J. appl. Bact. 1975,39, 1-13
Changes in the Extracellular Accumulation of Antibiotics duringiGrowth and Sporulation of Bacillus subtilis in Liquid Culture
J. G. BARR Agricultural and Food Bacteriology Research Division, Department of Agriculture for Northern Ireland, and The Queen’s University, Belfast, Northern Ireland Received 27 August I974 and accepted 3 March 197.5
Ten antimicrobial metabolites, produced by Bacillus subtilis NCIB 8872, have been categorized into 3 groups on the basis of their antimicrobial spectra, chromatographic mobility and solvent solubility. The maximum concentration of the different groups occurred at different times during fermentation. The accumulation of one antibiotic group was characteristic of the logarithmic growth phase. Groups also differed in their persistence. The intracellular antibiotics contributed little to the total antibiotic activity of the culture. The onset of production and the maintenance of the extracellular accumulation of the 3 antibiotic groups were linked to sporulation and associated specific changes in the intracellular and extracellular protein complement. Production of the antibiotics was not controlled by glucose catabolite repression, since the presence or absence of glucose in the medium did not affect the pattern of antibiotic accumulation.
THEREis considerable evidence that sporulation and antibiotic production by a number of Bacillus species are closely allied or interdependent processes (Bernlohr & Novelli, 1963; Schaeffer, 1969, Sadoff, 1972). However, Ray & Bose (1971) and Haavik & Thomassen (1973) have described mutants of Bacillus subtilis and B. lichenniformis, respectively, which retained the ability to sporulate without any accompanying antibiotic production. Antibiotic accumulation, either intracellular or extracellular, normally occurred after the end of the logarithmic growth phase and prior to endospore maturation (Woodruff, 1966; Bodansky & Perlman, 1969). It has been suggested that peptide antibiotics, like secondary metabolites, are only produced by organisms which have ceased to divide, after nutrient limitation (Bu’Lock, 1961). In cultures of Bacillus species, glucose may affect both sporulation and antibiotic production. Murrell (1967) and Schaeffer (1969) have reviewed the evidence that in B. subtilis cultured on a medium containing glucose, specific enzymes required for sporulation were repressed during exponential growth and suggested that these enzymes were controlled by glucose catabolite repression. Demain (1968) maintained that the production of many antibiotics, including bacitracin, may be controlled by glucose catabolite repression. Some strains of B. subtilis can produce several antibiotics (Majumdar & Bose, 1958; Schmitt & Freeze, 1968) and different strains can produce antibiotics of different antimicrobial spectra. (Koryzbski, Kowszk-Gindifer & Kurylowitz, 1967). In strains [I1
2
J. G . BARR
which can produce several antibiotics it has not been established how the accumulation and relative proportions of the different antibiotic compounds aIter during the development and sporulation of a culture. This paper describes studies of the pattern of antibiotic produc.ion in growing and sporulating cultures of a strain of B. subtilis, cultured in the presence or absence of glucose. The strain described is known to produce the antifungal antibiotic, bacillomycin (Landy, Warren, Rosenman & Colio, 1948; Babad, Pinsky & Turner-Graff, 1952) and uncharacterized metabolites possessing antibacterial activity (Landy et al., 1948). Derivative strains have been shown to produce 3 closely related antifungal polypeptide antibiotics (Burachek, Leardini & Paladini, 1964).
Materials and Methods Organisms
The organism used throughout the work was Bacillus subtilis NCIB 8872 (Landy et al., 1948). Antibacterial activity was evaluated against Staphylococcus aureus Oxford 209P and Erwinia carotovora NCIB 8097 and antifungal activity against a pathogenic strain of Nectria galligena (typed by Commonwealth Mycological Institute, Kew). Media
All media were prepared in glass distilled water. The inocula for fermentation studies with B. subtilis and for seed cultures for antibacterial antibiotic assay were grown on a nutrient medium (N) containing (g/l): peptone, 10.0 Lab Lemco (Oxoid L29), 8.0; NaCI, 5.0. For studies of antibiotic production by B. subtilis a chemically defined medium (F) of the following composition (g/l) was employed: glucose, 20.0; L-glutamic acid, 5 -0; MgS04.7H2O70-5; KCl, 0.5; KH2PO4,l-0; Fez (SO&. 6H2O7O.013;MnS04.Hz0, 0.005; CuS04.5H20,0.016. The pH value of the medium was 6.0 after sterilization at 121O for 20 min. In some experiments, glucose was omitted from medium F and the pH of this medium was 6.2 after sterilization. The nutrient agar used for the maintenance of bacterial cultures, for the enumeration of viable cells and for all antibiotic diffusion assays contained (g/l): peptone, 10.0; Lab Lemco (Oxoid L29), 10.0; NaCI, 5-0; agar (Difco No. 3), 10.0. Cultures of Nectria galligena were maintained on 2 % (w/v) malt agar. Growth conditions T%epreparation of inoculafor organisms used for microbiological assay. Bacteria were grown shaken in 250 ml Erlenmeyer flasks each containing 50 ml of medium. An orbital shaker (Mk. V, L. H. Engineering, Stoke Poges, Bucks., England) operating at 220 rev/min with a circular orbit of 25 mm was employed. Staphylococcus aureus and E. carotovora were incubated at 37" and harvested 24 h after inoculation. B. subtilis was incubated at 30" and harvested after 16 h. The culture conditions for conidial production of N. galligena were those described by Swinburne (1971).
ANTIBIOTICS OF BACILLUS SUBTILIS
3
The preparation of inoculafor fermentation studies Fifty ml of medium was inoculated from a 24 h surface colony on agar. The culture was grown shaken for 16 h at 30". The cells were harvested by centrifuging and resuspended in an equal volume of medium F. One ml of this suspension of logarithmic phase vegetative cells was added to each 50 ml of medium F. Measurement of growth and sporulation The growth of B. subtilis was measured as extinction at 620 nm (E620) in a Unicam SP8000 spectrophotometer. The frequency of sporulation (s/v) was measured by the method described by Ray & Bose (1971). Spore formation and sporangial lysis were confirmed microscopically after staining (Bartholemew & Mittwer, 1950), and by phase-contrast microscope examination. Quantitative microbiologicalassay Antibiotic activities were determined using NUNC @K-4000 Roskilde, Denmark) bioassay plates (235 mm x 235 mm) each containing 165 ml of agar of uniform depth. Molten assay agar was cooled to 50" and inoculated with bacterial cells to give a concentration of 107 organisms/ml of agar. For N. galligena the corresponding concentration was lo4 conidia/ml. Wells (8 mm diam) were made on an 8 x 8 template and a 50 pl sample dispensed in each well. Plates seeded with Staph. aureus were incubated at 16 h at 37"; plates seeded with N . galligena, 48 h at 25 '. The zones of inhibition were then measured. One unit of activity was defined as the amount of antibiotic which produced a zone of inhibition 1 mm in width around each 8 mm diam well. All solutions were diluted appropriately to produce a zone of this diameter. Extraction and assay of culturefluids Cell free fluids were obtained by centrifuging cultures through Hemming Seitz filters. Two ml volumes of fluid were extracted twice with 5 ml portions of n-butanol. The combined extracts were dried under reduced pressure in a rotary film evaporator and the residue reconstituted in 2 ml of phosphate buffer, 0.1 M, pH 7.4. Residual butanol was removed from the extracted fluid. Extraction and assay of intracellular antibiotics All extractions were from batches of cells harvested from 50 ml of culture by centrifuging and washed twice with phosphate buffer (0.1 M, pH 7.4). Whole cells were suspended in n-butanol for 16 h at 20", when the butanol-soluble fraction was evaporated to dryness under reduced pressure in a rotary film evaporator and the residue resuspended in 5 mI of phosphate buffer (0-1M, pH 7.4). Disintegrated cells were obtained by resuspending harvested cells in phosphate buffer (0 * 1 M, pH 7 -0)and treating in a Braun cell disintegrator for 3 min using glass beads of 0.10-0. I1 mm diam. After centrifuging, a part of the supernatant liquid was retained for antibiotic assay. The remainder was extracted twice with 5 ml portions of n-butanol fractions evaporated to dryness and reconstituted in 5 ml of phosphate buffer. A I0-fold concentration of the extracts derived from whole or disintegrated
4
J. G.BARR
cells was achieved using Amicon macrosolute concentrators (Amicon Corporation, Lexington, Mass.). Extracts derived from the cells and culture fluids were assayed and chromatographed by the same methods. Thin-layer chromatography and localization of antibiotics Extracts were adjusted to a standard volume and then 20 pl portions were applied to a cellulose chromatogram sheet (Eastman 6065) and to a silica gel chromatogram sheet (Eastman 6061). Cellulose chromatograms were developed in a solvent mixture of n-butanol : acetic acid : water (3 : 1 : 1) (System I) and silica gel chromatograms developed with chloroform : methanol : water (60 : 35 : 4) (System 11). Both systems were developed to a distance of 150 mm and residual solvent removed in an air stream. Cellulose and silica gel were fixed by a gentle spray with molten nutrient agar at 50 '. Antibacterial antibiotics were detected on chromatograms by an overlay technique. Fifty ml of seeded agar was poured over each chromatogram and allowed to set at uniform thickness. Localization of inhibition zones of Staph. aureus and E. carotovora was aided by the incorporation in the overlay agar of 0.002% (w/v) of2, 3, 5triphenyltetrazolium chloride. Agars seeded with Staph. aureus, E. carotovora or B. subtillis were incubated for 16 h at 37" prior to evaluation. Antifungal antibiotics were located on chromatograms by a similar overlay technique using N. galligena. Seeded agar was incubated for 72 h at 25 *. Starch gel electrophoresis Cell extracts were prepared by the disintegration method described above. After centrifuging, the supernatant liquids were concentrated 10-fold using Amicon macrosolute concentrators. Cell-free culture fluids were concentrated 50-fold by the same method. Total protein in cell extracts and in concentrated culture fluids was adjusted to an equivalent protein content by a standard turbidimetric method (Stadtman, Novelli & Lipmann, 1951). Starch gels were prepared and developed in the discontinuous buffer system of Poulik (1957) with the modifications described by Baillie & Norris (1963). Separations were carried out at a constant current of 30 mA for 4 h. Esterases were detected by immersion of the gel in Tris-maleate buffer (0.1 M, pH 6.5) containing 0.20 mg/ml of m-naphthyl acetate and 0.75 mg/ml of Fast Blue RR added from stock solutions maintained in 50 % acetone solution. For the detection of catalases, the gel was covered with a 1 (v/v) aqueous solution of hydrogen peroxide for a few moments, washed in running water and further immersed in a 2 % (w/v) potassium iodide solution acidified with acetic acid.
Differentiation of the antibiotic components produced by Bacillus subtilis N U B 8872 Bacillus subtilis produced 10 antimicrobial compounds during a 72 h fermentation in medium F. On the basis of their chromatographic mobility, solvent solubility and
Group*
10
2 3 4 5
1
I[i
No.
+ + + -+
-
+ + + +. + + + +-
Staph. aureus
+
-
-
-
-
-
+-
-
E. carotovora
A
-
-
-
-
-I-
+
-
+ + + +-
-
I
-
+ + + + + +
-
B subtilis
N. galligena
Inhibitory activity against
,
-
+ + + -
-
+ +-
-
-
Solubility in n-butanolf r
t Solubility in n-butanol based on extraction of antibiotic from aqueous solution at neutral pH.
* Three antibiotic groups distinguished on the basis of antimicrobial activity, solvent solubility and chromatographic mobility.
Bacitracin Tyrocidine Gramicidin Subtilin Polymyxin
ILI
I1
I
& (
An ti biotic
0-73 0.72 0.72 0.73 0.69 0.87 0.88 0.89 0.76 0.96 1.00 0.98 0.65 0.75
0.73
System I
A
0.89 0.38 0.00
0.00 0.27 0.92
0.00
0.04 0.08 0.10 0.19 0.24 0.32 0.90 0.96
System I1
chromatography
Rfvalues by thin-layer
TABLE 1 The distinguishing properties of 10 antibiotics produced by Bacillus subtilis NCZB 8872 and 5 standard Bacillus-derived antibiotics
1
2
2
3
s
b
2
R
% ?Q
8
8
E
5
J. G. BARR
6
antimicrobial activity these compounds fall into 3 groups (Table 1). Groups I and I1 exhibited antibacterial activity;Group I11 exhibited antifungal activity only. Group I was differentiated from Group I1 on the evidence of solvent solubility and chromatographic mobility. The number of antibiotic components in each group is also recorded in Table 1. On the basis of these properties, the antibacterial antibiotics differ from bacitracin, gramicidin, tyrocidine, subtilin and polymyxin (Table 1).
I5O
t
(b)
Age of culture ( h )
Fig. 1. The fermentation of Bueilfus subtilis in medium F. (a) The relationship between growth, p H value and thermoresistant spore formation. 0 ,growth; A, s/v ratio; 0,pH value. (b)The extracellular accumulation of antibiotic Groups I, I1 and 111. W, Group I, antibacterial antibiotics, not butanol soluble; 0 , Group 11, antibacterial antiobiotics, butanol soluble; A, Group 111, antifungal antibiotics. The arbitrary unit of activity of the Group I and Group I1 antibiotics was defined with Sruph. uureus as the sensitive organism. The arbitrary unit of antifungal activity (Group 111) was defined with N. guZligena as the test organism.
ANTIBIOTICS OF BACILLUS SCJBTZLZS
7
Growth, sporulation and total extracellular antibiotic accumulation during fermentation The progression of growth, sporulation and extracellular antibiotic accumulation in cultures of B. subtilis in medium F and medium F without glucose is recorded in Figs 1 and 2, respectively. Medium F. The end of the logarithmic growth phase occurred c. 20 h after inoculation [Fig. 1 ( a ) ] . Sporulation was complete at 30 h. Microscopic examination of samples taken at different time intervals showed that sporangial lysis and the release of free spores occurred after 40 h. The number of free spores increased between 40 and 60 h. The pH value did not change during the logarithmic growth phase while a steady rise in pH value was recorded after the onset of sporulation [Fig. 1 ( a ) ] . Figure 1 (b) shows that the concentration of the Group I antibacterial antibiotics and the Group I11 antifungal antibiotics began to increase before logarithmic growth had ceased. Activities reached a maximum between 25 and 28 h, during the period of active differentiation of the culture. Afterwards, the concentration of the Group I antibacterial antibiotics declined, at first rapidly and then more gradually. No comparable fall in the concentration of the antifungal metabolites was observed. The Group I1 antibacterial antibiotics were present only after 24 h. By then the concentration of the Group I antibacterial antibiotics was high. Subsequently the concentration of the Group IT antibiotics increased up to c. 48 h. The concentration of both antibacterial antibiotic groups declined significantly in the later stages of the fermentation. Medium F minus glucose. The end of the logarithmic growth phase occurred c. 26 h after inoculation [Fig. 2 (a)]. Although spores were detected after 30 h, sporulation was incomplete and an s/v ratio of 0.3 was recorded after 46 h. The pH value rose steadily throughout the fermentation [Fig. 2 (a)]. Figure 2 (b) shows that the pattern of accumulation of the 3 antibiotic groups paralleled the course of their accumulation in medium F. The 3 antibiotic groups were present in lower concentration, and their activities reached a maximum later than in medium F.
Theformation of specific antibiotic metabolites during fermentation Extracellular compounds. The compounds were detected by thin layer chromatography. In medium F and in medium F minus glucose, 2 antifungal components (detected by System I) were consistently present after 20 and 26 h, respectively. Throughout the fermentations the relative contributions of these 2 antifungal antibiotics to the total antifungal activity remained the same. Table 2 shows that the representation of the various antibacterial compounds (detected by System 11) changes with time. This is in accord with the fluctuationsin the concentration of the Group I and Group I1 compounds demonstrated in Figs 1 (b) and 2 (b). In medium F, the main Group I antibiotics, Rf 0.19 and Rf0.24, were detected during the logarithmic growth phase, although the peak in their accumulation did not occur until the sporulation phase. During the period of spore formation and maturation all 6 Group I antibiotics were detected. The concentration of the individual antibiotics diminished after 30 h, and only 1 component, Rf 0.19, could be detected
a
J. G . BARR
throughout, but during this period the less polar Group I1 antibiotics appeared and increased in concentration. Later, however, there was a reduction in the concentration of a11 the antibacterial antibiotics. In medium F minus glucose a similar pattern of antibiotic accumulation was found. The major Group I antibiotics, Rf 0.19 and Rf 0.24, were detected in the fermentation medium during the logarithmic growth phase, although the peak in their accumulation, and the appearance of other Group I antibiotics, did not occur until logarithmic growth had ceased after 26 h. The decrease in concentration of the Group I antibiotics after this time was accompanied by the appearance and increase in
I5O
t
10
.,
20
I 30 40 Age of culture ( h )
1
I
50
60
Fig. 2. The fermentation of Bacillus subtilis in medium F minus glucose. (u) The relationgrowth: A, ship between growth, pH value and thermoresistant spore formation. 0, s/v ratio; 0, pH value. (b)The extracellular accumulation of antibiotic Groups I, I1 and 111. Group I, antibacterial antibiotics, not butanol soluble; 0 , Group 11, antibacterial antibiotics, butanol soluble; A,Group 111, antifugal antibiotics. The arbitrary unit of activity of the Group I and Group I1 antibiotics was defined with Staph. aureus as the sensitive organism. The arbitrary unit of antifungal activity (Group 111) was defined with N. gulligenu as the test organism.
I1I
Group?
+ -
-
I7 8
-
10
4 5 6
3
No.
-
-
+
-
-
15
-
-
-
22
-
24
-
27
A
-
30
+ + ++ +++ ++ + ++ ++ ++ ++ ++ - - - - - + - - - - - +
20
- -
18
46
48
51
+ - - - ++ ++ ++ ++ -+ + + + ++ + ++ ++ ++
39
Time after inoculation 0)
+ -
-
60
++ +
-+
54
I
* Antibiotics, separated by chromatographicSystem II, are indicated as absent (-), present as a minor component (+), or present as a major component (+ +). Antibiotics were detected by a Staph. uureus overlay technique. t Groups I and I1 were distinguished by antimicrobial spectra, solvent solubility and chromatographic mobility.
F minus glucose
F
Medium
Antibiotic
TABLE 2 The occurrence* of antibacterial antibiotics at different times during the fermentation of Bacillus subtilis NCIB 2872 on medium F and on medium F minus glucose
W
10
J. G . BARR
concentration of the Group I1 antibiotics. Later, as in medium F, the concentration of all the antibacterial antibiotics decreased. Antibiotic production, in medium F minus glucose, was also distinguished by the absence of the antibiotic components at Rf0.04 and Rf0-08. Intracellular compounds. The Group I1 antibacterial antibiotics were detected in cell extracts between 20 and 26 h growth in medium F. After this time no antibiotic activity could be demonstrated in the butanol-soluble or water-soluble fractions of the cell extracts. The butanol-insoluble Group I antibiotics and the antifungal antibiotics could not be detected in the cell extracts at any point during the fermentation. During fermentation in the absence of glucose, the Group I1 antibacterial, antibiotics were detected in cell extracts after 30,40 and 46 h fermentation, but the Group I and Group 111 antibiotics could not be detected at any time. The intracellular concentration of antibiotics was always small relative to the extracellular antibiotic accumulation. The inhibition of B. subtilis by individual components of the total antibiotic complement
Culture samples containing each of the constituent antibiotic components were chromatographed in both development systems. The chromatograms were overiaid with agar seeded with logarithmic phase vegetative cells of the B. subtih strain itself. Those components of Rf 0.04, 0-08, 0*10,0-19 and 0.24 (detected by System 11) inhibited the growth of logarithmic phase vegetative cells, while the butanol-soluble antibiotics produced no zones of inhibition. The intracellular and extracellular levels of esterase and catalase activity during fermentation in medium F
The change in the extracellular and intracellular esterase and catalase activity were followed over a 12 h period which marked the end of the logarithmic growth phase and the formation of thermoresistant spores. Esterase activity was demonstrated in concentrates of culture fluid and in cell extracts. One major and one minor component with esterase activity were demonstrated by electrophoresis. Comparison of the intensities of the major band demonstrated that extracellular esterase activity increased rapidly between 24 and 28 h and intracellular activity markedly decreased between 20 and 32 h. The minor esterase component was present only in cell extracts. A change in catalase activity was demonstrated over the same period. The single catalase component could not be demonstrated in the cell extracts at any time. Catalase activity appeared in the culture fluid after 24 h and changes in the intensity of the band showed that the extracellular activity continued to increase until 32 h.
Discussion A total of 10 antimicrobial compounds was produced by B. sublilis NCIB 8872 when grown under the conditions described in a medium containing glucose. These antibiotics have been divided into 3 easily distinguished groups (Table 1) and may be further differentiated by their persistence in the culture fluid, by the correlation of antibiotic production with changes in the enzyme complement of the organism, by
ANTIBIOTICS OF BACILLUS SUBTILIS
11
their effect on the producing organism itself and by the pattern of their accumulation. The 2 antibacterial antibiotic groups accumulated and then declined. The decline could result from the action of antibiotic inactivating enzymes, and indeed Schmitt & Freeze (1968) have suggested that antibiotic degradation may be due to an enzyme(s) similar to the nisin inactivating enzymes of B. cereus and B. megaterium described by Jarvis (1967). The activity of such enzyme@)would appear to be associated with the sporulation and to be specific for the antibacterial antibiotics since a parallel decline in antifungal activity did not occur. Snoke & Cornell (1965) and Schmitt & Freeze (1968) demonstrated that the extracellular antibiotics of B. subtilis and B. licheniformis may limit the growth of the producing strain. Logarithmic phase cells of the B. subtilis strain investigated here were inhibited only by the Group I antibacterial antibiotics. Bott (1971) showed that changes in the specific activity of dehydrogenase enzymes of the tricarboxylic acid cycle and esterases of B. sublitis occurred after the end of the logarithmic phase and Baillie & Norris (1963) have shown changes in the properties of a catalase during early sporulation of B. cereus. Changes in the distribution of esterase and catalase have been demonstrated here to be associated with the phase of spore formation and maturation in a medium containing glucose. These changes were preceded, and not accompanied, by the initiation of antibiotic production. The present findings (Fig. 1) clearly indicate that the representation of individual component antibiotics varied considerably during the logarithmic growth phase, the sporulation and the post sporulation and lytic phase. The concentration of extracellular antibiotics may be taken as a measure of the total concentration, since concentrations of intracellular antibiotics were always low. In a medium containing glucose the accumulation of the Group I antibacterial antibiotics began during the logarithmic phase, the period of most rapid antibiotic accumulation preceding the initiation of sporulation. Group I1 antibacterial antibiotics accumulated only after the onset of sporulation, while the Group I11 antifungal antibiotics accumulated over a short period coincident with spore formation and maturation. The results indicate that the accumulation of the different antibiotic groups is related to the age of the culture. The accumulation of the Group I1 antibacterial antibiotics is preceded by the accumulation and decline of the Group I antibiotics. No evidence can be presented here, however, that these two antibiotic groups are biogenetically related. The evidence presented [Figs 1 (b) and 2 (b)] shows that in the presence or absence of glucose the pattern of accumulation of the different antibiotic groups is unchanged, although the quantity of antibiotic produced is altered. This suggests that, in this strain, antibiotic accumulation is not controlled by glucose catabolite repression, which Demain (1968) proposed may control the production of other antibiotics, including bacitracin. The time course of production of the Group I antibiotics, indeed, resembles that of bacitracin production by B. licheniformis (Snoke & Cornell, 1965; Haavik, 1974) where substantial antibiotic accumulation occurred during the growth phase. This similarity may be attributed to the fact that in all cases the pH does not decline during the growth phase. In these experiments the omission of glucose from the medium resulted in a large
12
J. G. BARR
reduction in spore formation without greatly influencing antibiotic production. The pattern of production of the Group I antibiotics, and the observation that the 3 antibiotic groups were produced when sporulation was minimal, indicated that antibiotic production was not confined to growth limited or sporulating cultures. This particular strain of B. subtilis appears to have valuable attributes for the study of the relationship between specific antibiotic metabolites and the age of the culture. The author would like to thank Mr G. Downey and Mr G. McKinley for valuable assistance.
References BABAD, J., PINSKY, A. & TURNER-GRAFF, R. (1952).An antifungal polypeptide produced by Bacillus subtilis. Nature, Lond. 170, 618. BAILLIE, A. & NORRIS,J. R. (1963).Studies of enzyme changes during sporulation of Bacillus cereus using starch gel electrophoresis.J. appl. Bact. 26, 102. BARTHOLEMEW, J. W. & MITTWER, T. (1950). A simplifiedbacterial spore stain. Stain Technol. 25, 153. BERNLOHR, T. W. & NOVELLI, G. D. (1963).Bacitracin biosynthesis and spore formation: the physiological role of an antibiotic. Archs Biochem. Biophys. 103,94. BODANSKY, M. & PERLMAN, D. (1969). Peptide antibiotics. Science, N. Y. 163,94. BOTT,K. R. (1971). Acrylamide gel electrophoresis of intracellular proteins during early stages of sporulation in Bacillus subtilis. J. Bact. 108,720. BU’LOCK, J. D.(1961). Intermediary metabolism and antibiotic synthesis. Adv. appl. Microbiol. 3, 293. BURACHEK, M., LEARDINI,N. A. & PALADINI, A. C. (1964). Three antifungal polypeptides from Bacillus subtilis. Experientia 20, 504. DEMAIN, A. L. (1968). Regulatory mechanism and the industrial production of microbial metabolites. LIoydia 31, 395. HAAVIK, H.I. (1974). Studies on the formation of bacitracin by Bacillus licheniformis: Effect of glucose. J. gen. Microbiol. 81, 383. HAAVIK, H. I. & THOMASSEN, S.(1973). A bacitracin-negative mutant of Bacillus Iicheniformis which is able to sporulate. J. gen. Microbiol. 76,451. JARVIS, B. (1967).Resistance to nisin and production of nisin-inactivatingenzymes by several Bacillus species. J. gen. Microbiol. 47, 33. KORYZBSKI, T., KOWSZYK-GINDIFER, Z. & KURYLOWITZ, W. (1967). In Antibiotics; Origin, Nature and Properties, Vol I. Translated from Polish by E. Paryski. First ed. London: Pergamon Press. LANDY,M., WARREN, G. H., ROSENMAN, S. B. & COLIO,L. G. (1948). Bacillomycin; an antibiotic from Bacillus subtilis active against pathogenic fungi. Proc. SOC.exp. Biol. New York 67,539. MAJUMDAR, S.K. & BOSE,S. K. (1958).Mycobacillin, a new antifungal antibiotic produced by Bacillus subtilis. Nature, Lond. 181, 134. MURRELL, W. G. (1967).The biochemistry of the bacterial endospore. In Advances in Microbial Physiology, Vol. I. Eds. A. H. Rose & J. R. Wilkinson. New York & London: Academic Press. POULIK, M. D. (1957).Starch gel electrophoresisin a discontinuous system of buffers. Nature, Lond. 180,1477. RAY,B. & BOSE,S. K. (1971). Polypeptide antibiotic-negative sporeformer mutants of Bacillus subtilis. J. gen. appl. Microbiol. 17,491. SADOFF, H. L. (1972). The antibiotics of Bacillus species: their possible role in sporulation. In Progress in Industrial Microbiology, Vol. 11. Ed. D. J. D. Hockenhull. Edinburgh & London : Churchill Livingstone. P. (1969). Sporulation and the production of antibiotics, exoenzymes and SCHAEFFER, exotoxins. Bact. Rev. 33, 48.
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SCHMITT, R. & FREEZE, E. (1968). Curing of a sporulation mutant and antibiotic activity of Bacillus subtilis. J. Bact. 96, 1255. SNOKE, J. E. & CORNELL, N. (1965). Protoplast lysis and inhibition of growth of Bacillus licheniformis by bacitracin. J. Bact. 89,415. STADTMAN, E. R., NOVELLI, G. D. & LIPMANN, R. (1951). Coenzyme A function in and acetyl transfer by phosphotrans-acetylase system. J . biol. Chem. 191, 365. SWINBURNE, T. R. (1971). The infection of apples cv. Bramley’s Seedling by Nectria gulligena Bres. Ann. appl. Biol. 68, 253. WOODRUFF, H. E. (1966). The physiology of antibiotic production: the role of the producing organism. Symp. SOC.gen. Microbiol. 16, 22.
Changes in the Extracellular Accumulation of Antibiotics duringiGrowth and Sporulation of Bacillus subtilis in Liquid Culture
J. G. BARR Agricultural and Food Bacteriology Research Division, Department of Agriculture for Northern Ireland, and The Queen’s University, Belfast, Northern Ireland Received 27 August I974 and accepted 3 March 197.5
Ten antimicrobial metabolites, produced by Bacillus subtilis NCIB 8872, have been categorized into 3 groups on the basis of their antimicrobial spectra, chromatographic mobility and solvent solubility. The maximum concentration of the different groups occurred at different times during fermentation. The accumulation of one antibiotic group was characteristic of the logarithmic growth phase. Groups also differed in their persistence. The intracellular antibiotics contributed little to the total antibiotic activity of the culture. The onset of production and the maintenance of the extracellular accumulation of the 3 antibiotic groups were linked to sporulation and associated specific changes in the intracellular and extracellular protein complement. Production of the antibiotics was not controlled by glucose catabolite repression, since the presence or absence of glucose in the medium did not affect the pattern of antibiotic accumulation.
THEREis considerable evidence that sporulation and antibiotic production by a number of Bacillus species are closely allied or interdependent processes (Bernlohr & Novelli, 1963; Schaeffer, 1969, Sadoff, 1972). However, Ray & Bose (1971) and Haavik & Thomassen (1973) have described mutants of Bacillus subtilis and B. lichenniformis, respectively, which retained the ability to sporulate without any accompanying antibiotic production. Antibiotic accumulation, either intracellular or extracellular, normally occurred after the end of the logarithmic growth phase and prior to endospore maturation (Woodruff, 1966; Bodansky & Perlman, 1969). It has been suggested that peptide antibiotics, like secondary metabolites, are only produced by organisms which have ceased to divide, after nutrient limitation (Bu’Lock, 1961). In cultures of Bacillus species, glucose may affect both sporulation and antibiotic production. Murrell (1967) and Schaeffer (1969) have reviewed the evidence that in B. subtilis cultured on a medium containing glucose, specific enzymes required for sporulation were repressed during exponential growth and suggested that these enzymes were controlled by glucose catabolite repression. Demain (1968) maintained that the production of many antibiotics, including bacitracin, may be controlled by glucose catabolite repression. Some strains of B. subtilis can produce several antibiotics (Majumdar & Bose, 1958; Schmitt & Freeze, 1968) and different strains can produce antibiotics of different antimicrobial spectra. (Koryzbski, Kowszk-Gindifer & Kurylowitz, 1967). In strains [I1
2
J. G . BARR
which can produce several antibiotics it has not been established how the accumulation and relative proportions of the different antibiotic compounds aIter during the development and sporulation of a culture. This paper describes studies of the pattern of antibiotic produc.ion in growing and sporulating cultures of a strain of B. subtilis, cultured in the presence or absence of glucose. The strain described is known to produce the antifungal antibiotic, bacillomycin (Landy, Warren, Rosenman & Colio, 1948; Babad, Pinsky & Turner-Graff, 1952) and uncharacterized metabolites possessing antibacterial activity (Landy et al., 1948). Derivative strains have been shown to produce 3 closely related antifungal polypeptide antibiotics (Burachek, Leardini & Paladini, 1964).
Materials and Methods Organisms
The organism used throughout the work was Bacillus subtilis NCIB 8872 (Landy et al., 1948). Antibacterial activity was evaluated against Staphylococcus aureus Oxford 209P and Erwinia carotovora NCIB 8097 and antifungal activity against a pathogenic strain of Nectria galligena (typed by Commonwealth Mycological Institute, Kew). Media
All media were prepared in glass distilled water. The inocula for fermentation studies with B. subtilis and for seed cultures for antibacterial antibiotic assay were grown on a nutrient medium (N) containing (g/l): peptone, 10.0 Lab Lemco (Oxoid L29), 8.0; NaCI, 5.0. For studies of antibiotic production by B. subtilis a chemically defined medium (F) of the following composition (g/l) was employed: glucose, 20.0; L-glutamic acid, 5 -0; MgS04.7H2O70-5; KCl, 0.5; KH2PO4,l-0; Fez (SO&. 6H2O7O.013;MnS04.Hz0, 0.005; CuS04.5H20,0.016. The pH value of the medium was 6.0 after sterilization at 121O for 20 min. In some experiments, glucose was omitted from medium F and the pH of this medium was 6.2 after sterilization. The nutrient agar used for the maintenance of bacterial cultures, for the enumeration of viable cells and for all antibiotic diffusion assays contained (g/l): peptone, 10.0; Lab Lemco (Oxoid L29), 10.0; NaCI, 5-0; agar (Difco No. 3), 10.0. Cultures of Nectria galligena were maintained on 2 % (w/v) malt agar. Growth conditions T%epreparation of inoculafor organisms used for microbiological assay. Bacteria were grown shaken in 250 ml Erlenmeyer flasks each containing 50 ml of medium. An orbital shaker (Mk. V, L. H. Engineering, Stoke Poges, Bucks., England) operating at 220 rev/min with a circular orbit of 25 mm was employed. Staphylococcus aureus and E. carotovora were incubated at 37" and harvested 24 h after inoculation. B. subtilis was incubated at 30" and harvested after 16 h. The culture conditions for conidial production of N. galligena were those described by Swinburne (1971).
ANTIBIOTICS OF BACILLUS SUBTILIS
3
The preparation of inoculafor fermentation studies Fifty ml of medium was inoculated from a 24 h surface colony on agar. The culture was grown shaken for 16 h at 30". The cells were harvested by centrifuging and resuspended in an equal volume of medium F. One ml of this suspension of logarithmic phase vegetative cells was added to each 50 ml of medium F. Measurement of growth and sporulation The growth of B. subtilis was measured as extinction at 620 nm (E620) in a Unicam SP8000 spectrophotometer. The frequency of sporulation (s/v) was measured by the method described by Ray & Bose (1971). Spore formation and sporangial lysis were confirmed microscopically after staining (Bartholemew & Mittwer, 1950), and by phase-contrast microscope examination. Quantitative microbiologicalassay Antibiotic activities were determined using NUNC @K-4000 Roskilde, Denmark) bioassay plates (235 mm x 235 mm) each containing 165 ml of agar of uniform depth. Molten assay agar was cooled to 50" and inoculated with bacterial cells to give a concentration of 107 organisms/ml of agar. For N. galligena the corresponding concentration was lo4 conidia/ml. Wells (8 mm diam) were made on an 8 x 8 template and a 50 pl sample dispensed in each well. Plates seeded with Staph. aureus were incubated at 16 h at 37"; plates seeded with N . galligena, 48 h at 25 '. The zones of inhibition were then measured. One unit of activity was defined as the amount of antibiotic which produced a zone of inhibition 1 mm in width around each 8 mm diam well. All solutions were diluted appropriately to produce a zone of this diameter. Extraction and assay of culturefluids Cell free fluids were obtained by centrifuging cultures through Hemming Seitz filters. Two ml volumes of fluid were extracted twice with 5 ml portions of n-butanol. The combined extracts were dried under reduced pressure in a rotary film evaporator and the residue reconstituted in 2 ml of phosphate buffer, 0.1 M, pH 7.4. Residual butanol was removed from the extracted fluid. Extraction and assay of intracellular antibiotics All extractions were from batches of cells harvested from 50 ml of culture by centrifuging and washed twice with phosphate buffer (0.1 M, pH 7.4). Whole cells were suspended in n-butanol for 16 h at 20", when the butanol-soluble fraction was evaporated to dryness under reduced pressure in a rotary film evaporator and the residue resuspended in 5 mI of phosphate buffer (0-1M, pH 7.4). Disintegrated cells were obtained by resuspending harvested cells in phosphate buffer (0 * 1 M, pH 7 -0)and treating in a Braun cell disintegrator for 3 min using glass beads of 0.10-0. I1 mm diam. After centrifuging, a part of the supernatant liquid was retained for antibiotic assay. The remainder was extracted twice with 5 ml portions of n-butanol fractions evaporated to dryness and reconstituted in 5 ml of phosphate buffer. A I0-fold concentration of the extracts derived from whole or disintegrated
4
J. G.BARR
cells was achieved using Amicon macrosolute concentrators (Amicon Corporation, Lexington, Mass.). Extracts derived from the cells and culture fluids were assayed and chromatographed by the same methods. Thin-layer chromatography and localization of antibiotics Extracts were adjusted to a standard volume and then 20 pl portions were applied to a cellulose chromatogram sheet (Eastman 6065) and to a silica gel chromatogram sheet (Eastman 6061). Cellulose chromatograms were developed in a solvent mixture of n-butanol : acetic acid : water (3 : 1 : 1) (System I) and silica gel chromatograms developed with chloroform : methanol : water (60 : 35 : 4) (System 11). Both systems were developed to a distance of 150 mm and residual solvent removed in an air stream. Cellulose and silica gel were fixed by a gentle spray with molten nutrient agar at 50 '. Antibacterial antibiotics were detected on chromatograms by an overlay technique. Fifty ml of seeded agar was poured over each chromatogram and allowed to set at uniform thickness. Localization of inhibition zones of Staph. aureus and E. carotovora was aided by the incorporation in the overlay agar of 0.002% (w/v) of2, 3, 5triphenyltetrazolium chloride. Agars seeded with Staph. aureus, E. carotovora or B. subtillis were incubated for 16 h at 37" prior to evaluation. Antifungal antibiotics were located on chromatograms by a similar overlay technique using N. galligena. Seeded agar was incubated for 72 h at 25 *. Starch gel electrophoresis Cell extracts were prepared by the disintegration method described above. After centrifuging, the supernatant liquids were concentrated 10-fold using Amicon macrosolute concentrators. Cell-free culture fluids were concentrated 50-fold by the same method. Total protein in cell extracts and in concentrated culture fluids was adjusted to an equivalent protein content by a standard turbidimetric method (Stadtman, Novelli & Lipmann, 1951). Starch gels were prepared and developed in the discontinuous buffer system of Poulik (1957) with the modifications described by Baillie & Norris (1963). Separations were carried out at a constant current of 30 mA for 4 h. Esterases were detected by immersion of the gel in Tris-maleate buffer (0.1 M, pH 6.5) containing 0.20 mg/ml of m-naphthyl acetate and 0.75 mg/ml of Fast Blue RR added from stock solutions maintained in 50 % acetone solution. For the detection of catalases, the gel was covered with a 1 (v/v) aqueous solution of hydrogen peroxide for a few moments, washed in running water and further immersed in a 2 % (w/v) potassium iodide solution acidified with acetic acid.
Differentiation of the antibiotic components produced by Bacillus subtilis N U B 8872 Bacillus subtilis produced 10 antimicrobial compounds during a 72 h fermentation in medium F. On the basis of their chromatographic mobility, solvent solubility and
Group*
10
2 3 4 5
1
I[i
No.
+ + + -+
-
+ + + +. + + + +-
Staph. aureus
+
-
-
-
-
-
+-
-
E. carotovora
A
-
-
-
-
-I-
+
-
+ + + +-
-
I
-
+ + + + + +
-
B subtilis
N. galligena
Inhibitory activity against
,
-
+ + + -
-
+ +-
-
-
Solubility in n-butanolf r
t Solubility in n-butanol based on extraction of antibiotic from aqueous solution at neutral pH.
* Three antibiotic groups distinguished on the basis of antimicrobial activity, solvent solubility and chromatographic mobility.
Bacitracin Tyrocidine Gramicidin Subtilin Polymyxin
ILI
I1
I
& (
An ti biotic
0-73 0.72 0.72 0.73 0.69 0.87 0.88 0.89 0.76 0.96 1.00 0.98 0.65 0.75
0.73
System I
A
0.89 0.38 0.00
0.00 0.27 0.92
0.00
0.04 0.08 0.10 0.19 0.24 0.32 0.90 0.96
System I1
chromatography
Rfvalues by thin-layer
TABLE 1 The distinguishing properties of 10 antibiotics produced by Bacillus subtilis NCZB 8872 and 5 standard Bacillus-derived antibiotics
1
2
2
3
s
b
2
R
% ?Q
8
8
E
5
J. G. BARR
6
antimicrobial activity these compounds fall into 3 groups (Table 1). Groups I and I1 exhibited antibacterial activity;Group I11 exhibited antifungal activity only. Group I was differentiated from Group I1 on the evidence of solvent solubility and chromatographic mobility. The number of antibiotic components in each group is also recorded in Table 1. On the basis of these properties, the antibacterial antibiotics differ from bacitracin, gramicidin, tyrocidine, subtilin and polymyxin (Table 1).
I5O
t
(b)
Age of culture ( h )
Fig. 1. The fermentation of Bueilfus subtilis in medium F. (a) The relationship between growth, p H value and thermoresistant spore formation. 0 ,growth; A, s/v ratio; 0,pH value. (b)The extracellular accumulation of antibiotic Groups I, I1 and 111. W, Group I, antibacterial antibiotics, not butanol soluble; 0 , Group 11, antibacterial antiobiotics, butanol soluble; A, Group 111, antifungal antibiotics. The arbitrary unit of activity of the Group I and Group I1 antibiotics was defined with Sruph. uureus as the sensitive organism. The arbitrary unit of antifungal activity (Group 111) was defined with N. guZligena as the test organism.
ANTIBIOTICS OF BACILLUS SCJBTZLZS
7
Growth, sporulation and total extracellular antibiotic accumulation during fermentation The progression of growth, sporulation and extracellular antibiotic accumulation in cultures of B. subtilis in medium F and medium F without glucose is recorded in Figs 1 and 2, respectively. Medium F. The end of the logarithmic growth phase occurred c. 20 h after inoculation [Fig. 1 ( a ) ] . Sporulation was complete at 30 h. Microscopic examination of samples taken at different time intervals showed that sporangial lysis and the release of free spores occurred after 40 h. The number of free spores increased between 40 and 60 h. The pH value did not change during the logarithmic growth phase while a steady rise in pH value was recorded after the onset of sporulation [Fig. 1 ( a ) ] . Figure 1 (b) shows that the concentration of the Group I antibacterial antibiotics and the Group I11 antifungal antibiotics began to increase before logarithmic growth had ceased. Activities reached a maximum between 25 and 28 h, during the period of active differentiation of the culture. Afterwards, the concentration of the Group I antibacterial antibiotics declined, at first rapidly and then more gradually. No comparable fall in the concentration of the antifungal metabolites was observed. The Group I1 antibacterial antibiotics were present only after 24 h. By then the concentration of the Group I antibacterial antibiotics was high. Subsequently the concentration of the Group IT antibiotics increased up to c. 48 h. The concentration of both antibacterial antibiotic groups declined significantly in the later stages of the fermentation. Medium F minus glucose. The end of the logarithmic growth phase occurred c. 26 h after inoculation [Fig. 2 (a)]. Although spores were detected after 30 h, sporulation was incomplete and an s/v ratio of 0.3 was recorded after 46 h. The pH value rose steadily throughout the fermentation [Fig. 2 (a)]. Figure 2 (b) shows that the pattern of accumulation of the 3 antibiotic groups paralleled the course of their accumulation in medium F. The 3 antibiotic groups were present in lower concentration, and their activities reached a maximum later than in medium F.
Theformation of specific antibiotic metabolites during fermentation Extracellular compounds. The compounds were detected by thin layer chromatography. In medium F and in medium F minus glucose, 2 antifungal components (detected by System I) were consistently present after 20 and 26 h, respectively. Throughout the fermentations the relative contributions of these 2 antifungal antibiotics to the total antifungal activity remained the same. Table 2 shows that the representation of the various antibacterial compounds (detected by System 11) changes with time. This is in accord with the fluctuationsin the concentration of the Group I and Group I1 compounds demonstrated in Figs 1 (b) and 2 (b). In medium F, the main Group I antibiotics, Rf 0.19 and Rf0.24, were detected during the logarithmic growth phase, although the peak in their accumulation did not occur until the sporulation phase. During the period of spore formation and maturation all 6 Group I antibiotics were detected. The concentration of the individual antibiotics diminished after 30 h, and only 1 component, Rf 0.19, could be detected
a
J. G . BARR
throughout, but during this period the less polar Group I1 antibiotics appeared and increased in concentration. Later, however, there was a reduction in the concentration of a11 the antibacterial antibiotics. In medium F minus glucose a similar pattern of antibiotic accumulation was found. The major Group I antibiotics, Rf 0.19 and Rf 0.24, were detected in the fermentation medium during the logarithmic growth phase, although the peak in their accumulation, and the appearance of other Group I antibiotics, did not occur until logarithmic growth had ceased after 26 h. The decrease in concentration of the Group I antibiotics after this time was accompanied by the appearance and increase in
I5O
t
10
.,
20
I 30 40 Age of culture ( h )
1
I
50
60
Fig. 2. The fermentation of Bacillus subtilis in medium F minus glucose. (u) The relationgrowth: A, ship between growth, pH value and thermoresistant spore formation. 0, s/v ratio; 0, pH value. (b)The extracellular accumulation of antibiotic Groups I, I1 and 111. Group I, antibacterial antibiotics, not butanol soluble; 0 , Group 11, antibacterial antibiotics, butanol soluble; A,Group 111, antifugal antibiotics. The arbitrary unit of activity of the Group I and Group I1 antibiotics was defined with Staph. aureus as the sensitive organism. The arbitrary unit of antifungal activity (Group 111) was defined with N. gulligenu as the test organism.
I1I
Group?
+ -
-
I7 8
-
10
4 5 6
3
No.
-
-
+
-
-
15
-
-
-
22
-
24
-
27
A
-
30
+ + ++ +++ ++ + ++ ++ ++ ++ ++ - - - - - + - - - - - +
20
- -
18
46
48
51
+ - - - ++ ++ ++ ++ -+ + + + ++ + ++ ++ ++
39
Time after inoculation 0)
+ -
-
60
++ +
-+
54
I
* Antibiotics, separated by chromatographicSystem II, are indicated as absent (-), present as a minor component (+), or present as a major component (+ +). Antibiotics were detected by a Staph. uureus overlay technique. t Groups I and I1 were distinguished by antimicrobial spectra, solvent solubility and chromatographic mobility.
F minus glucose
F
Medium
Antibiotic
TABLE 2 The occurrence* of antibacterial antibiotics at different times during the fermentation of Bacillus subtilis NCIB 2872 on medium F and on medium F minus glucose
W
10
J. G . BARR
concentration of the Group I1 antibiotics. Later, as in medium F, the concentration of all the antibacterial antibiotics decreased. Antibiotic production, in medium F minus glucose, was also distinguished by the absence of the antibiotic components at Rf0.04 and Rf0-08. Intracellular compounds. The Group I1 antibacterial antibiotics were detected in cell extracts between 20 and 26 h growth in medium F. After this time no antibiotic activity could be demonstrated in the butanol-soluble or water-soluble fractions of the cell extracts. The butanol-insoluble Group I antibiotics and the antifungal antibiotics could not be detected in the cell extracts at any point during the fermentation. During fermentation in the absence of glucose, the Group I1 antibacterial, antibiotics were detected in cell extracts after 30,40 and 46 h fermentation, but the Group I and Group 111 antibiotics could not be detected at any time. The intracellular concentration of antibiotics was always small relative to the extracellular antibiotic accumulation. The inhibition of B. subtilis by individual components of the total antibiotic complement
Culture samples containing each of the constituent antibiotic components were chromatographed in both development systems. The chromatograms were overiaid with agar seeded with logarithmic phase vegetative cells of the B. subtih strain itself. Those components of Rf 0.04, 0-08, 0*10,0-19 and 0.24 (detected by System 11) inhibited the growth of logarithmic phase vegetative cells, while the butanol-soluble antibiotics produced no zones of inhibition. The intracellular and extracellular levels of esterase and catalase activity during fermentation in medium F
The change in the extracellular and intracellular esterase and catalase activity were followed over a 12 h period which marked the end of the logarithmic growth phase and the formation of thermoresistant spores. Esterase activity was demonstrated in concentrates of culture fluid and in cell extracts. One major and one minor component with esterase activity were demonstrated by electrophoresis. Comparison of the intensities of the major band demonstrated that extracellular esterase activity increased rapidly between 24 and 28 h and intracellular activity markedly decreased between 20 and 32 h. The minor esterase component was present only in cell extracts. A change in catalase activity was demonstrated over the same period. The single catalase component could not be demonstrated in the cell extracts at any time. Catalase activity appeared in the culture fluid after 24 h and changes in the intensity of the band showed that the extracellular activity continued to increase until 32 h.
Discussion A total of 10 antimicrobial compounds was produced by B. sublilis NCIB 8872 when grown under the conditions described in a medium containing glucose. These antibiotics have been divided into 3 easily distinguished groups (Table 1) and may be further differentiated by their persistence in the culture fluid, by the correlation of antibiotic production with changes in the enzyme complement of the organism, by
ANTIBIOTICS OF BACILLUS SUBTILIS
11
their effect on the producing organism itself and by the pattern of their accumulation. The 2 antibacterial antibiotic groups accumulated and then declined. The decline could result from the action of antibiotic inactivating enzymes, and indeed Schmitt & Freeze (1968) have suggested that antibiotic degradation may be due to an enzyme(s) similar to the nisin inactivating enzymes of B. cereus and B. megaterium described by Jarvis (1967). The activity of such enzyme@)would appear to be associated with the sporulation and to be specific for the antibacterial antibiotics since a parallel decline in antifungal activity did not occur. Snoke & Cornell (1965) and Schmitt & Freeze (1968) demonstrated that the extracellular antibiotics of B. subtilis and B. licheniformis may limit the growth of the producing strain. Logarithmic phase cells of the B. subtilis strain investigated here were inhibited only by the Group I antibacterial antibiotics. Bott (1971) showed that changes in the specific activity of dehydrogenase enzymes of the tricarboxylic acid cycle and esterases of B. sublitis occurred after the end of the logarithmic phase and Baillie & Norris (1963) have shown changes in the properties of a catalase during early sporulation of B. cereus. Changes in the distribution of esterase and catalase have been demonstrated here to be associated with the phase of spore formation and maturation in a medium containing glucose. These changes were preceded, and not accompanied, by the initiation of antibiotic production. The present findings (Fig. 1) clearly indicate that the representation of individual component antibiotics varied considerably during the logarithmic growth phase, the sporulation and the post sporulation and lytic phase. The concentration of extracellular antibiotics may be taken as a measure of the total concentration, since concentrations of intracellular antibiotics were always low. In a medium containing glucose the accumulation of the Group I antibacterial antibiotics began during the logarithmic phase, the period of most rapid antibiotic accumulation preceding the initiation of sporulation. Group I1 antibacterial antibiotics accumulated only after the onset of sporulation, while the Group I11 antifungal antibiotics accumulated over a short period coincident with spore formation and maturation. The results indicate that the accumulation of the different antibiotic groups is related to the age of the culture. The accumulation of the Group I1 antibacterial antibiotics is preceded by the accumulation and decline of the Group I antibiotics. No evidence can be presented here, however, that these two antibiotic groups are biogenetically related. The evidence presented [Figs 1 (b) and 2 (b)] shows that in the presence or absence of glucose the pattern of accumulation of the different antibiotic groups is unchanged, although the quantity of antibiotic produced is altered. This suggests that, in this strain, antibiotic accumulation is not controlled by glucose catabolite repression, which Demain (1968) proposed may control the production of other antibiotics, including bacitracin. The time course of production of the Group I antibiotics, indeed, resembles that of bacitracin production by B. licheniformis (Snoke & Cornell, 1965; Haavik, 1974) where substantial antibiotic accumulation occurred during the growth phase. This similarity may be attributed to the fact that in all cases the pH does not decline during the growth phase. In these experiments the omission of glucose from the medium resulted in a large
12
J. G. BARR
reduction in spore formation without greatly influencing antibiotic production. The pattern of production of the Group I antibiotics, and the observation that the 3 antibiotic groups were produced when sporulation was minimal, indicated that antibiotic production was not confined to growth limited or sporulating cultures. This particular strain of B. subtilis appears to have valuable attributes for the study of the relationship between specific antibiotic metabolites and the age of the culture. The author would like to thank Mr G. Downey and Mr G. McKinley for valuable assistance.
References BABAD, J., PINSKY, A. & TURNER-GRAFF, R. (1952).An antifungal polypeptide produced by Bacillus subtilis. Nature, Lond. 170, 618. BAILLIE, A. & NORRIS,J. R. (1963).Studies of enzyme changes during sporulation of Bacillus cereus using starch gel electrophoresis.J. appl. Bact. 26, 102. BARTHOLEMEW, J. W. & MITTWER, T. (1950). A simplifiedbacterial spore stain. Stain Technol. 25, 153. BERNLOHR, T. W. & NOVELLI, G. D. (1963).Bacitracin biosynthesis and spore formation: the physiological role of an antibiotic. Archs Biochem. Biophys. 103,94. BODANSKY, M. & PERLMAN, D. (1969). Peptide antibiotics. Science, N. Y. 163,94. BOTT,K. R. (1971). Acrylamide gel electrophoresis of intracellular proteins during early stages of sporulation in Bacillus subtilis. J. Bact. 108,720. BU’LOCK, J. D.(1961). Intermediary metabolism and antibiotic synthesis. Adv. appl. Microbiol. 3, 293. BURACHEK, M., LEARDINI,N. A. & PALADINI, A. C. (1964). Three antifungal polypeptides from Bacillus subtilis. Experientia 20, 504. DEMAIN, A. L. (1968). Regulatory mechanism and the industrial production of microbial metabolites. LIoydia 31, 395. HAAVIK, H.I. (1974). Studies on the formation of bacitracin by Bacillus licheniformis: Effect of glucose. J. gen. Microbiol. 81, 383. HAAVIK, H. I. & THOMASSEN, S.(1973). A bacitracin-negative mutant of Bacillus Iicheniformis which is able to sporulate. J. gen. Microbiol. 76,451. JARVIS, B. (1967).Resistance to nisin and production of nisin-inactivatingenzymes by several Bacillus species. J. gen. Microbiol. 47, 33. KORYZBSKI, T., KOWSZYK-GINDIFER, Z. & KURYLOWITZ, W. (1967). In Antibiotics; Origin, Nature and Properties, Vol I. Translated from Polish by E. Paryski. First ed. London: Pergamon Press. LANDY,M., WARREN, G. H., ROSENMAN, S. B. & COLIO,L. G. (1948). Bacillomycin; an antibiotic from Bacillus subtilis active against pathogenic fungi. Proc. SOC.exp. Biol. New York 67,539. MAJUMDAR, S.K. & BOSE,S. K. (1958).Mycobacillin, a new antifungal antibiotic produced by Bacillus subtilis. Nature, Lond. 181, 134. MURRELL, W. G. (1967).The biochemistry of the bacterial endospore. In Advances in Microbial Physiology, Vol. I. Eds. A. H. Rose & J. R. Wilkinson. New York & London: Academic Press. POULIK, M. D. (1957).Starch gel electrophoresisin a discontinuous system of buffers. Nature, Lond. 180,1477. RAY,B. & BOSE,S. K. (1971). Polypeptide antibiotic-negative sporeformer mutants of Bacillus subtilis. J. gen. appl. Microbiol. 17,491. SADOFF, H. L. (1972). The antibiotics of Bacillus species: their possible role in sporulation. In Progress in Industrial Microbiology, Vol. 11. Ed. D. J. D. Hockenhull. Edinburgh & London : Churchill Livingstone. P. (1969). Sporulation and the production of antibiotics, exoenzymes and SCHAEFFER, exotoxins. Bact. Rev. 33, 48.
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SCHMITT, R. & FREEZE, E. (1968). Curing of a sporulation mutant and antibiotic activity of Bacillus subtilis. J. Bact. 96, 1255. SNOKE, J. E. & CORNELL, N. (1965). Protoplast lysis and inhibition of growth of Bacillus licheniformis by bacitracin. J. Bact. 89,415. STADTMAN, E. R., NOVELLI, G. D. & LIPMANN, R. (1951). Coenzyme A function in and acetyl transfer by phosphotrans-acetylase system. J . biol. Chem. 191, 365. SWINBURNE, T. R. (1971). The infection of apples cv. Bramley’s Seedling by Nectria gulligena Bres. Ann. appl. Biol. 68, 253. WOODRUFF, H. E. (1966). The physiology of antibiotic production: the role of the producing organism. Symp. SOC.gen. Microbiol. 16, 22.
