JOURNAL OF BACTERIOLOGY, Sept. 1975, p. 806-814 Copyright 0 1975 American Society for Microbiology
Vol. 123, No. 3 Printed in U.S.A.
Evidence for Extrusion of Unfolded Extracellular Enzyme Polypeptide Chains Through Membranes of Bacillus amyloliquefaciens R. L. SANDERS* AND B. K. MAY Department of Biochemistry, University of Adelaide, South Australia, 5001 Received for publication 21 April 1975
The production of extracellular a-amylase and protease by protoplasts of Bacillus amyloliquefaciens has been achieved. The production of enzymically active protease was totally dependent on a high concentration of either Mg'+, Ca2+, or spermidine, but production of active a-amylase was not. This cation dependence of protease production was seen immediately upon addition of lysozyme to intact cells. The cations could prevent the inactivation of protease and alter the cytoplasmic membrane configuration of protoplasts. Production of active a-amylase and protease by protoplasts was totally inhibited by proteolytic enzymes such as trypsin, a-chymotrypsin, or the organism's purified extracellular protease. The evidence suggests that these degradative enzymes act specifically on the emerging polypeptide of the extracellular enzyme and that the polypeptide emerges in a conformation different from that of the native molecule.
We are examining the synthesis and secretion of extracellular enzymes by Bacillus amyloliquefaciens as a model system for investigating how some proteins are selectively secreted through cellular membranes. This organism has the advantage that washed-cell suspensions rapidly produce large amounts of extracellular protease, a-amylase, and ribonuclease. No significant amounts of active protease, a-amylase, or inactive cross-reacting material can be detected inside secreting cells of B. amyloliquefaciens (4, 11). In addition, there exists in the cytoplasm an inhibitor specific for the extracellular ribonuclease (17), and since the formation of the enzyme-inhibitor complex is essentially irreversible (9), it seems unlikely that the native enzyme could ever have existed as such within the cell. We have therefore suggested that extracellular enzymes are synthesized on membrane-associated ribosomes and that the nascent polypeptides are extruded through the membrane to assume their active tertiary configuration only outside the permeability barrier (12). Compatible with this model are our findings, which suggest that within the cell there are pools of extracellular enzyme-specific messenger ribonucleic acid (2, 7). We have suggested that these apparent pools are the result of excessive transcription, possibly designed to saturate the membrane "translational-extrusion" sites with messenger ribonucleic acid
despite its rapid degradation en route from gene to membrane (6). Attempts to directly test the extrusion hypothesis in our system have been difficult because of the apparent inability of protoplasts of this organism to secrete active extracellular enzymes, even though intracellular protein and ribonucleic acid synthesis continue almost normally (12). However, it has now been shown that in the presence of sufficient concentrations of either Mg2+, Ca2+, or spermidine, protoplasts will secrete enzymatically active protease and a-amylase. During secretion these extracellular enzymes exist in a form that is sensitive to proteolytic attack, whereas the released enzymes are insensitive. This finding is compatible with our proposed model for enzyme secretion. A preliminary report of this work has appeared previously (16).
806
MATERIALS AND METHODS Organism used. The parent strain of the organism used in this work was an unclassified strain of Bacillus amyloliquefaciens (11). This strain produces an extracellular peptide-lipid molecule, "surfactin," that lyses protoplasts (13). A mutant unable to produce this compound was isolated as follows and used in the present work. Approximately 106 spores in 1 ml of glass-distilled water were treated with 0.1 ml of ethyl methyl sulfonate for 20 min at 37 C. The spores were washed once by centrifugation and suspension in 5 ml of 0.9% NaCl and were finally suspended in 1 ml of 0.9% NaCl. This suspension (0.1 ml) was used to
VOL. 123, 1975
inoculate 500 ml of culture medium (2), and the culture was shaken at 30 C for 25 h. Cultures were plated for single colonies on blood agar plates and incubated for 16 h at 37 C. Those colonies which did not produce a hemolyzed zone were picked off and purified by subculturing four times. The mutant strain used was cultured as described previously (2); cultures did not produce any lytic factor detectable by protoplast lysis (13), whereas the production of extracellular a-amylase and protease was normal compared with wild type. Washed-cell and protoplast suspension experiments. PMC medium contained tris(hydroxymethyl)aminomethane (Tris) (25 mM), (NH4)JHPO, (3.8 mM), KCl (5 mM), sodium citrate (4.25 mM), CaCl, (0.125 mM), ZnSO4 (0.0125 mM), 0.025% (wt/vol) Casamino Acids (Difco), 0.25 ml of trace metal solution (2) per liter, 1% maltose, and 22% (wt/vol) sucrose adjusted to pH 7.3 with HCI. Cells after 25 h of growth were harvested, washed once with PMC medium lacking sucrose, and finally suspended in the same volume of PMC medium. Protoplasts were prepared by shaking washed-cell suspensions at 30 C for 45 min with 133 jig of lysozyme per ml of cells. In some experiments, samples (0.8 ml) were removed at various times during protoplast formation and centrifuged, and the supernatants were assayed for a-amylase and protease activity. In other experiments, protoplasts were resuspended in fresh PMC media and further incubated at 30 C with shaking. Samples were withdrawn at appropriate times and centrifuged, and the supernatants were assayed for enzyme activity. Ribonuclease could not be measured in these experiments because of inhibitor released by lysis of a small proportion of protoplasts (17). Assay of enzymes. Protease activity was assayed by using a Remazol brilliant blue-hide powder assay (15). One unit of activity is defined as the amount of enzyme giving an increase in absorbance at 595 nm of 5.7 in 40 min at 37 C and corresponds to the unit defined earlier (11). a-Amylase was assayed as previously described (2). Measurement of total protein synthesis. The incorporation of L- ["C ]phenylalanine (460 mCi/ mmol) into total trichloroacetic acid-precipitable material was measured as described earlier (2). Total extracellular protein represents only about 5% of the total cellular trichloroacetic acid-precipitable material. Preparation of antibodies. B. amyloliquefaciens extracellular protease was purified to homogeneity as described earlier (2), and its antibody was prepared by inoculating New Zealand rabbits with weekly, multisite, subcutaneous injections of protease in complete Freund adjuvant. Rabbit antisera were fractionated by precipitation with 50% (wt/vol) ammonium sulfate, followed by dialysis of the precipitate to remove salt and fractionation on diethylaminoethylcellulose columns. The gamma globulin fractions were pooled and concentrated by using aquacide. Goat anti-rabbit gamma globulin was obtained by injecting goats with normal rabbit gamma globulin. Inactivation of trypsin and a-chymotrypsin. Trypsin (or a-chymotrypsin) in cell or protoplast
PROTEINS THROUGH MEMBRANES
807
supernatants was inactivated before the determination of B. amyloliquefaciens protease activity. Trypsin was inactivated by incubation for 4 h at 30 C with 1-chloro-3-tosylamide-7-amino-2-heptanone hydrochloride (TLCK) at a final concentration of 30 jig/ml. For a-chymotrypsin, L-1-tosylamide-2-phenylethyl chloromethyl ketone (TPCK) at 10 jig/ml was used (added as a methanolic solution of 1 mg/ml) and incubated at 30 C for 5 h. These procedures completely inactivated both enzymes but did not affect B. amyloliquefaciens protease. Polyacrylamide gel electrophoresis. Electrophoresis of protease was carried out as described earlier (2). To detect ptotease and 3H radioactivity, gels were frozen in solid CO2 and sliced longitudinally, and corresponding halves were cut into 1-mm slices. The enzyme was eluted overnight from the slices with 1.5 ml of 50 mM Tris-hydrochloride (pH 7.8), and the eluate was assayed for protease. Radioactivity was determined by dissolving gel slices for 16 h at 20 C in 2.5 ml of a solution of 0.3% 2,5-diphenyloxazole and 0.03% 1,4-bis-[2]-(4-methyl phenyloxazolyl)-benzene, 12% (vol/vol) NCS tissue solubilizer (Amersham/ Searle Corp.), and 0.08 M NH4OH, followed by liquid scintillation counting in a Packard Tri-Carb spectrometer. Covalent coupling of a-chymotrypsin to Sepharose 4B beads. a-Chymotrypsin was coupled to cyanogen bromide-activated Sepharose 4B in 0.1 M NaHCO,, and after coupling was completed the product was extensively washed with six cycles each of 0.1 M sodium acetate (pH 4.0) and 0.2 M NaHCO, (pH 8.5), each containing 1 M NaCl. The product was stored in distilled water with 0.1% (wt/vol) sodium azide and thoroughly washed before use. Using a casein assay (11), it was established that each 1.0 ml of the settled beads possessed proteolytic activity equivalent to 2.5 mg of free a-chymotrypsin. Materials. Radiochemicals were obtained from Schwarz/Mann. TLCK and TPCK were both purchased from Cyclo Chemical Corp., Los Angeles, Calif. TPCK-treated trypsin and TLCK-treated achymotrypsin were purchased from Worthington Biochemical Corp., Freehold, N.J. Chloramphenicol was a product of Parke-Davis and Co., Sydney. Egg white lysozyme, three times recrystallized, was a product of Sigma Chemical Co., St. Louis, Mo. The Remazol brilliant blue was a generous gift from Farbwerke Hoechst, AG, Frankfurt.
RESULTS Effect of lysozyme on extracellular enzyme secretion by washed-cell suspensions. Washed-cell suspensions were incubated in a 22% (wt/vol) sucrose medium (PMC medium) containing 1 mM Mg2+ (which is optimal for extracellular enzyme production by intact cells), and the effect of lysozyme on enzyme secretion was examined. Production of active protease was inhibited almost instantly (Fig. 1). The first protoplasts were not visible until 20 min, and electron microscopic examination
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SANDERS AND MAY
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showed that complete cell wall removal required 45 min of incubation. The inhibitory effect of lysozyme was reduced by about 50% when the medium contained 10 mM Mg2+ (Fig. 1). Although Mg2+ did have some effect on general protein synthesis (Fig. 2), incorporation of ["'Ciphenylalanine into total protein in the presence of lysozyme was not dependent on 10 mM Mg2+, as was protease synthesis. The selectivity of the Mg2+ effect for protease synthesis in the presence of lysozyme was further reinforced by the observation that extracellular
a-amylase synthesis after lysozyme addition the same in 1 or in 10 mM Mg2+. The inhibitory effect of lysozyme on protease production by cells in 1 mM Mg2+ was immediately alleviated by adding Mg2+ to 10 mM at was
any time up to at least 45 min. Similar results obtained with Ca2+ or spermidine. To test the possibility that cells treated with lysozyme in 1 mM Mg2+ secreted an inactive form of the protease molecule, a cell suspension (8 ml) was incubated in PMC medium containing 1 mM Mg2+ and lysozyme for 8 min, at which time 5 ltCi of L- [C "4]phenylalanine (460 mCi/mmol) and 15 gCi of L-[14CJleucine (312 mCi/mmol) were added. A control suspension did not contain lysozyme. Samples were taken at 8 min and again after 40 min of incubation and centrifuged, and the supernatants were assayed for material that cross-reacted with protease gamma globulin as described (8). The results suggest that lysozyme-treated cells in 1 mM Mg2+ produce a form of the protease molecule capable of reacting with protease gamma globulin (Table 1). Effect of cations on protease and a-amylase were
production by protoplasts. Protoplasts were incubated with shaking in PMC medium containing 10 mM Mg2+, and the production of protease was compared with that by intact cells
under conditi6ns identical except for the omission of lysozyme. The rate of synthesis of protease by protoplasts was about 30% of that of Incubation time (mm) intact cells, and production of enzyme was FIG. 1. Effect of Mg2+ concentration on production chloramphenicol sensitive (Fig. 3). (Separate of active protease by lysozyme-treated cell suspensions. Cells were incubated with lysozyme (added at experiments showed that the enzyme produced zero time) in the presence of I or 10 mM Mg2+ or without lysozyme in the presence of either
(O, A) 1
or
10
mM Mg,+ (O, 0). 3.
TABLE 1. Precipitation by protease gamma globulin of radioactively labeled material secreted by cells incubated with and without lysozyme in the presence of 1 mM Mg2+ Radioactivity precipitateda
0)
.2.
Cells
Time of incubation Net 8 min
Lysozyme treatedb Control .......... I,
Incubation time (min)
FIG. 2. Effect of Mg2+ concentration on the incorporation of L-["4CJphenylalanine into trichloroacetic acid-precipitable material by lysozyme-treated cell suspensions. Cells were incubated with lysozyme (added at zero time) in the presence of either I or 10 mM Mg2+ (A, 0) or without lysozyme in the presence of either 1 or 10 mM Mg2+ (0, A).
180 (244) 220 (411)
40 min
1,314 (1,624)
5,500 (4,328)
1,134 (1,380)
5,280 (3,917)
aRadioactivity precipitated by protease gamma globulin followed by goat anti-rabbit gamma globulin, expressed as counts per minute per milliliter of supernatant. All values have been corrected for nonspecificity by addition of normal rabbit gamma globulin instead of protease gamma globulin. Results are from two separate experiments. b Lysozyme was added at zero time and "4C-labeled amino acids were added after 8 min of incubation.
VOL. 123, 1975
by protoplasts was electrophoretically identical to that from cells.) The production of extracellular protease by the protoplasts could not be accounted for by the presence of intact cells since viable counts showed that there were less than 5 cells per ml of protoplast suspension and electron microscopy revealed negligible levels of cells. The protoplasts were devoid of both cell wall material and mesosomal structures as observed by electron microscopy of sectioned specimens. The lowered rate of extracellular protease secretion observed was not due to lysis of protoplasts or reduced general metabolism since incorporation of L- [IC ]phenylalanine into total trichloroacetic acid-precipitable material by protoplasts was essentially the same as that by intact cells. The production of active extracellular protease by protoplasts was dependent on the presence of 10 mM Mg2+ as found earlier with lysozyme-treated cells. When protoplasts prepared in PMC medium containing 10 mM Mg2+ were resuspended in the presence of 1 mM Mg2+, no active protease was produced (Fig. 4). A separate experiment established that this effect was selective since the incorporation by protoplasts of L- ["C ]phenylalanine into total protein was almost identical in the presence of 1 or 10 mM Mg2+. The Mg2+ required for active enzyme production by protoplasts could be replaced by Ca2+ at 10 mM or spermidine at 7.5 mM. The rate of production of a-amylase by protoplasts in PMC medium containing 10 mM Mg2+ was comparable with that of protease,
PROTEINS THROUGH MEMBRANES
809
Incubation tim.(n )
FIG. 4. Effect of Mg2+ concentration and chloramphenicol on the production of a-amylase and protease by protoplasts. Protoplasts were prepared in PMC medium containing 10 mM Mg2' and then resuspended in fresh PMC medium containing either I or 10 mM Mg2+. a-Amylase production in 1 mM Mg2+ (0), 10 mM Mg2+ (0), 1 mM Mg2+, and 20 ug of chloramphenicol per ml (A). (An identical result with chloramphenicol was observed in the presence of 10 mM Mg2+.) Protease production in 1 mM Mg2+ (0), 10 mM Mg2+ (U), 10 mM Mg2+, and 20 Mg of chloramphenicol per ml (A). (An identical result with chloramphenicol was observed in the presence of 1 mM Mg2+.)
representing approximately 25% that of intact cells (Fig. 4). This production was sensitive to chloramphenicol, and separate experiments showed that the enzyme was electrophoretically identical to that from cells. However, in marked contrast to protease, a-amylase production proceeded unimpaired in the presence of only 1 mM Mg2+. Effect of cations on protease inactivation. Performed protease molecules remained completely active in PMC medium containing 1 mM Mg2+, and it seemed possible that the addition of high concentrations of cations to protoplasts (or lysozyme-treated cells) was required to prevent inactivation of protease molecules during secretion. To see whether these cations prevent inactivation of protease, puri,fied B. amyloliquefaciens protease (2) was incubated in 25 mM Tris-hydrochloride (pH 7.3) at 30 C in the absence of cations. A 50% loss of activity occurred in 12 h, but this inactivation was totally prevented by either Mg2+, Ca2+, or spermidine at concentrations as low as 0.1 mM. electrophoresis on sodium dodecyl sulfate gels showed that no detectable autodigestion of protease occurred during this inactivation. Effect of proteolytic enzymes on extracelluIncubation t.m(mh) lar enzyme production by protoplasts. TrypFIG. 3. Effect of chloramphenicol on the production of protease by intact cells and protoplasts. Cells sin at 2 gg per ml of protoplast suspension in the presence and absence (A, 0) of chlorampheni- completely inhibited the production of a-amylase (Fig. 5). The appearance of protease (meacol. Protoplasts in the presence and absence (U, 0) of sured after 60 min of incubation with trypsin) chloramphenicol.
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SANDERS AND MAY
was also inhibited, 70% by 2 Ag and 98% by 10 jg of trypsin per ml. In these experiments, trypsin had no effect on total protein synthesis by protoplasts. A trivial explanation for this effect on a-amylase and protease elaboration was that the native enzymes were trypsin sensitive. However, this was not so since the activities of both a-amylase and protease, present in a protoplast supernatant, were not affected by incubation for 24 h at 30 C with 100 ,ug of trypsin per ml. In contrast to the protoplast findings, the production of a-amylase and protease by whole cells was unaffected by concentrations of up to 1 mg of trypsin per ml. However, their appearance was completely and almost immediately inhibited by trypsin upon the addition of lysozyme to cells suspended in PMC medium containing 10 mM Mg2+. The result for a-amylase is shown in Fig. 6. To distinguish whether trypsin affects the emerging enzyme or the protoplast itself, protoplasts were shaken with 2.5 Mg of trypsin per ml for 15 min at 30 C, centrifuged, and resuspended in the absence of trypsin. a-Amylase production recommenced immediately upon removal of trypsin, although at a slightly reduced rate, presumably because traces of trypsin remained (Fig. 7). Protease production similarly recovered. a-Chymotrypsin at 10 jg/ml gave results similar to those for trypsin; production of active a-amylase and protease was inhibited about 85%, although total protein synthesis was almost unaffected and the inhibition could be reversed immediately by centrifugation and resuspension of cells. It was of interest to examine the accessibility of the proteolytically sensitive forms of the extracellular enzymes to a-chymotrypsin cou-
pled to Sepharose 4B beads. The appearance of active extracellular a-amylase was inhibited 80% by the highest concentration of a-chymotrypsin-Sepharose 4B complex tested (Fig. 8). (The appearance of active protease was similarly inhibited at this concentration.) This effect was not due to the presence of unlinked a-chymotrypsin in the preparation since the inhibition of a-amylase appearance was immediately and completely reversed after the selec12
14
Incubation time (rnen)
FIG. 6. Effect of trypsin and lysozyme on a-amylase production by washed-cell suspensions. Cells were incubated in PMC medium containing 10 mM Mg2+ together with (0) lysozyme + trypsin; (A) lysozyme alone; (0) trypsin alone at 10 gg/ml; (A) no addition.
04-
~E
0-3-
a
Incubation time (min)
I0-1
Incubation time (min)
FIG.
5.
Effect of trypsin added
at
zero
time
on
a-amylase production by protoplasts. Protoplasts were incubated without (0) and with trypsin at 0.1 (0), 0.5 (0), and 2 ug of protoplasts (A) per ml.
FIG. 7. Recovery of a-amylase production by protoplasts after trypsin removal. Protoplasts in the presence of 10 mM Mg2+ were previously incubated with 2.5 jgg of trypsin per ml for 15 min. Suspensions were centrifuged and resuspended in fresh medium, and a-amylase production was followed in the presence and absence of 2.5 Mg of trypsin per ml (U, A). A control suspension of protoplasts, previously incubated for 15 min in the absence of trypsin, was similarly resuspended and incubated in the absence of trypsin (-), and a-amylase production was followed.
PROTEINS THROUGH MEMBRANES
VOL. 123, 1975
811
tive removal, by centrifugation, of the coupled enzyme from a protoplast suspension (Fig. 9). 0.25 The coupled enzyme had no effect on total protein synthesis by protoplasts nor extracellular enzyme production by intact cells, whereas uncoupled Sepharose 4B had no effect on enE zyme production by protoplasts. Effect of extracellular protease from B. *-amyloliquefaciens on extracellular enzyme I2, appearance by protoplasts. The appearance of X a-amylase was inhibited by treatment of protoplasts with the organism's own purified extracellular protease; 90 U of protease per ml was X , 20 15 10 5 0 needed for complete inhibition (Fig. 10). Simi(min) time Icubationl U of protease per ml inhibited the 90 larly, FIG. 9. Reversibility of a-chymotrypsin-Sepharose formation of active protease by protoplasts (Fig. 11). To determine the latter, protoplasts were 4B inhibition of a-amylase production by protoplasts. incubated with a-chymotrypsinincubated for 90 min with 50 M.Ci of L-[45- Protoplasts 4Bwere at a concentration equivalent to 200 eucine per ml together with 90 U of protease Sepharose HH ]leucine (0) per ml of protoplasts a-chymotrypsin free of ug per
ml. controget lerwith 9Uof rontease
per ml. A control sample did not contain and an equivalent concentration of unlinked Sephprotease. Supernatant samples were filtered arose 4B (a). At 5 min (arrow) the a-chymotrypsinthrough a membrane filter (0.45 gm, 47 mm; Sepharose 4B was rapidly removed by low-speed Millipore Corp.) and dialyzed for 4 h against 10 centrifugation for 50 s, and the protoplast suspension mM Tris-hydrochloride (pH 8.5) containing 1 was incubated fora further 15 min. mM CaCl2, and 50,l was subjected to polyacrylamide gel electrophoresis followed by pro04 tease and tritium determinations as described in Materials and Methods. Protease at 90 U/ml did not significantly O 3 affect total protein synthesis by protoplasts and E | intact cells, nor did it affect the secretion of A
02~~~~~~~~~~~~~~~~~~~~~ /l
0-2S
-
10
20
30
40
Incubation time (min)
sO
60
FIG. 10. Effect of purified extracellular protease from B. amyloliquefaciens on the production of aby protoplasts. Protoplasts were incubated //amylase without protease (0) and with 4 (0), 10 (A), 20 (A), 30 (0), and 90 U(0) of protease per ml. /
Vol. 123, No. 3 Printed in U.S.A.
Evidence for Extrusion of Unfolded Extracellular Enzyme Polypeptide Chains Through Membranes of Bacillus amyloliquefaciens R. L. SANDERS* AND B. K. MAY Department of Biochemistry, University of Adelaide, South Australia, 5001 Received for publication 21 April 1975
The production of extracellular a-amylase and protease by protoplasts of Bacillus amyloliquefaciens has been achieved. The production of enzymically active protease was totally dependent on a high concentration of either Mg'+, Ca2+, or spermidine, but production of active a-amylase was not. This cation dependence of protease production was seen immediately upon addition of lysozyme to intact cells. The cations could prevent the inactivation of protease and alter the cytoplasmic membrane configuration of protoplasts. Production of active a-amylase and protease by protoplasts was totally inhibited by proteolytic enzymes such as trypsin, a-chymotrypsin, or the organism's purified extracellular protease. The evidence suggests that these degradative enzymes act specifically on the emerging polypeptide of the extracellular enzyme and that the polypeptide emerges in a conformation different from that of the native molecule.
We are examining the synthesis and secretion of extracellular enzymes by Bacillus amyloliquefaciens as a model system for investigating how some proteins are selectively secreted through cellular membranes. This organism has the advantage that washed-cell suspensions rapidly produce large amounts of extracellular protease, a-amylase, and ribonuclease. No significant amounts of active protease, a-amylase, or inactive cross-reacting material can be detected inside secreting cells of B. amyloliquefaciens (4, 11). In addition, there exists in the cytoplasm an inhibitor specific for the extracellular ribonuclease (17), and since the formation of the enzyme-inhibitor complex is essentially irreversible (9), it seems unlikely that the native enzyme could ever have existed as such within the cell. We have therefore suggested that extracellular enzymes are synthesized on membrane-associated ribosomes and that the nascent polypeptides are extruded through the membrane to assume their active tertiary configuration only outside the permeability barrier (12). Compatible with this model are our findings, which suggest that within the cell there are pools of extracellular enzyme-specific messenger ribonucleic acid (2, 7). We have suggested that these apparent pools are the result of excessive transcription, possibly designed to saturate the membrane "translational-extrusion" sites with messenger ribonucleic acid
despite its rapid degradation en route from gene to membrane (6). Attempts to directly test the extrusion hypothesis in our system have been difficult because of the apparent inability of protoplasts of this organism to secrete active extracellular enzymes, even though intracellular protein and ribonucleic acid synthesis continue almost normally (12). However, it has now been shown that in the presence of sufficient concentrations of either Mg2+, Ca2+, or spermidine, protoplasts will secrete enzymatically active protease and a-amylase. During secretion these extracellular enzymes exist in a form that is sensitive to proteolytic attack, whereas the released enzymes are insensitive. This finding is compatible with our proposed model for enzyme secretion. A preliminary report of this work has appeared previously (16).
806
MATERIALS AND METHODS Organism used. The parent strain of the organism used in this work was an unclassified strain of Bacillus amyloliquefaciens (11). This strain produces an extracellular peptide-lipid molecule, "surfactin," that lyses protoplasts (13). A mutant unable to produce this compound was isolated as follows and used in the present work. Approximately 106 spores in 1 ml of glass-distilled water were treated with 0.1 ml of ethyl methyl sulfonate for 20 min at 37 C. The spores were washed once by centrifugation and suspension in 5 ml of 0.9% NaCl and were finally suspended in 1 ml of 0.9% NaCl. This suspension (0.1 ml) was used to
VOL. 123, 1975
inoculate 500 ml of culture medium (2), and the culture was shaken at 30 C for 25 h. Cultures were plated for single colonies on blood agar plates and incubated for 16 h at 37 C. Those colonies which did not produce a hemolyzed zone were picked off and purified by subculturing four times. The mutant strain used was cultured as described previously (2); cultures did not produce any lytic factor detectable by protoplast lysis (13), whereas the production of extracellular a-amylase and protease was normal compared with wild type. Washed-cell and protoplast suspension experiments. PMC medium contained tris(hydroxymethyl)aminomethane (Tris) (25 mM), (NH4)JHPO, (3.8 mM), KCl (5 mM), sodium citrate (4.25 mM), CaCl, (0.125 mM), ZnSO4 (0.0125 mM), 0.025% (wt/vol) Casamino Acids (Difco), 0.25 ml of trace metal solution (2) per liter, 1% maltose, and 22% (wt/vol) sucrose adjusted to pH 7.3 with HCI. Cells after 25 h of growth were harvested, washed once with PMC medium lacking sucrose, and finally suspended in the same volume of PMC medium. Protoplasts were prepared by shaking washed-cell suspensions at 30 C for 45 min with 133 jig of lysozyme per ml of cells. In some experiments, samples (0.8 ml) were removed at various times during protoplast formation and centrifuged, and the supernatants were assayed for a-amylase and protease activity. In other experiments, protoplasts were resuspended in fresh PMC media and further incubated at 30 C with shaking. Samples were withdrawn at appropriate times and centrifuged, and the supernatants were assayed for enzyme activity. Ribonuclease could not be measured in these experiments because of inhibitor released by lysis of a small proportion of protoplasts (17). Assay of enzymes. Protease activity was assayed by using a Remazol brilliant blue-hide powder assay (15). One unit of activity is defined as the amount of enzyme giving an increase in absorbance at 595 nm of 5.7 in 40 min at 37 C and corresponds to the unit defined earlier (11). a-Amylase was assayed as previously described (2). Measurement of total protein synthesis. The incorporation of L- ["C ]phenylalanine (460 mCi/ mmol) into total trichloroacetic acid-precipitable material was measured as described earlier (2). Total extracellular protein represents only about 5% of the total cellular trichloroacetic acid-precipitable material. Preparation of antibodies. B. amyloliquefaciens extracellular protease was purified to homogeneity as described earlier (2), and its antibody was prepared by inoculating New Zealand rabbits with weekly, multisite, subcutaneous injections of protease in complete Freund adjuvant. Rabbit antisera were fractionated by precipitation with 50% (wt/vol) ammonium sulfate, followed by dialysis of the precipitate to remove salt and fractionation on diethylaminoethylcellulose columns. The gamma globulin fractions were pooled and concentrated by using aquacide. Goat anti-rabbit gamma globulin was obtained by injecting goats with normal rabbit gamma globulin. Inactivation of trypsin and a-chymotrypsin. Trypsin (or a-chymotrypsin) in cell or protoplast
PROTEINS THROUGH MEMBRANES
807
supernatants was inactivated before the determination of B. amyloliquefaciens protease activity. Trypsin was inactivated by incubation for 4 h at 30 C with 1-chloro-3-tosylamide-7-amino-2-heptanone hydrochloride (TLCK) at a final concentration of 30 jig/ml. For a-chymotrypsin, L-1-tosylamide-2-phenylethyl chloromethyl ketone (TPCK) at 10 jig/ml was used (added as a methanolic solution of 1 mg/ml) and incubated at 30 C for 5 h. These procedures completely inactivated both enzymes but did not affect B. amyloliquefaciens protease. Polyacrylamide gel electrophoresis. Electrophoresis of protease was carried out as described earlier (2). To detect ptotease and 3H radioactivity, gels were frozen in solid CO2 and sliced longitudinally, and corresponding halves were cut into 1-mm slices. The enzyme was eluted overnight from the slices with 1.5 ml of 50 mM Tris-hydrochloride (pH 7.8), and the eluate was assayed for protease. Radioactivity was determined by dissolving gel slices for 16 h at 20 C in 2.5 ml of a solution of 0.3% 2,5-diphenyloxazole and 0.03% 1,4-bis-[2]-(4-methyl phenyloxazolyl)-benzene, 12% (vol/vol) NCS tissue solubilizer (Amersham/ Searle Corp.), and 0.08 M NH4OH, followed by liquid scintillation counting in a Packard Tri-Carb spectrometer. Covalent coupling of a-chymotrypsin to Sepharose 4B beads. a-Chymotrypsin was coupled to cyanogen bromide-activated Sepharose 4B in 0.1 M NaHCO,, and after coupling was completed the product was extensively washed with six cycles each of 0.1 M sodium acetate (pH 4.0) and 0.2 M NaHCO, (pH 8.5), each containing 1 M NaCl. The product was stored in distilled water with 0.1% (wt/vol) sodium azide and thoroughly washed before use. Using a casein assay (11), it was established that each 1.0 ml of the settled beads possessed proteolytic activity equivalent to 2.5 mg of free a-chymotrypsin. Materials. Radiochemicals were obtained from Schwarz/Mann. TLCK and TPCK were both purchased from Cyclo Chemical Corp., Los Angeles, Calif. TPCK-treated trypsin and TLCK-treated achymotrypsin were purchased from Worthington Biochemical Corp., Freehold, N.J. Chloramphenicol was a product of Parke-Davis and Co., Sydney. Egg white lysozyme, three times recrystallized, was a product of Sigma Chemical Co., St. Louis, Mo. The Remazol brilliant blue was a generous gift from Farbwerke Hoechst, AG, Frankfurt.
RESULTS Effect of lysozyme on extracellular enzyme secretion by washed-cell suspensions. Washed-cell suspensions were incubated in a 22% (wt/vol) sucrose medium (PMC medium) containing 1 mM Mg2+ (which is optimal for extracellular enzyme production by intact cells), and the effect of lysozyme on enzyme secretion was examined. Production of active protease was inhibited almost instantly (Fig. 1). The first protoplasts were not visible until 20 min, and electron microscopic examination
808
SANDERS AND MAY
J. BACTERIOL.
showed that complete cell wall removal required 45 min of incubation. The inhibitory effect of lysozyme was reduced by about 50% when the medium contained 10 mM Mg2+ (Fig. 1). Although Mg2+ did have some effect on general protein synthesis (Fig. 2), incorporation of ["'Ciphenylalanine into total protein in the presence of lysozyme was not dependent on 10 mM Mg2+, as was protease synthesis. The selectivity of the Mg2+ effect for protease synthesis in the presence of lysozyme was further reinforced by the observation that extracellular
a-amylase synthesis after lysozyme addition the same in 1 or in 10 mM Mg2+. The inhibitory effect of lysozyme on protease production by cells in 1 mM Mg2+ was immediately alleviated by adding Mg2+ to 10 mM at was
any time up to at least 45 min. Similar results obtained with Ca2+ or spermidine. To test the possibility that cells treated with lysozyme in 1 mM Mg2+ secreted an inactive form of the protease molecule, a cell suspension (8 ml) was incubated in PMC medium containing 1 mM Mg2+ and lysozyme for 8 min, at which time 5 ltCi of L- [C "4]phenylalanine (460 mCi/mmol) and 15 gCi of L-[14CJleucine (312 mCi/mmol) were added. A control suspension did not contain lysozyme. Samples were taken at 8 min and again after 40 min of incubation and centrifuged, and the supernatants were assayed for material that cross-reacted with protease gamma globulin as described (8). The results suggest that lysozyme-treated cells in 1 mM Mg2+ produce a form of the protease molecule capable of reacting with protease gamma globulin (Table 1). Effect of cations on protease and a-amylase were
production by protoplasts. Protoplasts were incubated with shaking in PMC medium containing 10 mM Mg2+, and the production of protease was compared with that by intact cells
under conditi6ns identical except for the omission of lysozyme. The rate of synthesis of protease by protoplasts was about 30% of that of Incubation time (mm) intact cells, and production of enzyme was FIG. 1. Effect of Mg2+ concentration on production chloramphenicol sensitive (Fig. 3). (Separate of active protease by lysozyme-treated cell suspensions. Cells were incubated with lysozyme (added at experiments showed that the enzyme produced zero time) in the presence of I or 10 mM Mg2+ or without lysozyme in the presence of either
(O, A) 1
or
10
mM Mg,+ (O, 0). 3.
TABLE 1. Precipitation by protease gamma globulin of radioactively labeled material secreted by cells incubated with and without lysozyme in the presence of 1 mM Mg2+ Radioactivity precipitateda
0)
.2.
Cells
Time of incubation Net 8 min
Lysozyme treatedb Control .......... I,
Incubation time (min)
FIG. 2. Effect of Mg2+ concentration on the incorporation of L-["4CJphenylalanine into trichloroacetic acid-precipitable material by lysozyme-treated cell suspensions. Cells were incubated with lysozyme (added at zero time) in the presence of either I or 10 mM Mg2+ (A, 0) or without lysozyme in the presence of either 1 or 10 mM Mg2+ (0, A).
180 (244) 220 (411)
40 min
1,314 (1,624)
5,500 (4,328)
1,134 (1,380)
5,280 (3,917)
aRadioactivity precipitated by protease gamma globulin followed by goat anti-rabbit gamma globulin, expressed as counts per minute per milliliter of supernatant. All values have been corrected for nonspecificity by addition of normal rabbit gamma globulin instead of protease gamma globulin. Results are from two separate experiments. b Lysozyme was added at zero time and "4C-labeled amino acids were added after 8 min of incubation.
VOL. 123, 1975
by protoplasts was electrophoretically identical to that from cells.) The production of extracellular protease by the protoplasts could not be accounted for by the presence of intact cells since viable counts showed that there were less than 5 cells per ml of protoplast suspension and electron microscopy revealed negligible levels of cells. The protoplasts were devoid of both cell wall material and mesosomal structures as observed by electron microscopy of sectioned specimens. The lowered rate of extracellular protease secretion observed was not due to lysis of protoplasts or reduced general metabolism since incorporation of L- [IC ]phenylalanine into total trichloroacetic acid-precipitable material by protoplasts was essentially the same as that by intact cells. The production of active extracellular protease by protoplasts was dependent on the presence of 10 mM Mg2+ as found earlier with lysozyme-treated cells. When protoplasts prepared in PMC medium containing 10 mM Mg2+ were resuspended in the presence of 1 mM Mg2+, no active protease was produced (Fig. 4). A separate experiment established that this effect was selective since the incorporation by protoplasts of L- ["C ]phenylalanine into total protein was almost identical in the presence of 1 or 10 mM Mg2+. The Mg2+ required for active enzyme production by protoplasts could be replaced by Ca2+ at 10 mM or spermidine at 7.5 mM. The rate of production of a-amylase by protoplasts in PMC medium containing 10 mM Mg2+ was comparable with that of protease,
PROTEINS THROUGH MEMBRANES
809
Incubation tim.(n )
FIG. 4. Effect of Mg2+ concentration and chloramphenicol on the production of a-amylase and protease by protoplasts. Protoplasts were prepared in PMC medium containing 10 mM Mg2' and then resuspended in fresh PMC medium containing either I or 10 mM Mg2+. a-Amylase production in 1 mM Mg2+ (0), 10 mM Mg2+ (0), 1 mM Mg2+, and 20 ug of chloramphenicol per ml (A). (An identical result with chloramphenicol was observed in the presence of 10 mM Mg2+.) Protease production in 1 mM Mg2+ (0), 10 mM Mg2+ (U), 10 mM Mg2+, and 20 Mg of chloramphenicol per ml (A). (An identical result with chloramphenicol was observed in the presence of 1 mM Mg2+.)
representing approximately 25% that of intact cells (Fig. 4). This production was sensitive to chloramphenicol, and separate experiments showed that the enzyme was electrophoretically identical to that from cells. However, in marked contrast to protease, a-amylase production proceeded unimpaired in the presence of only 1 mM Mg2+. Effect of cations on protease inactivation. Performed protease molecules remained completely active in PMC medium containing 1 mM Mg2+, and it seemed possible that the addition of high concentrations of cations to protoplasts (or lysozyme-treated cells) was required to prevent inactivation of protease molecules during secretion. To see whether these cations prevent inactivation of protease, puri,fied B. amyloliquefaciens protease (2) was incubated in 25 mM Tris-hydrochloride (pH 7.3) at 30 C in the absence of cations. A 50% loss of activity occurred in 12 h, but this inactivation was totally prevented by either Mg2+, Ca2+, or spermidine at concentrations as low as 0.1 mM. electrophoresis on sodium dodecyl sulfate gels showed that no detectable autodigestion of protease occurred during this inactivation. Effect of proteolytic enzymes on extracelluIncubation t.m(mh) lar enzyme production by protoplasts. TrypFIG. 3. Effect of chloramphenicol on the production of protease by intact cells and protoplasts. Cells sin at 2 gg per ml of protoplast suspension in the presence and absence (A, 0) of chlorampheni- completely inhibited the production of a-amylase (Fig. 5). The appearance of protease (meacol. Protoplasts in the presence and absence (U, 0) of sured after 60 min of incubation with trypsin) chloramphenicol.
810
J . BACTrERIOL.
SANDERS AND MAY
was also inhibited, 70% by 2 Ag and 98% by 10 jg of trypsin per ml. In these experiments, trypsin had no effect on total protein synthesis by protoplasts. A trivial explanation for this effect on a-amylase and protease elaboration was that the native enzymes were trypsin sensitive. However, this was not so since the activities of both a-amylase and protease, present in a protoplast supernatant, were not affected by incubation for 24 h at 30 C with 100 ,ug of trypsin per ml. In contrast to the protoplast findings, the production of a-amylase and protease by whole cells was unaffected by concentrations of up to 1 mg of trypsin per ml. However, their appearance was completely and almost immediately inhibited by trypsin upon the addition of lysozyme to cells suspended in PMC medium containing 10 mM Mg2+. The result for a-amylase is shown in Fig. 6. To distinguish whether trypsin affects the emerging enzyme or the protoplast itself, protoplasts were shaken with 2.5 Mg of trypsin per ml for 15 min at 30 C, centrifuged, and resuspended in the absence of trypsin. a-Amylase production recommenced immediately upon removal of trypsin, although at a slightly reduced rate, presumably because traces of trypsin remained (Fig. 7). Protease production similarly recovered. a-Chymotrypsin at 10 jg/ml gave results similar to those for trypsin; production of active a-amylase and protease was inhibited about 85%, although total protein synthesis was almost unaffected and the inhibition could be reversed immediately by centrifugation and resuspension of cells. It was of interest to examine the accessibility of the proteolytically sensitive forms of the extracellular enzymes to a-chymotrypsin cou-
pled to Sepharose 4B beads. The appearance of active extracellular a-amylase was inhibited 80% by the highest concentration of a-chymotrypsin-Sepharose 4B complex tested (Fig. 8). (The appearance of active protease was similarly inhibited at this concentration.) This effect was not due to the presence of unlinked a-chymotrypsin in the preparation since the inhibition of a-amylase appearance was immediately and completely reversed after the selec12
14
Incubation time (rnen)
FIG. 6. Effect of trypsin and lysozyme on a-amylase production by washed-cell suspensions. Cells were incubated in PMC medium containing 10 mM Mg2+ together with (0) lysozyme + trypsin; (A) lysozyme alone; (0) trypsin alone at 10 gg/ml; (A) no addition.
04-
~E
0-3-
a
Incubation time (min)
I0-1
Incubation time (min)
FIG.
5.
Effect of trypsin added
at
zero
time
on
a-amylase production by protoplasts. Protoplasts were incubated without (0) and with trypsin at 0.1 (0), 0.5 (0), and 2 ug of protoplasts (A) per ml.
FIG. 7. Recovery of a-amylase production by protoplasts after trypsin removal. Protoplasts in the presence of 10 mM Mg2+ were previously incubated with 2.5 jgg of trypsin per ml for 15 min. Suspensions were centrifuged and resuspended in fresh medium, and a-amylase production was followed in the presence and absence of 2.5 Mg of trypsin per ml (U, A). A control suspension of protoplasts, previously incubated for 15 min in the absence of trypsin, was similarly resuspended and incubated in the absence of trypsin (-), and a-amylase production was followed.
PROTEINS THROUGH MEMBRANES
VOL. 123, 1975
811
tive removal, by centrifugation, of the coupled enzyme from a protoplast suspension (Fig. 9). 0.25 The coupled enzyme had no effect on total protein synthesis by protoplasts nor extracellular enzyme production by intact cells, whereas uncoupled Sepharose 4B had no effect on enE zyme production by protoplasts. Effect of extracellular protease from B. *-amyloliquefaciens on extracellular enzyme I2, appearance by protoplasts. The appearance of X a-amylase was inhibited by treatment of protoplasts with the organism's own purified extracellular protease; 90 U of protease per ml was X , 20 15 10 5 0 needed for complete inhibition (Fig. 10). Simi(min) time Icubationl U of protease per ml inhibited the 90 larly, FIG. 9. Reversibility of a-chymotrypsin-Sepharose formation of active protease by protoplasts (Fig. 11). To determine the latter, protoplasts were 4B inhibition of a-amylase production by protoplasts. incubated with a-chymotrypsinincubated for 90 min with 50 M.Ci of L-[45- Protoplasts 4Bwere at a concentration equivalent to 200 eucine per ml together with 90 U of protease Sepharose HH ]leucine (0) per ml of protoplasts a-chymotrypsin free of ug per
ml. controget lerwith 9Uof rontease
per ml. A control sample did not contain and an equivalent concentration of unlinked Sephprotease. Supernatant samples were filtered arose 4B (a). At 5 min (arrow) the a-chymotrypsinthrough a membrane filter (0.45 gm, 47 mm; Sepharose 4B was rapidly removed by low-speed Millipore Corp.) and dialyzed for 4 h against 10 centrifugation for 50 s, and the protoplast suspension mM Tris-hydrochloride (pH 8.5) containing 1 was incubated fora further 15 min. mM CaCl2, and 50,l was subjected to polyacrylamide gel electrophoresis followed by pro04 tease and tritium determinations as described in Materials and Methods. Protease at 90 U/ml did not significantly O 3 affect total protein synthesis by protoplasts and E | intact cells, nor did it affect the secretion of A
02~~~~~~~~~~~~~~~~~~~~~ /l
0-2S
-
10
20
30
40
Incubation time (min)
sO
60
FIG. 10. Effect of purified extracellular protease from B. amyloliquefaciens on the production of aby protoplasts. Protoplasts were incubated //amylase without protease (0) and with 4 (0), 10 (A), 20 (A), 30 (0), and 90 U(0) of protease per ml. /
