Analysis of the loss in heat and acid resistance during germination of spores of Bacillus species

Abstract

A major event in the nutrient germination of spores of Bacillus species is release of the spores' large depot of dipicolinic acid (DPA). This event is preceded by both commitment, in which spores continue through germination even if germinants are removed, and loss of spore heat resistance. The latter event is puzzling, since spore heat resistance is due largely to core water content, which does not change until DPA is released during germination. We now find that for spores of two Bacillus species, the early loss in heat resistance during germination is most likely due to release of committed spores' DPA at temperatures not lethal for dormant spores. Loss in spore acid resistance during germination also paralleled commitment and was also associated with the release of DPA from committed spores at acid concentrations not lethal for dormant spores. These observations plus previous findings that DPA release during germination is preceded by a significant release of spore core cations suggest that there is a significant change in spore inner membrane permeability at commitment. Presumably, this altered membrane cannot retain DPA during heat or acid treatments innocuous for dormant spores, resulting in DPA-less spores that are rapidly killed.

FIG 1

Kinetics of germination, commitment, and loss of heat resistance during B. subtilis spore germination. (A) Spores of B. subtilis strains PS533 (wild type) (○, ●) and FB111 (cwlJ) (△, ▲) were germinated at an OD₆₀₀ of 0.5, and commitment (●, ▲) and germination (○, △) were determined as described in Materials and Methods. (B, C) PS533 (wild type) (B) and FB111 (cwlJ) (C) spores were germinated, and commitment (●), germination (○), and loss of heat resistance (△) were measured as described in Materials and Methods. The values shown are the averages of results from two (A) or three (B, C) replicate experiments, and between replicates there was ≤10% variation in values for commitment and germination and ≤20% variation in values for heat-resistant spores.

FIG 2

Kinetics of DPA release and loss of acid resistance during B. subtilis spore germination. Spores of B. subtilis strains PS533 (wild type) (○, ●) and FB111 (cwlJ) (△, ▲) were germinated at an OD₆₀₀ of 0.5, and germination (○, △) and loss of acid resistance (●, ▲) were determined as described in Materials and Methods. The values shown are the averages of results from three replicate experiments, and between replicates there was ≤10% variation in values for germination and ≤20% variation in values for acid-resistant spores.

FIG 3

Kinetics of DPA release during germination of B. subtilis spores with and without a heat (A) or acid (B) treatment. Spores of B. subtilis strains PS533 (wild type) (○, ●) and FB111 (cwlJ) (△, ▲) were germinated at an OD₆₀₀ of 5, and DPA release was measured at various times either without (○, △) or with (●, ▲) a heat treatment at 75°C for 30 min (A) or an acid treatment for 30 min at 23°C (B), as described in Materials and Methods. The values shown are averages of results from two independent replicate experiments, and values between replicates differed by ≤15%.

FIG 4

Kinetics of B. megaterium spore germination and commitment. Spores of B. megaterium strains QM B1551 (wild type) (○, ●) and PS4164 (cwlJ) (△, ▲) were germinated at an OD₆₀₀ of 0.5, and germination (○, △) and commitment (●, ▲) were determined as described in Materials and Methods. The values shown are averages of results from two independent experiments, and replicate values differed by ≤15%.

FIG 5

Kinetics of DPA release and loss of acid resistance during B. megaterium spore germination. Spores of B. megaterium strains QM B1551 (wild type) (○, ●) and PS4164 (cwlJ) (△, ▲) were germinated at an OD₆₀₀ of 0.5, and germination (○, △) and loss of acid resistance (●, ▲) were determined as described in Materials and Methods. The values shown are averages of results from three replicate experiments, and between replicates there was <10% variation in values for DPA release and <20% variation in values for acid-resistant spores. Note that the wild-type spore preparation used in this experiment was different from that used for the experiment whose results are presented in Fig. 4.

FIG 6

Kinetics of DPA release during germination of B. megaterium spores with or without a heat treatment. Spores of B. megaterium strains QM B1551 (wild type) (○, ●) and PS4164 (cwlJ) (△, ▲) were germinated at an OD₆₀₀ of 5, and DPA release was measured at various times without (○, △) or with (●, ▲) a heat treatment, as described in Materials and Methods. The values shown are averages of results from three independent experiments; replicate values for germination differed by ≤15%, and replicate values for the loss of spore heat resistance varied by ≤20%. Note that the wild-type spores used are from the same preparation used for the experiment whose results are presented in Fig. 4.