The possible involvement of trypsin-like enzymes in germination of spores of Bacillus cereus T and Bacillus subtilis 168

Abstract

Germination of spores of Bacillus cereus T and Bacillus subtilis 168 was inhibited by the trypsin inhibitors leupeptin and tosyllysine chloromethyl ketone (TLCK) and by the substrates tosylarginine methyl ester (TAME), benzoyl-Larginine-p-nitroanilide (L-BAPNA) and D-BAPNA. Potencies of these inhibitory compounds were estimated by finding the concentration which inhibited 50 % germination (IDs0), as measured by events occurring early (loss of heat resistance), at an intermediate stage [dipicolinic acid (DPA) release], and late in germination (decrease in optical density). In B. cereus T, all the compounds inhibited early and late events with the same IDs0. In B. subtilis, TAME inhibited early and late events at the same IDs0, but all other inhibitors had a lower IDs0 for late events than for early events. This suggests that a trypsin-like enzyme activity is involved at two sequential stages in the germination of B. subtilis spores, one occurring at or before the loss of heat resistance and one at or before the decrease in optical density. Different trypsin-like activities were detected in broken dormant spores and germinated spores of B. cereus T and in germinated spores of B. subtilis by means of three chromogenic substrates: benzoyl-L-phenylalanyl-L-valyl-L-arginine-~-nitroa~ide (L-PheVA), L-BAPNA and D-BAPNA. Separation of extracts of germinated spores on non-denaturing polyacrylamide gels showed that in both species the substrates were hydrolysed by three distinct enzymes with different electrophoretic mobilities. The three enzymes had different Ki values for the above inhibitors. The possibility that L-BAPNase from B. cereus T might be involved in the initial germination steps was suggested by the similarity of IDs0 values for germination and Ki values for inhibition of the enzyme in uitro by TAME and TLCK, and by the fact that both germination and the L-BAPNase were reversibly inhibited by TLCK.

HtrC Is Involved in Proteolysis of YpeB during Germination of Bacillus anthracis and Bacillus subtilis Spores

“…Chemicals that function as protease inhibitors have been shown to block spore germination at different stages, suggesting that protease activity is critical during germination (42)(43)(44)(45). While it has not been shown that this activity is actually due to inhibition of a protease rather than another protein function or what specific germination processes are affected, these results raise the possibility that initiation of cortex hydrolysis requires a protease activity.…”

Macromolecular synthesis during recovery of the marine Vibrio sp. S14 from starvation

“…DNAbinding proteins) and in the mechanisms of autolysis resistance. The initial events in the germination of Bacillus cereus spores have been suggested to involve such specific protease(s) (Boschwitz et al, 1985), which may be directly involved in autolysin activation (Mencher & Blankenship, 1971). However, these proteases are environmentally activated rather than being induced during the initial phase of germination (e.g.…”

Comparative Study of Pressure- and Nutrient-Induced Germination of Bacillus subtilis Spores

“…Aqueous spore suspensions, diluted fivefold with 50 mM phosphate buffer (pH 7) containing an inhibitor of nutrient-induced germination, were pressure treated (100 or 600 MPa at 40°C for 20 min). Inhibitors used were N␣-P-tosyl-L-arginine methyl ester (50 mM) (TAME) (3,18), HgCl 2 (3 mM) (8,18), and phenol (0.2% [wt/vol]) (16).…”