Disappearance of the Cooperative Effect of Glucose on L‐Alanine Binding during Heat Activation of Germination of Bacillus subtilis Spores

Abstract

Sublethal preheating of bacterial spores has been used to stimulate their germination. This treatment is known as "heat activation" or "heat shock." In our germination experiments of spores of Bacillus subtilis PCI219 (18-23), a sufficient and reproducible germination rate could be obtained with L-alanine and its analogs without preheating, and the heated spores required higher concentrations of Lalanine to germinate than the unheated spores in the presence of glucose. In the present study we examined the effect of heat treatment on the germination response, especially on the sites for the germinant and related compounds in the alanine receptor field of the spores (22). We found that heat treatment at 65 C for 30 min did not alter the binding sites for L-alanine, D-alanine, and the hydrophobic compounds, but it did affect the binding site for glucose. The results are described in some detail as follows.Actually, heat treatment of the spores of B. subtilis at various temperatures altered the germination rate (Fig. 1). At a sufficient concentration (1 mm) of L-alanine (21) the maximum germination rate was obtained by preheating at around 65 C for 30 min with or without glucose. The maximum germination rate of heated spores on L-alanine (Fig. 1A) or L-alanine plus glucose (Fig. 1B) was about 1.4 times more than that of the respective unheated ones, showing the heat activation. Heating at 70 C and higher temperatures caused a rapid decrease in germination rate. However, at lower concentrations of L-alanine only a little stimulation of the rate was observed between 50 and 60 C with L-alanine alone (Fig. 1A), and the germination rate decreased gradually as the preheating temperature in creased with L-alanine plus glucose (Fig. 1B). Germinability on glucose alone was not significant (Fig. 1B). These different responses of the germination rate to temperature indicated the complex effects of preheating.Preheating spores at 65 C for 30 min, a condition at which the maximum germination rate was obtained, was used in subsequent experiments. In the concentration-germination response curves the level of the maximum germination rate of heated spores in the absence of glucose was higher than that of unheated ones, but the apparent affinities of both spores for L-alanine, which were determined as the concentration of L-alanine showing 50 % of the maximum rate of each curve, were 101 1

Bacterial spores and chemical sporicidal agents

“…3a to d, respectively; Table 11). In our experiments, despite earlier claims to the contrary (112,(238)(239)(240)(241)(242), D-glucose has no significant effect on L-alanine uptake. These other workers, however, calculated the binding affinity of glucose solely on the loss of heat resistance and turbidity of germinated spores rather than by direct methods.…”

Bacterial spores and chemical sporicidal agents.

“…3a to d, respectively; Table 11). In our experiments, despite earlier claims to the contrary (112,(238)(239)(240)(241)(242), D-glucose has no significant effect on L-alanine uptake. These other workers, however, calculated the binding affinity of glucose solely on the loss of heat resistance and turbidity of germinated spores rather than by direct methods.…”