The results reported in this communication allow a number of new, significant conclusions about wet-heat inactivation of individual spores of Bacillus species, conclusions that could not be drawn from studies of the wet-heat treatment of spore populations. Most of these conclusions are the same for spores of three different Bacillus species, suggesting that these conclusions are general ones for spores of all Bacillus species.
The first conclusion is that during incubation in water at heat-killing temperatures, Bacillus
spores exhibit no significant change in their CaDPA content until Tlag
and then release the majority of their CaDPA in 1 to 2 min in ΔTrelease
. The mechanism that determines CaDPA release during ΔTrelease
is not clear. However, it seems likely that this rapid CaDPA release is due to a breakdown of the spore's major permeability barrier to small molecules, and this permeability barrier is most likely the spore's inner membrane (37
). In addition, it is not exactly clear how CaDPA leaves the spore core during heat treatment. During spore germination, CaDPA is also almost completely released in 1 to 2 min (5
), and while it is not known how this release is triggered, CaDPA is suggested to be released via inner membrane channels composed at least in part of SpoVA proteins (41
). Perhaps high-temperature treatment somehow triggers the rapid synchronous opening of these normal CaDPA channels, either by action on the channels themselves or indirectly by effects on the spore's inner membrane. Alternatively, there may be such a drastic change in inner membrane structure that all core small molecules, not just CaDPA, are rapidly released in parallel during wet-heat treatment. Indeed, all core small molecules are released during spore wet-heat treatment (21
), although the rates of rapid release of different core small molecules are not known for individual spores. In addition, not all core small molecules are released during spore germination (36
), so it may be possible to determine if small-molecule release during wet-heat treatment is similar in its selectivity to that seen with spore germination.
The second conclusion is that while values of ΔTrelease
during spore heat inactivation are relatively constant for different individual spores, either in one species or across species, the variation in Tlag
is extremely large, most notably between individual spores in the same spore preparation. The reason for these differences between individual spores in populations is not some heterogeneity in the physical microenvironment during wet-heat treatment, but a likely cause is spore heterogeneity due to differences in the microenvironments of different cells in the same sporulating culture, perhaps because different cells in a culture sporulate at different times. While the overall physical and chemical environments during sporulation are the same for all sporulating cells in a population, the microenvironments may vary significantly, in particular because of differences in the kinetics of sporulation of different cells in the population (18
). Such differences in spore formation may in turn result in slight differences in the levels of various components important in spore moist-heat resistance such as core water, divalent cations, and CaDPA, as well as slight differences in structures of important spore layers, such as the peptidoglycan cortex. A number of these slight differences might act synergistically to cause significant differences in the heat resistance of individual spores in populations, and a challenge for the future will be to identify those parameters responsible for the heterogeneity in spore wet-heat resistance and how this heterogeneity arises during sporulation.
The third conclusion is that during wet-heat treatment, some spore proteins are denatured or damaged in parallel with and even slightly prior to rapid CaDPA release during wet heat treatment, as some spore protein changes from an α-helical structure to one that is irregular and likely denatured. The state of phenylalanine in spore proteins also changes in parallel with and to some degree even before rapid CaDPA release, and this change may also be related to protein damage or denaturation. Given that protein damage can inactivate protein function, this suggests that protein damage is a major reason that wet heat kills spores of Bacillus
species, as was suggested recently (7
), although specific proteins to which damage causes spore death during wet-heat treatment have not been identified.
It was also notable with individual spores exposed to wet heat that there was significant change in overall spore protein structure prior to rapid CaDPA release. This suggests that during wet-heat treatment some spores should retain CaDPA for a short period, even if they are already dead from protein damage, and some B. subtilis
spores in populations treated with wet heat have been shown to do just that (7
). Presumably wet heat causes damage to some crucial protein or proteins such that while CaDPA is retained, the spores are not viable, and even if they can germinate, they cannot progress into outgrowth (7
). However, with further wet-heat treatment, more protein damage accumulates, including damage to proteins in the spore's inner membrane, and ultimately, this damaged membrane ruptures, leading to rapid CaDPA release. Unfortunately, we have no way at present to know exactly when individual spores being observed during wet-heat treatment actually die, although available evidence indicates that B. subtilis
spores that have lost all CaDPA during wet-heat treatment are definitely dead (7
). However, spores that retain CaDPA during wet-heat treatment may be dead, still alive, or only conditionally alive, depending on the conditions used for recovering viable spores (7
The fourth conclusion concerns correlations between the times for changes in spores' ELS intensities and CaDPA release during wet-heat treatment. For B. cereus
and B. megaterium
spores, ELS intensities decreased gradually during wet-heat treatment, reaching minimum values at T1
, when about 80% of the spores' CaDPA had been released, and then rose rapidly until T2
, when CaDPA release was complete. The ELS intensities of B. subtilis
spores during wet-heat treatment exhibited similar features, except that ELS intensities remained constant prior to T1
. Increases in ELS intensity are possibly related to abrupt and/or significant changes in the spore core refractive index due to CaDPA release and its replacement by water. Inhomogeneity of the refractive index inside the spore may also generate more scattering centers that scatter more incident photons (30
). Changes in the refractive index of spore core components also may result in minute transitions in spores' axial position in the optical trap, since changes in the ratio of the particle's refractive index to the refractive index of the medium leads to a reequilibration of scattering force and gradient force in the axial direction (28
). When the scattering force decreases or the gradient force increases, the spore will be trapped more closely to the focus of the laser. However, the reasons that the ELS intensities of B. cereus
and B. megaterium
spores decrease gradually prior to T1
and why this is not seen with B. subtilis
spores are not clear.
The last novel conclusion concerns the carotenoid or carotenoids present in high levels in B. megaterium
spores, supposedly in the spore inner membrane (26
). Carotenoid-specific Raman bands at 1,155 and 1,516 cm−1
decreased rapidly beginning as soon as these spores were exposed to elevated temperatures, with the 1,516-cm−1
peak disappearing completely and well before rapid CaDPA release. However, the intensity of the band at 1,155 cm−1
changed in two stages, an initially large decrease well before Tlag
and then a rapid decrease to zero accompanying rapid CaDPA release. The mechanism for the changes in the intensity of carotenoid-specific Raman bands is not clear but is possibly related to photo-oxidation of carotenoids induced by the trapping laser at elevated temperatures, although experiments carried out at room temperature indicated that this effect was not pronounced (data not shown). In addition, the residual intensity of the 1,155-cm−1
band prior to Tlag
may not be due to a carotenoid but to CaDPA (19
). However, if the carotenoid is indeed in the spore's inner membrane (26
), then changes in spores' carotenoid Raman spectra during wet-heat treatment may be a reflection of striking changes in the environment and structure of the spore inner membrane during this treatment.