How moist heat kills spores of Bacillus subtilis

Abstract

Populations of Bacillus subtilis spores in which 90 to 99.9% of the spores had been killed by moist heat gave only two fractions on equilibrium density gradient centrifugation: a fraction comprised of less dense spores that had lost their dipicolinic acid (DPA), undergone significant protein denaturation, and were all dead and a fraction with the same higher density as that of unheated spores. The latter fraction from heat-killed spore populations retained all of its DPA, but >/=98% of the spores could be dead. The dead spores that retained DPA germinated relatively normally with nutrient and nonnutrient germinants, but the outgrowth of these germinated spores was significantly compromised, perhaps because they had suffered damage to some proteins such that metabolic activity during outgrowth was greatly decreased. These results indicate that DPA release takes place well after spore killing by moist heat and that DPA release during moist-heat treatment is an all-or-nothing phenomenon; these findings also suggest that damage to one or more key spore proteins causes spore killing by moist heat.

FIG. 1.

Equilibrium density gradient centrifugation of spore populations with various percentages of spores killed by moist heat. Spores of strain PS533 (wild type) were either untreated (tube 1) or treated for increasing times at ∼89°C (40, 80, and 120 min for tubes 2 to 4, respectively), and viability was determined; spores were run on Nycodenz step gradients, and the spores in the upper (U) or lower (L) bands were isolated. The viability of the spores was determined as the number of CFU/0.5 ml of spores at an OD₆₀₀ of 1, as described in Materials Methods. The values for the viability of untreated spores before centrifugation or from the lower band in tube 1 were each set at 100%, and the absolute viabilities for these two samples were almost identical. Values for the viability of spores from upper and lower bands in tubes 2 to 4 are expressed relative to the value for the lower band from untreated spores in tube 1. Based on the OD₆₀₀ of spores recovered from the upper and lower bands, the upper band comprised 4%, 20%, and 60% of total spores in tubes 2, 3, and 4, respectively. Since the upper band had lost DPA and this decreases the OD₆₀₀ of spore suspensions ∼25% (28), the ratios of the amounts of spores in the upper to the lower bands are 0.05, 0.3, and 3 in tubes 2 to 4, respectively. This experiment was repeated three times with essentially identical findings.

FIG. 2.

Mean Raman spectra of individual untreated spores and fractionated moist-heat-treated spores and of individual spores from moist-heat-killed spore populations that retained DPA. (A) The Raman spectra of 20 strain PS533 (wild type) spores of various types were collected, and the mean Raman spectra were determined as described in Materials and Methods. The individual spectra are from untreated spores (a) or spores from a population in which 97% were killed by moist heat at 87 to 90°C, as follows: spores that retained DPA isolated from the lower band on an equilibrium density gradient (b), spores that had lost DPA isolated from the lower band on an equilibrium density gradient (c), and spores from the upper band on an equilibrium density gradient (d). (B) Ninety-seven percent of strain PS533 spores (wild type) were killed by moist heat at 87 to 90°C; the spores were separated by equilibrium density gradient centrifugation, and Raman spectra of nine individual spores (curves c to k) that retained DPA from the lower, denser band as well as the mean Raman spectrum (curve b) for these nine spores were determined as described in Materials and Methods. The different individual spectra are offset vertically for clarity. The mean Raman spectrum of 10 individual untreated spores (curve a) is also shown for comparative purposes.

FIG. 3.

GFP fluorescence of untreated spores and fractions from moist-heat-treated spore populations. Spores of strain PS3518 that accumulate significant amounts of GFP in the spore core were treated with moist heat at 87 to 90°C so that 99% of the spores were killed; the treated spores were run on an equilibrium density gradient, and the spores in the lower and upper bands were isolated and examined by fluorescence and DIC microscopy as described in Materials and Methods. The DIC images (a, c, and e) and fluorescence images (b, d, and f) in the various frames are all at the same magnification and are as follows:: untreated spores (a and b), spores from the lower band (c and d), and spores from the upper band (e and f). Scale bar, 20 μm. Images from unfixed spores looked identical to those shown in this figure (data not shown).

FIG. 4.

Rates of moist-heat killing of spores of various strains determined by survival on plates with or without antibiotics. Spores of various strains in water were heated at 87 to 90°C, and at various times aliquots were taken and cooled, and spore survival on plates with or without antibiotics was determined as described in Materials and Methods. (A) Survival of spores of strain PS4006 (Tc^r) measured on plates without (○) or with (•) tetracycline. (B) Survival of spores of strains PS2586 (Em^r; circles) or PS3379 (Em^r; triangles) measured on plates without (open symbols) or with (filled symbols) the appropriate antibiotic. Values shown are averages from duplicate determinations on at least two different serial dilutions and were ±25%.

FIG. 5.

l-Alanine and dodecylamine germination of spores from the lower band on equilibrium density gradients from heat-treated spores. Ninety-seven percent of strain PS533 spores (wild type) were killed by moist heat at 87 to 90°C, and these spores were fractionated by equilibrium density gradient centrifugation. The spores in the lower band and untreated spores were germinated with l-alanine or dodecylamine, and spore germination was quantitated by measuring DPA release as described in Materials and Methods. (A) l-Alanine germination of untreated spores (○) or spores from the lower band (•). (B) Dodecylamine germination with either 0.25 mM dodecylamine at 45°C (circles) or 1 mM dodecylamine at 50°C (triangles) of untreated spores (open symbols) or spores from the lower band (filled symbols).

FIG. 6.

Outgrowth of untreated and heat-treated spores. Ninety-nine percent of strain PS533 spores (wild type) were killed by moist-heat treatment at 87 to 90°C. The spores were separated by equilibrium density gradient centrifugation; the spores in the lower, denser band were isolated, and untreated spores and spores from the lower band were germinated or outgrown in LB medium plus l-alanine as described in Materials and Methods. After incubation in LB medium plus l-alanine for either 75 min (untreated spores) or 5 h (spores from the lower band), aliquots were harvested, fixed with formaldehyde, suspended in 200 μl of PBS, and examined by DIC microscopy as described in Materials and Methods. The images of untreated spores (a to c) and spores from the lower band (d) are all at the same magnification. Scale bar, 20 μm.

FIG. 7.

Light production and ATP accumulation during outgrowth of untreated and heat-killed spores. (A) In populations of PS3379 (luxAB) spores that contained luciferase, 80 or 99% of spores were killed by incubation at 87 to 90°C in water. For the spore population killed at the 99% level, the lower band containing spores that retained DPA but were dead was isolated by equilibrium density gradient centrifugation. For the spore population killed at the 80% level, essentially all spores retained DPA, so these were not fractionated by density gradient centrifugation. The killed spores that retained DPA were germinated in LB medium plus l-alanine, and light production was measured at various times as described in Materials and Methods. ○, untreated spores; •, spores from the lower band in which 97% of spores were dead; ▴, spore population in which 80% of spores were dead. (B) ATP accumulation during outgrowth of untreated and heat-killed spores. In a population of PS533 (wild type) spores, 94 to 99% of spores were killed by incubation at 87 to 90°C in water. For the heat-treated spores, the lower band containing spores that retained DPA but were dead was isolated by equilibrium density gradient centrifugation. The untreated spores and the heat-killed spores that retained DPA were germinated at an OD₆₀₀ of 5 in LB medium plus l-alanine, and samples were extracted, processed, and assayed for ATP as described in Materials and Methods. ○, untreated spores; •, spore population in which ∼94% of spores were dead; ▴, spore population in which ∼99% of spores were dead. Examination of cultures by phase-contrast microscopy indicated that ∼80% of untreated spores had germinated and many were elongating after 60 min of incubation and that ∼80% of heat-treated spores had germinated after 2 h but none were elongating.