Acta path. microbiol. scand. Sect. B, 83: 513-518, 1975
T H E EFFECT OF BACITRACIN AND Mn(I1)IONS UPON T H E PRODUCER STRAIN BACILLUS LlCHENIFORMIS HANSI. HAAVIK Department of Research and Development, A/S Apothekernes Laboratorium for Specialpraeparater, Oslo 2, Norway
Haavik, H. I. T h e effect of bacitracin and Mn(I1)ions upon the producer strain Bacillus licheniformis. Acta path. microbiol. scand. Sect. B, 83: 513-518, 1975. The peptide antibiotic bacitracin is inhibitory to growth of the producer strain Bacillus licheniformis only in the presence of excess manganese (11)ions. Both the early and the late growth are inhibited in a similar manner upon addition of bacitracin and manganese (11)ions. Thus, B. licheniformis does not develop resistance to its own antibiotic during growth. Added bacitracin is stimulatory to growth of B. Zicheniformis in media with a very low content of manganese (11) ions. These results support the hypothesis that bacitracin participates in the manganese transport of the producer strain B. licheniformis. Key words: Bacillus licheniformis; bacitracin ; Mn (11)ions.
H.I. Haavik, A/S Apothekernes Laboratorium for Specialpmparater, Skoyen, Oslo 2, Norway.
Received 5.ii.75
Accepted 9.iv.75
Since many antibiotics are very potent antimicrobial agents, the question as to how the antibiotic producing organisms are able to survive their own abundant production has been given some attention (2, 9). The microbial production of antibiotics is conveniently separated into a growth phase (Trophophase) followed by a production phase (Idiophase), (1, 8, 10). During the growth phase producer organisms are usually sensitive to their own antibiotic, whereas during the production phase even large amounts of the antibiotic produced have no effect (2, 9, 12). It is therefore suggested that microorganisms can grow and produce antibiotics because the latter are not formed until after growth (2, 9). We have recently reported, however, that
the antibiotic bacitracin was produced after rapid growth of Bacillus licheniformis due to an incidental inhibitory effect of low pH and organic acids (4,6). The low pH and the acids arose from the metabolism of glucose which usually is present in media for antibioitic production. Thus in media without glucose or when p H was properly controlled, bacitracin production paralleled growth. The bacitracin produced during the early growth phase was not inhibitory to growth of B. licheniformis ( 6 ) . This does not support the suggestion that microorganisms can grow and produce antibiotics because the latter are not formed until after growth. The aim of the present work was to investigate the effect of bacitracin and M n ( 11)ions upon the growth of the producer strain R. licheniformis ATCC 10716.
513
MATERIALS AND METHODS
RESULTS
Reagents All reagents used were of analytical grade. Bacitracin was kindly supplied by Mr. K . Ulverud, A/S Apothekernes Laboratorium for Specialprieparater, Oslo 2, Norway. The purity of the bacitracin used was according to the rules given in Ph.Nord and U.S.P. XVIII.
Fig. l a shows that the addition of 25 i.u. bacitracin/ml or less had no effect upon the early growth of Bacillus licheniformis in the M2 medium. The addition of 50 i.u. bacitracin/ml, however, resulted in a significant inhibition of growth. By increasing further the amounts of bacitracin, an increasing growth inhibition was observed (Fig. l a ) . The inhibitory effect of 50 i.u. bacitracin/ ml upon the growth of B. licheniformis markedly increased as the amount of manganese (11)ions in the medium was increased (Fig. l b ) . In the presence of 200 mg MnSO,/I the early growth was significantly inhibited when only 5 i.u. bacitracin/ml was added to the medium (Fig. lc) . The bacitracin added did not disappear from the medium during the incubation period, an observation also made by others ( 9 ) . As seen from Fig. l b high amounts of manganese(11)ions were somewhat inhibitory to growth when added alone. Fig. 2 shows that the addition of 50 i.u. bacitracin/ml later in growth had no effect upon the succeeding growth. However, when 5 mg MnSO,/I was added together with bacitracin late in growth, a marked inhibition was observed. The addition of 5 mg MnSO,/l alone resulted in a slight stimulation of growth. When larger amounts of Mn (11)ions were added alone late in growth, lysis of the bacteria occurred. This lytic activity was enhanced by the addition of bacitracin (Fig. 2). T h e amounts of manganese (11)ions which resulted in lysis when added late in growth, had no effect upon growth when added to the early growth phase. It was further found that the addition of 50 i.u. bacitracin/ml together with 5 mg MnSO,/I was inhibitory whenever added during growth. B. licheniformis grew well when no manganese(I1)ions were added to the M2 medium. The traces of manganese( 1I)ions present were apparently sufficient for growth. When bacitracin was added to this medium a significant stimulatory effect upon the early
Media and Growth Conditions The chemically defined medium (M2) for growth of B. licheniformis ATCC 10716 and conditions for incubation have been described previously (4).The effect of bacitracin upon the early growth was examined by the addition of sterile filtered bacitracin (Millipore) to the incubation flasks just before inoculation. The effect of bacitracin upon later growth was examined by aseptically adding bacitracin to the flasks after 9 hours of incubation. Manganese( 11)ions were added to the cultures during growth as sterile solutions, or were included in the medium before autoclaving. The inoculum consisted of 1 per cent of an overnight culture in the M2 medium which resulted in an E,,,, of 0.04 when added to the medium in the shaking flasks. The amounts of Mn(1I)ions present in the inoculum may give a contribution to the medium of 0.1 mg MnSO,/I which is negligible as compared to the 10 mg MnSO,/I usually present in the M2 medium. When the bacteria was grown in the M2 medium without Mn(I1)ions added, the inoculum was also grown in the same medium. The traces of Mn(1I)ions present in this medium was due to impurities from the other ingredients and might amount to about 0.01-0.001 mg Mn (11)ions/l. The amount of Mn (11)ions in the inoculum in this case will be about 1 per cent of the amount in the medium. Growth Bacterial growth was measured as extinction at 650 nm using a Spectronic 20 spectrophotometer as described elsewhere (5). Assay of Bacitracin The bacitracin used was determined microbiologically by an agar diffusion method described previously ( 3 ) . Inactivation of Bacitracin A bacitracin solution with a known potency (1000 i.u. bacitracinJm1) was heated at 90" C for 48 hours. Upon reanalyzing no antibiotic activity was found.
514
Fig. 1 . Growth of B. licheniformis in the M2 medium with the following additions:
Fig. la: no bacitracin added ( 0 ) , 1, 5, 10, and 25 i.u. bacitracin/ml added 50 i.u. bacitracin/ml added (O), and 100 i.u. bacitracinlml added (0).
(v),
Fig. l b : 50 mg MnSO,/I added ( 0 ,)50 mg MnSOJl and 50 i.u. bacitraciuml added (0) , 200 mg MnSO,/I added ( A ) , and 200 mg MnSO,/I and 50 i.u. bacitraciuml added ( A ) .
Fig. l c : 200 mg MnSO,/I added ( 0 ), 200 mg MnSO,/I and 1 i.u. bacitraciuml added (A), 200 mg MnSOJl and 5 i.u. bacitracin/ml added (0) ,and 200 mg MnSO,/I and 10 i.u. bacitracinJm1 added
(V). 515
Fig. 2. Growth of B . licheniformis in the M2 medium with the following additions later in growth. Arrow indicate time of addition: Nothing added 50 i.u. bacitracin/ml added 5 mg MnSO,/l added ( O ) , 10 mg MnSO,/l added ( W ) , 5 mg MnSOJl and 50 i.u. bacitracid ml added ( A ) , and 10 mg f MnSOJl and 50 i.u. bacitracin/ 5 ml added (0). I
7
(a),
6
(v),
-
5
4
; 2
3
U
2
I
INCUBATION TIME (h)
growth was observed (Fig. 3a). The addition of only 2 i.u. bacitracin/ml resulted in the most pronounced stimulation. When the inoculum consisted of spores this was somewhat different. By the addition of spores and bacitracin to the M2 medium with no Mn (11)ions added, a marked stimulatory effect upon growth was observed. The addition of 50 i.u. bacitracin/ml resulted in the most pronounced stimulation (Fig. 3b). When 50 i.u. bacitracin/ml and 100 mg MnSO,/I was added together with the spore inoculum, growth was inhibited (Fig. 3b). When corresponding amounts of inactivated bacitracin was added to bacteria or spores no stirnulatory effect upon growth wns observed. DISCUSSION
Our results indicate that the effect of bacitracin upon the growth of the producer strain Bacillus licheniformis is dependent upon the amount of manganese (11)ions present in the medium. Both stimulatory, inhibitory, and lytic effects upon the cells have been observed. In media with a very low content of this cation, bacitracin seems to be
516
stimulatory to growth, whereas in media with a high amount of manganese( 11)ions, bacitracin is inhibitory to growth or induces lysis depending upon the amount of bacitracin added. Snoke & Cornell (9) have previously reported that small amounts of bacitracin markedly inhibited the early growth of B. licheniformis ATCC 10716. They suggested that bacitracin could accumulate during later stages of growth because the inhibitory effect of bacitracin was restricted to the early growth phase only. Our results, however, show bacitracin is able to inhibit both the early and the late growth of B. licheniformis in the presence of manganese (11)ions. It is further proposed that antibiotic producers can survive their own production because they develop resistance to their own antibiotic during later stages of growth (2, 12). This resistance is suggested to be phenotypic because the growth of these resistant cells in a fresh medium containing the antibiotic, will be inhibited once again ( 2 ) . Our results, however, indicate that B. licheniformis does not develop resistance to bacitracin during growth since both the early and the late growth of the organism are in-
Fig. 3. Growth of B. licheniformis in the M2 medium with no manganese( 1I)ions added and with the following additions:
Fig. 3a: Bacteria as inoculum with no bacitracin added with 1 i.u. bacitracidml added (O), with 2 i.u. bacitracidml added ( A ) , with 5 i.u. bacitracin/ml added and with 10 i.u. bacitracidml added ( A ) .
(a),
(a),
Y
Fig. 36: Spores as inoculum with ,.. no bacitracin added with 50 i.u. bacitracin/ml added (0) , and with 50 i.u. bacitracin/ml and 100 mg MnSOJl added
(a),
(V). I
2
hibited in a similar manner, i.e., only in the presence of sufficient amount of manganese(1I)ions. I t appears that B. licheniformis is “resistant” to its own antibiotic during later stages of growth because there are normally very low concentrations of this cation left in the medium towards end of growth. The addition of 10 mg MnS04/l late in the growth had a marked toxic effect upon the culture, whereas the same amounts added to the early growth phase had no such effect. This supports our suggestion, since it is possible that the relatively high amounts of bacitracin produced late in growth (4)act
3
4 5 6 INCLIBATION TIME l h )
7
8
P
10
It
inhibitorily when sufficient amounts of manganese( 1I)ions are added. Since the activity of many antibiotics is influenced by metal ions ( l l ) , it is possible that the frequently observed inhibition of the early growth of producer strains by their own antibiotic may by due to the presence of excess metal ions during early growth. This may explain why “resistant” cells from the production phase are inhibited once again when inoculated in a fresh medium containing the antibiotic. We have recently put forward the hypothesis that bacitracin may participate in the
517
transport of manganese (11)ions in B. licheniformis ( 7 ) . This hypothesis may explain why bacitracin acts inhibitorily in media with high amounts of manganese (11)ions, and stimulatorily in media with a very low content of the same cation. In the first case too much manganese (11)ions are transported into the cells upon the addition of bacitracin, and toxic concentrations are reached. In the latter case bacitracin stimulates growth by increasing the uptake of Mn(I1)ions which now are needed by the bacteria. When bacitracin was added to spores in low Mn (11)ions environments, the stimulatory effect upon growth was more pronounced. The spores must probably germinate and grow out before bacitracin is produced. The addition of bacitracin to spores will therefore probably result in a more rapid uptake of sufficient amounts of the desired cation. Since an inactivated bacitracin solution did not stimulate growth, the stimulatory effect of bacitracin was not due to impurities present in the bacitracin used. To the authors knowledge this is the first example of a stimulatory effect of an antibiotic upon the producer cells. The author would like to thank Mr. T . Hoyland, Director of Research and Development, for his support in this work, and Dr. H . P. Throndsen and Mr. S . Thomassen for valuable discussions during the work and in preparing the text. The skilled technical assistance of Mrs. Inger Austang and Mrs. Linda Horn is greatly appreciated.
518
REFERENCES 1. Demain, A . L.: Regulatory mechanisms and
2. 3.
4.
5.
6.
7. 8.
9.
10.
11.
12.
the industrial production of microbial metabolites. Lloydia. 31: 395-418, 1968. Demain, A . L.: How do antibiotic-producing microorganisms avoid suicide? Annal. N.Y. Acad. Sci. 235: 601-612, 1974. Haavik, H . I . & Thomassen, S.: A bacitracinnegative mutant of Bacillus licheniformis which is able to sporulate. J. gen. Microbiol. 76: 451-454, 1973. Haavik, H . I.: Studies on the formation of bacitracin by Bacillus licheniformis: effect of glucose. J. gen. Microbiol. 81: 383-390, 1974. Haavik, H . I . : Studies on the formation of bacitracin by Bacillus licheniformis: effect of inorganic phosphate. J. gen. Microbiol. 84: 226-230, 1974. Haavik, H . I.: Studies on the formation of bacitracin by Bacillus licheniformis: role of catabolite repression and organic acids. J. gen. Microbiol. 84: 321-326, 1974. Haavik, H . I . & Froyshov, 0.:O n the function of peptide antibiotics in the producer organisms. Nature 254: 79-82, 1975. Sadoff, H . L.: Sporulation antibiotics of Bacillus species. Spores 5 : 157-166, 1972. Snoke, J . E. & Cornell, N.: Protoplast lysis and inhibition of growth of Bacillus licheniformis by bacitracin. J. Bacteriol. 89: 415420, 1965. Woodruff, H . B.: The physiology of antibiotic production: the role of the producing organism. SOC. Gen. Microbiol. Symp. 26: 22-46, 1966. Weinberg, E. D.: The mutual effects of antimicrobial compounds and metallic cations. Bact. Rev. 21: 46-68, 1957. Yoshida, T., Weissbach, H . & Katz, E.: Inhibitory effect of actinomycin upon the producing organism. Arch. Biochem. Biophys. 114: 252-255, 1966.
T H E EFFECT OF BACITRACIN AND Mn(I1)IONS UPON T H E PRODUCER STRAIN BACILLUS LlCHENIFORMIS HANSI. HAAVIK Department of Research and Development, A/S Apothekernes Laboratorium for Specialpraeparater, Oslo 2, Norway
Haavik, H. I. T h e effect of bacitracin and Mn(I1)ions upon the producer strain Bacillus licheniformis. Acta path. microbiol. scand. Sect. B, 83: 513-518, 1975. The peptide antibiotic bacitracin is inhibitory to growth of the producer strain Bacillus licheniformis only in the presence of excess manganese (11)ions. Both the early and the late growth are inhibited in a similar manner upon addition of bacitracin and manganese (11)ions. Thus, B. licheniformis does not develop resistance to its own antibiotic during growth. Added bacitracin is stimulatory to growth of B. Zicheniformis in media with a very low content of manganese (11) ions. These results support the hypothesis that bacitracin participates in the manganese transport of the producer strain B. licheniformis. Key words: Bacillus licheniformis; bacitracin ; Mn (11)ions.
H.I. Haavik, A/S Apothekernes Laboratorium for Specialpmparater, Skoyen, Oslo 2, Norway.
Received 5.ii.75
Accepted 9.iv.75
Since many antibiotics are very potent antimicrobial agents, the question as to how the antibiotic producing organisms are able to survive their own abundant production has been given some attention (2, 9). The microbial production of antibiotics is conveniently separated into a growth phase (Trophophase) followed by a production phase (Idiophase), (1, 8, 10). During the growth phase producer organisms are usually sensitive to their own antibiotic, whereas during the production phase even large amounts of the antibiotic produced have no effect (2, 9, 12). It is therefore suggested that microorganisms can grow and produce antibiotics because the latter are not formed until after growth (2, 9). We have recently reported, however, that
the antibiotic bacitracin was produced after rapid growth of Bacillus licheniformis due to an incidental inhibitory effect of low pH and organic acids (4,6). The low pH and the acids arose from the metabolism of glucose which usually is present in media for antibioitic production. Thus in media without glucose or when p H was properly controlled, bacitracin production paralleled growth. The bacitracin produced during the early growth phase was not inhibitory to growth of B. licheniformis ( 6 ) . This does not support the suggestion that microorganisms can grow and produce antibiotics because the latter are not formed until after growth. The aim of the present work was to investigate the effect of bacitracin and M n ( 11)ions upon the growth of the producer strain R. licheniformis ATCC 10716.
513
MATERIALS AND METHODS
RESULTS
Reagents All reagents used were of analytical grade. Bacitracin was kindly supplied by Mr. K . Ulverud, A/S Apothekernes Laboratorium for Specialprieparater, Oslo 2, Norway. The purity of the bacitracin used was according to the rules given in Ph.Nord and U.S.P. XVIII.
Fig. l a shows that the addition of 25 i.u. bacitracin/ml or less had no effect upon the early growth of Bacillus licheniformis in the M2 medium. The addition of 50 i.u. bacitracin/ml, however, resulted in a significant inhibition of growth. By increasing further the amounts of bacitracin, an increasing growth inhibition was observed (Fig. l a ) . The inhibitory effect of 50 i.u. bacitracin/ ml upon the growth of B. licheniformis markedly increased as the amount of manganese (11)ions in the medium was increased (Fig. l b ) . In the presence of 200 mg MnSO,/I the early growth was significantly inhibited when only 5 i.u. bacitracin/ml was added to the medium (Fig. lc) . The bacitracin added did not disappear from the medium during the incubation period, an observation also made by others ( 9 ) . As seen from Fig. l b high amounts of manganese(11)ions were somewhat inhibitory to growth when added alone. Fig. 2 shows that the addition of 50 i.u. bacitracin/ml later in growth had no effect upon the succeeding growth. However, when 5 mg MnSO,/I was added together with bacitracin late in growth, a marked inhibition was observed. The addition of 5 mg MnSO,/l alone resulted in a slight stimulation of growth. When larger amounts of Mn (11)ions were added alone late in growth, lysis of the bacteria occurred. This lytic activity was enhanced by the addition of bacitracin (Fig. 2). T h e amounts of manganese (11)ions which resulted in lysis when added late in growth, had no effect upon growth when added to the early growth phase. It was further found that the addition of 50 i.u. bacitracin/ml together with 5 mg MnSO,/I was inhibitory whenever added during growth. B. licheniformis grew well when no manganese(I1)ions were added to the M2 medium. The traces of manganese( 1I)ions present were apparently sufficient for growth. When bacitracin was added to this medium a significant stimulatory effect upon the early
Media and Growth Conditions The chemically defined medium (M2) for growth of B. licheniformis ATCC 10716 and conditions for incubation have been described previously (4).The effect of bacitracin upon the early growth was examined by the addition of sterile filtered bacitracin (Millipore) to the incubation flasks just before inoculation. The effect of bacitracin upon later growth was examined by aseptically adding bacitracin to the flasks after 9 hours of incubation. Manganese( 11)ions were added to the cultures during growth as sterile solutions, or were included in the medium before autoclaving. The inoculum consisted of 1 per cent of an overnight culture in the M2 medium which resulted in an E,,,, of 0.04 when added to the medium in the shaking flasks. The amounts of Mn(1I)ions present in the inoculum may give a contribution to the medium of 0.1 mg MnSO,/I which is negligible as compared to the 10 mg MnSO,/I usually present in the M2 medium. When the bacteria was grown in the M2 medium without Mn(I1)ions added, the inoculum was also grown in the same medium. The traces of Mn(1I)ions present in this medium was due to impurities from the other ingredients and might amount to about 0.01-0.001 mg Mn (11)ions/l. The amount of Mn (11)ions in the inoculum in this case will be about 1 per cent of the amount in the medium. Growth Bacterial growth was measured as extinction at 650 nm using a Spectronic 20 spectrophotometer as described elsewhere (5). Assay of Bacitracin The bacitracin used was determined microbiologically by an agar diffusion method described previously ( 3 ) . Inactivation of Bacitracin A bacitracin solution with a known potency (1000 i.u. bacitracinJm1) was heated at 90" C for 48 hours. Upon reanalyzing no antibiotic activity was found.
514
Fig. 1 . Growth of B. licheniformis in the M2 medium with the following additions:
Fig. la: no bacitracin added ( 0 ) , 1, 5, 10, and 25 i.u. bacitracin/ml added 50 i.u. bacitracin/ml added (O), and 100 i.u. bacitracinlml added (0).
(v),
Fig. l b : 50 mg MnSO,/I added ( 0 ,)50 mg MnSOJl and 50 i.u. bacitraciuml added (0) , 200 mg MnSO,/I added ( A ) , and 200 mg MnSO,/I and 50 i.u. bacitraciuml added ( A ) .
Fig. l c : 200 mg MnSO,/I added ( 0 ), 200 mg MnSO,/I and 1 i.u. bacitraciuml added (A), 200 mg MnSOJl and 5 i.u. bacitracin/ml added (0) ,and 200 mg MnSO,/I and 10 i.u. bacitracinJm1 added
(V). 515
Fig. 2. Growth of B . licheniformis in the M2 medium with the following additions later in growth. Arrow indicate time of addition: Nothing added 50 i.u. bacitracin/ml added 5 mg MnSO,/l added ( O ) , 10 mg MnSO,/l added ( W ) , 5 mg MnSOJl and 50 i.u. bacitracid ml added ( A ) , and 10 mg f MnSOJl and 50 i.u. bacitracin/ 5 ml added (0). I
7
(a),
6
(v),
-
5
4
; 2
3
U
2
I
INCUBATION TIME (h)
growth was observed (Fig. 3a). The addition of only 2 i.u. bacitracin/ml resulted in the most pronounced stimulation. When the inoculum consisted of spores this was somewhat different. By the addition of spores and bacitracin to the M2 medium with no Mn (11)ions added, a marked stimulatory effect upon growth was observed. The addition of 50 i.u. bacitracin/ml resulted in the most pronounced stimulation (Fig. 3b). When 50 i.u. bacitracin/ml and 100 mg MnSO,/I was added together with the spore inoculum, growth was inhibited (Fig. 3b). When corresponding amounts of inactivated bacitracin was added to bacteria or spores no stirnulatory effect upon growth wns observed. DISCUSSION
Our results indicate that the effect of bacitracin upon the growth of the producer strain Bacillus licheniformis is dependent upon the amount of manganese (11)ions present in the medium. Both stimulatory, inhibitory, and lytic effects upon the cells have been observed. In media with a very low content of this cation, bacitracin seems to be
516
stimulatory to growth, whereas in media with a high amount of manganese( 11)ions, bacitracin is inhibitory to growth or induces lysis depending upon the amount of bacitracin added. Snoke & Cornell (9) have previously reported that small amounts of bacitracin markedly inhibited the early growth of B. licheniformis ATCC 10716. They suggested that bacitracin could accumulate during later stages of growth because the inhibitory effect of bacitracin was restricted to the early growth phase only. Our results, however, show bacitracin is able to inhibit both the early and the late growth of B. licheniformis in the presence of manganese (11)ions. It is further proposed that antibiotic producers can survive their own production because they develop resistance to their own antibiotic during later stages of growth (2, 12). This resistance is suggested to be phenotypic because the growth of these resistant cells in a fresh medium containing the antibiotic, will be inhibited once again ( 2 ) . Our results, however, indicate that B. licheniformis does not develop resistance to bacitracin during growth since both the early and the late growth of the organism are in-
Fig. 3. Growth of B. licheniformis in the M2 medium with no manganese( 1I)ions added and with the following additions:
Fig. 3a: Bacteria as inoculum with no bacitracin added with 1 i.u. bacitracidml added (O), with 2 i.u. bacitracidml added ( A ) , with 5 i.u. bacitracin/ml added and with 10 i.u. bacitracidml added ( A ) .
(a),
(a),
Y
Fig. 36: Spores as inoculum with ,.. no bacitracin added with 50 i.u. bacitracin/ml added (0) , and with 50 i.u. bacitracin/ml and 100 mg MnSOJl added
(a),
(V). I
2
hibited in a similar manner, i.e., only in the presence of sufficient amount of manganese(1I)ions. I t appears that B. licheniformis is “resistant” to its own antibiotic during later stages of growth because there are normally very low concentrations of this cation left in the medium towards end of growth. The addition of 10 mg MnS04/l late in the growth had a marked toxic effect upon the culture, whereas the same amounts added to the early growth phase had no such effect. This supports our suggestion, since it is possible that the relatively high amounts of bacitracin produced late in growth (4)act
3
4 5 6 INCLIBATION TIME l h )
7
8
P
10
It
inhibitorily when sufficient amounts of manganese( 1I)ions are added. Since the activity of many antibiotics is influenced by metal ions ( l l ) , it is possible that the frequently observed inhibition of the early growth of producer strains by their own antibiotic may by due to the presence of excess metal ions during early growth. This may explain why “resistant” cells from the production phase are inhibited once again when inoculated in a fresh medium containing the antibiotic. We have recently put forward the hypothesis that bacitracin may participate in the
517
transport of manganese (11)ions in B. licheniformis ( 7 ) . This hypothesis may explain why bacitracin acts inhibitorily in media with high amounts of manganese (11)ions, and stimulatorily in media with a very low content of the same cation. In the first case too much manganese (11)ions are transported into the cells upon the addition of bacitracin, and toxic concentrations are reached. In the latter case bacitracin stimulates growth by increasing the uptake of Mn(I1)ions which now are needed by the bacteria. When bacitracin was added to spores in low Mn (11)ions environments, the stimulatory effect upon growth was more pronounced. The spores must probably germinate and grow out before bacitracin is produced. The addition of bacitracin to spores will therefore probably result in a more rapid uptake of sufficient amounts of the desired cation. Since an inactivated bacitracin solution did not stimulate growth, the stimulatory effect of bacitracin was not due to impurities present in the bacitracin used. To the authors knowledge this is the first example of a stimulatory effect of an antibiotic upon the producer cells. The author would like to thank Mr. T . Hoyland, Director of Research and Development, for his support in this work, and Dr. H . P. Throndsen and Mr. S . Thomassen for valuable discussions during the work and in preparing the text. The skilled technical assistance of Mrs. Inger Austang and Mrs. Linda Horn is greatly appreciated.
518
REFERENCES 1. Demain, A . L.: Regulatory mechanisms and
2. 3.
4.
5.
6.
7. 8.
9.
10.
11.
12.
the industrial production of microbial metabolites. Lloydia. 31: 395-418, 1968. Demain, A . L.: How do antibiotic-producing microorganisms avoid suicide? Annal. N.Y. Acad. Sci. 235: 601-612, 1974. Haavik, H . I . & Thomassen, S.: A bacitracinnegative mutant of Bacillus licheniformis which is able to sporulate. J. gen. Microbiol. 76: 451-454, 1973. Haavik, H . I.: Studies on the formation of bacitracin by Bacillus licheniformis: effect of glucose. J. gen. Microbiol. 81: 383-390, 1974. Haavik, H . I . : Studies on the formation of bacitracin by Bacillus licheniformis: effect of inorganic phosphate. J. gen. Microbiol. 84: 226-230, 1974. Haavik, H . I.: Studies on the formation of bacitracin by Bacillus licheniformis: role of catabolite repression and organic acids. J. gen. Microbiol. 84: 321-326, 1974. Haavik, H . I . & Froyshov, 0.:O n the function of peptide antibiotics in the producer organisms. Nature 254: 79-82, 1975. Sadoff, H . L.: Sporulation antibiotics of Bacillus species. Spores 5 : 157-166, 1972. Snoke, J . E. & Cornell, N.: Protoplast lysis and inhibition of growth of Bacillus licheniformis by bacitracin. J. Bacteriol. 89: 415420, 1965. Woodruff, H . B.: The physiology of antibiotic production: the role of the producing organism. SOC. Gen. Microbiol. Symp. 26: 22-46, 1966. Weinberg, E. D.: The mutual effects of antimicrobial compounds and metallic cations. Bact. Rev. 21: 46-68, 1957. Yoshida, T., Weissbach, H . & Katz, E.: Inhibitory effect of actinomycin upon the producing organism. Arch. Biochem. Biophys. 114: 252-255, 1966.
