Function and Location of a "Germination Enzyme" in Spores of Bacillus Cereus

Abstract

SUMMARYAn enzyme extracted from Bacillus cereus spores caused to germinate spores of this organism which had been sensitized by reagents which rupture disulphide bonds. Inactivation of the enzyme by thiol-blocking agents and by oxidation, and reactivation by reduction suggested that the enzyme's ability to germinate spores depended on thiol groups. No evidence was obtained to support the hypothesis that the enzyme was present in dormant spores in the oxidized inactive form and became reduced and active during germination. When spores were disrupted at pH 3 the enzyme was found to be bound to debris; probably on or within the central core of the spore. At pH 5.8 or below the enzyme remained bound to the debris, but a t pH 7.0 the enzyme was irreversibly released when the ionic strength of the medium was high. I n solutions of sodium phosphate less than 0.05 M, the enzyme remained mostly bound even at pH 7-0. It was largely released when the concentration of sodium phosphate was increased to 0.2 M. Extracts of germinated spores contained more unbound and less bound enzyme than extracts of ungerminated spores, suggesting that release of enzyme from a bound form occurred during germination of the spores.

Extraction of Spore-lytic Enzyme from Clostridium perfringens Spores

“…Since then, the spore-lytic enzymes of Bacillus cereus have been isolated and extensively studied by Strange & Dark (1957), Gould et al (1966), Warth (1972) and Brown et al (1975Brown et al ( , 1977Brown et al ( , 1978. In most cases, this lytic system was found to hydrolyse the peptidoglycan of the spore cortex, resulting in germination-like changes in the spore.…”

Mechanism of the Inhibition of Germination of Bacterial Spores by -radiation in the Presence of Iodoacetamide and Iodate

“…The enzyme was assayed by its ability to cause a decrease in the extinction of suspensions of spores which had been sensitized by incubation in thioglycollic (mercaptoacetic) acid (25 %, v/v) plus urea (6 M) at 70" for 30 min. (Gould et al 1966). The enzyme reaction was carried out in sodium phosphate buffer (100 mM, pH 8.0) at 37"; the initial extinction read at 580 nm.…”

“…to 20 mg. dry wt/ml.) which had been disrupted by shaking with glass beads as described by Gould, Hitchins & King (1966). The enzyme was assayed by its ability to cause a decrease in the extinction of suspensions of spores which had been sensitized by incubation in thioglycollic (mercaptoacetic) acid (25 %, v/v) plus urea (6 M) at 70" for 30 min.…”

Sequence of Events during Rapid Germination of Spores of Bacillus cereus

“…Although the mechanism initiating these changes is not known, Strange & Dark (1957) and Gould, Hitchins & King (1966) suggested that they result more or less directly from depolymerization of peptidoglycan in the spore cortex, catalysed by a lytic enzyme which can be extracted from spores of some species and which can be shown to cause germination-like changes in chemically sensitized spores (Gould & King, 1969). If this is so then the excretion of peptidoglycan fragments would be expected to be one of the earliest determinable events during germination.…”