Studies on the Bacterial Spore Coat: IX. The Role of Surface Charge in Germination of Bacillus megaterium Spores

Abstract

The surface charge of Bacillus megaterium QM B 1551 spores was estimated to be negative, -0.2 and -0.4 t-teq(mg by colloidal titration using glycol chitosan (GCh) and methylglycol chitosan (MGCh), respectively, as positive colloids. MGCh, which reacts with all of the negatively charged groups including carboxylate, inhibited the second stage of the germination to result in semirefractile spores, but GCh, which reacts only with strong acidic groups such as phosphate, did not. The spores produced in a medium with limited phosphate had coats with low phosphate content and carried less negative charge, and they were induced to germinate with 0.4 msr KN03, which is one-tenth of the minimum concentration required for the germination of the control spores. A similar increase in germinability was observed in spores incubated with calcium acetate.. These results suggest that the role of the surface charge in germination is as follows. Strong acidic groups (such as phosphate) in the coat may block the action of ionic germinants and act as a barrier against the initiation of ionic germination. Positively charged compounds (such as calcium) may compensate for this blocking effect. Weak acidic groups (such as carboxylate) may be involved in the later stage of germination.The spore coat, composed of proteins with phosphate, is the outermost layer of Bacillus megaterium spores (5), and is the first contact of the spores with information in their environment, such as germinants. Therefore, the coat has been a subject for the study of the properties of spores, especially the mechanism of germination. On the spore surface are exposed many reactive groups of the coat, including such components as sulfhydryl, hydroxyl, carboxylic, phosphoric, and amino groups. We reported that sulfhydryl and hydroxyl groups might playa role in the recognition of a germinant in the initiation of germination (14). We have now studied the role of the negatively charged groups of the spore surface in germination by using positive colloids for the colloid titration.

Bacterial spores and chemical sporicidal agents

“…Methods of removing one or both spore coats have been described in detail by Nishihara et al (141)(142)(143). Coats may be extracted by using 2-mercaptoethanol, sodium lauryl (dodecyl) sulfate, dithiothreitol (DTT), and urea.…”

Bacterial spores and chemical sporicidal agents.

“…Probably the most detailed approach to studying resistance of spores has involved the use of spore coatless forms (46, 62, 63, 69, 82, 88-91, 93, 124, 126, 132, 189, 194, 212, 224-228, 231, 237). Methods of removing one or both spore coats have been described in detail by Nishihara et al (141)(142)(143). Coats may be extracted by using 2-mercaptoethanol, sodium lauryl (dodecyl) sulfate, dithiothreitol (DTT), and urea.…”

The Role of Surface Charge in Ionic Germination of Clostridium perfringens Spores

“…The slightly modified method of Watanabe & Takesue (1976) was used. Instead of protamine sulphate, GCh and MGCh (Wako Pure Chemical Corp.) were used as positive colloids, since Nishihara et al (1981) reported that titration values were estimated to be higher with protamine sulphate than with MGCh. An excess amount of the colloid (0.005 M, 5 ml) was added to an appropriate concentration of spores (20-30 mg per 2 ml) and the mixture was adjusted to the required pH with 0.05 M-HCI or 0.05 M-NaOH.…”

“…After incubation at room temperature for 20 min, the uncombined colloid was back-titrated with 0-0025 M-potassium polyvinyl sulphate (Wako Pure Chemical Corp.) using toluidine blue as the indicator. A sharp end-point with MGCh was obtained by removing the spores by centrifuging the titration mixture and adjusting the pH of the supernatant to 2.5 with 0.05 M-HCI before titration, according to the method of Nishihara et al (1981).…”

“…Little or no charge was observed between these pH values when the spores were titrated with GCh. This implies that weak acidic groups such as carboxyl groups are the only charged species on the spore surface because MGCh reacts with all negatively charged groups including carboxyl groups, while GCh dissociates at pH < 6.5 and reacts only with strong acidic groups dissociable even at low pH (Nishihara et al, 1981). Table 1 shows variations in colloidal charge at three different pHs for various ionic forms of the spores, along with the ability of these spores to germinate in the KCl medium.…”