Effect of inhibitors of trypsin-like proteolytic enzymes Bacillus cereus T spore germination

Abstract

The germination of Bacillus cereus T spore suspensions is partially prevented by several inhibitors of trypsin-like enzymes. Leupeptin, antipain, and tosyl-lysine-chloromethyl ketone are effective inhibitors, whereas chymostatin, elastatinal, and pepstatin are inactive. A synthetic substrate of trypsin, tosyl-arginine-methyl ester, also inhibits germination. Its inhibitory effect decreases as a function of incubation time in the presence of spores and is abolished by previous hydrolysis with trypsin. Germinating, but not dormant, spore suspensions hydrolyze tosyl-arginine-methyl ester; its hydrolysis is insensitive to chloramphenicol, sulfhydryl reagents, and EDTA. A crude extract of germinated B. cereus spores contains a trypsin-like enzyme whose activity, as measured by hydrolysis of benzoyl-arginine p-nitroanilide, is sensitive to germination-inhibitory compounds such as leupeptin, tosyl-arginine-methyl ester, and tosyl-lysine-chloromethyl ketone. Spore suspensions exposed to the above inhibitors under germination conditions lose only part of their heat resistance and some 10 to 30% of their dipicolinic acid content. Part of the germinating spore population becomes "phase grey" under phase optics. Based on a study of the inhibition of germination by protease inhibitors and the activity of a protease in germination spores and spore extracts, it is suggested that the activity of a trypsin-like enzyme may be involved in the mechanism of the breaking of dormancy in spores of B. cereus T.

Analysis of the action of compounds that inhibit the germination of spores of Bacillus species

“…A number of enzyme inhibitors, including protease inhibitors and sulphydryl reagents significantly inhibit nutrient germination of spores of a number of Bacillus species (Boschwitz et al 1983(Boschwitz et al , 1985Foster and Johnstone 1986;Venkatasubramanian and Johnstone 1989 6 ; Boschwitz et al 1991;Wuytack et al 1998Wuytack et al , 2000. Of the protease inhibitors tested, PMSF, had a significant effect on germination of B. megaterium and B. subtilis spores with all nutrients (Table 3), and the effect of this inhibitor was reversible (data not shown), in agreement with previous work (Venkatasubramanian and Johnstone 1989).…”

“…The reversibility of the NEM inhibition of either nutrient or Ca 2+ -DPA germination is also inconsistent with this compound inhibiting germination by reacting with an essential protein sulphydryl group. The inhibition of spore germination by TAME and PMSF was taken as evidence of a role for trypsin-like serine proteases in spore germination (Boschwitz et al 1983(Boschwitz et al , 1985Foster and Johnstone 1986;Boschwitz et al 1991). However, no such protease has ever been identified as playing a role in the initial steps of germination of spores of Bacillus species (e.g.…”

“…Of the protease inhibitors tested, PMSF, had a significant effect on germination of B. megaterium and B. subtilis spores with all nutrients (Table 3), and the effect of this inhibitor was reversible (data not shown), in agreement with previous work (Venkatasubramanian and Johnstone 1989). The protease inhibitor tosyl-L Larginine methyl ester (TAME), a compound that inhibits germination of spores of Bacillus species (Boschwitz et al 1983(Boschwitz et al , 1991Foster and Johnstone 1986;Wuytack et al 1998), also gave significant inhibition of germination of B. subtilis and B. megaterium spores with all nutrients (including KBr with B. megaterium spores). However, the maximal inhibition of germination with nutrients observed with TAME was only 60-70% when germination was measured by the fall in O.D.…”

“…High pressures either activate the nutrient receptors or Ca 2+ -DPA release ( ‡450 MPa), while alkylamines activate Ca 2+ -DPA release and perhaps release of other ions by effects at the spore's inner membrane. Exogenous Ca 2+ -DPA activates CwlJ that then catalyzes cortex hydrolysis, and this process may be amplified by consequent release of endogenous Ca 2+ -DPA Laible 1970 ;Vinter 1970;Cassone and Simonetti 1971;Gould and Dring 1972;Dring and Gould 1975;Enfors and Molin 1975;Watanabe and Takesue 1976;Yasuda-Yasaki et al 1978a;Smoot and Pierson 1981;Yasuda et al 1982;Boschwitz et al 1983Boschwitz et al , 1985Yasuda and Tochikubo 1985;Foster and Johnstone 1986;Mitchell et al 1986;Shyu and Foegeding 1991b;Ababouch et al 1992). With few exceptions, these studies have examined the effects only on nutrient germination and often only on a single nutrient.…”

Germination requirements of an aerobic spore-forming marine bacterium

“…Germination experiments were performed at 30°C in 3 ml of germination medium which, unless otherwise indicated, contained 170 mM NaC1, 1.9 mM adenosine, and 0.07 M NH 4 Hepes buffer (N-2-hydroxyethylpiperazine-N l-2-ethanesulfonic acid), pH 7.8. Germination was monitored as described elsewhere [11]. The rate of germination was calculated as the % decrease in absorbance units per rain.…”