Kinetics of Germination of Individual Spores of Geobacillus stearothermophilus as Measured by Raman Spectroscopy and Differential Interference Contrast Microscopy

Geobacillus stearothermophilus is a gram-positive, thermophilic bacterium, spores of which are very heat resistant. Raman spectroscopy and differential interference contrast microscopy were used to monitor the kinetics of germination of individual spores of G. stearothermophilus at different temperatures, and major conclusions from this work were as follows. 1) The CaDPA level of individual G. stearothermophilus spores was similar to that of Bacillus spores. However, the Raman spectra of protein amide bands suggested there are differences in protein structure in spores of G. stearothermophilus and Bacillus species. 2) During nutrient germination of G. stearothermophilus spores, CaDPA was released beginning after a lag time (T lag) between addition of nutrient germinants and initiation of CaDPA release. CaDPA release was complete at T release, and ΔT release (T release – T lag) was 1–2 min. 3) Activation by heat or sodium nitrite was essential for efficient nutrient germination of G. stearothermophilus spores, primarily by decreasing T lag values. 4) Values of T lag and T release were heterogeneous among individual spores, but ΔT release values were relatively constant. 5) Temperature had major effects on nutrient germination of G. stearothermophilus spores, as at temperatures below 65°C, average T lag values increased significantly. 6) G. stearothermophilus spore germination with exogenous CaDPA or dodecylamine was fastest at 65°C, with longer Tlag values at lower temperatures. 7) Decoating of G. stearothermophilus spores slowed nutrient germination slightly and CaDPA germination significantly, but increased dodecylamine germination markedly. These results indicate that the dynamics and heterogeneity of the germination of individual G. stearothermophilus spores are generally similar to that of Bacillus species.


Introduction
Many components of the spore germination machinery are conserved between spore forming members of the Bacillales [1]. Bacillus subtilis spore germination can be initiated by a variety of chemicals, including nutrients, cationic surfactants, and enzymes, as well as by hydrostatic pressure [2]. Nutrient germinants for spore germination generally include amino acids, purine derivatives, and sugars, and are species and strain specific. These nutrient germinants interact with germination receptors (GRs) located in the inner spore membrane [2], stimulating the release of the spore core's large (,10% of spore dry wt) depot of pyridine-2,6-dicarboxylic acid (dipicolinic acid [DPA]) and divalent cations, predominantly Ca 2+ , which are likely present as a 1:1 chelate (CaDPA) [3]. CaDPA in the core is released and replaced by water in stage I of spore germination, and CaDPA release then triggers stage II of germination, a major event which is the hydrolysis of spores' peptidoglycan cortex by cortex lytic enzymes (CLEs) [2,4].
Concomitant with cortex hydrolysis, the core's full rehydration ultimately leads to resumption of enzyme activity, and initiation of metabolism and macromolecular synthesis in the core, and thus spore outgrowth [2,5].
B. subtilis spores contain three major GRs, termed GerA, GerB and GerK, each of which contains A, B and C subunits all of which are required for GR function [2]. These GRs are encoded by three tricistronic operons, each of which appears to encode a single GR [1,2]. The GerD protein is also essential for proper GR function, and the proteins encoded by the spoVA operon are essential for DPA uptake in sporulation and probably CaDPA release during germination as well [1,6,7]. Geobacillus stearothermophilus is a Gram-positive spore-forming thermophile. Genomic analysis suggests that G. stearothermophilus has clear homologs of the B. subtilis GR genes as well as gerD and spoVAB, C, D genes, and genes encoding the cortex lytic enzymes CwlJ and SleB [1,8].
G. stearothermophilus spores are the most wet heat-resistant among spores of aerobic spore-forming bacteria, and can spoil a variety of types of foodstuffs [9][10][11][12]. These spores are also commonly used as a biological indicator to evaluate the effectiveness of sterilization processes, in particular wet heat. However, the germination of spores of G. stearothermophilus species is much less well studied than that of spores of Bacillus species. Limited studies have shown that G. stearothermophilus spores germinate in response to low mol wt nutrient germinants including amino acids, purine and pyrimidine nucleosides, and sugars. However, the kinetics of the germination of individual G. stearothermophilus spores and the heterogeneity among individual spores in a population has not been studied.
In this study, we investigated the nutrient and non-nutrient germination of multiple individual intact and decoated G. stearothermophilus spores at various temperatures. We also measured the CaDPA level and Raman spectra of individual G. stearothermophilus spores and compared these with those of spores of several Bacillus species, as well as effects of different activation methods on kinetics of germination of individual G. stearothermophilus spores. This work has provided new information on the dynamics of and the heterogeneity in the germination of G. stearothermophilus spores.

Bacterial Species Used and Spore Preparation
Spores of G. stearothermophilus NGB101 were prepared and purified as described previously [13]. The Bacillus species used in this work were Bacillus subtilis PS533 [14] and Bacillus cereus T (originally obtained from H.O. Halvorson). Spores of these species were prepared and stored as described [15,16]. All spores used in this work were free (.98%) of growing or sporulating cells, as determined by phase contrast microscopy.

Measurement of CaDPA Level and Raman Spectra of Individual Spores by Laser Tweezers Raman Spectroscopy
The CaDPA levels of individual spores of various species were determined by laser tweezers Raman spectroscopy at 25uC [17]. Briefly, an individual spore was captured with laser tweezers, and its Raman spectrum was acquired with an integration of 20 s and a laser power of 20 mW at 780 nm. Spectra of 30 individual spores were measured and averaged. The CaDPA level in an individual spore was determined from the peak intensity at 1,017 cm 21 in its Raman spectrum relative to the peak intensity of the same Raman band from a CaDPA solution of known concentration (50 mM) and by multiplying this concentration value by the excitation volume of 1 fl to obtain attomoles of CaDPA/spore [17]. Raman spectra of 30 individual spores of G. stearothermophilus, B. subtilis and B. cereus at 25, 65, and 95uC were also averaged for analysis of heat-induced changes in spores' molecular components.

Activation of G. stearothermophilus Spores
Unless noted otherwise, prior to germination experiments, G. stearothermophilus spores were activated by one of three methods: 1) incubation in water at 100uC for 30 min followed by cooling in ice water for 15 min; 2) incubation in water at 30uC for 120 h; or 3) incubation in 0.2 M sodium nitrite (pH 8.0) at 30uC for 17 h. Germination of unactivated G. stearothermophilus spores was also carried out in a few experiments.  Monitoring Germination of Single Spores by Raman Spectroscopy and Differential Interference Contrast (DIC) Microscopy The germination of an individual G. stearothermophilus spore with 0.1 mM L-valine in 10 mM sodium phosphate buffer (pH 8.0) at 65uC was monitored simultaneously by Raman spectroscopy and DIC microscopy, as described [18,19]. Briefly, a single G. stearothermophilus spore was optically captured immediately after the addition of 65uC 0.1 mM L-valine/10 mM sodium phosphate buffer. Both the Raman spectra and DIC microscopy images of the trapped spore were recorded simultaneously for a period of 45 min with intervals of 30 s per spectrum and 15 s per image frame, respectively. Note that the low concentration of L-valine used in this experiment was to slow spore germination sufficiently to allow its measurement by Raman spectroscopy.

Monitoring Germination of Multiple Individual Spores by DIC Microscopy
The germination of a number of individual spores was simultaneously monitored with DIC microscopy [18]. Prior to germination, the spores were routinely activated at 100uC for 30 min unless noted otherwise. Briefly, 1 ml of heat-activated spores (10 8 spores/ml in water) was spread on the surface of a glass coverslip glued to a clean and sterile sample container. The spores on the container were quickly dried in a vacuum chamber at room temperature so that they adhered to the coverslip. The spore container was then mounted on a microscope heat stage kept at the appropriate temperature. Preheated germinant / buffer solution was then added to the container, and a digital CCD camera (12 bits; 1600 by 1200 pixels) was used to record the DIC images at a rate of 1 frame per 15 s for 60-120 min. These DIC images were analyzed with a computation program in Matlab to  Table 2. Effect of activation methods on G. stearothermophilus spore germination*. locate each spore's position and to calculate the summed pixel intensity. The DIC image intensity of each spore was plotted as a function of the incubation time (with a resolution of 15 s). Unless noted otherwise, G. stearothermophilus spores were germinated at various temperatures in: (i) 1 mM L-valine in 10 mM sodium phosphate buffer (pH 8.0); (ii) 1 mM AGFK (a mixture of 1 mM each of L-asparagine, D-glucose, D-fructose, and potassium ions) in 10 mM sodium phosphate buffer (pH 8.0); (iii) 60 mM CaDPA made to pH 7.4 with Tris base; and (iv) 1 mM dodecylamine in 10 mM sodium phosphate buffer (pH 8.0). Except for dodecylamine and CaDPA germination, spores were routinely activated for 30 min at 100uC prior to germination experiments unless noted otherwise.

Chemical Decoating of G. stearothermophilus Spores and Germination of Decoated Spores
Spores of G. stearothermophilus at an optical density at 600 nm of ,10 were decoated by treatment with 1% sodium dodecylsulfate (SDS)-0.1 M NaOH-0.1 M NaCl-0.1 M dithiothreitol for 30 min at 65uC [20]. This procedure removes much of the spore's coat protein as well as the spore's outer membrane [20]. The decoated spores were washed at least 10 times with 0.1 M NaCl by centrifugation to remove all traces of the decoating solution and suspended in water. The decoated spores were then germinated with various agents with or without activation treatment as described above.

Data Analysis
The DIC microscope that monitored individual spores was set such that the polarizer and analyzer were crossed, and thus the DIC bias phase was zero. After adding pre-heated germinant/ buffer solution to spores on the coverslips, a digital CCD camera was used to record the DIC images. These images were analyzed with a Matlab program to locate each spore's position and to calculate the averaged pixel intensity of an area of 20620 pixels that covered the whole individual spore on the DIC image. The DIC image intensity of each individual spore was plotted as a function of the incubation time and the initial intensity (the first DIC image recorded after the addition of the germinant) was normalized to 1 and the intensity at the end of measurements was normalized to zero. Invariably, the latter value had been constant for $10 min at the end of measurements.
From the time-lapse DIC image intensity, we can determine the time of completion of the rapid fall of ,75% in spore DIC image intensity, which is concomitant with the time of completion of spore CaDPA release (T release ). CaDPA release kinetics during germinationof individual spores were described by the parameters T lag , T release and DT release [7,18]. We also defined the additional germination parameters, T lys and DT lys where T lys is the time when spore cortex hydrolysis is completed as determined by the completion of the fall in the spore's DIC image intensity, and DT lys = (T lys -T release ).

Raman Spectra and Average CaDPA Level of Individual G. Stearothermophilus Spores
CaDPA dominates the Raman spectra of individual spores of Bacillus species [21], and this was also the case for spores of G. stearothermophilus (Fig. 1). The intensity of the CaDPA-specific 1,017 cm 21 Raman band in the average spectrum from 30 individual spores indicated that the CaDPA level in the core of G. stearothermophilus spores was , 382 mM, and this value was only slightly higher than the values for B. subtilis and B. cereus spores ( Table 1).  [22][23][24]. Raman spectra of B. subtilis and B. cereus spores ( Fig. 1(B, C)) show that as the temperature was increased from 25 to 95uC, the intensity of the 1653 cm 21 band was slightly decreased and the intensity at 1667 cm 21 slightly increased. Similarly, the peak of the 1253 cm 21 band (protein amide III) was slightly shifted to the left at the higher temperatures. This suggests that the structure of proteins in B. subtilis and B. cereus spores had changed significantly from an ahelical structure to a nonregular structure at high temperature, indicative of significant denaturation of proteins in these spores as found previously [24][25][26]. Indeed, when incubated at 95uC, some B. cereus spores lost their CaDPA, the 1653c m 21 band shifted to 1667 cm 21 , and the 1253 cm 21 band shifted to 1235 cm 21 (curve d in Fig. 1(C)), suggesting that significant protein denaturation took place after CaDPA release at 95uC. In contrast to results with B. subtilis and B. cereus spores, the Raman bands of protein amide I (1653/1667 cm 21 ) were unchanged for G. stearothermophilus spores at 95uC (Fig. 1(A)), indicating that these spores' proteins are stable even at 95uC, consistent with these spores' extremely high wet heat resistance. The amide III band (1230-1300 cm 21 ) region centered  at 1253 cm 21 shifted to a lower wavenumber at 65uC and at 95uC for B. cereus spores, but for G. stearothermophilus and B. subtilis spores, this change was less prominent. The Raman band at 783 cm 21 seen at 25uC is attributed to ring breathing of cytosine/thymine/ uracil and the O-P-O symmetric stretch of the phosphodiester bond in DNA and RNA [27,28]. At 95uC, the Raman band at 783 cm 21 was nearly unchanged, suggesting that the double helical structure of nucleic acids in G. stearothermophilus spores is stable at elevated temperature. Fig. 2 shows dynamics of an optically trapped individual G. stearothermophilus spore during L-valine germination at 65C, as monitored by Raman spectroscopy and DIC microscopy. After the addition of the germinant the CaDPA level as measured by the 1017 cm 21 band [17] and the DIC image intensity were nearly unchanged before T lag at , 2.2 min. The intensity of the 1017 cm 21 band then quickly dropped to zero and the spore's DIC image intensity decreased ,70% by T release at , 3.2 min. In this experiment, the DIC image intensity of the G. stearothermophilus spore usually continued to fall (but see below) until T lys at , 9.6 min, corresponding to the completion of spore cortex hydrolysis, and then remained constant. As seen with the germination of Bacillus spores [19], the termination point of the rapid fall in DIC image intensity precisely corresponded to the completion of CaDPA release for G. stearothermophilus spores.

Effect of Different Activation Methods on G. stearothermophilus Spore Germination
Previous studies [29,30] have shown that germination of G. stearothermophilus spores becomes much more rapid if the spores are first given an activation treatment such as incubation in water for short times at a high temperature, long times in water at a moderate temperature, or incubation in sodium nitrite at a moderate temperature for intermediate times. The current work demonstrated that these different activation regimens led to different kinetics of L-valine germination of individual G. stearothermophilus spores at 65uC (Table 2; Fig. 3). All three activation regimens increased the overall rates of spore germination, almost completely by decreasing average T lag values with minimal if any effects on values for DT release and DT lys . Note also that for a number of the individual spores activated by various regimens, following the initial rapid fall in DIC image intensity of , 60%, there was an lag of 5-20 min following T release and before the further fall in DIC image intensity. This was also seen in many other germination experiments (see below), although the reason for this lag period is not clear.

Kinetics of Germination of Multiple Individual G. stearothermophilus Spores with L-valine or AGFK
Previous work [29] has shown that G. stearothermophilus spores are able to germinate in the presence of L-valine or AGFK. Consequently we used DIC microscopy to analyze the germination of multiple individual G. stearothermophilus spores with L-valine or AGFK at multiple temperatures ( Fig. 4 and 5; Table 3; and data not shown). G. stearothermophilus spores germinated faster with AGFK than L-valine at 65uC, and as expected, germination with these nutrients was faster at 65uC than at 55uC or 45uC, while no germination was observed when G. stearothermophilus spores were incubated with 1 mM L-valine at 37uC or 25uC (Table 3; and data not shown). The slower germination of these spores at lower temperatures was due primarily to longer T lag values as: i) many spores did not even germinate in the observation times at the lower temperatures, and thus have very long T lag values; ii) DT release values increased only slightly at lower temperatures; and iii) DT lys values were essentially unchanged at low and high temperatures.

Kinetics of Non-nutrient Germination of Individual G. stearothermophilus Spores
In addition to nutrients, spores can germinate with a variety of non-nutrients [2,3], including lysozyme, CaDPA, cationic surfactants, high pressures and some salts. Unlike the case with nutrient germination, exogenous CaDPA induced germination of G. stearothermophilus spores at 25uC (Fig. 6; Table 3). However, CaDPA germination of G. stearothermophilus spores was faster at 65uC due largely to a much shorter average T lag value than at 25uC, although the DT release values were almost identical at these two temperatures. The average DT lys value for CaDPA germination at 25uC was also much longer than for CaDPA germination at 65uC. Another group of non-nutrient germinants is cationic surfactants, with dodecylamine being the one that has been best studied [31].With 1 mM dodecylamine at 65uC, only , 50% of G. stearothermophilus spores germinated in 120 min, a slow germination compared to those with other germinants, and dodecylamine germination was minimal at 45uC ( Fig. 7; Table 3). As seen with CaDPA germination at low and high temperatures, most of the decrease in the rate of germination with dodecylamine at the lower temperature was due to much longer average T lag values.

Kinetics of Germination of Individual Decoated G. stearothermophilus Spores
Since at least some proteins involved in spore germination in Bacillus species are located in the spore coats, in particular the CLE CwlJ [2], we also examined the effect of chemical decoating on G. stearothermophilus spores' germination with nutrient and nonnutrient germinants, all at 65uC (Fig. 8; Table 3). With L-valine and AGFK, the rate of germination of decoated G. stearothermophilus spores decreased by , 15%, while T lag values increased ,1.5 fold. However, the amount and rate of CaDPA germination of the decoated spores were markedly lower than with intact spores, as the average T lag value increased .6-fold while the average T lys value increased,13-fold, although the average DT release value was essentially unchanged from that for intact spores. Decoating also greatly increased the rate of dodecylamine germination of G. stearothermophilus spores markedly, largely by decreasing the average T lag value (Table 3).

Discussion
The work in this communication has revealed a number of similarities in the properties of spores of G. stearothemophilus and Bacillus species, in particular the nearly identical DPA concentrations in these spores' core. However, there were some differences. One was the lack of change in the Raman spectrum of proteins in  G. stearothermophilus spores upon incubation at 95uC. This behavior, as well as the high temperature needed for heat activation of G. stearothermophilus spores, is undoubtedly a reflection of G. stearothermophilus being a thermophile, and is consistent with both the high temperature optimum for germination of spores of this species and their extremely high wet heat resistance compared to spores of B. cereus and B. subtilis [10]. The second difference, and a more intriguing one was the highly variable lag period between T release and the initiation of the second fall in G. stearothermophilus spores' DIC image intensity during spore germination with all germinants tested, as this has been seen only rarely in germination of spores of Bacillus species [19,32]. While we have no good explanation for this difference, it is as if there is a much higher threshold for the signal event that begins G. stearothermophilus spore cortex degradation by CLEs following CaDPA release than with spores of Bacillus species. However, these signaling mechanisms are not well understood, so we have no good mechanistic explanation for this difference between spores of these two genera.
While there were the notable differences between G. stearothermophilus and Bacillus spore properties noted above, the overall features of the nutrient and non-nutrient germination kinetics of individual spores of this species were very much like those of Bacillus species. Thus CaDPA release for all G. stearothermophilus spore germinations examined began only after a highly variable T lag period but DT release took only a few min, with T release followed by cortex hydrolysis that was completed at T lys . Almost always, DT lys was longer than DT release , and most of the heterogeneity in the germination between individual spores was in DT lag values, as seen previously with spores of Bacillus species [18,32,33]. The effects of activation treatments on the germination G. stearothermophilus were also largely, if not completely on T lag values, as average DT release and DT lys values in nutrient germination of unactivated and maximally activated spores were essentially identical. Optimal heat activation also decreases average T lag values for nutrient germination of spores of Bacillus species [34]. Since a major factor determining the T lag period for nutrient germination of spores of Bacillus species is spores' levels of functional GRs [33], this further suggests that heat activation of G. stearothermophilus spores for 30 min at 100uC makes these spores' GRs optimally functional, perhaps by some conformational protein changes as has been suggested for spores of Bacillus species [35]. The mechanism of nitrite activation of spores has never been analyzed in detail, but could be due to covalent modification of the spore cortex by  nitrous acid [36]. However, this could equally well be due to nitrous acid modification of GRs.
It was also notable that germination at suboptimal temperatures greatly increased T lag values for nutrient germination of G. stearothermophilus spores, especially given that lower percentages of these spores germinated at lower temperatures in the observation periods used. In contrast, there was essentially no effect on DT lys values as the germination temperature was lowered, indicating that the temperature sensitive step in nutrient germination of G. stearothermophilus spores is in T lag , and probably is on the GRs themselves, although there was also a small increase in DT release times as germination temperature was lowered. The effect of temperature on kinetics of the germination of individual spores has not been studied with spores of Bacillus species.
Decoating of G. stearothermophilus spores had only a minimal effect on their nutrient germination, with the biggest effect being 1.5 to 2-fold increases in DT lys values. The G. stearothermophilus genome has the genes for the two redundant CLEs, CwlJ and SleB, involved in cortex hydrolysis during spore germination in Bacillus species. With Bacillus spores, decoating largely removes or inactivates CwlJ [37], and presumably a decrease in CwlJ level is the reason for the increased DT lys values in decoated G. stearothermophilus spores. However, we do not know if all G. stearothermophilus CwlJ is inactivated by the decoating regimen we used. Indeed, decoating or loss of CwlJ by mutation increases values of DT release in nutrient germination of spores of several Bacillus species 6-to 10-fold [38,39], while the increase in decoated G. stearothemophilus spores was at most 2-fold. Thus with G. stearothermophilus spores either CwlJ is not essential for rapid CaDPA release in spore germination, or some active CwlJ survives the decoating regimen used. We favor the latter possibility, since CwlJ is essential for CaDPA germination of spores of Bacillus species [37,40], while significant CaDPA germination still took place with decoated G. stearothermophilus spores. However, the average T lag value for CaDPA germination increased , 7-fold in decoated G. stearothermophilus spores. Thus it seems most likely that CwlJ is also the primary target of CaDPA in triggering germination of G. stearothermophilus spores.
Along with nutrient germination, G. stearothermophilus spore germination with the non-nutrients CaDPA and dodecylamine also decreased markedly at suboptimal temperatures, again largely due to effects on T lag . However, the latter effect is almost certainly not on GRs, which are not involved in CaDPA and dodecylamine germination of spores of Bacillus species [31,37]. Indeed, as noted above, CaDPA probably triggers G. stearothermophilus spore germination by activating the CLE CwlJ, while in Bacillus spores dodecylamine likely triggers germination by triggering the opening of the CaDPA channel in the spores' inner membrane that is composed at least in part of SpoVA proteins [41]. Interestingly, decoating of G. stearothermophilus spores significantly increased these spores' germination with dodecylamine primarily by decreasing T lag values, just as with spores of Bacillus species [31]. Why this should be is not completely clear, but decoating may allow easier access of dodecylamine to the SpoVA CaDPA channel than in an intact spore.
In summary, the analysis of the dynamics of the germination of multiple individual G. stearothermophilus spores with a variety of germinants indicates that the general features of the germination of these spores appear to be quite similar to those of spores of Bacillus species.