Isolation and characterization of Bacillus megaterium mutants ...

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ing 20 to 30% of the normal level of spore proteolytic activity have been isolated. Partial purification of the protease from wild-type spores by a revised procedure.

Vol. 135, No. 3

JOURNAL OF BACTERIOLOGY, Sept. 1978, p. 841-850 0021-9193/78/0135-0841$02.00/0

Copyright X) 1978 American Society for Microbiology

Printed in U.S.A.

Isolation and Characterization of Bacillus megaterium Mutants Containing Decreased Levels of Spore Protease CYNTHIA J. POSTEMSKY, SUSAN S. DIGNAM, AND PETER SETLOW* Department of Biochemistry, University of Connecticut Health Center, Farmington, Connecticut 06032 Received for publication 16 June 1978

A proteolytic activity present in spores of Bacillus megaterium has previously been implicated in the initiation of hydrolysis of the A, B, and C proteins which are degraded during spore germination. Four mutants of B. megaterium containing 20 to 30% of the normal level of spore proteolytic activity have been isolated. Partial purification of the protease from wild-type spores by a revised procedure resulted in the resolution of spore protease activity on the A, B, and C proteins into two peaks-a major one (protease II) and a minor one (protease I). The protease mutants tested lacked active protease II. All of the mutants exhibited a decreased rate of degradation ofthe A, B, and C proteins during spore germination at 30°C, but degradation of the proteins did occur. Degradation of the A, B, and C proteins during germination of the mutant spores was decreased neither by blockade of ATP production nor by germination at 44°C. Initiation of spore germination was normal in all four mutants, and all four mutants went through outgrowth, grew, and sporulated normally in rich medium. Similarly, outgrowth of spores of two of the four mutants was normal in minimal medium at 30°C. In the two mutants studied, the kinetics of loss of spore heat resistance and spore UV light resistance during germination were identical to those of wild-type spores. This indicates that the A, B, and C proteins alone are not sufficient to account for the heat or UV light resistance of the dormant spore.

Approximately 15 to 20% of the protein in resistant to UV light. These findings have led to dormant spores of B. megaterium and other the suggestion that the A, B, and C proteins Bacillus and Clostridium species consists of a might be involved in the reistance of the dorgroup of low-molecular-weight proteins (10, 11, mant spore to UV light (10, 11). To evaluate this 15). Three proteins, termed A, B, and C proteins, possibility, as well as the possibility that the A, comprise the majority (>85%) of these proteins B, and C proteins might be involved in some in B. megaterium spores. These proteins are not other dormant spore property, we decided to found in log phase or young sporulating cells, isolate mutants which were defective in their but appear late in sporulation within the devel- degradation of the A, B, and C proteins during oping forespore (10). All three proteins are de- spore germination. Clearly, a spore protease mugraded to amino acids in the early minutes of tant should show decreased protein degradation spore germination, and these amino acids sup- during spore germination. This paper describes port much of the protein synthesis during this the isolation and characterization of such spore period (9, 14). Most of the degradation of these protease mutants. proteins in vivo appears to be initiated by an MATERAULS AND METHODS endoprotease(s) with absolute specificity for the Materials and organism used. L-[4,53Hlleucine, A, B, and C proteins (12). This enzyme(s) is present only in developing forespores and dor- L-[2,3-3H]valne, and [5-3H]uridine were obtained from mant spores and is lost during spore germination New England Nuclear Corp. Leucine-p-nitranilide, lysozyme, and nitrosoguanidine were obtained from and outgrowth (12). Co. Aminopeptidase was partially The A, B, and C proteins are localized in the Sigma Chemical from ge ted spores of B. megaterium, core or central region of the dormant spore (the purified concentrated, and stored as previously described (12). site of spore DNA) where they comprise up to A crude mixture of the A, B, and C proteins for use as 50% of the total protein (10). The proteins are a protease substrate was obtained from dormant basic, with isoelectric points greater than 9.8, spores of B. megaterium, partially purified, and stored and all three species bind to spore DNA in vitro. as previously described (12). All work described in this communication utilized These proteins appear during sporulation at the time during which the developing spore becomes as a parent strain B. megaterium QM B1551 originally 841



obtained from Hillel Levinson (U. S. Army Laboratories, Natick, Mass.). Spores of this organism, as well as those of all of the mutant strains, were obtained by growth in supplemented nutrient broth (SNB) at 300C (13). Spores were harvested, washed, lyophilized, and stored as previously described (13). Mutagenesis. Cells were grown in SNB to an optical density at 600 nm of 0.7 (mid-log phase). The cells (24 ml) were centrifuged, washed once with an equal volume of tris(hydroxymethyl)aminomethane (Tris)-maleate buffer (0.05 M Tris-maleate [pH 6.0]), and suspended in an equal volume of Tris-maleate buffer at 300C to which was added nitrosoguanidine (75 ,g/ml). The mixture was shaken at 300C for 1 h, centrifuged (5 min, 10,000 x g), washed three times with 20 ml of prewarmed SNB, and suspended in 10 ml of SNB. Approximately 30% of the cells survived this procedure. Aliquots (2 ml) of the final suspension were inoculated in three flasks containing 25 ml of SNB, the cultures were allowed to sporulate, and the spores were harvested and washed as previously described (13). The spores were stored in 50% glycerol (2 ml) at -20°C. The effectiveness of the mutagenesis procedure was tested by analysis for streptomycinresistant organisms using SNB agar plates (SNB plates) with or without streptomycin sulfate (100 yg/ml). Streptomycin-resistant organisms were present in spores from the parent culture at a frequency of 1.7 x 1i-' and in the spores derived from the mutagenized cultures at a frequency of 2 x 10-4. Screening procedure. The only known function of the spore protease in B. megaterium is to degrade the A, B, and C proteins during spore germination (12). Similarly, the only function known for the A, B, and C proteins is to be degraded to free amino acids early in spore germination (14). Because generation of amino acids in this manner is essential for rapid protein synthesis during germination and outgrowth (14), the only phenotype that we could reasonably ascribe a priori to a spore protease mutant was that, in a medium devoid of amino acids, spores of a protease mutant might go through outgrowth more slowly than wild-type spores. Consequently, the first step in our mutant isolation was the screening of -10' colonies of spores for slow colony formation on Spizizen minimal plates containing no Casamino Acids (SMM plates) at 400C (16). The high temperature was chosen to maximize changes of isolation of a temperature-sensitive mutant. Dilutions of spore stocks were plated on SNB plates (60 to 150 colonies per plate), and after 13 h at 300C, single colonies were transferred to fresh SNB plates (52 colonies per plate). The latter were then incubated for 3 days at 300C to allow sporulation and sporangial lysis, and these plates were replica plated onto an SMM plate. The replica was heated (700C, 60 min) to kill any remaining cells and to heat activate spores and then transferred to a 400C incubator. After -8 h (outgrowth plus up to four cell divisions) the plate was examined, and colonies which gave slower colony formation than wild-type spores were scored as positive. Positives were further tested by a second screening exactly as described above. Colonies which passed both screenings were then tested for vegetative growth at 30 and 400C. Plates were treated as described above,

but the replica was made on SNB plates and incubated at 300C. After significant growth of all colonies had occurred, the plate was replica plated on two SMM plates. These were incubated at 30 and 40°C, respectively, for about 6 h. The plates were then compared, and colonies which were temperature sensitive for growth were discarded. Identification of protease mutants. Because a large number of colonies (-1.8 x 103) survived the screening procedure, we required a rapid assay which could quantitate spore protease in single colonies on plates. Additionally, these assays had to be carried out on spores, because only this stage of growth contains spore-type protease (12). Consequently, colonies which were to be assayed for protease were first allowed to sporulate on SNB plates (52 colonies per plate) for 3 days at 300C. Colonies were then scraped from the plate with a small glass rod and incubated in 500 Ai of pH 8.0 stripping buffer (8 M urea, 1.5% sodium dodecyl sulfate, 50 mM Trs-hydrochloride [pH 8.0], and 100 mM 2-mercaptoethanol) at 370C for 45 min. This treatment inactivates extracellular proteases as well as those in vegetative and sporulating cells (but not spores) and removes sufficient coat protein from dormant spores to allow them to be disrupted with lysozyme (12). The treated colonies were washed two times by centrifugation with 8 M urea (2 ml) and then three times with 0.1 M Trishydrochloride (pH 7.4). The final pellet was suspended in 100 pl of 0.1 M Tris-hydrochloride (pH 7.4) and 5 mM CaCl2 containing lysozyme (30 Mg), a crude mixture of the A, B, and C proteins (60 jug), and 1 to 2 U of spore amnmopeptidase. After incubation for 60 mmn at 46°C (all colonies had lysed by the first 5 to 15 min), 400 p1 of 3% acetic acid was added, and the reaction mix was centrifuged (5 min, 10,000 x g). The supernatant fluid was assayed for ninhydrin-positive material by its addition to 2 ml of ninhydrin reagent (8), boiling for 3 min, and measuring the optical density at 505 nm (12). The optical densities of the blank values in this assay were -0.25, whereas wild-type colonies gave values of -1.0. Colonies were scored as potential mutants if they gave 3 times higher than wild-type values were classified as mutants. Germination and outgrowth. Standard conditions for spore germination were as follows unless otherwise noted. Spores were germinated after a heat shock (10 min, 6000) of spores (20 mg/ml) in water. After cooling in ice, spores were germinated at 300C, and 1 mg/ml in glucose-phosphate (0.05 M KPO4, pH 7.4, and 0.1 M glucose). In cases in which outgrowth was to be followed, germination was in the minimal medium of Spizizen (16) with or without Csammino Acids (0.1%) at a spore concentration of 0.15 mg/ml. The initiation of spore germination was followed either


VOL. 135, 1978


by the release of dipicolinic acid (DPA) or the fall in optical density of the culture at 600 nm as previously described (12, 14). The percentage of spores which had germinated at the end of an experiment was determined by examination of 100 to 200 spores in a phasecontrast microscope. Preparation of spore extracts. Extracts of mutant or wild-type spores grown in liquid culture were prepared by a number of different methods. In method 1, dormant spores (-10 mg/ml) were extracted with pH 8.0 stripping buffer and washed as described above, and the spores were suspended at a concentration of about 20 mg/ml in buffer A (50 mM Tris-hydrochloride, pH 7.4, and 5 mM CaCL6). Spore lysis was initiated by addition of lysozyme (800 pg/ml), and after 10 min at 37°C the mix was centrifuged (10 min, 15,000 x g), and the supernatant fluid was dialyzed against buffer B (10 mM Tris-hydrochloride [pH 7.4], 5 mM CaCl2, and 20% glycerol) for 16 h at 4°C. In method 2, dormant spores (-25 mg/ml) in buffer A were disrupted by sonic treatment with glass beads as previously described (12), centrifuged, and dialyzed as described above. In method 3, spores were germinated under standard conditions for 10 min, centrifuged (10 min, 10,000 x g), and suspended at 25 mg/ml in cold buffer A. The germinated spores were disrupted by sonic treatment with glass beads, centrifuged, and dialyzed as described above. In method 4, spores were germinated and centrifuged as described above and washed with 10 volumes of cold water. The final pellet was suspended at a spore concentration of 25 mg/ml in 0.1 M Tris-hydrochloride (pH 8.0) and 5 mM CaCl2. Lysozyme was added to 1 mg/ml, and the mix was incubated for 15 min at 370C. This treatment does not cause complete spore disruption but makes spores very sensitive to sonic treatment (12). The mix was then chilled, and spore disruption was completed by sonic treatment (-8 min) with glass beads. The sonic extract was centrfuged (10 min, 12,000 x g), the pellet was again sonically disrupted after addition of more buffer and recentrifuged, and all supernatant fluids were combined and dialyzed against buffer B. Purification of protease from wild-type or mutant spores. A 5-g amount of spores was germinated under standard conditions for 10 min and extracted by using method 4 but with lysozyme added to 2 mg/ml and a spore concentration of 50 mg/ml. Nucleic acid was removed from the pooled supernatant fluid with streptomycin sulfate (1 g in 10 ml of water). Subsequent purification of the protease was as described previously (12), but the gradient for the diethylaminoethyl (DEAE)-cellulose column was from 75 mM in NaCl to 250 mM in NaCl. Assay procedures. Aminopeptidase was determined by hydrolysis of leucine-p-nitroanilide as previously described (12). DNA and RNA were determined by the diphenylamine and orcinol procedures, respectively (6). Protein was determined by the Lowry method (3), and DPA was determined by the method of Rotman and Fields (4). Unless otherwise noted, spore protease in extracts was determined at 30°C by conversion of proteins A, B, and C into ninhydrinpositive material by using spore aminopeptidase to amplify the protease reaction (assay procedure 3 [12]). The units are as previously described (12). NaCl


concentrations were determined with a conductivity meter calibrated with NaCl solutions of known concentration. Protein degradation during germination and outgrowth. Degradation of proteins A, B, and C during spore germination was measured as previously described (12). A quantitative estimate of the rate of degradation of the A and C (A+C) or B proteins during germination was defined as a At. This value was determined by measuring the At in minutes between curves giving the percentage loss during germination of DPA and a particular protein or proteins (see Fig. 2). These At values were determined at 20, 40, 60, and 80% los of DPA, and all values were averaged to give At. The higher the At., the slower the protein is degraded. By the use of the t, as a measure of protein degradation during germination, we hoped to minim differences in spore stocks due to differences in rates of initiation of germination. Degradation of newly synthesized protein during germination was measured as previously described (9). Spores were germinated at 1 mg/ml in Spizizen's minimal medium with no Casamino Acids and with [3H]leucine (50 nM) present from 0 to 10 min of germination. The [3H]leucine was removed, the spores were resuspended in fresh medium containing 2.5 mM cold leucine, and protein degradation was measured (9). Protein and RNA synthesis. Spores were germinated at 0.5 mg/ml in glucose-phosphate. Protein synthesis was measured with 1.0 mM [3H]leucine (&103 cpm/nmol) and RNA synthesis with 50 IAM [3H]uridine (- 104 cpm/nmol) present from zero time. At various times, aliquots (0.5 ml) were taken and mixed with 1.5 ml of 6.7% trichloroacetic acid. This mixture was filtered through Whatman GF/C filters, washed five times with 5 ml of 5% trichloracetic acid and three times with 5 ml of ethanol and then dried and counted. Changes in spore heat and UV light resistance during germination. Spores were germinated under standard conditions, and heat-resistant or UV-resistant forms were determined as described previously (2, 12).

Lcalization of the A, B, and C proteins in lysates of dormant or germinated spores. Dormant spores of wild-type or mutant strains were treated with pH 8.0 stripping buffer and washed as described above. Lysis was as described above, but with no CaC12 and with ethylenediaminetetraacetic acid (25 mM) and phenylmethylsulfonyl fluoride (0.1 mM) present. Lysates were then fractionated as previously described (10) by the method of Chambon et aL (1) which separates spore DNA from ribosomes in insoluble proteins. Lysates of spores germinated for 15 min were prepared and fractionated similarly.

RESULTS Mutant isolation. Using the screening and assay procedures described above, we isolated four spore protease mutants starting with -1-W colonies (Table 1). At least two groups of these mutants (B set and C set) are independent isolates. One of the mutants (B-2) was found to contain an unidentified auxotrophic mutation




levels wid-type and mutant spore? TABLz 1. Protease and aminopeptidase Protease activity at 4OC/aminopeptidase activity at 30°C Aminopeptim Protease activity at Spores extracted

Wild type B-2 B-41


Method lb

Method 2r

Method 3"



6.6 (

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