1. Location and the dynamics of SpoVAEa protein in dormant spores
By using widefield microscopy, we found that the spore surface area did not have a uniform level of SpoVAEa-SGFP2. Next, we employed RCM with a scanning time of 2 sec per frame to obtain more detailed structural information . Surprisingly, clusters of GFP fluorescence were observed, which clearly stood out from the background in dormant PS832 SpoVAEa-SGFP2 spores (Fig. 1A, left panel). By enlarging one of the spores, two spots were observed in the spore labelled ‘a’ (Fig. 1A, right panel). The full width at half maximum (FWHM) of the pronounced spot of spore ‘a’ was 220 nm (Fig. 1C). Due to the fact that the visualization of germination proteins can be interfered with by heavy autofluorescence from the spore coat, we also imaged SpoVAEa-SGFP2 in spores with a severely defective coat . Similar fluorescent spot structures were found in the coat defective PS4150 SpoVAEa-SGFP2 spores (Fig. 1B, C). These spots were, however, not more pronounced indicating that our observations in wild-type spores were not majorly obscured by coat layers.
Previously, we showed that GerKB-SGFP2 proteins, one class of GRs, also presented themselves as 1-3 spots per spore . These spots ‘oscillated’ at high frequency, but were confined only to a small area of the IM . Here, the crucial question is whether SpoVAEa-SGFP2 is also at least somewhat mobile in the IM. To address this query, RCM microscopy was applied for high frequency imaging with a scanning time of 22.2 milliseconds per frame. Even more surprisingly, a single spot, traveling around the IM, i.e. not confined to a small area, was observed in the PS832 SpoVAEa-SGFP2 dormant spores when the spore was tracked for 1000 frames. We presume that we are the first to see this movement, as we imaged at very high time resolution. The SpoVAEa-SGFP2 fluorescent spot in the spore ‘randomly’ appeared in different time frames and different locations of the spore. Fig. 1D shows eight frames with a pronounced fluorescent spot, and the location differs in each frame. The measured FWHM of the spot in different frames varied from 96 nm to 263 nm. Hence, the observed high frequency movement with longer exposure time, could explain why not all the spores in Fig. 1A and Fig. 1B, showed a spot structure, as well as the uniform distribution of SpoVAE, most likely SpoVAEa, in previous work . It is, however, not clear how SpoVAEa is able to move at such a high frequency in an otherwise ‘rigid’ inner membrane [19, 23], although SpoVAEa appears to be on the outer surface of the IM . Moreover, we cannot exclude that there are some ‘free’ SpoVAEa proteins distributed over the IM outside of the spot.
2. Dynamics of SpoVAEa in the IM of B. subtilis spores during GR-triggered spore germination
Previously, we found that during spore germination GerKB-SGFP2 spots gradually increased in fluorescence intensity . This phenomenon is potentially related to the change of the spore’s physical state in general and the IM in particular upon germination, because no new protein synthesis was observed . It could be linked, for instance, to the increase in core water content and core pH due to the release of Ca2+-DPA and cortex hydrolysis. Here we used widefield microscopy to track the overall mean intensity of SpoVAEa-SGFP2 in spores during germination via GerB and GerK GRs by supplying the AGFK nutrient germinant cocktail (L-asparagine, glucose, fructose, and potassium chloride).
The phase brightness and SpoVAEa-SGFP2 fluorescence history of a single spore is shown in the time-lapse image montage in Fig. 2A and 2C. By analyzing the brightness and fluorescence profiles of this spore, we observed that the peak fluorescence intensity of SpoVAEa-SGFP2 was reached before the appearance of the phase dark spore, followed by a slow decline of fluorescence intensity in the phase dark spore (Fig. 2A-D). The initiation of SpoVAEa-SGFP2 fluorescence increase was at the same time as the start of the spore’s brightness rapid decline, which is considered as the start of rapid Ca2+-DPA release (Fig. 2B, D, E). In order to confirm the observed dynamics in the population, we synchronized SpoVAEa-SGFPs fluorescence profiles by defining the t=0 as the ‘time to germination’, which is the time needed for the spore to complete half of its rapid decline in phase brightness (Fig. 2E). In the averaged trace of 418 germinating spores, the SpoVAEa-SGFP2 intensity increased sharply and the peak was around the ‘time to germination’ (Fig. 2F), while no SpoVAEa-SGFP2 synthesis was detected by western blot analysis (Fig. S1). Two independent batches of spores were analysed in this experiment. Thus, the increase of SpoVAEa-SGFP2 fluorescence intensity during germination is correlated with the rapid release of Ca2+-DPA. Western blot analysis in the current work, as well as previous work, did not detect a significant decrease of SpoVAEa level in germinated spores compared to dormant spores (Fig. S1), however, a decrease of SpoVAEa-SGFP2 fluorescence was observed in germinated spores (Fig. 2D, 2F) . Subsequently, we found that photobleaching had a role in the observed fluorescence decrease (Fig. S2, S3), and photochemical alteration of both spore coat and SGFP2 potentially contributed to the photobleaching. A spore coat defective strain with no autofluorescence will likely be necessary to study the dynamics of SpoVAEa-SGFP2 in germinated spores [22, 24].
3. Changes in IM structure upon triggering spore germination via GRs
It is known that lipids in the B. subtilis dormant spore IM are largely immobile [2, 19]. Still, this IM is capable of increasing its surface area ~1.3 fold upon the spore swelling due to spore core water uptake and cortex hydrolysis . As indicated above, SpoVAEa-SGFP2 fluorescence reached peak intensity around ‘time to germination’. Consequently, we decided to probe for a putative correlation between the SpoVAEa-SGFP2 peak fluorescence intensity and the change in mobility of the IM lipids. To that end, the IM of PS832 spores was stained with either the carbocyanine dye DiIC12 or the styryl dye FM5-95, which were added to a sporulating culture and hence incorporated into the IM upon the formation of the forespore, as has been shown in previous studies [16, 19]. Any lipid probe present in the OM could be removed by extensive washing during spore purification .
We again used widefield microscopy to track the change in IM staining during germination. The fluorescence intensity of DiIC12 and FM5-95 stained spores dropped dramatically upon the start of the rapid decline in spore brightness, and the drop was completed around the ‘time to germination’ (Fig. 3, 4). In a word, just as with the dynamics of SpoVAEa-SGFP2, the change of the IM during germination is also highly correlated with the rapid Ca2+-DPA release and cortex hydrolysis, which lead to the increase of spore core pH, and water content [26, 27]. The FM5-95 stained spore also had a fluorescent spot, whereas DiIC12 spores had relative uniform staining (Fig. 3C, 4C). Regarding the fact that the FM5-95 dye was almost invisible in the germinated spore (Fig. 4C), we note that the hydrophobicity and affinity of FM5-95 for the IM lipids seems lower than that of DiIC12. This implies also that FM5-95 prefers to bind in lipid domains with higher fluidity. Hence, the FM5-95 spot area could well be a fluid membrane domain, instead of the compressed IM. The disappearance of the FM5-95 spot in the phase dark spore, suggests that this region might be a germination related functional membrane microdomain (Fig. 4C).
4. Response of SpoVAEa and probe stained IM to thermal treatments
Our previous work showed that heating B. subtilis spores at 40-65°C promotes spore germination in a positive time/temperature dependent manner, higher heat temperatures resulted in both heat activation and heat damage, and eventually led to heat inactivation at 80°C . The germination proteins and their surrounding IM are potential targets of the heat treatments. Here we studied whether heat treatments change the states of the spore’s, SpoVAEa and the IM after 5 hours of heat treatment at 40-80°C.
Significant drops of spore’s absorbance at the population (Fig. 5A, 7A, 7E) and refractive index at single spore level (Fig. 5C, 5E, 7C, 7G) were detected in spores treated at 80°C, and in some cases also in 75°C treated spores. Spores treated at 40-70°C morphologically looked similar upon the microscopical examination. Hence, we only present images of 65, 75, and 80°C heated spores as representative images (Fig. 6, 8). As shown in the phase contrast images (Fig. 6, 8), a subpopulation of phase-grey-like spores appeared in 80°C treated groups. These phase-grey-like spores are potentially spores that have lost Ca2+-DPA, as reported previously [28, 29].
Heat treatments at 40-80°C led to a decrease of fluorescence intensity of coat defective PS4150 SpoVAEa-SGFP2 spores (Fig. 5F, 6D). However, we cannot exclude the possibility that the decreased SpoVAEa-SGFP2 spore fluorescence was caused by heat denaturation of SGFP2 . This denaturation seems reversible at temperatures ≤65°C, due to the fact that 65°C treated PS832 SpoVAEa-SGFP2 spores still exhibited similar fluorescence profiles as untreated spores during spore germination (Fig. S2, 2). However, it is not clear how spores maintain functionally active channel proteins during the heat activation process. Clearly, a pH and thermal stable fluorescent reporter will be important for future spore related research [31–33]. Notably, we tested the response of SpoVAEa-SGFP2 to heat in coat defective spores, because the change of SpoVAEa-SGFP2 fluorescence was masked by the enhanced green autofluorescence of the spore coat (Fig. 5, 6). The increase of autofluorescence had a positive correlation with the heat treatment temperature. We speculate the enhanced autofluorescence is likely related to coat protein denaturation, since studies showed that a lethal thermal treatment induced significant protein denaturation in B. subtilis, B. cereus, and B. megaterium spores [28, 29]. Besides, the observed increased autofluorescence in PS4510 spores treated at 80°C is considered due to the response of either the residual outer coat or the inner coat layers to heat (Fig. 6C).
Compared to the untreated (ut) DiIC12 labeled spores, spores heated at 40-80°C exhibited slightly decreased fluorescence intensity (Fig. 7B, 7D, 8A). However, no correlation was detected between the drop of the phase contrast image brightness and changes in DiIC12 fluorescence (Table.S2). In contrast, FM5-95 stained spores showed a continuous fluorescence decrease upon treatment at 70, 75, and 80°C (Fig. 7F, 7H, 8B). The FM5-95 fluorescence had a positive correlation with the spore brightness of 70 and 75°C heated spores (Table.S2), Pearson correlation coefficients of 0.54 and 0.76, respectively, and it was minimal in spores heated at 80°C. As mentioned above, we speculate that spores with decreased brightness are potentially spores that have lost Ca2+-DPA. Hence, the decrease of FM5-95 intensity presumably correlated with the release of Ca2+-DPA, and the subsequent physical state changes in spores.