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.

A stereospecific enzyme activity capable of cleaving the amide bond of the synthetic substrate N-benzoyl-Darginine-p-nitroanilide (D-BAPA) has been found in all aerobic and anaerobic members of the family Bacillaceae tested by us. Cells of nonsporeforming gram-positive or gram-negative bacteria contain a hydrolase activity stereospecific to N-benzoyl-L-arginine-p-nitroanilide. The D-BAPA-hydrolyzing enzymes (DBAPAases) of mid-logarithmic-phase cells of Bacillus subtilis 168 and B. cereus T were compared. These enzymes had the same molecular weight of approximately 66,000 in gel filtration and the same electrophoretic mobility after electrophoresis on polyacrylamide gels. The D-BAPAases of B. subtilis 168 and B. cereus T differed in the effect of inhibitors on enzymatic activity. While both hydrolases were inhibited by tosyl-L-lysine chloromethyl ketone and tosyl-L-arginine-methyl ester as well as leupeptin, only the D-BAPAase of B. Most proteolytic enzymes are capable of discriminating between the D and L forms of molecules. Naturally occurring amino acids found in proteins are generally L stereochemical isomers. Synthetic protease substrates were used as L forms also. One of these substrates, benzoyl-L-arginine-p-nitroanilide (L-BAPA), was found to be hydrolyzed by trypsin (10), trypsinlike enzymes (7), various peptidases of plants (6,7,9,15,18,21,26,28), and the peptidase of Corynebacterium matruchotii (11). The D isomer, D-BAPA, is known as a competitive inhibitor for trypsin (10) and plant peptidases (5, 7). It has been shown that germinated spores of Bacillus cereus T hydrolyzed the racemate DL-BAPA more effectively than the L isomer of BAPA alone (3). This result suggested hydrolysis of the D isomer of BAPA. The present investigation revealed that the D-BAPA-hydrolyzing enzyme is more active in vegetative cells than in germinating spores of bacilli. As no peptide-hydrolyzing enzyme stereospecific to the D isomer of BAPA has been described in the literature, we decided to study the prevalence and some properties of this enzyme in a variety of bacteria. In the following paper we refer to the D-BAPA-hydrolyzing enzymes as D-BAPAase. MATERIALS AND METHODSBacterial strains and culture conditions. Bacillus subtilis 168 trpC2 and B. subtilis 43.4 (spoOA43 leu-8) were used throughout this study. Additional strains tested were B. cereus T; B. thuringiensis var. israelensis; 6A10, an aerobic sporeforming marine bacterium (31) The majority of the work was carried out with B. subtilis 168 and B. cereus T. B. subtilis 168 cells were grown at 30°C in LB medium (11) and B. cereus T cells were grown at 37°C in brain heart infusion (BHI) medium (Difco Laboratories), both with shaking. Starter inocula were prepared from individual colonies on nutrient agar followed by cultivation of B. subtilis 168 in nutrient broth (Difco Laboratories) medium at 30°C and cultivation of B. cereus T in BHI medium at 37°C. The log-phase starters were used as 1% (vol/vol) of the final cultivation medium. To obtain semisynchronized cultures, cu...

Three out of the four described halophilic obligately anaerobic bacteria of the family Haloanaerobiaceae hydrolyze d‐BAPA (N′‐benzoyl‐d‐arginine‐p‐nitroanilide), while showing no or little l‐BAPA hydrolyzing activity. This property was shown earlier to be characteristic only of non‐halophilic Gram‐positive endospore‐forming bacteria of the genera Bacillus and Clostridium. These results suggest that Haloanaerobium praevalens, which has never been shown to produce endospores, but was shown to be related to the endosphere‐forming representatives of the Haloanaerobiaceae on the basis of 16S rRNA nucleotide sequence data, shares other properties characteristics of the endospore‐forming bacteria. Neither significant d‐BAPA nor l‐BAPA hydrolyzing activity was found in Sporohalobacter lortetii.