Some characteristics of bacitracin production by Bacillus licheniformis

ARCHIVES

OF

BIOCHEMISTRY

Some

AND

Characteristics by Bacillus

ROBERT From

the Biology

87, 232-238

BIOPHYSICS

of Bacitracin licheniformis

W. BERNLOHRl

Division,

Oak

Ridge

Received

(1960)

AND

National September

Production

G. D. NOVELL1

Laboratory,g

Oak

Ridge,

Tennessee

21, 1959

The production of bacitracin by Bacillus licheniformis is a function of the cells in the stage of their life cycle after growth and before sporulation. The antibiotic is released from cells only under cultural conditions that will support spore formation. The time required for production of bacitracin has been shortened from 3 to 6 days to 24 hr., and a working hypothesis for the mechanism of biosynthesis of this polypeptide antibiotic has been proposed.

by Biffi et al. (4) for chlortetracycline production; by Corum et al. (5) for erythromycin; and by Waksman for streptomycin (6) and neomycin (7), in the actinomycete group. An emphasis on antibiotic production during the nongrowing phase in true bacterial fermentations has not been specifically indicated, although it can be inferred from the literature. Polymyxin is produced in sporulating, 5-day cultures of Bacillus polymyxa (8), and the same general time considerations and cultural morphology are true of circulin (9), polypeptin (lo), and subtilin (11). Dubos (12) demonstrated the release of tyrothricin from autolyzing cells of Bacillus brevis that had been suspended in water. It is also interesting that all the antibiotic producers mentioned thus far are spore formers; in one case, streptomycin production (6), an asporogenous strain of the responsible microorganism, Streptomyces griseus, produces no antibiotic. We shall indicate the time during the life cycle of Bacillus licheniformis that bacitracin is produced and suggest some relation between spore formation and antibiotic production.

INTRODUCTION

During an investigation on the biosynthesis of bacitracin, it became apparent that the antibiotic was produced by the cells concurrently with active spore formation (1). Production of other antibiotics by several different microorganisms seems to be a function of the cells or mycelium in the postlog phase. Bhuyan and Johnson (2) observed that production of synnematin by Cephalosporum salmosynnematin occurs after growth is almost complete. They define specific media for either growth or antibiotic production, stressing a rapid growth phase followed by a fermentation period of very slow growth. Halliday and Arnstein (3) reported biosynthesis of penicillin by washed mycelial pads of 70-hr. cultures of Penicillium chrysogenum. These pads, when supplied only with the carbohydrate precursor of the penicillin side chain, released the antibiotic for 5 hr., during which time mycelial nitrogen remained constant. In addition to these fungal systems, identical observations on the fast growth-slow fermentation effect were made 1 Postdoctoral Fellow of the Life Insurance Medical Research Fund. Present address: Department of Agricultural Biochemistry, Ohio State University, Columbus, Ohio. * Operated by Union Carbide Corporation for the U. S. Atomic Energy Commission.

METHODS

AND

MATERIALS

Bacillus licheniformis, A-5, obtained from Dr. E. P. Abraham, Sir William Dunn School of Pathology, University of Oxford, Oxford, England, was used exclusively in these experiments. Spores 232

BACITRACIN

PRODUCTION

were prepared by B-day growth with shaking in 1% tryptone broth, followed by suspension in distilled water at 3°C. The spores were then separated from lysed cell wall material by centrifugation at 3000 X g for 20 min. and were pasteurized at 60°C. for 90 min. The salt mixture used in all experiments consisted of (per liter of tap water): MgS04.7Ht0, 1.0 g.; MnS0,.4Hz0, 6.0 mg.; FeS0,.(NHJr!S01.6H20, 25.0 mg.; NaCI, 0.4 g.; KCl, 0.4 g.; IlaP , 0.45 g.; and citric acid, 0.312 g. The pH was adjusted to 7.2 with NH,QH for the experiments described in Fig. 1 and Tables I-IV, and with KOH for experiments described in Figs. 2 and 3. The salts mixture was sterilized separately from the carbohydrate solutions. Carbohydrate additions to this salts mixture are noted in the text. The inoculum (always 8%) consisted of cells from germinated spores after overnight incubation at 37°C. in an Eberbach water-bath shaker with a l-in. stroke. Bacitracin was measured by an antibiotic assay, using Bacillus megaterium as test organism. The inoculum was grown at 30°C. in Bactopenassay broth before seeding into nutrient agar. Approximately 0.25 ml. of logarithmic phase cells (Klett reading of 200 at 540 rnp) was added to 150 ml. of sterile nutrient agar, and this was poured into a sterile 6 X 10 in. glass dish fitted with an aluminum cover. Test solutions were pipetted onto Schleicher and Schuell No. 740-E filter-paper disks, dried, and placed on the agar surface. Incubation was overnight at 37°C. One unit of antibiotic activity is defined as the amount of bacitratin in 0.2 ml. of culture supernatant solution that will cause a l-mm. inhibition zone outside the filter-paper disk. The bacitracin unit values reported are averaged from at least six replicate determinations, the variation being not more than 10%. Authentic bacitracin (Nutritional Biochemicals Corporation, Cleveland, Ohio), rated at 55 Staphylococcus aureus units/mg., will assay ~750 units/mg., under these conditions. In addition to routine antibiotic assays on culture supernatants, we made occasional checks to verify that bacitracin was responsible for the antibacterial activity. The polypeptide was isolated by the method of Sharp et al. (13) and was chromatographed in the t-butanol-water, 80:20, NH3 atmosphere, Isolvent of Snell et al. (14). After the paper was developed, it was dried and applied to a seeded agar rsurface. An inhibition zone was observed only a.t the position assumed by authentic bacitracin. Growth was determined by optical density in the Klett-Summerson calorimeter at 540 q. A Beckman model G pH meter was used in pH determinations and titrations. Glucose was measured by the glucostat preparation of the Worthington

BY

B.

233

LICHENIFORMIS

50

25

25

100 !A 0

A/ 24

48

0 72 TIMEIhr)

96

420

FIG. 1. Growth, pH change, and bacitracin production by B. Zicheniformis. Cells were grown at 37°C. in 25-ml. lots in a medium of: salts mixture, 220 pmoles/ml. glucose, 50 pmoles/ml. ammonium lactate, and 11.3 rmoles/ml. ammonium chloride. The three cultures used were: A, stagnant; B, fast shake; and C, slow shake. Biochemicals Corp., lactic acid by the procedure of Barker and Summerson (15), and ammonia after microdiffusion into acid by the Nessler technique as described by Vanselow (16). Relative numbers of bacterial cells and spores were estimated by observation with a Bausch and Lomb phase-contrast microscope after the necessary dilutions, except where noted. RESULTS

Hills et al. (17) and Hendlin (18) reported the production of bacitracin from growing cultures of B. lichenifornzis. No separation of growth from antibiotic production was mentioned in either work. Hills et al. (17) used stagnant cultures and did not compare the increase in cell population with bacitracin release. When this is done (Fig. 1A) it can be seen that the release of antibiotic lags behind the growth of the cells by about 2 days. Bacitran is not detected until the third day and increases for the following 4 days, whereas growth, which was initiated by the first day, is complete in 5 days.. Submerged aerated cultures can produce bacitracin according to Hendlin (18), but in his

234

BERNLOHR

TABLE RELEASE SPORE Time

I

OF BACITRACIN

DURING

GERMINATION”

Spores/ml. x 10’0

c’“;~~l.

x

Bacitracin units/ml.

hr.

0 1 2 3 4 5 6 7 23

7.5 7.6 7.0 6.6 6.2 5.3 4.4 3.5 3.5

0.0 0.0 1.8 3.4 4.3 5.7 7.6 10.5 33.0

0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0

a B. lichenijormis, germinated in the salts mixture, 50 pmoles/ml. ammonium lactate, 220 pmoles/ml. glucose, and 11.3 pmoles/ml. ammonium chloride. Cells and spores were counted in a Petroff-Hauser counting chamber.

work, different nutritional conditions were used (i.e., a salts mixture, glucose, and glutamic acid). Under these conditions antibiotic release follows the growth curve closely. This may be caused by a strain difference or the nutritional conditions themselves. When strain A-5 is grown in shake culture in the medium of Hills et al. (17) containing salts, 220 pmolesglucose, and 50 pmoles ammonium lactate, growth and antibiotic release follow the patterns shown in Fig. 1B and 1C. At 140 strokes/min. (fast shake), acid is formed at an accelerated rate, the pH drops to 4.5 where it remains, and bacitracin is not formed. At 90 strokes/min. (slow shake), growth is complete in 48 hr. ; after a drop in pH to about 5.5 the pH returns to 7.0, and bacitracin begins to appear in the medium. Bacitracin cannot be found, either inside the cells or in the medium during growth under these conditions. Experiments of this type were quite variable, and on occasion, no bacitracin was observed after 120 hr. It was soonfound,however, that experiments using freshly germinated spore suspensions as the inoculum always gave good bacitracin production. A culture carried in* the vegetative state for several days produced very little bacitracin. An examination of the spore content of A-5 cultures showed that every bacitracin-pro-

AND

NOVELL1

ducing culture was sporulating. On the other hand, the cultures in which the pH dropped below 5.0 and remained there produced no spores and no bacitracin. Attempts were thus made to correlate bacitracin production with spore metabolism. After cell growth in Hills’ medium is complete, a sufficient amount of carbon- and nitrogen-containing compounds remain to permit germination of spores. The possibility existed, therefore, that the polypeptide antibiotic was released during germination of the spores formed in the incubation period, so we tested this hypothesis directly. A suspensionof 7.5 X lOlo spores/ml. was introduced into fresh growth medium. Table I indicates that 4.0 X lOlo spores/ml. germinated over a period of 7 hr., but no bacitracin was detected in the medium. Attention was then directed toward spore formation, in an attempt to correlate a change in the amount of bacitracin produced with the degree of sporulation. Experiments were designed along lines similar to those used by others (19) for increasing spore production. B. licheniformis cells were grown in 25-ml. lots by slow shaking until the pH of the medium had risen to 7.0 (about 36 hr.). After standing at 3°C. for 2 hr. (cold shock), the cultures were sedimented, washed once with distilled water at 3”C., resuspended, and shaken at 37°C. under the conditions shown in Table II. Control flasks were submitted to the cold shock only, and returned to the water-bath shaker at 37°C. After an additional 18 hr., the pH was measured and the medium was assayed for bacitracin. Cells resuspended in fresh medium continued to grow, the pH dropped, and no bacitracin could be detected, even though the control flasks (in old medium) produced a small but significant amount of bacitracin. Cells resuspended in fresh medium from which ammonium ions were omitted did not grow but released a small amount of bacitracin. In culture medium without glucose, growth of resuspendedcells was also stopped, but a slight stimulation of bacitracin production over the controls was observed. In all of these cultures, spores were produced in 70 hr. The most striking result (Table II) was the fivefold increase in spore production and

BACITRACIN

PRODUCTION

2.5-fold increase in bacitracin release exhibited by cells resuspendedin distilled water. This effect is shown more clearly in Table III. Buffering the water with Tris [tris(hydroxymethyl)aminomethane] at pH 7.4 doubles the antibiotic production but does not double the amount of sporulation. The presence of Mn+k and Ca++ enhances sporulation to a much greater extent than it does the appearance of bacitracin. These observations lead to the following conclusions. First, under conditions of controlled growth, bacitracin appears in the medium after growth is complete. Second, and most important, this n-amino acid-containing polypeptide antibiotic is released only under cultural conditions that will permit eventual sporulation. Third, there does not seem to be a direct proportionality between the extent of sporulation and the amount of bacitracin produced. Foster (20) introduced the term endotrophic sporulation “as a convenient means of alluding to spores produced in the absence of exogenous nutrition and where growth is precluded, in distinction to those formed in growth media.” In our studies, under endotrophic conditions both sporulation and baci.tracin releaseare observed only in culturesthat have completed logarithmic growth and in which the pH of the medium has risen above 6.5. In the medium of Hills et al. (17), glucose is present at a concentration of 220 clmoles/ml. and is not the limiting factor for growth. Under these conditions, considerable glucose is still present in the medium when the pH is rising toward 7.0. If acid is still being produced from glucose, an excess of base must be generated in order to return the medium to pH 7.0, thus iucreasing the time required to reach the latter pH and subsequent bacitracin release. The

return to pH 7 and bacitracin releasein these high glucose cultures occurs only in stagnant or slowly shaken cultures. If such cultures are vigorously aerated, they continue to

produce acid in excess of base and the pH remains low and bacitracin is never produced. In order to decreasethe time required to return to neutral pH, the glucose concentration was reduced to the point where it became limiting. Table IV indicates the effect of glucose concentration on the pH of

BY

B.

235

LICHENIFORMIS

TABLE II EFFECT

OF MEDIA

ON BACITRACIN

AND

APPEARANCE

SPORULATIONQ

I

Time

Treatment (suspending medium)

18 hr.

70 hr.

BX. units/ml.

PH

Sprllla&n

% Control Fresh medium at twofold dilution Fresh medium minus NHa+ Fresh medium minus glucose Distilled water

7.5

10

1

5.2

0

0

5.1 6.7

5 15

1 1

7.2

25

5

a B. lichenijormis, grown for 36 hr. at 37°C. in a medium of: salts mixture, 50 pmoles/ml. ammonium lactate, 11.3 rcmoles/ml. ammonium chloride, and 220 pmolesjml. glucose. Spore counting was done under the phase-contrast microscope.

TABLE III BACITRACIN APPEARANCE DURING ENDOTROPHIC SPORULATIONQ Time

I Treatment (suspending medium)

2 hr.

5 hr.

20 hr.

I

_-~ Control Distilled water 0.03 M Tris 0.03M MnS04 0.03 M MnSOh and CaClz

6.0

0

6.6

5

7.4 6.2

5 0

6.55

0

6.0 6.6 7.4 6.2 6.1

0

% 0

10

10

20 5 5

5 30 50

a B. bichenijormis, grown for 48 hr. at 37°C. in a medium of: salts mixture, 50 rmoles/ml. ammonium lactate, 11.3 pmoles/ml. ammonium chloride, and 220 pmoles/ml. glucose. Sporulation was estimated by examination in the phase-contrast microscope. Other conditions identical with Table II; see text.

the culture, the appearance of bacitracin, and the formation of spores. At a concentration of 27 pmoles/ml. growth is complete in 18 hr., the pH has risen, and bacitracin is released. It is evident that, although the cells grow well on glucose, the removal of

236

BERNLOHR

EFFECT

OF GLUCOSE

AND TABLE PH.

CONCENTRATION

ON

NOVELL1 IV

BACITRACIN

APPEARANCE

AND

SPORULATION~

Time Glucose concentration

18 hr.

42

%

#mioles/ml. 220 110 54 27

5.5 5.9 7.3 7.4

Q B. licheniformis, pmoleslml. ammonium contrast microscope.

0

5

0 0 0 5 grown in chloride,

IO

15 TIMElk)

20

0 0 0 0.25

5.5 6.9 7.4 7.7

% 0 0 0.25 0.5

0 0 5 10

6.0 7.7 7.8 7.3

hr.

a medium of: salts mixture, 50 rmoles/ml. ammonium and glucose. Sporulation was estimated by examination

25

30

% 0.0 0.5 2.5 5.0

0 5 15 20

lactate, 11.3 in the phase-

rise in pH is not exclusively the result of the utilization of the organic acids formed from glucose since substitution of ammonium chloride for ammonium lactate does not prevent this rise. In the latter case, an amount of base, equivalent to the moles of ammonia utilized during growth, must be provided by the cell in order to facilitate the observed pH change. After glucose utilization is complete, the cells begin to oxidize lactic acid and for

FIG. 2. Time course of growth, pH ‘change, bacitracin production, and spore formation. Cells were grown at 37°C. in a medium of: salts mixture, 14 /rmoles/ml. glucose, 50 ~moles/ml. ammonium lactate. The number of spores was estimated under the phase contrast microscope.

this fermentable substrate facilitates the rise in pH and the subsequent differentiation of the microorganism. It should be mentioned that the rate of shaking of these lowglucose cultures has no effect on the formation of bacitracin or spores, in contrast to cultures grown in the high-glucose concentration. In the presence of a very limited amount of glucose, 14 pmoles/ml. growth, pH changes, bacitracin release, and sporulation have been observed. In addition, the utilization of glucose, ammonia, and lactic acid was measured. These data are plotted in Figs. 2 and 3. It can be seen that, while glucose is being utilized (Fig. 3), the pH drops to 5.6-6.0 and does not rise until this carbohydrate is essentially removed. This

‘,

4

8

I2 TIME(hr)

I6

20

24

FIG. 3. The utilization of nutrients by B. lich.eniformis. Additions to the salts mixture were ii. pmoles/ml. glucose and 56 pmoles/ml. ammonium lactate.

BACITRACIN

PRODUCTION

about 5 hr. grow at the expense of this acid. It is probable that another acid is formed during this time because the pH and titratable acidity remain constant. Bacitracin can be detected in small quantities after 10 hr., but spores cannot be observed (Fig. 2). After growth is complete, at about 13 hr., there is no further change in the concentration of ammonium ions in the medium. Lactic acid is slowly utilized with a concomitant rise in pH. Bacitracin is present in large quantities by the time free spores are first observed in the culture (-20 hr.). Sporulation is completed at -32 hr., and the quantity of bacitracin begins to decrease, probably because of its instability at pH’s higher than 7.5-8.0. After about 8 hr., when logarithmic growth is completed, the culture can be placed under endotrophic conditions and subsequent sporulation can be observed. Thus, by restricting the glucose concentration, the ti.me required for bacitracin production wa;s reduced from 72 to 20 hr., and the time required for cells to acquire the ability to initiate endotrophic sporulation was reduceld from 48 to 8 hr. In an attempt to dissociate the release of bacitracin from the phenomenon of spore formation, 50 clmoles/ml. ethyl malonate, a known inhibitor of sporulation (al), was added to the culture when the pH of the medium was beginning to rise from its lowest point. Vegetative cell growth was not altered, but sporulation was inhibited to an extent that allowed for formation of only 2-5 % of sporangia. Bacitracin production in these cultures was inhibited 60-80 %.

BY

B.

LICHENIFORMIS

237

points out that a great part of the cehwall is dissolved during sporulation, and, that much of the peptide material is then released into the medium. From these reports, together with our own data, it is tempting to speculate that bacitracin might arise as a result of the hydrolysis of cell wall material by a lytic activity developed by the cell during the time of special metabolic activity eventually leading to sporulation. It should be pointed out however that there is no evidence available that indicates that bacitracin ever is a cell wall component. The antibiotic is released from nongrowing cells only under cultural conditions that will eventually support spore formation. The relation between the two phenomena is not clear, however, but the endotrophic sporulation experiments may suggest a possible explanation. Foster (20), who worked with cells of Bacillus megaterium under conditions of endotrophic sporulation, reported that once the enzymes for sporogenesis have been synthesized, the induction of fl-galactosidase or maltase can no longer be demonstrated and methionine-S35 is incorporated into total protein at a greatly reduced rate. He suggests that there is a competition for the free amino acid pool between the systems involved in spore formation and those required for new protein synthesis and that this competition is not directly related to sporulation but yet is involved in the metabolism of the differentiated cell. He considers that the amino acid pool is not large enough to provide the necessary building blocks for either of these systems and invokes the activity of proteases to supply the cell with amino acids DISCUSSION during this phase in its life cycle. Nomura Bacitracin is a polpyeptide that contains and Hosoda (25) demonstrated production 12 amino acids, four of which are the Dof protease and amylase by Bacillus subtilis isomers of glutamic acid, aspartic acid, under endotrophic conditions. Optimal prophenylalanine, and ornithine (22). n-Amino duction of these enzymes proceeds in a culacids seem to be localized primarily in cell ture that is sporulating to a very limited exwalls of a large variety of microorganisms. tent. In this culture, however, the cells must Studies related to the release of peptides con- be protected from lysis by compounds providtaining these isomers from the cell walls of ing a high external osmotic pressure, owing bacilli have been reviewed by Strange (23). presumably to alterations in the cell wall at The lytic enzymes, apparently responsible this time in the life cycle. If bacitracin is for the hydrolysis of the cell walls and thereproduced as a result of the activity of a fore the exudation of the peptides, are isoproteolytic or lytic enzyme synthesized in lated from cultures that are in the process competition with spore formation, a reverse of snore flormation (24. \I, 25). Strangev (23) I \ T relation between sporulation and bacitracin

238

BERNLOHR

AND

release would be expected and is observed (Table III). Thus, it is shown that, although bacitracin is produced only in cultures that will support sporulation, conditions in which the cells produce optimal amounts of antibiotic provide for poor spore production. Also, conditions that will allow a high proportion of the cells to sporulate seem to inhibit the production of bacitracin. This phenomenon is exhibited only when the cells are resuspended under endotrophic conditions. From a number of considerations, it seems evident that a vegetative cell must undergo profound changes in its metabolic activities in preparation for sporulation. Our observations suggest that similar metabolic changes are required for the synthesis and release of bacitracin, as well as other peptides. It is possible that, at some point in the life cycle of this spore former, the metabolic paths diverge so that antibiotic production eventually competes with sporulation. This suggested dichotomy of a relation between spore formation and antibiotic production does not exclude other alternatives. Further study of the biosynthesis of bacitracin in living cells and in cell extracts is under way to elucidate additional details of the mechanism. REFERENCES 1. BERNLOHR, R. W., AND NOVELLI, ture, in press. 2. BHUYAN, B. K., AND JOHNSON, M. teriol. 76, 376 (1958). 3. HALLIDAY, W. J., AND ARNBTEIN, Biochem. H. 64,380 (1956). 4. BIFFI, G., BORETTI, G., DIMARCO, PENELLA, P., Appl. Microbiot. 2,

G. D.,

Na-

J., J. BacH.

R.

V.,

A., AND 288 (1954).

NOVELL1

5. CORUM, C. J., STARK, W. M., WILD, G. M., AND BIRD, H. L., JR., Appl. Microbial. 2, 326 (1954). 6. WAKSMAN, S. A., “Streptomycin.” The Williams and Wilkins Co., Baltimore, Md., 1949. 7. WAKSMAN, S. A., “Neomycin.” Rutgers University Press, New Brunswick, N. J., 1953. 8. BENEDICT, R. G., AND STODOLA, F. H., Ann. N. Y. Acad. Sci. 61, 866 (1949). 9. MURRAY, F. J., TETRAULT, P. A., KAUFMANN, 0. W., KOFFLER, H., PETERSON, D. H., AND COLLINGSWORTH, D. R., J. Bacterial. 67, 305 (1948). 10. MCLEOD, C. M., J. Bacterial. 66, 749 (1948). 11. JANSEN, E. F., AND HIRSHMAN, D. J., Arch. Biochem. 4, 297 (1944). 12. DUBOS, R. J., J. Exptt. Med. 70. 1 (1939). 13. SHARP, V. E., ARRIAGEDA, A., NEWTON, G. G. F., AND ABRAEAM, E. P., Brit. J. Exptl. Pathol. 30,444 (1949). 14. SNELL, N., IJICHI, K., AND LEWIS, J. C., Appl. Microbial. 4, 13 (1956). 15. BARKER, S. B., AND SUMMERSON, W. H., J. Biol. Chem. 138, 535 (1941). 16. VANSELOW, A. P., Ind. Eng. Chem., Anal. Ed. 12, 516 (1940). 17. HILLS, G. M., BELTON, F. C., AND BLATCHLEY, E. D., Brit. J. Exptl. Pathol. 30. 427 (1949). 18. HENDLIN, D., Arch. Biochem. 24, 435 (1949). 19. ORDAL, Z. J., in “Spores” (H. 0. Halvorson, ed.), pp. le26. Monumental Printing Company, Baltimore, Md., 1957. 20. FOSTER, J. W., Quart. Rev. Biol. 31, 102 (1956). 21. NAKATA, H. M., AND KRISHNAMURTY, G. G., Bacterial. Proc. (Sot. Am. Bacteriologists) 69, 39 (1959). 22. ABRAHAM, E. P., “Biochemistry of Some Peptide and Steroid Antibiotics,” p. 7. John Wiley and Sons, New York, N. Y., 1957. 23. STRANGE, R. E., Bacterial. Rev. 23. 1 (1959). 24. STRANGE, R. E., AND DARK, F. A., J. Gen. Microbiol. 17, 525 (1957). 25. NOMURA, M., AND HOSODA, J., J. Bacterial. 72, 573 (1956).