectra. Heat activation treatment of spores was reflected by TEM but no effects were measured by solid-state nuclear magnetic resonance spectroscopy.

Acknowledgements The authors thank Mr. A.H. Darke for expert NMR analysis and Mr. A.C. Weaver for support during electron microscopy. This work was supported by an European Community Marie Curie Research Training Grant in the Framework of the FAIR Program (FAIR CT 975014).

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