Replication in Bacillus subtilis - NCBI

JOURNAL OF BACTERIOLOGY, Jan. 1992,

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Vol. 174, No. 2

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0021-9193/92/020477-09$02.00/0 Copyright C 1992, American Society for Microbiology

Temporal Expression of a Membrane-Associated Protein Putatively Involved in Repression of Initiation of DNA Replication in Bacillus subtilis BETHANIE EIDENT-WILKINSON,1 LORETTA MELE,1 JOHN LAFFAN,2 AND WILLIAM FIRSHEIN1* Department of Molecular Biology and Biochemistry, Wesleyan University, Middletown, Connecticut 06459,1 and Department of Molecular, Cellular, and Developmental Biology, University of Colorado, Boulder, Colorado 803092 Received 13 August 1991/Accepted 18 November 1991

A Bacillus subtilis membrane-associated protein that binds specifically to the origin region of DNA replication may act as an inhibitor of DNA replication (J. Laffan and W. Firshein, Proc. Natl. Acad. Sci. USA 85:7452-7456, 1988). This protein, originally estimated to be 64 kDa, had a slightly lower molecular size (57 kDa), as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis during these studies. The size difference may be due to processing that results in modification of the protein. The protein can be extracted from both cytosol and membrane fractions, and the amounts in these fractions vary during the developmental cycle of B. subtilis. A complex pattern of expression in which significant levels were detected in spores was revealed; levels decreased dramatically during germination and increased after the first round of DNA replication. The decrease during germination was due to protease activity, as demonstrated by the addition of protease inhibitors and radioactive-labeling chase experiments. During vegetative growth, the protein levels increased until stationary phase, after which there was another decrease during sporulation. The decrease during sporulation may be partially due to sequestering of the protein into forespores, since as the putative repressor protein decreased in the mother cell, it increased in the forespores. However, protease activity was also involved in the decrease in the mother cell. The changes in expression of this protein are consistent with its role as a repressor of initiation of DNA replication. Additional studies, including sequence analysis and further antibody analysis, show that this protein is not a subunit of the pyruvate dehydrogenase complex. This relationship had been a possibility based upon the results of others (H. Hemila, A. Pavla, L. Paulin, S. Arvidson, and I. Palva, J. Bacteriol. 172:5052-5063, 1990).

these studies reacted with a protein which has an altered mobility. To avoid confusion, we will refer to the previously called 64-kDa protein as the putative repressor (PR) protein. To investigate the activity of the PR protein further, studies of the PR protein during the developmental life cycle of B. subtilis were undertaken. The aim was to determine relative amounts of this protein during dormancy (spores), exponential growth (vegetative cells), stationary phase (TO), and sporulation (T1 to T20). Tn is defined as n hours after the end of logarithmic growth, and To is defined as the end of logarithmic growth which designates the initiation of sporulation. Results presented in this paper show a temporal regulation of the PR protein which is consistent with the model of repression of initiation of replication suggested by the in vitro replication studies. This paper also investigates the possibility that the PR protein may be related to a subunit of the pyruvate dehydrogenase complex. Hemila et al. (6) discovered that an apparent 64-kDa protein was, in fact, one of the enzymes of the pyruvate dehydrogenase complex in B. subtilis, the E2 subunit dihydrolipoamide acetyltransferase. Its actual molecular size, however, as determined by sequencing the operon, was 48 kDa. Antibody prepared by P. C. Tai (The Boston Medical Institute) which reacted with the PR protein also reacted with the 48- to 64-kDa subunit of the pyruvate dehydrogenase complex. The results here will show that there are two distinct proteins to which the antibody reacted

Germination and sporulation of Bacillus subtilis involve a temporally regulated set of changes in response to a variety of signals which produce changes in growth rate and DNA replication. Sporulating cells have a decreased rate of cell growth and DNA replication, while germinating spores have a sudden increased rate of growth and DNA replication compared with the dormant spore. The exact mechanisms that repress or derepress the rate of growth and DNA replication are not known. Here, we investigate the pattern of temporal expression of one protein that may be involved in these control phenomena, a repressor of DNA replication that is membrane associated. Analysis of DNA-membrane complexes from B. subtilis have revealed membrane proteins that bind to the origin region of replication (for a recent review, see reference 2). One membrane protein of interest had an apparent molecular size of 64 kDa. The selective binding of this protein to the region of origin could be blocked by a specific antibody. Furthermore, in an in vitro replication system, in which specific anti-64-kDa-protein antibody was added, there was a significant enhancement of initiation of DNA replication (2, 15). Other antibodies to different membrane proteins did not inhibit or enhance initiation (3, 14). The enhancement suggested that the protein may act as a repressor of replication. It should be noted that the 64-kDa protein antibody used in *

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and that the PR protein is not identical to the pyruvate dehydrogenase subunit.

MATERIALS AND METHODS Bacterial strains and phages. A B. subtilis prototrophic strain, 1A2 (Bacillus Genetic Stock Center, Ohio State University, Columbus, Ohio), and a B. subtilis proteasedeficient mutant strain, DB104 (his nprE18 hprR2 AaprA3, kindly provided by R. H. Doi, University of California, Davis, Calif.), were used in these studies. The latter strain was a double mutant deficient in extracellular neutral (nprE) and serine proteases (aprA) (9). Echerichia coli E600 cells were used for infection with various AgtlO clones, including Sc2, Sc4 and an unrelated X CE6 control. SC2 and SC4 are S-complex clones containing the entire pyruvate dehyrogenase operon (6), kindly supplied by H. Hemila (Genesit Ltd., Helsinki, Finland). Growth conditions. BBL Microbiology Systems Penassay or Difco antibiotic no. 3 medium was used for germination experiments with B. subtilis. Germination and outgrowth were followed by the drop in optical density as described by Oishi et al. (19). A sporulation medium described by Gould (5) was used to purify spores. Stationary phase or vegetative cells were grown in a Spizizen minimal medium (22) and supplemented with 0.1% yeast extract. Nonsporulating cultures were obtained by twice plating cells on tryptose-bloodbased agar plates and inoculating Spizizen minimal medium. Cultures were induced to sporulate by the addition of 500 ,ug of decoyinine per ml (kindly provided by the Upjohn Co.) to the Spizizen minimal medium supplemented with following concentrations of minerals: 5 mM FeCl3, 2 mM MnCl2, 1 mM ZnCl2, and 0.7 mM CaCl2, at the end of exponential growth (25). Exponential growth was monitored by determining the optical density at 660 nm. The extent of sporulation was followed by the appropriate proportion of refractile spores observed microscopically or by heating serial dilutions of cell cultures at 80°C for 10 min and determining the number of surviving colonies on Difco AK agar plates. To express XgtlO clones, E. coli C600 cells were infected (Klett = 60 to 70) with the lambda clones by using a multiplicity of approximately 2 (-108). The cells were grown in NZC medium (per liter, 10 g of NZ amine A, 5 g of NaCl, 2 g of MgCl2 6H20, 0.1% Casamino Acids) supplemented with 0.2% maltose and 5 mM CaCl2. Infected cells were shaken vigorously and allowed to lyse, and equivalent amounts of protein were subjected to immunoblot procedures as described below. Preparation of extracts. Disrupted spores were obtained from spores harvested at 72 h. Cultures were grown in sporulation medium, and spores were purified by a method described by Vold (26), with the following modification: after the cells were lysed, spores were obtained by centrifugation at 10,000 x g and washed extensively with sterile distilled water to remove cell debris. They were then plated on Difco AK agar plates to obtain a viable spore count. The final suspension contained approximately 101l spores per ml which were stored in distilled water at 4°C. Spores were heated to 80°C for 10 min prior to disruption to remove any germinated spores and cells and then chilled to 0°C. They were washed in a lysis buffer (10 mM Tris-hydrochloride [pH 7.6], 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride [PMSF], 1 FiM leupeptin, 1 ,uM pepstatin) and disrupted by blending 5 g (wet weight) of spores with 4 volumes of 0.1-mm-diameter glass beads in a high-speed Sorvall omnimixer (Ivan Sorvall, Inc., Norwalk, Conn.) for 10 min at 0°C.

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The glass beads were removed by decantation, and the disrupted spore suspension was centrifuged at 10,000 x g to remove undisrupted spores and cell debris. The disrupted spore extract was analyzed for protein concentration by the bicinchoninic acid method of Pierce Chemical Co. A vegetative cell extract was obtained by centrifuging mid-log-phase cells at 30,000 x g for 10 min, resuspending pellets in lysis buffer, and washing the pellet once in the same buffer. Cells were treated with 0.3 mg of lysozyme per ml and subjected to a French press at 10,000 lb/in2 for 2 min to lyse the cells. The protein concentration was analyzed as stated above. Germinating spores were collected by centrifugation as described above, resuspended in the lysis buffer, treated with 0.3 mg of lysozyme per ml, and subjected to freezethaw three times to lyse the cells. The cell extract was filtered through a 0.2-,um-pore-size membrane filter to remove ungerminated spores and analyzed for protein content. Antibodies. Purified immunoglobin G fraction of rabbit antisera specific for a 64-kDa protein of B. subtilis was kindly donated by P. C. Tai. Another immunoglobin G fraction was prepared from the PR protein which was separated and partially purified on gradient sodium dodecyl sulfate (SDS)-polyacrylamide gels as described by Horiuchi et al. (7), except that the protein obtained for immunization was derived from the cytosol fraction. Cocalico Biologicals, Inc. (Reamstown, Pa.) carried out the immunization of rabbits and the purification of the immunoglobulin G fraction. This antibody preparation is called WEl to distinguish it from the first antibody preparation. Immunoblotting. After the concentration of each extract was adjusted to equivalent amounts of protein, it was heated to 90°C for 5 min, quickly cooled, and subjected to SDS-9% polyacrylamide gel electrophoresis by the methods of Laemmli (13). After electrophoresis, usually for 17 to 19 h at 40 V, the proteins were transferred electrophoretically at 20 V for 1 h at 10°C to nitrocellulose filters (0.45-,um pore size; Schleicher & Schuell). The filters were stained with Ponceau S to determine the efficiency of transfer and washed with distilled water to remove the stain. The filters were then probed with antibody specific for the PR protein, using BLOTTO (8) as a blocking agent. The presence of PR protein was determined by the color reaction of secondary antibody conjugated to peroxidase (Cappel Research Products). Immunoprecipitation. Prelabeled spores were prepared in sporulation medium in which 5 pLCi of [35S]methionine per ml (ICN Radiochemicals, Inc.) was added at the end of exponential growth, and the spores were harvested at 48 h. Extracts were obtained from germination of prelabeled spores as described above except that they were prepared in 1 ml of 0.5% Triton X-100 NET buffer (50 mM Trishydrochloride [pH 8.0], 5 mM EDTA, 400 mM NaCl) and incubated with anti-PR-protein antibody for 60 min. The immunocomplexes were absorbed by Panasorbin (Calbiochem Corp.) Staphylococcus aureus cells and centrifuged to pellet the complexes, and the pellets were washed three times with NET buffer. The proteins were analyzed by gel electrophoresis as described for immunoblots and were visualized by autoradiography. Measurements of rate of DNA synthesis. Germinating spores were pulsed for 1 min with 5 ,uCi of [3H]thymidine per ml (ICN Radiochemicals) at 10 min intervals. Incorporation was stopped by the addition of 0.2 N NaOH; the samples were then incubated at 80°C for 30 min, precipitated with

PUTATIVE PROTEIN REPRESSOR OF B. SUBTILIS DNA REPLICATION

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FIG. 1. Detection of the PR protein during various stages of B. subtilis growth cycle. Purified spores were harvested from 72-h cultures. A portion of the spores was disrupted as described in Materials and Methods, and a portion was used for germination. Germinated spores (107/ml) were prepared by heat activating the spores at 80°C for 10 min and inoculating sidearm flasks containing BBL Penassay medium. Germination was monitored by the change in optical density using a Klett-Summerson colorimeter, and the sample was harvested 30 min after the onset of germination. Vegetative cells were grown in BBL Penassay medium, growth was monitored by optical density, and the cells were harvested at mid-log phase. All extracts were prepared as described in Materials and Methods and adjusted to 2.5 mg of total protein per ml, and 250 pg of each sample per cell was loaded onto an SDS-9% polyacrylamide gel. The proteins were transferred to nitrocellulose and probed with anti-64-kDa-protein antibody obtained from P. C. Tai. The protein was visualized by a peroxidase reaction. Lanes: V, vegetative cells; S, disrupted spores; G, germinating spores.

10% (wt/vol) trichloroacetic acid, and analyzed for acidinsoluble radioactivity by liquid scintillation counting. RESULTS

Changes in levels of the PR protein during germination and possible identity to E2 subunit of pyruvate dehydrogenase complex. The relative amounts of the PR protein in B. subtilis strains in disrupted vegetative cells, spores, and germinating spores were compared. In all strains analyzed, there was a significant amount of the PR protein in vegetative cells and spores. However, the amount in germinating spores (30 min after the onset of germination) was dramatically reduced (Fig. 1). A larger complex (185 kDa, relative molecular size) containing the PR protein appeared in vegetative cell extracts. This complex was partially resistant to SDS dissociation. When a higher concentration (2% instead of 1%) of SDS was used in the electrophoresis sample buffer, the complex was not detected. These results were obtained by using antibody prepared by P. C. Tai. Recent findings of Hemila et al. (6) demonstrated that this same antibody reacted with an apparent 64-kDa protein that was identified as the E2 subunit of the pyruvate dehydrogenase complex. To determine whether the E2 subunit was related to the PR protein, several experiments were undertaken. First, the initial sequence of 12 amino acids from the N-terminal end of the PR protein purified as described previously (3) was determined by the Yale protein sequencing laboratory (New Haven, Conn.). This sequence was compared with that reported by Hemila et al. (6). The comparison (Fig. 2A) showed that no similarity

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FIG. 2. Comparison of PR protein and E2 subunit of the pyruvate dehydrogenase complex. (A) N-terminal amino acid sequence comparison between the PR protein and the E2 subunit. The former sequence was obtained by using the Applied Biosystems Model 470A Protein Peptide Sequencer at the Yale School of Medicine Protein and Nucleic Acid Chemistry facility. The latter sequence was reported by Hemila et al. (6). X indicates a residue which could not be assigned with certainty. (B) Western blot analysis of expression products from XgtlO clones with two different preparations of antibodies (immunoglobulin G fractions) against a 64-kDa protein. E. coli C600 cells were infected and lysed as described in Materials and Methods. As control, AgtlO and unrelated X CE6 were used; SC2 and SC4 are S-complex clones containing the entire pyruvate dehydrogenase operon (6). Samples were incubated with 1:1,000 dilutions of the respective antibodies (antibody I, Tai's 64-kDa antibody, and antibody II, WE1). Lanes M, prestained molecular weight markers (in thousands, as given on the right).

between the N-terminal sequences of either protein existed. Nor was there similarity to any sequence in the entire PDH operon (data not shown). Another antibody was prepared against the PR protein (as described in Materials and Methods). This new antibody preparation, called WE1, and the anti-64-kDa protein antibody preparation obtained from P. C. Tai were used in Western blot (immunoblot) experiments against the XgtlO clones containing the pyruvate dehydrogenase operon. The latter antibody preparation did, as expected from Hemila's results (6), react with the subunit produced in the lysate, but the new antibody, WE1, reacted only barely with clone SC4 and not at all with clone SC2 (Fig. 2B). When WEl antibody was tested in the in vitro synthesis assay previously described (2, 3, 14, 15), the WEl antibody had the same stimulatory effect on initiation of DNA replication as the P. C. Tai antibody (data not shown). All the rest of the experiments shown in this paper utilize the WEl

antibody. To analyze in more detail the decrease in PR protein levels shown in Fig. 1, samples were taken every 15 min during germination (Fig. 3A). The PR protein appeared to decrease at the onset of germination, reached a minimum at 30 min, and then increased. The DNA synthesis activity of the cultures was inversely

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FIG. 3. Changes in PR protein levels during germination and outgrowth correlated to DNA synthetic activity. (A) Immunoblot showing PR protein levels during germination. Purified spores were germinated as described for Fig. 1 and allowed to continue into outgrowth. Samples were treated with 0.1 M sodium cyanide to stop growth and lysed. The extracts were adjusted to 2.0 mg of total protein per ml, and 200 pg of each sample per cell was subjected to electrophoresis on an SDS-9% polyacrylamide gel and immunoblotted as described in Materials and Methods with WEl antibody. Lanes: 0 to 90, minutes after germination; 0, proteins extracted from disrupted spores; M, prestained molecular weight markers (in thousands as indicated on the right). (B) Densitometer scan of immunoblot panel (A) correlated with incorporation of [3H]thymidine during germination and outgrowth, as described in Materials and Methods. The incorporation of [3H]thymidine is depicted by the line graph. The densitometer scan of the immunoblot is depicted as a bar graph, and the percentage of PR protein was determined by the amount of PR protein in each lane compared with the percentage of PR protein in vegetative cells, which was valued at 100%.

correlated with the percentage of PR protein, as determined by densitometer scans of immunoblots (Fig. 3B). Since DNA replication is synchronous during the beginning of germination (19), it is possible to approximate the time of initiation of DNA replication by determining the sudden increase in the incorporation of [3H]thymidine. The lowest concentration of the protein occurred just prior to initiation of DNA replication, and the percentage of the PR protein did not increase significantly until after a round of initiation of replication had occurred. To determine if the decrease in levels of PR protein during germination was due to degradation, the PR protein was immunoprecipitated from samples of spores germinated in cold medium. The spores were prelabeled with 35[S]methionine as described in the legend of Fig. 4A. It can be seen that a significant amount of degradation products appears at 20 min after germination (Fig. 4A). A densitometer scan of this blot shows that the majority of the degradation is completed by 25 min (Fig. 4B). To further analyze the effects of protease activity on the levels of the PR protein, samples were taken from spores germinated in the presence of protease inhibitors (0.1 mM PMSF, 1 ,uM leupeptin, and 1 ,M pepstatin). Such inhibitors were added after 15 min of germination, at which time 90% of the spores had germinated. In a duplicate experiment, the inhibitors were washed off 30 min later. Figure 5 shows immunoblots of the experiments. It can be seen that protease inhibitors substantially reduce the degradation of the PR protein (Fig. 5, protease inhibitors-1). However, after removal of the protease inhibitors, the PR protein is subsequently degraded (see 60 min in Fig. 5, protease inhibitors2). To determine whether the addition of protease inhibitors could reveal further insight into the relationship between levels of the PR protein and DNA replication, the same experiment was performed as described in the legend to Fig. 5 except that the samples were pulsed with [3H]thymidine every 10 min (Fig. 6). It can be seen in Fig. 6A that in the

absence of protease inhibitors, the greatest reduction of the PR protein occurs just prior to initiation of replication (at 30 min). However, in the presence of protease inhibitors, there was (as shown in Fig. 6B) no significant decrease in PR protein, but more importantly, there was no significant incorporation of [3H]thymidine (no initiation of DNA replication). Finally, when the protease inhibitors were removed after 45 min of incubation, PR protein levels decreased 15 min later, and this decrease was followed by a burst of DNA replication as demonstrated by the sudden increase in incorporation of [3H]thymidine (Fig. 6C). It was observed that the PR protein levels increased during the burst of DNA synthesis activity (Fig. 6C). However, this increase occurred well after initiation of DNA replication and could reflect other

variables.

Dancer and Mandelstam (1) have demonstrated that PMSF is not toxic to growing or vegetative cells. Pepstatin and leupeptin also do not seem to interfere with growth (unpublished observations). However, their addition does delay cell division when added during germination (unpublished observations). Nevertheless, it was observed that cells recovered quite readily from the effects of protease inhibitors after removal. Even when the protease inhibitors were not removed, the cells eventually did overcome their effects. Thus, if the experiment is extended to 120 min, cells will begin to divide, as observed by an increase in optical density (unpublished observations). Changes in levels of the PR protein during sporulation. To continue the analysis of PR protein levels throughout the vegetative and sporulation cycles, experiments similar to those described in the legend to Fig. 3A were performed through logarithmic growth into stationary phase and sporulation. In an attempt to synchronize the cells to initiate sporulation, the drug decoyinine (Upjohn Co.) was added in the middle of exponential growth. It was found that the addition of minerals (5 mM FeCl3, 2 mM MnCl2, 1 mM ZnCl2, and 0.7 mM CaCl2) was also required for sporulation in Spizizen minimal medium (see Materials and Methods).


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FIG. 4. Degradation of the PR protein during germination. (A) Autoradiogram of radioactively labeled PR protein. Prelabeled spores were prepared as described in Materials and Methods and were then germinated in cold BBL Penassay medium supplemented with excess unlabeled methionine (50 ,ug/ml). The samples were taken at 5 min intervals after 15 min of germination, treated with 0.1 M sodium cyanide to stop growth, and lysed. The extracts were adjusted to 1.0 mg of total protein per ml, and 100 pg of each sample per cell was immunoprecipitated with WEl antibody, as described in Materials and Methods. (Prior to 15 min, not enough spores had germinated to produce 1.0 mg of total protein per ml.) Lanes: C, control did not contain antibody; 15 to 45, minutes after the onset of germination. Molecular weight markers (in thousands) are indicated on the left. (B) Densitometer scans of the immunoprecipitated PR protein. The percentage of protein was determined as described in the Fig. 3 legend. Conrda

When the minerals were not added, cells remained in the stationary phase for an extended period, with no evidence of sporulation (not shown). PR protein levels continued to increase throughout vegetative growth (V, which represents mid-log phase) and reached a peak at the end of exponential growth or the initiating of sporulation, designated To (Fig. 7A). Unexpectedly, the relative amount of PR protein decreased as sporulation continued (Fig. 7A, T4 to T0o). When cultures remained for an extended period in the stationary phase, PR protein levels did not decrease; only upon initiation of sporulation was a decrease in the PR protein observed (data not shown). The decoyinine samples showed results with respect to the PR protein levels similar to those of control samples in which no decoyinine was added (data not shown). Since the protein was shown to be present in disrupted mature spores, its unexpected disappearance from total-cell extracts during sporulation prompted us to determine whether it was present in developing forespores (Fig. 7B). Forespores were separated by a procedure of Panzer et al. (20), and as anticipated, the PR protein was detected in developing forespores; a smaller percentage of PR protein was present at T4 but increased at T6 as the forespore developed. However, the level does not seem to increase beyond T6. Thus, although the PR protein decreased in the mother-cell extract, it increased in the forespore for a period of time and then remained stable (T8). The percentage of PR protein in forespores was still much less than the amount previously seen at the end of exponential growth (TO). To determine if the PR protein was degraded in the mother cell during sporulation, experiments were conducted in which protease inhibitors were added at To. In addition, a protease mutant was analyzed for protease activ-

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FIG. 5. Immunoblots of PR protein from samples germinated in the presence of protease inhibitors. Spores were germinated as described for Fig. 3. Protease inhibitors (0.1 mM PMSF, 1 ,M leupeptin, 1 ,uM pepstatin) were added 15 min after the onset of germination, except to one set of samples to which no protease inhibitors were added (control). All samples were filtered at 45 min and resuspended in fresh medium to keep the conditions consistent. The protease inhibitors were added again at 45 min to the fresh medium for samples labeled Protease inhibitors-1. To samples labeled Protease inhibitors-2, the protease inhibitors were removed at 45 min by filtering the germinated cells, rinsing with fresh medium, and resuspending them in medium without protease inhibitors. Samples were lysed, and extracts with equivalent amounts of protein as described for Fig. 3 were taken for immunoblotting with WEl antibody at the time (in minutes) listed above each lane. Lane 0, proteins extracted from disrupted spores; lane M, prestained molecular weight markers in thousands as indicated on the left. Control, no protease inhibitors; protease inhibitors-1, protease inhibitors added at 15 min and maintained throughout the experiment; protease inhibitors-2, protease inhibitors added at 15 min and washed off at 45 min.

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500 p.g of decoyinine per ml at the end of exponential growth, as described in Materials and Methods. Samples were harvested at various times and immunoblotted with WEl antibody as described in Materials and Methods with equivalent amounts of protein as described in the legend to Fig. 3. Lanes: M, standard molecular markers (in thousands as indicated on the left); V, vegetative cells or cells harvested at mid-log phase; To, cells harvested at the end of exponential growth; T4 to T1o, cells harvested at 4, 6, 8, and 10 h, respectively, after the end of exponential growth. Lanes labeled Forespores were samples of cells harvested at T4, T6, T8, and T1o that were fractionated into extracts enriched for forespores as described by Panzer et al. (20).

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FIG. 6. Measurement of DNA synthesis and the level of PR protein without (A) or with (B) protease inhibitors and with protease inhibitors washed off at 45 min (C). Spores were germinated and treated as described for Fig. 5 except that the samples were also pulsed with [3H]thymidine every 10 min, as described in Materials and Methods. The incorporation of [3H]thymidine is depicted by the line graph showing the means and standard deviations of data points from several experiments with sample sizes of 5 (panel A) and 2 (panels B and C). Samples containing equivalent amounts of protein, as described in the legend to Fig. 3, were immunoblotted and scanned with a densitometer, the percentage of protein was determined, and the average and standard deviation of data points were calculated. The percentage of PR protein is depicted by stippled vertical bars.

ity in comparison to that of a wild-type control. Figure 8 shows that there was less decrease in the amount of PR protein levels in the presence of protease inhibitors, or when the protease mutant was examined, compared with that of the control. These results suggest that the PR protein in the mother cell was degraded during sporulation. Analysis of membrane and cytosolic forms of the PR protein

during germination and sporulation. The PR protein has both a cytosolic and a membrane form. It was of interest to determine which form was present during various stages of the developmental cycle. The only form found in dormant spores was the membrane-associated form (Fig. 9). The cytosolic form did not appear until 90 min after the onset of germination or after the first round of replication had been completed (compare Fig. 3B with Fig. 9). The membrane form continued to increase until the end of exponential growth and the initiation of sporulation (TO), while the cytosolic form first increased then decreased during this time period. During sporulation, the membrane form of the PR protein decreased while the cytosolic form of the protein increased slightly and was the only form seen in whole-cell extracts at T8. This suggests that the PR protein may be cycled off the membrane to be degraded in the mother cell during sporulation. DISCUSSION

During early stages of germination, there is reasonable synchronization which allows the study of the initiation of macromolecular synthesis in cells. The timing of protein, RNA, and DNA syntheses has been determined (4, 21, 23, 27). RNA synthesis occurs first, within minutes of onset of germination; protein synthesis follows 2 to 5 min later; and then DNA replication occurs about 30 to 60 min after the onset of germination. The precursors for protein synthesis are believed to be supplied by turnover of the proteins during germination and sporulation. This accounts for the massive degradation that has been observed during such stages (1, 4, 10, 21, 23). The massive degradation might, however, have another important function in addition to supplying amino acid precursors, namely, removing repressors of various


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16 8 12 2( TIMe (hour) FIG. 8. Effects of protease inhibitors on levels of PR protein during sporulation. Various strains of B. subtilis were induced to sporulate as described in the legend to Fig. 7. The treatment with protease inhibitors was the same as that described for Fig. 5; the inhibitors were added at To. Samples were subjected to immunoblotting as described in Materials and Methods with equivalent amounts of protein as described for Fig. 3. T-4, represents 4 h before the end of exponential growth. Designations of To, T4, and T8 are the same as those for Fig. 4. The densitometer scans of the immunoblotted PR protein are depicted by the following: control, 1A2 prototrophic strain (El); 1A2 strain in the presence of protease inhibitors (-); and DB104 protease mutant strain (0). The percentage of protein was determined by the amount of PR protein in each sample compared with the value at the end of exponential growth, which was set at 100%. 4

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metabolic processes. For example, with dormant spores, it might be expected that there are many repressors keeping metabolic activity to a minimum. The great increase in protease activity during germination could therefore be a global response to eliminate these repressors from the dormant spore to allow newly synthesized proteins to participate in cellular activities of the actively growing cell. The degradation of the PR protein just prior to a round of DNA replication during germination is consistent with this possibility.

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A point should be made concerning the determination of PR protein levels by immunotechniques. The decrease in protein levels as detected by interaction with antibody does not necessarily indicate degradation of the protein, since the antigenic sites may be blocked by a variety of modifications of the protein, by conformational changes of the protein, or by complexing with other proteins. However, the fact that the levels of PR protein remained high in samples containing protease inhibitors and in the protease mutant strain strongly suggests that the observed decreases are, indeed, due to degradation. An important consideration in this respect is the effect of protease inhibitors on the levels of PR protein and DNA replication. Not only do the protease inhibitors prevent the degradation of the PR protein, but initiation of DNA replication is delayed as well. Removal of the protease inhibitors allows degradation of the PR protein followed by DNA replication (Fig. 6C). It could be expected from these results that such protease inhibitors would ultimately be detrimental for cell growth because of the deleterious effects on DNA replication. However, as reported by others (1), the addition of protease inhibitors does not affect cell growth during the vegetative phase. One explanation is that the PR protein may function differently during vegetative growth than it does during germination. Also, it is possible that the PR protein is not the only component involved in the repressor effect. The effects of protease inhibitors on those other possible components are unknown. What can be stated definitively is that protease inhibitors prevent the degradation of PR protein during germination and that there is a direct correlation between this and a depression of DNA replication consistent with the repressor hypothesis. The turnover of proteins at sporulation has been explained by the depletion of nutrients at initiation of sporulation, which causes the bacterium to recycle its own components (11). It is unclear why the PR protein is also degraded at this point except that it may simply be part of this turnover. However, while the protein is decreasing in the mother cell, it appears to be increasing in the developing spore. These results could suggest that the PR protein is sequestered into the forespore. However, it should also be noted that this increase in PR protein levels may also be due to differential gene expression in the mother cell and the forespore.

0 Cytosol N *mlembrane

IL

: 60 0 .-

40

-

8

&

20_ 0

1

2

8

12

T0 T4 Time (hours) Sporulatlon Germination FIG. 9. Detection of the cytosolic and membrane forms of PR protein during the life cycle of B. subtilis. Spores were germinated as described in the legend to Fig. 1 and cell extracts were fractionated by a method described by Marty-Mazars et al. (17). The membrane and cytosol fractions were assayed for protein and subjected to immunoblotting as described in Materials and Methods using equivalent amounts of protein as described for Fig. 3. The samples were taken at various times after germination as listed on the bottom of the graph; zero time represents disrupted spores since none of the cells have germinated at this time. Cells were induced to sporulate as described for Fig. 4 and fractionated as described for Fig. 7. Cells were harvested at mid-log phase or 4 h before the end of exponential growth, T-4 (TO, T4, and T8 are the same as described in the legend to Fig. 7). The percentage of protein was determined as described for Fig. 8.

484


It appears that protease activity during germination is different from protease activity occurring during sporulation. While all the protease mutants tested demonstrated the same rate of PR protein degradation as wild-type strains during germination (data not shown), rates of PR protein degradation differed during sporulation between a protease mutant and wild-type strain, as shown in Fig. 8. Another observation which supports this difference in protease activity is the increase in the cytosolic form of PR protein during sporulation as it is being degraded (Fig. 9). The PR protein may be cycled off the membrane to be degraded during sporulation. However, no cytosolic form of the PR protein is seen during germination. Either the protein is degraded while in the membrane form or the protein is degraded much faster and cannot be seen in the cytosolic form during germination. These results suggest that specific proteases act differentially during germination and sporulation to affect the PR protein. The identity of the PR protein was questioned when a report by Hemila et al. (6) showed that an apparent 64-kDa protein was the E2 subunit of the pyruvate dehydrogenase complex. Since the results obtained in the present experiments originally utilized the same antibody preparation as that used by Hemila et al. (6) (P. C. Tai antibody), it became important to determine whether the PR protein was the same as the E2 subunit. On the basis of sequence and Western blot analyses and use of another antibody preparation (WE1) against the PR protein which did not react with the E2 subunit in the present experiments, it can be concluded that the PR protein is not identical to the E2 subunit. One explanation as to why the P. C. Tai antibody reacted with the E2 subunit and the WEl antibody did not could be that the former polyclonal antibody contained antibodies to both the E2 subunit and the PR protein, while the WEl polyclonal antibody did not. Another explanation is that the first antibody preparation recognized similar epitopes on the PR protein and the pyruvate dehydrogenase subunit, whereas the WEl antibody recognized a separate site unique to the PR protein. In either case, it is still puzzling that there was no antibody reaction to the E2 subunit by the first antibody observed for B. subtilis, especially during those instances in which the PR protein was extensively degraded. It could be that the reaction with the E2 subunit is weak and not detectable under the Western blot conditions used. In addition, it must be pointed out that the tests of the two antibody preparations were directed against proteins produced by AgtlO expression vector clones containing the pyruvate dehydrogenase operon in E. coli and not in B. subtilis (Fig. 2B). It is unlikely that the level of expression of this operon would be as great, even in B. subtilis, as it is in the X expression vectors used in E. coli. Nevertheless, it is important to point out that a relationship between the PR protein and other known proteins could conceivably exist. Commonality between two presumably unrelated functions may be of significance, as pointed out by Srere (24). For example, thioredoxin, a protein involved in a number of metabolic reactions, including ribonucleotide reductase activity, is also found to act as a subunit of bacteriophage T7 DNA polymerase (16, 18). Another enzyme, dihydrofolate reductase, related to thymidylate synthetase metabolism, has also been found to act as a structural subunit of the bacteriophage T4 base plate (12). In summary, based on the results described in this report, the temporal regulation of the PR protein during the developmental growth cycle is consistent with its action as a repressor for initiation of DNA replication. Thus, in dormant spores, in which metabolic activity is repressed, there are

J. BACTERIOL.

significant levels of the PR protein. However, during spore germination, when DNA synthesis must be initiated to allow the outgrowth of cells, levels of the PR protein decrease dramatically from the level present in dormant spores. As germination ceases and the cells enter the normal vegetative growth (in which the population is no longer synchronous in regard to DNA replication), the PR protein level remains constant when measured per cell, reflecting its role as a functional inhibitor of the cyclical DNA replication in individual cells. Finally, as the cells enter stationary phase and begin sporulation, PR protein levels increase in the forespore while decreasing in the mother cell, which will eventually lyse to release the mature dormant spore. The regulation of the PR replication-repressor protein during B. subtilis life cycle is very complex. It involves differential protease activity during germination and sporulation and also may involve compartmentalization of the protein into forespores or differential gene regulation in the mother cell and forespore during sporulation. A totally different mechanism that involves the cycling of the protein on and off the membrane may be operating during vegetative growth, as demonstrated by the changes in the levels of PR protein in the cytosol and membrane. Further studies as well as attempts to clone the PR protein are planned to explore these possibilities. ACKNOWLEDGMENTS

This work was supported by NSF grant DCB-894657 and U.S. Army grant 28090LS to W.F., by the Connecticut High Technology Scholarship to B.E.-W. and L. Mele, and by biomedical funds from Wesleyan University. Special thanks to Jose Tori and Astrid Wilkie for technical assistance. REFERENCES 1. Dancer, B. N., and J. Mandelstam. 1975. Production and possible function of serine protease during sporulation of Bacillus subtilis. J. Bacteriol. 121:406-410. 2. Firshein, W. 1989. Role of the DNA/membrane complex in procaryotic DNA replication. Annu. Rev. Microbiol. 43:89-120. 3. Firshein, W., J. Laffan, D. A. Kostyal, L. A. Mele, B. EidentWilkinson, A. M. McCabe, Z. Mei, M. P. Farrell, and I. L. Scolnik. 1990. Membrane associated replisomes of Bacillus subtilis and plasmid RK2, p. 83-111. In P. A. Srere, M. E. Jones, and C. K. Mathews (ed.), Structural and organizational aspects of metabolic regulation, vol. 133. Alan R. Liss, Inc., New York. 4. Garrick-Silversmith, L., and A. Torriani. 1973. Macromolecular synthesis during germination and outgrowth of Bacillus subtilis spores. J. Bacteriol. 114:507-516. 5. Gould, G. W. 1971. Methods for studying bacterial spores, p. 327-381. In J. B. Norris and D. W. Ribbons (ed.), Methods in microbiology, vol. 6A. Academic Press, Inc., New York. 6. Hemila, H., A. Palva, L. Paulin, S. Arvidson, and I. Palva. 1990. Secretory S complex of Bacillus subtilis: sequence analysis and identity to pyruvate dehydrogenase. J. Bacteriol. 172:50525063. 7. Horiuchi, S., D. Marty-Mazars, P. C. Tai, and B. D. Davis. 1983. Localization and quantitation of proteins characteristic of the complex membrane of Bacillus subtilis. J. Bacteriol. 154:12151221. 8. Johnson, D. A., J. W. Gautsch, J. R. Sportsman, and J. H. Eider. 1984. Improved technique utilizing nonfat dry milk for analysis of proteins and nucleic acids transferred to nitrocellulose. Gene Anal. Tech. 1:3-8. 9. Kawamura, F., and R. H. Doi. 1984. Construction of a Bacillus subtilis double mutant deficient in extracellular alkaline and neutral proteases. J. Bacteriol. 160:442-444. 10. Kohler, E., and G. Antranikian. 1989. Covalent modification of

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