Kinetics of germination of wet-heat-treated individual spores of Bacillus species, monitored by Raman spectroscopy and differential interference contrast microscopy

Abstract

Raman spectroscopy and differential interference contrast (DIC) microscopy were used to monitor the kinetics of nutrient and nonnutrient germination of multiple individual untreated and wet-heat-treated spores of Bacillus cereus and Bacillus megaterium, as well as of several isogenic Bacillus subtilis strains. Major conclusions from this work were as follows. (i) More than 90% of these spores were nonculturable but retained their 1:1 chelate of Ca²+ and dipicolinic acid (CaDPA) when incubated in water at 80 to 95°C for 5 to 30 min. (ii) Wet-heat treatment significantly increased the time, T(lag), at which spores began release of the great majority of their CaDPA during the germination of B. subtilis spores with different nutrient germinants and also increased the variability of T(lag) values. (iii) The time period, ΔT(release), between T(lag) and the time, T(release), at which a spore germinating with nutrients completed the release of the great majority of its CaDPA, was also increased in wet-heat-treated spores. (iv) Wet-heat-treated spores germinating with nutrients had higher values of I(release), the intensity of a spore's DIC image at T(release), than did untreated spores and had much longer time periods, ΔT(lys), for the reduction in I(release) intensities to the basal value due to hydrolysis of the spore's peptidoglycan cortex, probably due at least in part to damage to the cortex-lytic enzyme CwlJ. (v) Increases in T(lag) and ΔT(release) were also observed when wet-heat-treated B. subtilis spores were germinated with the nonnutrient dodecylamine, while the change in I(release) was less significant. (vi) The effects of wet-heat treatment on nutrient germination of B. cereus and B. megaterium spores were generally similar to those on B. subtilis spores. These results indicate that (i) some proteins important in spore germination are damaged by wet-heat treatment, (ii) the cortex-lytic enzyme CwlJ is one germination protein damaged by wet heat, and (iii) the CaDPA release process itself seems likely to be the target of wet-heat damage which has the greatest effect on spore germination.

Fig. 1.

(a to c) Viability, CaDPA retention, and Raman spectra of wild-type B. subtilis spores with and without wet-heat treatment. (a and b) B. subtilis PS832 (wild-type) spores were incubated at either 90°C (○) or 95°C (▴) for various times, and spore viability (a) and the percentage of individual spores that retained CaDPA (b) were determined as described in Materials and Methods, by the examination of ∼100 individual spores. (c) Raman spectra of individual B. subtilis PS832 spores without heat treatment (curve a), wet heat treated at 95°C for 30 min with retention of CaDPA (curve b), and wet heat treated at 95°C for 30 min with loss of CaDPA (curve c). The short dotted lines show the positions of the amide I Raman bands from protein in α-helical (1,650 cm⁻¹) or irregular (1,665 cm⁻¹) structures.

Fig. 2.

l-Alanine germination of untreated and wet-heat-treated wild-type B. subtilis spores. B. subtilis PS832 (wild-type) spores were left untreated (▪), incubated at 90°C for 15 (•) or 30 min (▴), or incubated at 95°C for 15 min (▾). Spore killing was determined as described in Materials and Methods and was as follows: 90°C for 15 min, 90%; 90°C for 30 min, 93%; 95°C for 15 min, 97%. After cooling, spores were heat activated at 70°C for 30 min and germinated with l-alanine and percentages of spore germination were determined by the intensity of DIC images of 300 to 400 individual spores that retained CaDPA as described in Materials and Methods.

Fig. 3.

(a to d) l-Alanine germination of 10 individual untreated or wet-heat-treated wild-type B. subtilis spores. B. subtilis PS832 (wild-type) spores were either left unheated (a) or incubated at 90°C for 15 (b) or 30 (c) min or at 95°C for 15 min (d), spores were germinated with l-alanine, and the germination of the individual spores was followed by DIC microscopy as described in Materials and Methods. a.u., arbitrary units.

Fig. 4.

(a to c) Typical kinetics of l-alanine germination of untreated and wet-heat-treated individual wild-type B. subtilis spores. (a) Normalized intensity of DIC images of individual B. subtilis PS832 (wild-type) spores that were germinated with l-alanine, with germination followed by DIC microscopy as described in Materials and Methods. Symbols: □, untreated spore; ▵ and ○, individual spores wet heat treated at 90°C for 30 min that retained CaDPA. (b and c) Normalized intensities of DIC images and height of the Raman band at 1,017 cm⁻¹ of an untreated spore (b) or a wet-heat-treated (15 min; 90°C) spore that retained CaDPA (c), both germinating with l-alanine and with spore germination monitored as described in Materials and Methods. Symbols: ○, germination kinetics monitored by Raman spectroscopy; □, germination kinetics monitored by DIC microscopy; ⋄, normalized DIC image intensities with the image intensity at T_release set to zero. a.u., arbitrary units.

Fig. 5.

(a to d) Germination of untreated and wet-heat-treated spores of various B. subtilis strains. PS832 (wild-type) spores were germinated with AGFK (a), FB111 (cwlJ) spores (b) and FB112 (sleB) spores (c) were germinated with l-alanine, PS3415 (↑GerB*) and FB10 (gerB*) spores were germinated with l-asparagine (d), and the germination of ∼300 individual spores was followed by DIC microscopy as described in Materials and Methods. Symbols in panels a and b: ▪, unheated spores; •, spores incubated at 90°C for 15 min; ▴, spores incubated at 90°C for 30 min. Symbols in panel c: ▪, unheated spores; •, spores incubated at 85°C for 15 min; ▴, spores incubated at 85°C for 30 min. Symbols in panel d: ▪, unheated PS3415 spores; •, PS3415 spores incubated at 90°C for 15 min; ▴, PS3415 spores incubated at 90°C for 30 min; ▾, unheated FB10 spores; ◂, FB10 spores incubated at 90°C for 30 min.

Fig. 6.

(a to d) Dodecylamine germination of unheated and wet-heat-treated wild-type B. subtilis spores. Unheated or wet-heat-treated B. subtilis PS832 (wild-type) spores were germinated with dodecylamine, and spore germination was followed by DIC microscopy as described in Materials and Methods. In panel a, the germination of ∼300 spores was followed whether or not they retained CaDPA, and in panels b to d, the germination of 10 randomly chosen spores that were left untreated (b), treated at 90°C for 15 min (c), or treated at 90°C for 30 min with retention of CaDPA (d) were followed. Symbols in panel a: ▪, unheated spores; •, spores incubated at 90°C for 15 min; ▴, spores incubated at 90°C for 30 min. a.u., arbitrary units.

Fig. 7.

(a and b) Nutrient germination of wet-heat-treated B. megaterium and B. cereus spores. Heat-activated B. megaterium spores were germinated with glucose (a), and heat-activated B. cereus spores were germinated with l-alanine (b), and the germination of ∼300 unheated or wet-heat-treated spores that retained CaDPA was followed by DIC microscopy as described in Materials and Methods. Symbols in panel a: ▪, unheated spores; •, spores incubated at 80°C for 15 min. Symbols in panel b: ▪, unheated spores; •, spores incubated at 90°C for 15 min. The viability of the B. megaterium and B. cereus spores was reduced >90% by the wet-heat treatment used, but ≥90% of these wet-heat-treated spores retained CaDPA.