JOURNAL OF BACrERIOLOGY, Apr. 1975, p. 34-40 Copyright i 1975 American Society for Microbiology

Vol. 122, No. 1

Printed in U.S.A.

Release of Extracellular Enzymes from Bacillus amyloliquefaciens A. R. GOULD, B. K. MAY,* AND W. H. ELLIOTT Department of Biochemistry, University of Adelaide, Adelaide, South Australia 5001

Received for publication 30 December 1974

Washed-cell suspensions of Bacillus amyloliquefaciens secrete significant amounts of the extracellular enzymes a-amylase and protease for about 15 min in the almost complete absence of protein synthesis. This apparently represents release of preformed enzyme en route to secretion. The release was independent of energy but was affected by temperature. Pulse-labeling experiments showed that newly synthesized enzyme molecules are either immediately released into the external medium or equilibrate with the preformed enzyme prior to eventual secretion. The results are compatible with a model of secretion whereby enzyme molecules emerging from the cell membrane become temporarily restricted by the cell wall so that a small pool of active enzyme accumulates in this region. mined by a casein digestion method (5). However, to measure low levels of the enzyme, a Remazol brilliant blue-hide powder assay (7) was used and protease activity was converted to the units used in the casein digestion assay. a-Amylase and ribonuclease were assayed as previously described (1). Measurement of total protein synthesis. Incorporation of either L-['4C]leucine (312 mCi/mmol), L[l4C]phenylalanine (460 mCi/mmol), or reconstituted "IC-labeled protein hydrolysate into total cellular protein was measured as previously described (1). Preparation of gamma globulin fractions. Adult New Zealand rabbits were immunized with purified a-amylase or protease incorporated in complete Freund adjuvant and were administered subcutaneously at the base of the neck at multiple sites. Subsequent injections of extracellular enzyme in incomplete adjuvant were administered subcutaneously at intervals of 2 weeks. Rabbit antisera to the extracellular enzymes were fractionated on diethylaminoethyl-cellulose columns and the gamma globulin fractions were collected and concentrated by vacuum dialysis in collodion bags. Goat anti-rabbit gamma globulin was similarly purified after injecting goats with normal rabbit gamma globulin. MATERIALS AND METHODS Materials. Radiochemicals were obtained from Washed-cell suspensions. B. amyloliquefaciens Schwarz/Mann. Chloramphenicol was obtained from cells were grown from a spore inoculum in a salts-malt- Parke Davis and Co., Sydney, and lysozyme (3 times ose-Casamino Acids growth medium as described crystallized from egg white) from Sigma Chemical Co. previously (1). After growth for 25 h, the cells were Remazol brilliant blue was a generous gift from harvested and washed twice in a suspending medium, Farbwerke Hoechst AG, Frankfurt. which was the same as the growth medium except RESULTS that FeCl3 and yeast extract were omitted. A washedcell suspension sample (20 ml) was shaken at 30 C Presence of a preformed pool of a-amylase and samples (1.0 ml) were removed at appropriate time intervals. The cells were removed by centrifuga- and protease in B. amyloliquefaciens cells. tion and the supernatant fluids (or culture fluids) When protein synthesis was stopped by addition of chloramphenicol to washed-cell suspenwere assayed for extracellular enzymes. Assay of enzymes. Protease activity was deter- sions of B. amyloliquefaciens, release of a-amy-

We are examining the mechanism of the vectorial transport of proteins through membranes, using as a model system the secretion of the extracellular enzymes a-amylase and protease by washed-cell suspensions of Bacillus amyloliquefaciens. In previous studies (3) it was consistently noted that small amounts of the enzymes were released into the external medium in the presence of inhibitors of protein synthesis such as chloramphenicol. It was of importance to define what this release of enzyme represented. The most likely possibilities were that it was a trivial phenomenon representing elution of enzyme adsorbed to the cells or that it represented preformed enzyme en route to secretion. The results presented in this paper are compatible with the latter and with a model of secretion (1) in which newly synthesized extracellular enzymes can be secreted directly from the cell or become cell associated (but external to the permeability barrier) prior to release.

34

VOL. 122, 1975

35

RELEASE OF EXTRACELLULAR ENZYMES

lase and protease continued for approximately 15 min (Fig. 1, 2). This was due to the release of preformed enzymes from the cell, since chloramphenicol at 10 gg/ml inhibited protein synthesis immediately as measured by ["4C ]phenylalanine incorporation (Fig. 3). The same release occurred in the presence of 100 ,g of chloramphenicol per ml, puromycin (20 gg/ ml), or sodium fusidate (22.5 gg/ml), all of which inhibited protein synthesis immediately and almost completely. Cell lysates were examined for enzyme; washed-cell suspensions were treated with chloramphenicol (10 ug/ml) and immediately cooled to 0 C (at which temperature release does not occur). The cells were disrupted in the French pressure cell at 0 C and the lysate was

2

5~~~~~~~~~

5

10-

10

15

20

Incubation tim(mi,)

FIG. 2. Effect of chloramphenicol on the production of protease by normal washed-cell suspensions and washed-cell suspensions prepared from culture cells preexposed to chloramphenicol. Experimental details are given in Fig. 1. Sym Lols: 0, no addition of drug; 0, 10 sg of chloramphenicol per ml added at time zero to a normal washed-cell suspension; A, 10 Ag of chloramphenicol per ml added at zero time to a washed-cell suspension prepared from culture cells preexposed to chloramphenicol.

E

centrifuged at 150,000 x g for 3 h at 0 C. Protease and a-amylase were found in the t0 supernatant fluid in amounts porresponding to Incubation time (min) those released by the same quantity of cells FIG. 1. Effect of chloramphenicol on the produc- incubated at 30 C in the presence of chloramtion of a-amylase by normal washed-cell suspensions phenicol. (The protease value was 98% of the and washed-cell suspensions prepared from culture expected and the a-amylase 97%.) The remaincells preexposed to chloramphenicol. Cell cultures treated with chloramphenicol (10 pg/ml) for 15 min ing activity was detected in the pellet fraction. The cell-associated enzyme pools were not were harvested, washed twice in suspending medium, and resuspended in this medium. Chloramphenicol due to simple adsorption to the cell walls. When (10 Ag/ml) was added and the release of extracellular cell cultures containing large amounts of extraa-amylase was followed for 20 min. This was com- cellular protease (about 700 U/ml) and a-amypared with the release of enzyme from cells which had lase (about 600 U/ml) were treated with chlornot been exposed to chloramphenicol in the culture amphenicol (10 ug/ml) for 15 min before harmedium but which were treated with the drug at zero vesting of the cells, subsequent incubation of time in the washed-cell suspension experiments. the cells in the presence of chloramphenicol (10 Symbols: *, no addition of drug; 0, 10 1g of chloramphenicol per ml added at time zero to a normal Mg/ml) gave no release of enzyme (Fig. 1, 2). washed-cell suspension; A, 10 stg of chloramphenicol The release is not therefore due to simple per ml added at zero time to a washed-cell suspension desorption of contaminating enzymes. 2,4-Dinitrophenol (2 mM) and sodium azide prepared from culture cells preexposed to chloram(20 mM) did not affect the chloramphenicolphenicol. ec

5

15

20

GOULD, MAY, AND ELLIOTT

36

.2

I

20 _

W

Incubation time(mmin)

FIG. 3. Effect of chloramphenicol on L- ["C]phenylalanine incorporation into total protein by a washed-cell suspension. Chioramphenicol and L['4C]phenylalanine (0.5 MCi/mI) were both added at zero time. Symbols: 0, no addition;O*, chloramphenicol (10 yg/ml). The 100%X value for L-['4C]phenyLalanine incorporation was 1,000 counts/mmn.

J . BACTrERIOL.

the exterior, the last released being the last synthesized. Alternatively, newly synthesized molecules may feed into and equilibrate with a pool of enzyme, the release of which occurs at a rate requiring 15 min for completion. In the latter case an individual molecule has a statistical chance of being released immediately after its synthesis. The following experiment was designed to see whether there is a significant delay in the time of appearance of newly synthesized extracellular enzymes in the external medium. Cells from 40 ml of culture were harvested at 25 h and washed twice in suspending medium which lacked Casamino Acids. The cells were resuspended in this same medium and pulselabeled for 90 s using reconstituted "4C-labeled protein hydrolysate (20 uCi) and the incorporation was stopped with 2.0 ml of a 40% (wt/vol) Casamino Acids solution. To measure total protein synthesis, samples (0.1 ml) were directly added to 3.0 ml of 10% trichloroacetic acid containing 1% Casamino Acids and processed for measuring incorporation of isotope into protein. To measure the amount of radioac0

insensitive appearance of the extracellular protease by washed-cell suspensions (Fig. 4), although it was shown that these drugs separately 0 10 inhibited general protein synthesis by greater than 95% (Fig. 5). a-Amylase release was similarly unaffected. Enzyme was not released from the cells in the presence or absence of chloramphenicol at 0 C. This was apparently not due to the necessity for an enzymic reaction in the release process; if cells in the presence of chloramphenicol were incubated at 10 or 20 C, then release occurred until 25 and 50%o, respectively, of the maximum had appeared and then no further release was observed. Since it seemed likely that the preformed pools of enzyme are en route to secretion, it seemed that a study of the release of these enzymes would give information about the normal release process in extracellular enzyme production. The experiments below are concerned with this. Time of appearance of pulse-labeled protease and az-amylase in the external mekiubation time (nn.) dium. If the chloramphenicol-insensitive reFIG. 4. of sodium azide or 2,4-dinitroEffect lease represented preformed enzyme en route to on the release of protease by washed-cell the external medium, the question arises as to phenol suspensions in the presence of chloramphenicol. Symwhy 15 min is needed before all the enzyme bols: 0, no addition of drugs; 0, sodium azide (20 becomes extracellular. An enzyme molecule mM) and chloramphenicol (10 ug/ml); A, 2,4-dinitromay require 15 min to complete its passage to phenol (2 mM) and chloramphenicol (10 ug/ml).

OF EXTRACELLULAR ENZYMES VRELEASE VOL. 122, 1975

c,cbatiWon tun (mi) FIG. 5. Effect of sodium azide (20 mM) and 2,4dinitrophenol (2 mM) on the incorporation of L[I4Clleucine (0.5 sg/ml) into total protein by washedcell suspensions. Symbols: 0, no addition; 0, sodium azide; A, 2,4-dinitrophenol. The 100% value for L[I4C]leucine incorporation was 2,000 counts/min.

tive protein accumulating in the external medium, 3.5-ml samples were centrifuged and any remaining bacterial cells were removed by filtering through membrane filters (0.45 Am; 47 mm; Millipore Corp.). Samples (1.0 ml) of the supernatant fluid were then added to 6.0 ml of 10% trichloroacetic acid containing 1% Casamino Acids and processed. The addition of the Casamino Acids after the 90-s pulse period inhibited incorporation of "4C-labeled amino acids into total proteins after about 4 min (Fig. 6a), although radioactive material continued to accumulate in the external medium for about 15 min (Fig. 6b). Using an immunological precipitation assay it was possible to determine how much of the radioactive material in the external medium was due to the extracellular enzymes, a-amylase and protease. To supernatant samples (2.0 ml) previously filtered through membrane filters, a fixed amount of either anti-protease or anti-amylase rabbit gamma globulin was added at a twofold excess to complex each exoenzyme. After incubation at 37 C for 60 min, a predetermined amount of goat anti-rabbit gamma globulin was added to precipitate maximally the previous complex. Incubation was continued for a further 60 min at 37 C and then 16 h at 4 C. The precipitate was washed three times with 5.0-ml

37

lots of cold 0.9% (wt/vol) saline by centrifugation and resuspension and was collected onto 47-mm membrane filters (Millipore Corp.), and the filters were washed five times with 10.0-ml portions of cold saline. The filters were then dried and the radioactivity was determined by liquid scintillation. Controls in which antibody was replaced by nonimmune rabbit gamma globulin were treated identically to correct for the nonspecific precipitation of radioactive material. It can be seen that both protease and a-amylase appeared in the external medium of the washed-cell suspension and that there was no apparent delay before this pulse-labeled enzyme emerged (Fig. 7). After the pulse period it takes about 15 min before all of the radioactive a-amylase or protease synthesized in 90 s is extracellular corresponding with the time for the chloramphenicol release of the enzymes described above. Equilibration of new enzyme molecules with the preformed pool of enzyme. If the newly synthesized enzyme equilibrates with a pool of preformed enzyme awaiting release, then pulse-labeled cells will secrete enzyme of a higher specific activity in the absence of the pool than in normal cells containing the pool. To test this prediction, a washed-cell suspension was exposed to chloramphenicol (10lg/ml) for 15 min at 30 C and incubated to release the preformed enzyme. A control sample of washed-

FIG. 6. Incorporation of reconstituted '4C-labeled protein hydrolysate into (a) total protein and (b) extracellular protein. Cells were pulsed for 90 s with 20 gCi of radioisotope and the incorporation was quenched with 2.0 ml of 40%0 (wt/vol) Casamino Acids. The incorporation into total protein was measured by the addition of samples (0.1 ml) of washedcell suspension to trichloroacetic acid. The incorporation into extracellular protein was measured by the addition to trichloroacetic acid of supernatant fluids (1.0 ml) from which cells had been removed.

GOULD, MAY, AND ELLIOTT

38

Icubation tinw (min)

FIG. 7. The accumulation of radioactive protease and a-amylase in the external medium of a washedcell suspension after a 90-s pulse (arrow) with protein [I4C]hydrolysate (as determined by immune precipitation). The pulse labeling and immune precipitation conditions were as described in the text. Symbols: *, protease; 0, a-amylase; 0, nonspecific precipitation by nonimmune rabbit gamma globulin measured at 0 and 30 min for protease; 0, nonspecific precipitation by nonimmune rabbit gamma globulin measured at 0 and 30 min for a-amylase.

cell suspension, not exposed to chloramphenicol, was also incubated at 30 C for 15 min. After 15 min, a 1.0-ml sample from each of the two suspensions was removed and diluted 15 times in suspending medium lacking Casamino Acids at 30 C. A 10.0-ml sample of these diluted cells was then filtered on a 47-mm membrane filter (Millipore Corp.) and washed five times with 10.0 ml of the same medium at 30 C. This procedure removes chloramphenicol from cells so that general protein synthesis and extracellular enzyme synthesis resume immediately (2). The cells on the filter were then placed into a flask containing 10 ml of suspending medium lacking Casamino Acids but containing 0.5 ACi of reconstituted "4C-labeled protein hydrolysate per ml. The flask was then shaken at 30 C for 90 s and 1.0 ml of a 40% (wt/vol) Casamino Acids solution was added. After the addition of the Casamino Acids, a 5.0-ml sample was taken immediately and again after 5 min of incubation at 30 C. Cells were removed by centrifuga-

J . BACTrERIOL.

tion followed by membrane filtration. The filtrates were assayed for protease using the Remazol brilliant blue-hide powder assay. The radioactivity in the protease was measured by the immunological precipitation procedure described earlier. In three separate experiments, the specific activity of the protease (counts per minute per unit of enzyme activity) from cells pre-incubated with chloramphenicol was approximately 2.5 times greater than that from control cells (Table 1). To check the possibility that the internal amino acid pools were reduced by chloramphenicol treatment, the specific activities of the amino acids of the control and chloramphenicol-treated cells after the pulselabeling period were determined. The cells were treated and labeled for 90 s as in the previous experiment and then quickly transferred to 47-mm membrane filters (Millipore Corp.) and rapidly washed four times with suspending medium lacking Casamino Acids. The filters with the retained cells were then plunged into 10.0 ml of distilled water at 100 C and kept at this temperature for 10 min. Cell debris was removed by centrifugation (25,000 x g, 30 min) and the supernatant fluid was added to five volumes of saturated (1%) picric acid and left for 10 min at room temperature. After centrifugation, the supernatant fluid was passed through a Dowex 2 x 8 (100 to 200 mesh) column (1.0 by 4 cm). The column was washed with five bed volumes of 0.02 N HCl and the eluate was collected and freeze dried. The samples thus collected were divided into equal portions; one portion of the sample was analyzed on the Technicon amino acid analyzer and the other was similarly loaded except that fractions of the column effluent were directly collected. These fractions were put into Brays scintillation fluid and the radioactivity was determined. Owing to the low levels of amino acids present and the problems of incomplete separation in some instances, the specific activity (expressed as total counts per minute per micromole of amino acid) of only seven amino acids could be determined (Table 2). However, the results do not support the idea that the chloramphenicol-treated cells contain internal amino acids, the specific activities of which were 2.5 times greater than those of the control cells. It therefore is concluded that the chloramphenicol-insensitive fraction and newly synthesized enzyme are secreted simultaneously. Time course of accumulation of the cell-associated pool of enzyme. As shown above, chloramphenicol-treated cells, after removal of the drug, immediately synthesize and secrete extracellular enzymes into the external me-

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RELEASE OF EXTRACELLULAR ENZYMES

VOL. 122, 1975

associated with the cells. It can be seen from Fig. 8 that the chloramphenicol-treated cells began to accumulate a pool almost immediately after the removal of the drug and increased to a maximum in about 20 min. This final level was approximately the same as that in normal dilute cell suspensions. DISCUSSION We have shown in this paper that B. amyloliquefaciens cells contain a preformed pool of a-amylase and protease that is not due to nonspecific adsorption of the enzymes to the cell surface. The pool is released in approximately 15 min (in the presence of chloramphenicol) and this energy-independent release provides a means of studying the final process of

dium. It was of interest to see whether such cells re-accumulated the protease cell-associated pool and at what rate. A washed-cell suspension was treated with chloramphenicol (10,ug/ml) for 15 min to deplete the cells of enzyme and then the drug was washed away as described previously. The dilute cell suspensions were shaken in suspending medium in a number of separate flasks and, after various times of incubation, the amount of enzyme pool accumulated in the cells was determined. To do this, chloramphenicol was added at different times and the extracellular enzyme was determined in samples taken immediately after chloramphenicol addition and again after 20 min further incubation. The difference between these values represented the amount of the preformed enzyme

TABLE 1. Specific activities from three separate experiments of radioactive protease secreted by untreated washed-cell suspensions and by chloramphenicol-treated cells after removal of the drug from the cells Chloramphenicol-treated cells Net Net 0-min 5-min 0-min 5-min incubation incubation increase incubation incubation increase Untreated cells

Determinations

126 86 102

206 183 196

93 73 78

219 201 211

Radioactivity precipitated by nonimmune (counts/min per ml)

88 60 70

84 59 74

84 70 76

85 60 76

Corrected values (counts/min per ml)

38 26 32

122 124 121

84 98 89

9 3 2

134 141 135

125 138 133

8 7 7

32 38 39

24 31 32

3 2 2

17 21 18

14 19 16

3,500

3,160

2,780

8,930

7,260

8,310

Radioactivity in protease-immune precipitate (counts/min per ml)

Protease activity (U/ml x 103)

Specific activities of protease (counts/min per U of activity) Ratio of specific activities

2.3

2.6

3.0 _

TABLE 2. The specific activities of internal amino acids present in chloramphenicol-treated and untreated cells after a 90-s pulse with protein ['4C]hydrolysate. Untreated cells

Chloramphenicol-treated cells Amino acid

Total Mmnol

Glutamic .................. Glycine ................... Alanine ................... Valine .................... Isoleucine ................. Leucine ................... Ornithine .................

0.092 0.005 0.022 0.007 0.003 0.002 0.002

Total

counts/ min

21,816 259

1,261 237 558 553 261

Sp act (counts/ min per umol) X

Total

103

'Umol

237 52 57 34 186 277 131

0.122 0.011 0.025 0.004 0.002 0.004 0.005

Total

counts/ min

26,329 336

1,419 270 580 732 663

Sp act (counts/ min per mol) X 103

216 31 57 68 290 183 133

40

GOULD, MAY, AND ELLIOTT

0.1

0

,a X, e

0-05r

10

0

20

30

40

(men) of protease re-accumula-

.b.tio tfme

FIG. 8. The time course tion by pool depleted dilute washed-cell suspensions. The experimental details are given in the text.

enzyme secretion. That the pool enzyme molecules en route to the

extracellular represents

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protease and a-amylase do not possess any preformed pools. The curious effects of temperature in altering the absolute proportion of the pool capable of release may possibly be due to different proportions of the enzyme adsorbing to or complexing with the cell membrane and/or cell wall. A temperature-dependent, energy-independent release of penicillinase from chloramphenicol-treated B. subtilis and B. licheniformis cells has been previously reported (4, 6). There are three forms of the penicillinase, one form tightly bound to the cell membrane and another form associated with the vesicles, with the latter being a precursor to the extracellular form (8). It has been shown that in the presence of chloramphenicol penicillinase associated with the vesicular fraction is released, but not that bound to the cell membrane. This situation seems to be different from the present one since all of the cell-associated extracellular enzyme is released in the presence of chloramphenicol and no particulate association of the enzymes was established.

exterior is indicated by several observations; it is constantly present in normally secreting cells, it is exhausted when cells are allowed to release it in the absence of further protein synthesis, and it is restored again when such depleted cells are permitted to resume enzyme synthesis. The pulse-labeling experiments provide some information about the nature of the pool and its LITERATURE CITED position in the secretory process. Since newly synthesized enzyme molecules begin to emerge 1. Both, G. W., J. L. McInnes, J. E. Hanlon, B. K. May, and W. H. Elliott. 1973. Evidence for an accumulation of from the cell within the shortest time measured messenger RNA specific for extracellular protease and (5 min), it seems that there is not an obligatory its relevance to the mechanism of enzyme secretion in delay of at least 15 min before a completed bacteria. J. Mol. Biol. 67:199-217. molecule is secreted. That is, the pool is not a 2. Glenn, A. R., G. W. Both, J. L. McInnes, B. K. May, and W. H. Elliott. 1973. Dynamic state of the messenger "pipeline" from which enzyme molecules RNA pool specific for extracellular protease in Bacillus emerge in the order that they were synthesized. amyloliquefaciens: its relevance to the mechanism of The fact that enzyme molecules labeled during enzyme secretion. J. Mol. Biol. 73:221-230. a 90-s pulse period continue to emerge (in the 3. Gould, A. R., B. K. May, and W. H. Elliott. 1973. Accumulation of messenger RNA for extracellular enpresence of chloramphenicol) at a constant rate zymes as a general phenomenon in Bacillus for 15 min suggest that newly synthesized amyloliquefaciens. J. Mol. Biol. 73:213-219. enzyme molecules immediately equilibrate with 4. Lampen, J. 0. 1967. Cell-bound penicillinase of Bacillus licheniformis. Properties and purification. J. Gen. Mithe entire enzyme pool. Compatible with this is crobiol. 48:249-259. the finding that pulse-labeled enzyme emerges B. K., and W. H. Elliott. 1968. Characteristics of with a higher specific activity from pool-de- 5. May, extracellular protease formation by Bacillus subtilis and pleted cells than from control cells containing its control by amino acid repression. Biochim. Biophys. Acta 157:607-615. the pool. 1961. The mechanism of liberation of The simplest explanation of our findings is 6. Pollock, M. R.from Bacillus subtilis. J. Gen. Microbiol. penicillinase that enzyme molecules emerge from the cell 26:267-276. membrane and final release from the cell is to 7. Rinderknecht, H., M. C. Geokas, P. Silverman, and B. J. Haverback. 1968. A new ultrasensitive method for the some degree restricted by diffusion through the determination of proteolytic activity. Clin. Chim. Acta cell wall resulting in the accumulation of a 21:197-203. small pool underneath the cell wall. This agrees 8. Sargent, M. G., and J. 0. Lampen. 1970. A mechanism for that data) with recent findings (unpublished penicillinase secretion in Bacillus licheniformis. Proc. Natl. Acad. Sci. U.S.A. 65:962-969. protoplasts capable of secreting extracellular