Eur. J. Biochem. 79, 329-338 (1977)

Characterization of an Intracellular Protease Isolated from Bacillus thuringiensis Sporulating Cells and Able to Modify Homologous RNA Polymerase Marguerite-M. LECADET, Monique LESCOURRET, and Andre KLlER Institut de Recherche en Biologie Moleculaire, Centre National de la Recherche Scientifique et UniversitC Paris VII (Received January 24/May 17, 1977)

The level of intracellular proteolytic activities was measured in Bacillus thuringiensis sporulating cells and it is shown that the maximum is reached after 4 h from onset of sporulation ( t 4 ) . A purification procedure using cells harvested at t5 is reported, which leads to the isolation of an intracellular protease totally different from the extracellular enzymes and from another intracellular fraction detected in crude extracts. The homogeneity of the purified enzyme was verified using immunological techniques, electrophoretic migration and kinetics of thermodenaturation. Its molecular weight was estimated to b’e about 23 000. This enzyme was characterized as a seryl protease, totally inhibited by phenylmethylsulfonyl fluoride or EDTA and requiring Ca2+ ions for activity. From the esterolytic activity of the enzyme we deduce a chymotrypsin-like specificity. The pure protease was able to cleave specifically the p’ subunit of the form I1 of B. thuringiensis RNA polymerase purified from t5 sporulating cells. The vegetative holoenzyme and core enzyme appear much less susceptible to the action of the protease. Moreover the and c( subunits do not seem to be altered by similar treatments. The existence of a mechanism regulating the level of intracellular proteases during the sporulation phase is postulated. During the last several years many studies have been devoted to the characterization of proteases, especially of extracellular proteases, produced during the sporulation phase of Bacilli [l - 61, but no relationship has been clearly demonstrated between protease excretion and the differentiation process. Another important aspect concerns the existence of intracellular proteases and their possible role in the biochemical events associated with sporogenesis. The first characterization of such proteases was reported by Millet [7] in B. meguterium and by Reysset and Millet [S] in B. subtilis. In addition, Millet et ul. [9] have shown that the intracellular protease of B. meguterium was able to cleave in vitro a p subunit of B. subtilis RNA polymerase. It should be noted that the earlier studies of Losick et al. [10,11] and Maia et al. [IZ], on the modification of RNA polymerase subunits associated with sporulation, has focused the interest of workers on the problem of endocellular proteases. More recently it was demonstrated for B. subtilis that a protease was responsible Enzyme. DNA-dependent RNA polymerase or ribonucleoside triphosphate :RNA nucleotidyltransferase (EC 2.7.7.6).

for the artefactual degradation of the p subunit during the polymerase purification procedure [13,14]. Using another sporulating bacterium, B. tlzuringiensis, we have previously shown [15] the existence of two different RNA polymerases resulting from sequential modifications of the subunits and demonstrated that such modifications occur in vivo during the sporulating phase [16,17]. Consequently it was important to estimate the level of intracellular proteolytic activities in B. thuringiensis sporulating cells, in order to determine whether such modifications, especially the j‘ modification, can result from proteolysis in vivo. To this end we have tried to isolate an intracellular protease and to carry out cleavage of the p’ subunit in vitro. Until now, no study bearing on this problem has been described in the case of B. thuringiensis. Such an approach might also help to relate the production of proteases inside the cell to spore formation. In the present paper we report the purification and general properties of an intracellular seryl protease isolated from B. thuringiensis cells in the middle of the stationary phase.

330

MATERIALS AND METHODS Chemicals

Alumina (bacterial grade A-305) was purchased from Alcoa; dextran T-500 and Sephadex G-200 from Pharmacia ; poly(ethyleneglyco1) 6000 from Merck, DEAE-cellulose DE 52 (1.O mequiv/g) from Whatman, hydroxyapatite C from Clarkson, and disc-electrophoresis reagents from Eastman-Kodak. Dithiothreito1 (Clealand's reagent), Azocoll 50 - 100 mesh, Azocasein and phenylmethylsulfonyl fluoride were from Calbiochem. Substrates for esterase activity were from Sigma. All other chemicals were reagent grade. Casein hydrolysate and tryptone were purchased from Oxoid. Buffers

Buffer A contained: 20 mM Tris-HCI pH 7.4, 2 mM CaC12, 1 M NaCI. Buffer B contained: 20 mM Tris-HCI pH 7.4, 2 mM CaC12. Buffer C contained: 200 mM Tris-HC1 pH 7.4, 2 mM CaCI2. Buffer D contained: 50 mM Tris-HC1 pH 7.4, 1 mM CaCI2, 0.1 mM dithiothreitol. Buffer E contained: 20 mM Tris-HC1 pH 8.0, 1 mM CaCl2, 0.1 mM dithiothreitol, 10% (v/v) glycerol. Buffer F, 200 mM Tris-HC1 pH 7.4, 1 mM CaCI2, 0.1 mM dithiothreitol. Buffer G : 10 mM sodium phosphate pH 6.5, 0.1 mM dithiothreitol. Buffer H: 10 mM Tris-HCI pH 8.0, 1 mM CaC12,0.5 mM dithiothreitol, 0.1 mM EDTA, 10 mM MgCl2. Bucteriul Strain and Culture Conditions B. thuringiensis Berliner 1715, wild-type strain (serotype I) was from the Pasteur Institute Collection. Cultures were grown as previously described [15,18]. Sporulating cells were harvested at given times after the to of the sporulation, which is defined as the moment when growth ceases to be exponential, and washed successively with buffers A and B, according to Reysset and Millet [S] in order to eliminate extracellular proteases. Preparation of Crude Extracts Throughout Sporogenesis

For kinetic studies of intracellular proteolytic activity during sporogenesis, 100-ml aliquots of culture were harvested at l-h intervals and crude extracts were prepared as previously reported [17]. At the same time aliquots of culture supernatants were kept for the estimation of extracellular proteolytic activities. Enzyme Assuys

Proteolytic activity was measured with two subtrates: Azocoll and Azocasein. For the Azocoll assay [19] the reaction mixture contained: 10 mg Azocoll

Intracellular Protease of B. thuvingiensis and RNA Polymerase

in buffer C and aliquots of extracts in a final volume of 2 ml. After a 60-min incubation at 37 "C, the mixture was filtered and the absorbance determined at 520 nm. The Azocasein assay was carried out as previously described [17]. Units of activity were expressed as pg Azocoll or Azocasein hydrolized/min at 37 "C. Purification of Intracellular Proteases

All operations were carried out at 0-4 "C. Step I : Preparation of the Crude Extract. 100 g sporulating (t s )cells, exhaustively washed, were ground with 230 g alumina for 30 min and extracted with 250 ml buffer C. After centrifugation for 20 min at 10000 x g , the pellet was re-extracted with 150 ml of the same buffer and centrifuged again. The supernatants (fraction P1) were pooled and submitted either to streptomycin sulfate treatment or to phase partition. Step 2: Streptomycin Sulfate Treatment and Ammonium Sulfate Fractionation. Precipitation of nucleic acids was carried out by adding streptomycin sulfate (1 mg/mg protein) to the crude extract. After 1 h at 4 "C with mild stirring, the preparation was centrifuged 15 min at 12000 x g. The resulting supernatant was treated with deoxyribonuclease (20 pg/ml) at 4 "C for 20 min and then precipitated with ammonium sulfate. The fraction precipitating between 40 and 75 "/, saturation contains the proteolytic activity. Phase Partition and Ammonium Sulfate Fractionation. Nucleic acids can be more efficiently eliminated with the phase partition procedure initially reported by Babinet [20]. The crude extract was treated according to an adaptation of the method previously described for the purification of RNA polymerase [ 151 except that proteases were recovered in the first poly(ethyleneglyco1) phase. The poly(ethyleneglyco1) fraction was dialysed against buffer D, and then submitted to ammonium sulfate fractionation as reported [15]. Step 3: DEAE-Cellulose Chromatography. Fraction P2 (80 ml) resulting from ammonium sulfate fractionation was dialysed 3 h against two changes 2 I buffer E, then diluted to 300 ml and passed through a DEAE-cellulose column (3.5 cm x 20 cm) freshly equilibrated with the same buffer. After washing the column with 50 ml buffer E, a linear gradient (600 ml) from 0 to 0.5 M NaCl was started. The active fractions eluted with the gradient were pooled and precipitated with ammonium sulfate at 75 "/, saturation ; the resulting precipitate was solubilized in buffer F to give the fraction P3 (20 ml). Step 4 :Sephadex G-200 Chromatography.Fraction P3, divided into two parts, was filtered through a Sephadex G-200 column (1.6cm x 50 cm) equilibrated

331

M.-M. Lecadet, M. Lescourret, and A. Klier

with buffer F. The active fractions were pooled (fraction P4) and dialysed overnight against two changes of 2 1 of buffer G. Step 5 : Hydroxyapatite Chromatography. Fraction P4 was passed through a column of hydroxyapatite C (1.2cm x 10cm) equilibrated with buffer G. The column was washed with 30 ml buffer G, then elution was carried out by steps of increasing phosphate concentration 0.05 M, 0.07 M, 0.1 M, 0.2 M and 0.4 M. Active fractions were pooled (P5) and kept frozen at - 22 "C. Polyacrylamide Gel Electrophoresis

The intracellular proteases can be further purified by electrophoresis on polyacrylamide gels. The disc gels containing 7 % acrylamide were prepared and run according to Ornstein [22] and then cut in 1.5-mm slices. In order to localize the activity, each slice was ground in 0.5 ml buffer C and incubated with 5 mg Azocoll at 37 "C,during at least 2 h. Gels (6.3 or 7.5 % acrylamide) containing sodium dodecyl sulfate (0.1 %) were prepared and run as described by Kamen et al. [23] using discontinuous Tris buffer solutions.

Controls without protease or with protease but in the presence of phenylmethylsulfonyl fluoride were run in the same way. After incubation at either 23 "C or 37 "C for given times (generally 40 min and 10 min respectively) the reaction was stopped by the addition of phenylmethylsulfonyl fluoride (2 mM final) and glycerol (10% final) followed by rapid cooling. Then the different mixtures were dialysed overnight at 4 "C against distilled water containing 0.05 mM phenylmethylsulfonyl fluoride and then lyophilized and submitted to sodium dodecyl sulfate gel electrophoresis as described above. After staining, the gels were scanned at 515 nm using a Gilford spectrophotometer recorder. RESULTS Kinetics of Proteases Accumulation

Fig.1 shows the evolution of the level of the intracellular proteolytic activities measured with the two substrates in crude extracts prepared thoughout sporogenesis. As in the case of extracellular proteases [18,26] we observe an increasing activity between to and t3, especially with Azocoll, the maximum activity being reached around t3- t4; in contrast hydrolysis of Azocasein did not increase in the same proportion and remains at a low level after tz; as a consequence, the ratio of the two activities Azocoll/ Azocasein increases from 0.85 to 8.0 between to and t3.

Immunology

The preparation of globulins against the different enzymatic fractions was performed as previously described [ 161. Immunoprecipitation, using the double-diffusion technique in agarose gel, was carried out either in Ouchterlony plates or after electrophoresis according to Grabar and William [24]. In some cases activity at the level of the precipitin lines was detected according to Uriel and Avrameas [25] using acetyltyrosine ethyl ester as a substrate. After washing for two days in 0.15 M NaC1, the plates were partially dried and then recovered with agarose solution containing the substrate and neutral red as an indicator. Along the precipitin lines possessing the enzymatic activity the coloured indicator turns from yellow to red [25]. Modfieation of RNA Polymerase in vitro

Purified vegetative RNA polymerase and the sporulation form I1 RNA polymerase were obtained as previously described [15]. The preparation of intracellular proteases used for the reaction with RNA polymerase in vitro is from the hydroxyapatite chromatography followed by an acrylamide electrophoresis step. The reaction mixture contained: 100 pl RNA polymerase (5070 pg, about 30 units), 10 Azocoll units of protease in a final volume adjusted to 600 pl with buffer H.

E 0 2 100

m

2.0

0 Time from start of sporulation, f (h)

Fig. 1. Kineiics of intracellular proteases accumulation in B. thuringiensis sporulating cells. Proteolytic activity was measured with or Azocasein (0-4) as substrates and Azocoll (t-) expressed as units/mg proteins in crude extracts. Other symbols: (04) ratio of the two activities Azocoll/Azocasein; ( x x) absorbance of the cultures measured at 650nm using a Zeiss PMQ2 spectrophotometer ; (A----A) heat-resistant spores (80 "C, 10 min) ~~

Intracellular Protease of B. thuringiensis and RNA Polymerase

332

Table 1. Purification procedure of an intracellular protease of B. thuringiensis The amounts indicated refer to the purification of 100 g cells. The activity was measured with Azocoll as a substrate. The final steps 4 and 5 were performed on the peak I1 fraction resulting from step 3 Fraction

AzsoIAz60

Step 1: Crude extract Step 2 : Streptomycin sulfate treatment Step 3: DEAE-cellulose Peak I Peak I1 Step 4: Sephadex G-200 Step 5: Hydroxyapatitc

0.6 0.8

Total protein

Total activity

Specific activity

Purification

mg

U

U/mg protein

-fold

4775 1620

778 500 646 SO0

150 417

43 000 445 so0 270000 170 300

210 1332 2 520 23 500

205 334 107 7.2

0.96 1.72 1.9

B

A

Fig. 2. Acrylamide gel electrophoresis of the purified intracellular protease resulting,from the hydroxyupatite chromatography. (A) Gel obtained with 30 pg native purified enzyme ( R F 0.8). (B) Gel obtained with 30 pg denatured enzyme run in 7.5 acrylamide gels containing 0.1 sodium dodecyl sulfate, according to Kamen et al. ~ 3 1

x

Such results might suggest not only qualitative changes as regards the nature of the protease, but also a modulation of the intracellular activities in the first hours of the stationary phase. In this connection other results, namely an increase of the global activities in crude extracts observed after prolonged dialysis or after dilution of the extracts, might be explained by the presence of an inhibitor allowing a regulation of the level of proteases inside the cell.

Purijication of an Intracellular Protease Until now, no purification of intracellular proteases from B. thuringiensis has been reported, so we used

2.75

9

17 156

Yield

x 83

5.5 57 34 21

the first step of a purification procedure previously described for B. subtilis [8] and B. megaterium [7]. Two final steps were added : gel filtration on Sephadex G-200 and hydroxyapatite chromatography. Table 1 summarizes the different steps of the purification. As can be seen, the partial elimination of nucleic acids by streptomycin sulfate precipitation gave a good yield of the global activity, but was not efficient with regard to the ratio of absorbances at 280 nm and 260nm. Another more efficient process may be used instead : viz. the phase partition system followed by ammonium sulfate fractionation, but the yield in this case was less than 50%. The subsequent steps of the purification gave the same results whatever the technique chosen for step 2. On DEAE-cellulose chromatography the major part of the activity (peak 11)was eluted at 0.25- 0.3 M NaCl while a lower but significant activity was observed in the flowthrough of the column (peak I). The further purification was carried out with the peak I1 fraction; the nature of the peak I activity will be discussed later. After a gel filtration on Sephadex G-200, the active fractions retained on the gel were pooled and submitted to hydroxyapatite chromatography. The major part of the proteolytic activity was eluted at 0.05 M phosphate. A low affinity of the protease for hydroxyapatite might explain the trailing edge of the peak observed in some cases. As seen in Table 1, a high factor of purification was obtained after the last step; moreover the final yield was rather satisfactory as compared with results of partial purification of intracellular proteases reported for B. subtilis [8] or E. coli [27].

Analysis of the Purified Protease Fig. 2A shows disc gel electrophoresis carried out at the end of the purification. It has been verified that the single major band (which represents more than 90% of total protein as seen by scanning of the gel) corresponds precisely ( R F 0.80) to the proteolytic

333

M.-M. Lecadet, M. Lescourret, and A. Klier

A

I

I-I '

I

0

' II

6

Fig. 3. Immunoelectrophoresis ojthe purified intracellulurproteaseand localization of activity. (A) Plates showing immunodiffusion after electrophoresis of the antigen. The upper trench contained globulins against the purified fraction, the lower trench contained globulins against the step 4 fraction. (B) Pattern of the precipitin lines after revelation of the enzymatic activity using acetyltyrosine ethyl ester as described in the Methods

activity estimated after slicing of the gel. The localization of activity was exactly the same when the gels were loaded with fractions corresponding to the preceding steps of the purification. It should be noted that the localization of the proteolytic activity corresponding to peak I of the DEAE-cellulose chromatography was quite different (RFless than 0.1 similar to the migration of extracellular proteases). Immunological characterization provides further proof of the purity of the preparation. In Fig. 3 A the pattern of immunoelectrophoresis on agarose gel is shown; only one line of precipitation was observed with globulins against purified enzyme. The same line and another very faint one were seen with globulins against the step 4 fraction. As shown in Fig.3B the main line corresponds to the active enzyme revealed with specific substrates. These results suggest that the purified preparation contains essentially one protease. In order to determine the relationship between this enzyme and other fractions (peak I of the DEAEcellulose chromatography and extracellular proteases), immunodiffusion experiments were undertaken using globulins against the purified intracellular fraction (Fig.4). Fig.4A shows that these globulins do not react with the peak I fraction or with the extracellular fraction. On the other hand, it appears that these two last preparations present cross-reacting materials but only partial homology, when compared in an experiment using globulins against the peak I fraction (Fig. 4B). From these results we can reasonably conclude that our purified intracellular protease differs from the

extracellular enzymes and from the other proteolytic fraction of the intracellular extract. Properties of the Purified Intracellular Fractions Thermostability. In order to obtain more accurate indications with regard to the purity of our preparations the kinetics of thermal inactivation at 62 "C were determined for two different preparations corresponding to the last steps of purification (4 and 5). From the results given in Fig. 5 we conclude: (a) the rate of inactivation was similar for the two preparations and followed first-order reaction kinetics ; (b) the activities with the two substrates, Azocoll and Azocasein, decreased at the same rate, thus indicating that in both preparations a unique molecular species was responsible for the two activities. Molecular Weight. Preliminary indications concerning the molecular weight of the native protease may be deduced from the localization of the activity after chromatography on Sephadex G-200, as compared with proteins of known molecular weights. The elution pattern is identical to that of trypsin, suggesting a molecular weight of about 24000. However, it must be borne in mind that proteolytic selfdegradation can occur, so the validity of the results obtained by such techniques has recently been questioned [28]. Another approach, based on acrylamide gel electrophoresis under dissociating conditions, was used (Fig.2B). A major band was observed; comparison with known protein markers run under the same conditions indicates a molecular weight of

334

Intracellular Protease of B. rhuringiensis and RNA Polymerase

A

B

Fig. 4. Relationships between the extracellular fraction and the different intracellular fractions as revealed by immunodQ'jusion reactions. (A) In the center well: globulins against the purified intracellular fractions. (1 and 4) Intracellular fraction peak I of the DEAE-cellulose chromatography; (2 and 5 ) partially purified intracellular protease step 3; (3 and 6) extracellular fraction. The extracellular fraction corresponds to partially purified preparation of extracellular proteases obtained after two successive ammonium sulfate fractionations according to Leighton et al. [21]. (B) In the center well : globulins against the intracellular fraction peak I. (1 and 3) Homologous fraction peak I ; (2 and 4) extracellular fraction

150 I

I

I

I

I

I

I

30

40

B

"0

10

20

30 40 0 10 20 Preincubation time (min)

Fig. 5. Kinetics ojthermal inactivation qftwo preparations (step 4 in A , step 5 in B ) of the intracellular protease. Aliquots of enzyme preparations were diluted in buffer C, in prewarmed tubes, to give about 100 pg protein/ml and then heated at 62 "C. At intervals between 5 and 60 min, samples were withdrawn, cooled in ice and O), or Azocasein then assayed for activity with: Azocoll (e-~ (A-A) as substrates

23000. Recently a molecular weight of 21000 was reported for protease I from E. coli [31]. The results suggest that the purified intracellular protease consists of a unique polypeptide chain. Esterolytic Activity and Specijkity. As for many proteolytic enzymes the range of pH for optimal activity was between pH 7.5 and pH 8.0. No activity could be detected using a variety of synthetic substrates (substituted dipeptides) commonly employed for the

determination of specificity. However, a weak but significant esterolytic activity was observed with several substituted esters (Table 2). The specificity is of the same order as that of chymotrypsin. The different results, particularly the absence of hydrolysis of short peptides and of some proteins, strongly suggest that the purified protease has a requirement for a minimum peptide size and probably manifests a restricted specificity. Action of Inhibitors. In order to clarify the nature of this protease we have examined the effects of classical inhibitors on its activity. Results are summarized in Table 3. We observe a total inhibition of the activity by phenylmethylsulfonyl fluoride added at concentrations higher than 0.5 mM, whatever the step of the purification, thus allowing us to consider the enzyme a seryl protease. The addition of EDTA at a concentration higher than the usual concentration of calcium ions (2 mM) in the buffers completely abolished the activity, thus suggesting a catalytic role of Ca2+ in the enzymatic reaction. In addition, results given in Table 4 confirm that Ca2+ ions are necessary for the activity, but do not appear essential for the stability of the enzyme. Moreover no detectable activity was found when EDTA (1 mM) and phenylmethylsulfonyl fluoride (0.5 mM) were added together in a buffer deprived of Ca2+ ions.

Modification of R N A Polymerase by the Purgied Protease in vitro In line with our initial purpose we examined the effect of the purified seryl protease on different

335

M.-M. Lecadet, M. Lescourret, and A. Klier Table 2. Esterolytic activity of the purfied intracellular protease of B. thuringiensis The esterolytic activity was measured with two preparations corresponding to step 4 and step 5 of the purification procedure. The assay was carried out according to Martin et al. [30]. One unit of activity corresponds to the hydrolysis of 1 pmol of substrate per min at 25 "C. Z = cdrbobenzoxy; ONp = p-nitrophenyl ester Specific activity

Substrate

step 5

step 4 U/mg protein

0.26 0.15 0

Z-Tyr-ONp Z-Phe-ONp Z-Lysz-ONp

4.20 1.08 0

Table 3. Effect of inhibitors on the proteolytic activity at different steps ofthe purification The activity was measured with Azocoll as substrate in the presence of the various inhibitor concentrations. In (d) the assay buffer contained the usual concentrations (2 mM) of CaZ+ ions; in (b) Ca2+was omitted in the buffer. Results are given as a percentage of the initial activity of the preparation Inhibitor

Concn

Residual activity

mM

step 1

Phenylmethylsulphonyl fluoride 0.5 1 2 EDTA

step 4

step 5