Bacillus megaterium spore protease. Synthesis and processing of precursor forms during sporulation and germination.

The protease which initiates the rapid protein degradation during germination of Bacillus megaterium spores was synthesized during sporulation as a Mr = 46,000 polypeptide (P46) which was found in the developing forespore. P46 was processed during sporulation to a Mr = 41,000 species (P41) 2-3 h after P46 synthesis and at the time of or slightly before accumulation of dipicolinic acid. P41 was the predominant form of the protease in the dormant spore, with smaller amounts of unprocessed P46. In the first minutes of spore germination P41 was processed (t1/2 less than 10 min) to a Mr = 40,000 species (P40), which appeared identical to the subunit of the purified active enzyme. The latter processing reaction did not require metabolic energy, but P40 disappeared completely during further germination (t1/2 approximately 40 min) in a reaction which did require metabolic energy. It seems probable that precursors P46 and P41 of the spore protease are involved in the regulation of the activity of this spore enzyme.

The first minutes of germination of spores of various Bacillus and Clostridium species are accompanied by the degradation of up to 25% of the spore's protein (1). The major substrates for this proteolysis are two to three low molecular weight proteins (termed A, B, and C proteins in Bacillus megaterium) which are synthesized during sporulation (1). In B. megaterium the degradation of these proteins during germination is initiated by an amino acid sequence specific endoprotease which acts only on the A, B, and C proteins and analogous proteins from spores of other species (2,3). Mutants have been isolated with low levels of this protease, and all exhibit a decreased rate of proteolysis during germination, but no other phenotypic defect (4). The protease has been purified from spores of B. megaterium, and is a tetramer of Mr = 40,000 subunits; only the tetramer is enzymatically active (5). Use of a radioimmunoassay for the spore protease has demonstrated that: 1) the protease antigen disappears during spore germination in parallel with the loss in protease enzyme activity; 2) the protease antigen is absent from log phase and young sporulating cells; and 3) the protease antigen appears within the developing forespore midway through sporulation at about the time of or even slightly before synthesis of the enzyme's substrates (5). Since the A, B, and C proteins are not attacked by the protease within the developing or dormant * This work was supported by a grant from the Army Research Office. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
t Present address, Department of Clinical Chemistry, Hartford Hospital, Hartford, CT.
§ Author to whom correspondence and requests for reprints should be addressed. spore (1), there must be some regulatory mechanism such that this enzyme is inactive in the developing forespore and dormant spore yet becomes active upon spore germination. While many mechanisms could effect the required regulation, a frequently observed regulatory mechanism for proteolytic enzymes is their synthesis as an inactive precursor or zymogen which has a higher molecular weight than the active enzyme. Consequently, we undertook to determine the molecular weight of the spore protease subunit throughout sporulation and germination in order to detect possible zymogen forms.

MATERIALS AND METHODS
Bacterial strain-Most of the work described in this report was carried out with B. megaterium QM B1551 (originally obtained from H. S. Levinson, U. S. Army Natick Laboratories, Natick, MA). Some experiments also utilized a spontaneous streptomycin-resistant derivative of this strain, which grew and sporulated in the presence of streptomycin sulfate (100 g/ml). The four protease mutant strains (B-2, B-41, C-1, and C-44) of B. megaterium QM B1551 were described previously (4), and spontaneous streptomycin-resistant derivatives of each of these strains were used throughout this work. These strains are designated B-2-1, B-41-2, C-1-5, and C-44-1.
Radiochemicals, Enzymes, and Immunological Reagents-[ 3 5 S] Methionine (1000 Ci/mmol) was obtained from ICN, and a uniformly labeled [1 4 C]amino-acid mixture (>50 mCi/mg of atom carbon) was obtained from Amersham Corp. The fluor for enhancement of autoradiographic detection of 14C or 35S was obtained from New England Nuclear (ENHANCE). Trypsin was obtained from Worthington. The purified B. megaterium spore protease, the spore protease iodinated with 125 I-Bolton-Hunter reagent, the rabbit anti-spore protease y-globulin, control rabbit y-oobulin, and goat anti-rabbit yglobulin were prepared and stored as described previously (5). Freezedried cells of Staphylococcus aureus were used as a source of protein A for precipitation of rabbit y-globulin and were obtained from the Enzyme Center as IgGsorb. Peroxidase-coupled goat anti-rabbit yglobulin was obtained from Cappel and normal goat serum from GIBCO. Nitrocellulose paper for transfer of proteins from acrylamide gels was obtained from Millipore.
Growth, Sporulation, and Spore Germination-All bacterial strains were grown at 30 C in supplemented nutrient broth (6). The medium for streptomycin-resistant strains also contained 100 pg/ml of streptomycin sulfate. Developing forespores were isolated from sporulating bacteria as described previously (7). Spores of all strains were harvested, washed, and stored as previously described (6). For germination spores (5-25 mg/ml) were first heat-shocked (60 C, in 15 min) in water and cooled in ice. Germination was at 30 C and 1-3 mg/ml of spores in 50 mM Tris-HCI (pH 7.4) and 50 mM glucose. In this medium the initiation of spore germination was 95% complete in 10 min as measured by release of the dormant spore's DPA.' Some experiments also used a KBr germination medium containing 50 mM KPO 4 (pH 7.4) and 50 mM KBr.
Pulse-labeling and Pulse-Chase Experiments-Cells were grown in supplemented nutrient broth and were routinely pulse-labeled by addition of 2. S]methionine-containing tryptic peptides of P 4 6 and P 4 1 . Cells (10 ml) were pulse-labeled with I mCi of [35S]methionine slightly before the time for peak synthesis of P 4 6 . After 60 min, 5 ml were harvested immediately while 5 ml were chased for 5 h with unlabeled methionine. The cells were broken and extracted with 2.5 ml of buffer A, and the samples were processed for immunoprecipitation. In this experiment, the whole Time in minutes sion of P 4 1 to P 4 0 in spore extracts is presumably the reason that P 41 is not found in preparations of the purified spore protease. In contrast to P 41 and P 40 , any P 46 present in the dormant spore did not disappear rapidly during germination or in dormant spore extracts (Fig. 10). This was true both with wild type spores (Fig. 10) and with spores of C-1-5 which contained levels of P 46 3-4 times higher than wild type spores (data not shown).

DISCUSSION
The data presented in this communication indicate that the B. megaterium spore protease is synthesized during sporula-sample was treated with immune y-globulin (10 pl). The solubilized protein from the immunoprecipitates was run on four 4-mm lanes of a 1.5-mm SDS slab gel, stained, and autoradiographed. Labeled P 46 and P 4 1 were cut out, processed, and digested, and the tryptic peptides were resolved by HPLC and the [35S]methionine counted for 20 min as described under "Materials and Methods." All counts have been corrected for background (16 cpm). -100 280 g of purified spore protease (P40) , were run on 10 4-mm lanes of a 1.5-mm X SDS slab gel, and stained. The stained bands were cut out and mixed with the 3 P 46 band from 5 ml of pulse-labeled cells ' obtained as described in the legend to -50 ? Fig. 8. The mixture was then processed and digested with trypsin, and peptides were resolved by HPLC and counted as described under "Materials and Methods." The optical density tracing at 214 has been corrected for absorbtion due to -0 impurities in the buffers and gel itself. This correction was minor.

45
tion as a Mr = 46,000 polypeptide (P 4 6 ). P 4 6 is processed to a Mr = 41,000 (P 41 ) form 2-3 h later and the dormant spore contains predominantly P 41 with small amounts of unprocessed P 46 . In the first minutes of spore germination P 41 is converted to a Mr = 40,000 species (P 40 ) which appears identical with the subunits of the active enzyme purified from germinated spores. P 40 then disappears during further germination in an ATP-dependent process. However, any P 46 present in dormant spores is not significantly altered during germination.
The identification of P 46 as a spore protease precursor was made initially by its immunoprecipitation from extracts of is certainly possible that the precursors have no significant function, it is more attractive to imagine that they have some regulatory role. This is particularly attractive, since the spore protease must be inactive in the developing forespore and dormant spore, and become active only upon germination. Consequently, if P 41 and/or P 46 were catalytically inactive, or were unable to form an enzymatically active tetramer (5), this would explain the lack of protease action in developing and dormant spores. However, it would then imply that the system(s) involved in the P 4 6 to P 41 and P 41 to P 40 conversions must themselves be very tightly regulated. This would further suggest that the understanding of the control of these conversion processes may bring us one step closer to an understanding at the molecular level of the controls involved in the onset and maintenance of the enzymatic dormancy of the bacterial spore.