both tetrameric, but that only P41 is enzymatically active. The identification of a zymogen form (P46) of this protease explains in part the regulation of the activity.
JOURNAL OF BACTERIOLOGY, Jan. 1983, p. 375-378
Vol. 153, No. 1
0021-9193/83/010375-04$02.00/0 Copyright C 1983, American Society for Microbiology
Enzymatic Activity of Precursors of Bacillus megaterium Spore Protease REBECCA H. HACKETT AND PETER SETLOW* Department ofBiochemistry, University of Connecticut Health Center, Farmington, Connecticut 06032 Received 21 June 1982/Accepted 27 September 1982
The protease that initiates rapid proteolysis during germination of Bacillus megaterium spores is synthesized during sporulation as a 46,000-molecularweight polypeptide (P46) and is processed later in sporulation to a 41,000molecular-weight polypeptide (P41), which is converted to a 40,000-molecularweight polypeptide (P40) early in spore germination. P40 is known to be both tetrameric and enzymatically active. In this work, we show that P46 and P41 are both tetrameric, but that only P41 is enzymatically active. The identification of a zymogen form (P46) of this protease explains in part the regulation of the activity of this enzyme.
Approximately 20% of the protein of dormant spores of Bacillus megaterium is degraded in the first 30 min of spore germination (7). The proteins degraded are a group of low-molecularweight species unique to the spore stage of the life cycle (7). The proteolysis is initiated during germination by an amino acid sequence-specific endoprotease also unique to the spore (4, 7, 8). This protease disappears rapidly (t1/2, 40 min) during spore germination and appears only late in sporulation within the developing spore at the same time and in the same compartment as its low-molecular-weight protein substrates (2, 6). Since the enzyme acts on its substrates only during spore germination, the activity of this enzyme must be regulated in developing and dormant spores (10). Recent work has shown that the enzymatically active protease purified from germinated spores is a tetramer with subunits of 40,000 molecular weight (P4,o) (2, 3). However, the enzyme as first synthesized during sporulation has a polypeptide of 46,000 molecular weight (P46) (3). The majority (but not all) of the P46 is processed later in sporulation to a form with a 41,000-molecular-weight polypeptide (P41), and it is only early in spore germination that P41 is converted to P40 (3). P40 is degraded further as germination proceeds; however, any P46 which has not been processed to P41 late in sporulation is neither processed nor degraded during germination (3). Given the precedents for zymogen forms of proteolytic enzymes, it is possible that P46 and perhaps P41 are enzymatically inactive, thus effecting the regulation of this key proteolytic enzyme. This report deals with the enzymatic activity and subunit structure of the spore protease precursors P46 and P41.
MATERIALS AND METHODS Growth and isolation of spores and enzyme assays and analyses. All work was carried out with B. megaterium QMB1551 (originally obtained from H. S. Levinson, U.S. Army Natick Laboratories, Natick, Mass.). Bacteria were grown at 30°C in supplemented nutrient broth, and spores were isolated, washed, and stored as previously described (9). Spores were germinated after a heat shock (15 min, 60°C) of spores (25 mg/ml) in water. Germination was at 30°C with 5 mg of spores per ml of 50 mM Tris-hydrochloride (pH 7.4)-50 mM glucose. In some germination experiments, KCN (10 mM) was added to prevent the ATP-dependent loss of the protease (2). As determined by dipicolinic acid release, spore germination was >90%o complete in 6 mm.
Protease enzyme activity was assayed by using a mixture of its in vivo substrates as described previously (2, 6). Different protease forms were separated by electrophoresis on a sodium dodecyl sulfate-acrylamide gel, the proteins were transferred to nitrocellulose, and protease forms were detected with anti-spore protease immunoglobulin (immunoblotting) as described previously (3, 11). The ratios of different protease forms were determined by scanning appropriate immunoblots as described previously (3). Extraction of protease forms from forespores, sporulating cells, or germinated spores by using sonication. Sporulating cells from 5 ml of culture were harvested by centrifugation, and the pellet was frozen and lyophilized. The dry sample was disrupted by shaking in a dental amalgamator with glucose crystals as the abrasive for 2 to 3 min. Protease was extracted with 250 ,ul of 50 mM Tris-hydrochloride (pH 7.4)-20 mM EDTA0.1 mM phenylmethylsulfonyl fluoride-20% glycerol (buffer A) as described previously (3). This extraction has been shown to preserve protease antigen and allows no processing of precursor forms (3). Forespores were isolated from 50 ml of culture as described previously (10), and purified forespores were disrupted by sonic oscillation with glass beads (500 mg) in 2 ml of 50 mM Tris-hydrochloride (pH 7.4-S5 mM CaCl2 375
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(buffer B) followed by centrifugation for 5 min at 10,000 x g. A small portion of the supernatant fluid was dialyzed at 4°C against buffer B containing 20% glycerol (buffer C), and the remainder was partially purified by passage over a 1-ml column of hemoglobinSepharose (1) in buffer B. The flow-through fraction was pooled and made to 60% of saturation in ammonium sulfate. After 30 min at 4°C, the precipitate was isolated by centrifugation, dissolved in 0.3 to 0.6 ml of buffer B, and dialyzed overnight against buffer C. Control experiments showed that 50 to 80% of the specific spore protease was recovered in this procedure, whereas >99%o of protease activity on azocasein and azocoll (5) was removed. An extract from spores (50 mg) germinated for 15 min with KCN present was also prepared by sonication and partially purified as described above. Extraction and purification of P46 and P40 from germinated spores. For partial purification of P40, spores (2 g) were germinated for 10 min with KCN to prevent degradation of P40 (3). The spores were then extracted with lysozyme, and P4, was purified through a DEAE-Sephadex column as described previously (2). For partial purification of P46, spores (2 g) were germinated for 120 min without KCN, and P46 was extracted and purified as described above. P40 was detected by its enzymatic activity, using a mixture of its in vivo substrates as previously described (2, 6). P46 was analyzed by gel electrophoresis followed by transfer of proteins to nitrocellulose and finally detection
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with anti-protease immunoglobulin as described previously (3, 11). Appropriate fractions from the DEAESephadex columns were pooled and concentrated as described previously (3). Isolation of P41 from dormant spores. Dormant spores (30 to 50 mg) were disrupted by shaking in a dental amalgamator with glucose (75 mg) as the abrasive. Two to three 1-min periods of shaking sufficed to disrupt >80o of the spores, and control experiments showed that there was no destruction of protease antigen. A portion of the powder was extracted with cold buffer A, and the remainder was extracted with cold buffer B, using a small homogenizer. The suspensions were immediately centrifuged for 4 min at 4°C in an Eppendorf microcentrifuge. A portion of the supernatant fluid in buffer B was dialyzed overnight against cold buffer C, and the remainder in buffer B was partially purified, using hemoglobin-Sepharose and ammonium sulfate as described above.
RESULTS
Two different methods were used to isolate P46 free of high levels of P40 and P41. The first method took advantage of the lag between synthesis of P46 and its processing to P41 (Fig. 1, lanes 1 and 2) (3). Forespores were isolated when significant P46 but no P41 had accumulated; the forespores were extracted, and the extract was rapidly purified to remove nonspecific
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FIG. 1. Immunoblot analysis of P46, P41, and P40 from sporulating cells and dormant and germinated spores. (A) Samples were extracted from sporulating cells by using dry breakage and from forespores or germinated spores by using sonication, treated, and analyzed as described in the text. (B) Samples were isolated from germinated spores by using lysozyme, purified, and analyzed as described in the text. (C) Samples were isolated from dormant spores, treated, and analyzed as described in the text. Lane 1, sporulating cells at time offorespore isolation; lane 2, sporulating cells 2.5 h after the time of forespore isolation; lane 3, protease partially purified from forespores; lane 4, protease partially purified from germinated spores; lane 5, protease extracted from forespores without further purification and incubated for 2 h at 37°C; lane 6, protease purified through DEAESephadex from spores germinated for 10 min with KCN; lane 7, protease purified through DEAE-Sephadex from spores germinated for 120 min without KCN; lane 8, unpurified dormant-spore extract in buffer A within 5 min of preparation, lane 9, unpurified dormant-spore extract in buffer B dialyzed for 4 h against buffer C at 4°C; lane 10, unpurified dormant-spore extract in buffer B dialyzed for 16 h against buffer C at 4°C; lane 11, dormant-spore extract in buffer B partially purified and dialyzed for 16 h against buffer C at 4°C; lane 12, 1:1 mixture of samples from lanes 10 and 11. The positions of P46, P41, and P4, were determined with reference to appropriate markers as described previously (3). The unlabeled bands visible on the immunoblots all reacted equally well with nonimmune serum with the exception of the band labeled 1, which was a contaminant in the protease preparation used to raise the antiserum (3). This protein is not antigenically related to the protease (3).
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VOL. 153, 1983
proteases. This procedure resulted in a preparation with a ratio of P46 to P41 greater than 20 (Fig. 1, lane 3; Table 1). In contrast, spores
germinated for 10 min which were extracted and purified similarly had a ratio of P46 to P40 of 0.2 (Fig. 1, lane 4; Table 1). The second method for P46 isolation took advantage of the degradation of P41 and P40 during spore germination, whereas any P46 which is not processed during sporulation is not degraded (3). Thus, extracts from spores germinated for 2 h have ratios of P46 to P41 of >3 (3). Consequently, P40 and P46 were purified from spores germinated for 10 min with KCN or for 2 h without KCN, respectively. This resulted in preparations with ratios of P46 to P40 of 0.2 (10min germinated) and 3.2 (2-h germinated), respectively (Fig. 1, lanes 6 and 7; Table 1). Strikingly, the P46 in extracts from either germinated spores or forespores was stable, and neither P41 nor P40 was generated upon incubation of even an unpurified extract at 37°C for up to 2 h (Fig. 1, lane 5; data not shown). Conversion of P46 to P40 was also not seen when forespore extracts were supplemented with small amounts of purified P40 or when purified forespore or germinated-spore extracts were incubated at 37°C for up to 2 h (data not shown). In contrast to the results noted above for P46, P41 was labile in spore extracts as shown previously (3). Dormant spores extracted with the protease
inhibitors EDTA and phenylmethylsulfonyl fluoride present contained essentially no P40 (Fig. 1, lane 8) (3). However, preparation of spore extracts without these inhibitors and incubation at 4°C for 4 h resulted in 50% conversion of P41 to P40, with almost 100% conversion in 16 h (Fig. 1, lanes 9 and 10). Similarly, crude dormant-spore extracts incubated for 30 min at 37°C showed almost complete conversion of P41 to P40 (data not shown). In contrast, when the dormantspore extract was first partially purified to remove nonspecific proteases, the ratio of P41 to P40 in the partially purified extract was 7 (Table 1), and P41 was stable either at 4°C overnight (Fig. 1, lane 11) or at 37°C for up to 2 h (data not shown). The key step in the partial purification of the P41 was the hemoglobin-Sepharose column since after this step the P41 in the extract was stable (data not shown). As noted previously, gel electrophoresis of a 1:1 mixture of an extract containing only P40 and one containing predominantly P41 resolved these two species (lane 12, Fig. 1), indicating that P41 and P40 are two different species. Comparison of the enzymatic activity of a forespore preparation containing P46 with that of a germinated spore preparation showed that P46 had less than 2% of the specific activity of P40 (Table 1, experiment 1). Similarly, P46 preparations purified from spores germinated for 120 min had only 10% of the specific activity of
TABLE 1. Relative specific activities of various protease formsa Source of protease
Lane in Fig. 1
Spores germinated for 10 min Forespores
4 3
P46/P40 0.2 >20
2e
Spores germinated for 10 min Spores germinated for 120 min
6 7
0.23 3.2
39
Dormant spores, unpurified Dormant spores, purified
Expt
10 11
Ratio of:
P41P4
Relative sp act
