THE JOURNAL OF BIOLOGICAL CHEMISTRYVol. 257, No. 18, Issue of September 25, pp. 10838-10845, 1982Printed in U.S.A.

Bacillus megaterium Spore ProteaseSYNTHESIS AND PROCESSING OF PRECURSOR FORMS DURING SPORULATION AND GERMINATION*

(Received for publication, March 2, 1982)

Charles A. Loshon:, Bonnie Massey Swerdlow, and Peter Setlow§From the Department of Biochemistry, University of Connecticut Health Center, Farmington, Connecticut 06032

The protease which initiates the rapid protein deg-radation during germination of Bacillus megateriumspores was synthesized during sporulation as a Mr =46,000 polypeptide (P4 6) which was found in the devel-oping forespore. P4 6 was processed during sporulationto a Mr = 41,000 species (P41) 2-3 h after P4 6 synthesisand at the time of or slightly before accumulation ofdipicolinic acid. P41 was the predominant form of theprotease in the dormant spore, with smaller amountsof unprocessed P46. In the first minutes of spore ger-mination P41 was processed (tl/ 2 <10 min) to a Mr =40,000 species (P40), which appeared identical to thesubunit of the purified active enzyme. The latter proc-essing reaction did not require metabolic energy, butP40 disappeared completely during further germination(t1/ 2 ~40 min) in a reaction which did require metabolicenergy. It seems probable that precursors P4 6 and P4 1of the spore protease are involved in the regulation ofthe activity of this spore enzyme.

The first minutes of germination of spores of various Bacil-lus and Clostridium species are accompanied by the degra-dation of up to 25% of the spore's protein (1). The majorsubstrates for this proteolysis are two to three low molecularweight proteins (termed A, B, and C proteins in Bacillusmegaterium) which are synthesized during sporulation (1). InB. megaterium the degradation of these proteins during ger-mination is initiated by an amino acid sequence specific en-doprotease which acts only on the A, B, and C proteins andanalogous proteins from spores of other species (2, 3). Mutantshave been isolated with low levels of this protease, and allexhibit a decreased rate of proteolysis during germination, butno other phenotypic defect (4). The protease has been purifiedfrom 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 dem-onstrated that: 1) the protease antigen disappears during sporegermination in parallel with the loss in protease enzymeactivity; 2) the protease antigen is absent from log phase andyoung sporulating cells; and 3) the protease antigen appearswithin the developing forespore midway through sporulationat about the time of or even slightly before synthesis of theenzyme's substrates (5). Since the A, B, and C proteins arenot attacked by the protease within the developing or dormant

* This work was supported by a grant from the Army ResearchOffice. The costs of publication of this article were defrayed in partby the payment of page charges. This article must therefore be herebymarked "advertisement" in accordance with 18 U.S.C. Section 1734solely to indicate this fact.

t Present address, Department of Clinical Chemistry, HartfordHospital, Hartford, CT.

§ Author to whom correspondence and requests for reprints shouldbe addressed.

spore (1), there must be some regulatory mechanism suchthat this enzyme is inactive in the developing forespore anddormant spore yet becomes active upon spore germination.While many mechanisms could effect the required regulation,a frequently observed regulatory mechanism for proteolyticenzymes is their synthesis as an inactive precursor or zymogenwhich has a higher molecular weight than the active enzyme.Consequently, we undertook to determine the molecularweight of the spore protease subunit throughout sporulationand germination in order to detect possible zymogen forms.

MATERIALS AND METHODS

Bacterial strain-Most of the work described in this report wascarried out with B. megaterium QM B1551 (originally obtained fromH. S. Levinson, U. S. Army Natick Laboratories, Natick, MA). Someexperiments also utilized a spontaneous streptomycin-resistant deriv-ative of this strain, which grew and sporulated in the presence ofstreptomycin sulfate (100 g/ml). The four protease mutant strains(B-2, B-41, C-1, and C-44) of B. megaterium QM B1551 were describedpreviously (4), and spontaneous streptomycin-resistant derivatives ofeach of these strains were used throughout this work. These strainsare 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 uni-formly labeled [14C]amino-acid mixture (>50 mCi/mg of atom carbon)was obtained from Amersham Corp. The fluor for enhancement ofautoradiographic detection of 14C or 35S was obtained from NewEngland Nuclear (ENHANCE). Trypsin was obtained from Worth-ington. The purified B. megaterium spore protease, the spore proteaseiodinated with 125I-Bolton-Hunter reagent, the rabbit anti-spore pro-tease y-globulin, control rabbit y-oobulin, and goat anti-rabbit y-globulin were prepared and stored as described previously (5). Freeze-dried cells of Staphylococcus aureus were used as a source of proteinA for precipitation of rabbit y-globulin and were obtained from theEnzyme Center as IgGsorb. Peroxidase-coupled goat anti-rabbit y-globulin was obtained from Cappel and normal goat serum fromGIBCO. Nitrocellulose paper for transfer of proteins from acrylamidegels was obtained from Millipore.

Growth, Sporulation, and Spore Germination-All bacterialstrains were grown at 30 C in supplemented nutrient broth (6). Themedium for streptomycin-resistant strains also contained 100 pg/mlof streptomycin sulfate. Developing forespores were isolated fromsporulating bacteria as described previously (7). Spores of all strainswere harvested, washed, and stored as previously described (6). Forgermination spores (5-25 mg/ml) were first heat-shocked (60 C, in15 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. Inthis medium the initiation of spore germination was 95% complete in10 min as measured by release of the dormant spore's DPA.' Someexperiments also used a KBr germination medium containing 50 mMKPO4 (pH 7.4) and 50 mM KBr.

Pulse-labeling and Pulse-Chase Experiments-Cells were grownin supplemented nutrient broth and were routinely pulse-labeled byaddition of 2.5-5 IpCi of the [14C]amino-acid mixture or 50-100 pCi of[35S]methionine/ml of culture. After a labeling period of 20-60 min,

'The abbreviations used are: DPA, dipicolinic acid; SDS, sodiumdodecylsulfate; PMSF, phenylmethylsulfonyl fluoride; HPLC, highpressure liquid chromatography.

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B. megaterium Spore Protease

Time in mi

FIG. 8. HPLC analysis of the [3 6S]methionine-containingtryptic peptides of P4 6 and P4 1 . Cells (10 ml) were pulse-labeledwith I mCi of [35S]methionine slightly before the time for peaksynthesis of P4 6. After 60 min, 5 ml were harvested immediately while5 ml were chased for 5 h with unlabeled methionine. The cells werebroken and extracted with 2.5 ml of buffer A, and the samples wereprocessed for immunoprecipitation. In this experiment, the whole

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sion of P4 1 to P40 in spore extracts is presumably the reasonthat P41 is not found in preparations of the purified sporeprotease. In contrast to P41 and P40, any P46 present in thedormant spore did not disappear rapidly during germinationor in dormant spore extracts (Fig. 10). This was true both withwild type spores (Fig. 10) and with spores of C-1-5 whichcontained levels of P46 3-4 times higher than wild type spores(data not shown).

DISCUSSION

The data presented in this communication indicate that theB. megaterium spore protease is synthesized during sporula-

sample was treated with immune y-globulin (10 pl). The solubilizedprotein from the immunoprecipitates was run on four 4-mm lanes ofa 1.5-mm SDS slab gel, stained, and autoradiographed. Labeled P46and P41 were cut out, processed, and digested, and the tryptic peptideswere resolved by HPLC and the [35S]methionine counted for 20 minas described under "Materials and Methods." All counts have beencorrected for background (16 cpm).

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FIG. 9. Comparison of tryptic pep-tides from P40 with [3 5 S]methionine-

0 containing tryptic peptides from P4 6.-100 280 g of purified spore protease (P40)

, were run on 10 4-mm lanes of a 1.5-mmX SDS slab gel, and stained. The stained

bands were cut out and mixed with the3 P46 band from 5 ml of pulse-labeled cells' obtained as described in the legend to

-50 ? Fig. 8. The mixture was then processedand digested with trypsin, and peptideswere resolved by HPLC and counted asdescribed under "Materials and Meth-ods." The optical density tracing at 214has 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 (P4 6). P4 6 is processed to aMr = 41,000 (P41) form 2-3 h later and the dormant sporecontains predominantly P41 with small amounts of unproc-essed P46. In the first minutes of spore germination P41 isconverted to a Mr = 40,000 species (P40) which appears iden-tical with the subunits of the active enzyme purified fromgerminated spores. P40 then disappears during further germi-nation in an ATP-dependent process. However, any P46 pres-ent in dormant spores is not significantly altered during ger-mination.

The identification of P46 as a spore protease precursor wasmade initially by its immunoprecipitation from extracts of

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B. megaterium Spore Protease

is certainly possible that the precursors have no significantfunction, it is more attractive to imagine that they have someregulatory role. This is particularly attractive, since the sporeprotease must be inactive in the developing forespore anddormant spore, and become active only upon germination.Consequently, if P41 and/or P46 were catalytically inactive, orwere unable to form an enzymatically active tetramer (5), thiswould explain the lack of protease action in developing anddormant spores. However, it would then imply that the sys-tem(s) involved in the P46 to P41 and P41 to P40 conversionsmust themselves be very tightly regulated. This would furthersuggest that the understanding of the control of these conver-sion processes may bring us one step closer to an understand-ing at the molecular level of the controls involved in the onsetand maintenance of the enzymatic dormancy of the bacterialspore.

Acknowledgments-We are grateful to Robert Nelson and Dr.Juris Ozols for assistance in the experiments utilizing HPLC, and toRebecca Hawes Hackett for the experiment on the in vitro conversionof P41 to P40.

REFERENCES

1. Setlow, P. (1981) in Sporulation and Germination (Levinson, H.S., Sorenshein, A. L., and Tipper, D. J., eds) pp. 13-28, Ameri-

can Society for Microbiology, Washington, D. C.2. Setlow, P., Gerard, C., and Ozols, J. (1980) J. Biol. Chem. 255,

3624-36283. Yuan, K., Johnson, W. C., Tipper, D. J., and Setlow, P. (1981) J.

Bacteriol. 146, 965-9714. Postemsky, C. J., Dignam, S. S., and Setlow, P. (1978) J. Bacte-

riol. 135, 841-8505. Loshon, C. A., and Setlow, P. (1981) J. Bacteriol. 150, 303-3116. Setlow, P., and Kornberg, A. (1969) J. Bacteriol. 100, 1155-11607. Singh, R. P., Setlow, B., and Setlow, P. (1977) J. Bacteriol. 130,

1130-11388. Setlow, P., and Primus, G. (1975) J. Biol. Chem. 250, 623-6309. Towbin, H., Staehelin, T., and Gordon, J. (1979) Proc. Natl.

Acad. Sci. U. S. A. 79, 4350-435410. Setlow, P., and Ozols, J. (1980) J. Biol. Chem. 255, 8413-841611. Dignam, S. S., and Setlow, P. (1980) J. Biol. Chem. 255, 8417-

842312. Lowry, O. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J.

(1951) J. Biol. Chem. 193, 265-27513. Bradford, M. M. (1976) Anal. Biochem. 72, 248-25414. Rotman, Y., and Fields, M. L. (1967) Anal. Biochem. 22, 16815. Laemmli, U. K. (1970) Nature (Lond.) 227, 680-68516. Setlow, P., and Kornberg, A. (1970) J. Biol. Chem. 245, 3637-

364417. Gould, G. W. (1969) in The Bacterial Spore (Gould, G. W., and

Hurst, A. W., eds) pp. 397-444, Academic Press, New York18. Setlow, B., and Setlow P. (1980) Proc. Nat. Acad. Sci. U. S. A.

77, 2474-247619. Singh, R. P., and Setlow, P. (1979) J. Bacteriol. 139, 889-898

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C A Loshon, B M Swerdlow and P Setlowsporulation and germination.

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

1982, 257:10838-10845.J. Biol. Chem. 

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