ANTIMICROBIAL AGENTS AND CHmO4rHzRAPY, Jan. 1977, P. 34-37 Copyright © 1976 American Society for Microbiology
Vol. 11, No. 1 Printed in U.S.A.
Production of Megacins C and Cx: Presumptive Evidence of Extrachromosomal Control HELEN D. DONOGHUE' Bacteriology Department, The Medical School, Bristol University, Bristol BS8 lTD, England
Received for publication 3 June 1976
Exposure of growing cultures of Bacillus megaterium C4A- to ethidium bromide or an elevated growth temperature was found to eliminate megacin C production. Ethidium bromide resulted in a cure rate of up to 13%. Growth at 430C gave a cure rate of up to 99%. Megacin C production was lost spontaneously at a rate of 4% or less. There was a greater rate of spontaneous loss of megacin Cx production by B. megaterium 337, up to 14%. Growth at 430C resulted in a cure rate of up to 24% in this organism. Reversion to the Meg+ state by cured clones has never been demonstrated. These observations support the hypothesis that production of megacins C and Cx is plasmid mediated. Meg- clones adsorbed more megacin than either parent strain and were more susceptible to megacin action.
Megacins are bacteriocins produced by strains of Bacillus megaterium. "Megacine" was first described by Ivanovics and Alfoldi (16). Later it was realized that the term covered a number of different agents. Megacin C appears to be similar in its effect to certain colicins (22), acting on deoxyribonucleic acid (DNA) synthesis in susceptible organisms (1315). Megacin Cx, first thought to be similar to megacin C (6, 8), is now known to inhibit protein synthesis in susceptible bacteria (7). Little is known of the genetic regulation of bacteriocin production by gram-positive organisms, although some staphylococcins are apparently plasmid mediated and can be cured by an elevated growth temperature or by ethidium bromide (3, 4, 17, 24). Kawakami and Landman (18) noted that megacin C production could not be cured by acriflavine treatment, yet Donoghue (5) observed that production of megacins C and Cx could be eliminated by incubating Meg+ cultures at 4300. This paper examines the effect of chemical curing agents or an elevated initial incubation temperature on the production of megacin C or Cx by two strains of B. megaterium. (This paper is taken in part from a Ph.D. thesis submitted by H.D.D. to Bristol University.) MATERIALS AND METHODS
netics, University of Leicester, England. B. megaterium 337 (MegCx+) was obtained from G. Ivinovics, Institute of Microbiology, University Medical School, Szeged, Hungary. B. megaterium 162u is a streptomycin-resistant indicator strain that was originally isolated from soil. Media. Oxoid blood agar base no. 2 (4%, wt/vol; NA) was used for routine subculture and counts of bacteria. The liquid medium used was peptone-water, which contained 1% (wt/vol) each of peptone and tryptone (Difco) and 0.05% (wt/vol) NaCl. Soft agar used for overlayering plates contained 1.6% (wt/vol) Difco nutrient broth, 0.5% (wt/vol) NaCl, and 0.7% (wt/vol) agar. Peptone-glucose broth (12) was used for megacin production and broth cultures. Megacin. Megacins C and Cx were produced according to the method described by Donoghue (5). Crude culture supernatants were used as a source of both megacins. Demonstration of megacin production. Cultures were diluted in saline, and 0.1 ml of appropriate dilutions was spread on NA plates. After overnight incubation at 370C, plates showing well-distributed colonies were replicated onto NA plates seeded with 162u (0.1 ml of an overnight 162u peptone-water culture per 4 ml of soft agar). The replicated plates were kept at 5°C overmight and then incubated at 37°C for 8 to 12 h. Meg- colonies were easily identified by the absence of an inhibition zone (Fig. 1). Elimination by chemical agents. A growing C4Aculture in peptone-glucose broth was diluted 1:10 and 1:100 in warm broth, and an aqueous acriflavine solution added to give a final concentration of 10.0, 1.0, or 0.1 pg/ml. The organisms were incubated at 370C overnight, together with control cultures without acriflavine. To examine the effect of culture age, C4A- was inoculated into peptone-glucose broth and samples were taken at various times after incubation. The optical density of samples was adjusted with warm broth to give a reading of 0.06 to 0.07
Organisms. B. megaterium C4A- (MegA-C+) was obtained from I. B. Holland, Department of GeX Present address: School of Medical Sciences, University of Bradford, Bradford, West Yorkshire BD7 lDP, England. 34
35
PRODUCTION OF MEGACINS C AND CX
VOL. 11, 1977
(Unicam SP600, 610 nm). Volumes (10 ml) of diluted sample were mixed with various concentrations of aqueous acriflavine or ethidium bromide solutions. These and control cultures were incubated overnight at 37°C and then tested for megacin production. Effect of temperature. Two peptone-water cultures of the test organism, inoculated from the same colony, were set up. One was incubated overnight at 37°C and the other at 43°C. Samples were then tested for megacin production. All subsequent incubation was at 37C. Adsorption of megacin and susceptibility to megacin action. The organisms to be used for megacin adsorption were grown as a confluent layer on the surface of a large (304.8 mM2 or 12 by 12 inch) NA plate. After overnight incubation, they were scraped off the agar, suspended in saline, and washed twice with saline. A sample from each cell suspension was counted by the Miles and Misra method (21), using triplicate plates. Equal volumes of sterile megacin solution and either undiluted or diluted cell suspen-
FIG. 1. Demonstration of megacin production by replica plating on to agar seeded with the indicator strain B. megaterium 162u. (a) Meg+ colony; (b) Meg' colony; (c) Meg- colony.
sion were mixed and left for 1 to 2 h at 5°C to allow adsorption to occur. The bacterial cells were then spun down, resuspended in saline, and counted. The supernatant was assayed for residual megacin by a punch-hole assay (20). The percentage of megacin adsorbed was calculated, and when different strains were compared, the figures were corrected to allow for differences in cell numbers.
RESULTS
A summary of the results is shown in Table 1. Chemical curing agents. Acriflavine, at a concentration of 0.1, 1.0, or 10.0 ,ug/ml, had little curing effect on C4A- cultures containing 1.3 x 106, 2.5 x 107, or 2.8 x 108 viable organisms per ml. A concentration of 10 ug of acriflavine per ml was bactericidal except in the undiluted C4A- culture. Acriflavine had no curing effect on C4A- cells obtained from 1- to 5-h-old cultures. In a similar experiment, there was an increase in the proportion of Meg- colonies when 1 x 10-5 M ethidium bromide was added to a 3-h (exponential phase) culture of C4A-. The cure rate was 13% (15/116), compared with 2% (4/196) in the control. Effect of high temperature. An initial incubation temperature of 43°C was highly effective in eliminating megacin C production. A maximal cure rate of 99% (110/111) could be obtained by this procedure. Using data from several experiments, an average cure rate of 85% (468/ 550) was realized after initial growth at 43°C, compared with a rate of 1.7% (17/975) in the controls. Using strain 337, there was a slight increase in cure rate of megacin Cx production. In one experiment a rate of 24% (44/184) was noted, compared with 2% (4/198) in the corresponding control. Average rates, however, were 10.6% (125/1,174) at 430C and 6.3% (54/854) at 370C. Spontaneous loss of megacin Cx production. Because MegCx- clones appeared to arise so readily, individual MegCx+ colonies were examined to see whether they too contained
TABLE 1. Comparison of the effect of various curing agents on megacin production Treatment
Ethidium bromide (1 x 10-5 M) Control
Acriflavine (1.0 ,ug/ml) Control
Growth at 43°C Control a NT, Not tested.
Megacin C Megacin Cx No. of colonies cured/ Maximum No. of colonies cured/ Maximum no. no. tested no.tested percent cured percent cured
15/116
4/196
12.9 2.0
NT NT
NT NT
3/111
0/89
2.7 0
NT NT
NT NT
110/111 2/152
99.1 1.3
44/184 4/196
23.9 2.0
36 DONOGHUE MegCx- cells. Both MegCx+ colonies and those colonies surrounded by a hazy zone, termed MegCx+, contained a significant proportion of viable Meg- cells, 13.5% (8/133) and 9.8% (19/ 193), respectively. Reversion from Meg- to Meg+. No Megclone has yet been shown to have reverted to the Meg+ state. From the data currently available, if reversion does occur it must be at a rate less than 0.22% (0/456) for MegCx and 0.073% (0/1,371) for MegC. Activity spectrum of Meg+ and Meg- organisms. Using an overlay technique, both Meg+ strains were apparently nonsusceptible to megacins C and Cx. Meg- clones were susceptible to both megacins. This was the only difference in activity spectrum observed. In broth cultures, Meg+ strains could be shown to be megacin susceptible when high multiplicities of megacin were used. A typical result as shown in Fig. 2. Adsorption of megacin. Meg- cultures adsorbed more megacin than Meg+ cells (Fig. 2). In this example, cells of the indicator organism 162u adsorbed about five times more megacin
00
1.0. megacin
100
100.0
miltiplicity
FIG. 2. Adsorption of megacin Cx to MegCx+ and MegCx- cells of B. megaterium and their subsequent survival. Symbols: (-) Survival curve after an "infinite" time for megacin adsorption. Log,, survivor ratio plotted against megacin multiplicity. (A) Adsorption curve. Log,. percent megacin adsorbed per cell plotted against megacin multiplicity.
ANTIMICROB. AGENTS CHEMOTHER.
than did MegCx- cells of strain 337. The Megcells adsorbed approximately 50 times more megacin Cx than did the MegCx+ cells. DISCUSSION An elevated incubation temperature has been shown to eliminate megacin C production highly effectively from B. megaterium C4A-. Ethidium bromide has a lesser effect. These observations, when compared with staphylococcal studies (1, 3, 17, 19, 24), strongly suggest that the determinant for megacin C production is situated on a plasmid. Failure to eliminate megacin C production with acriflavine is not unexpected. Curing of plasmids is highly unpredictable, and agents active against one organism may have no effect even on another strain of the same species (3, 10). Too little is known of the genetics ofB. megaterium to enable plasmid linkage to be established by the other criteria suggested by Novick (23). It is known that a high proportion of the DNA in B. megaterium is in the form ofheterogeneous circular DNA segments (2, 11). Although an additional element could not be distinguished in one Meg+ strain examined, the existence of a megacin plasmid has not been disproved. The megacin Cx determinant is probably also plasmid bome, but since its high rate of spontaneous loss masks the effect of possible curing agents, B. megaterium 337 cultures were not tested with chemical curing agents. The demonstration of mixed clones of strain 337 is rather surprising, since it would be expected that a Meg- cell arising in a Meg+ colony would be killed immediately by the megacin being produced by adjacent cells. Possibly most of this megacin is adsorbed to the cell walls of the producing organisms or rapidly diffiuses away into the surrounding agar. If Meg- organisms are being killed, their proportions as reported in this paper may be underrepresented. Previous work (5) has demonstrated the presence of megacin on the surface of Meg+ cells. This may explain the low levels of megacin adsorption found in Meg+ cultures. However, the difference in megacin susceptibility between Meg+ and Meg- cultures of the same bacterial strain suggests that, as with other bacteriocins (9), there may be an immunity substance present in the cytoplasm of Meg+ organisms that can be overwhelmed by high megacin levels. B. megaterium is a particularly suitable organism for future investigations along these lines since its cell wall can be completely removed by treatment with lysozyme. Kawakami
VOL. 11, 1977
and Landman (18) have already shown that the capacity of C4A- to produce megacin C is not lost when cultures are converted into protoplasts. The study of megacin production and its action on protoplasts should, therefore, prove most rewarding. ACKNOWLEDGMENTS The advice and helpful discussions of the late Anna Mayr-Harting, formerly of this Department, are gratefully acknowledged. Vilma Stanisich is thanked for her constructive criticism of the manuscript. Most of the work was carried out while I was in receipt of an M.R.C. scholarship for training in research methods. LITERATURE CITED 1. Bouanchaud, D. H., M. R. Scavizzi, and Y. A. Chabbert. 1968. Elimination by ethidium bromide of antibiotic resistance in enterobacteria and staphylococci. J. Gen. Microbiol. 54:417-425. 2. Carlton, B. C., and D. R. Helinski. 1969. Heterogeneous circular DNA elements in vegetative cultures ofBacillus megaterium. Proc. Natl. Acad. Sci. U.S.A. 64:592-599. 3. Dajani, A. S., and Z. Taube. 1974. Plasmid-mediated production of staphylococcin in bacteriophage type 71 Staphylococcus aureus. Antimicrob. Agents Chemother. 5:594-598. 4. Dastidar, S. G., S. Mitra, S. N. Sarkar, and A. N. Chakrabarty. 1974. Transformation with bacteriocin factors in staphylococci. J. Gen. Microbiol. 84:245252. 5. Donoghue, H. D. 1972. Properties and comparative starch-gel electrophoresis of megacins from several Bacillus megaterium strains. J. Gen. Microbiol. 72:473-483. 6. Durner, K. 1970. Anreicherung, Reinigung und Charakterisierung eines Bacteriocins ausBacillus megaterium 337. Z. Allg. Mikrobiol. 10:93-102. 7. Durner, K. 1970. Untersuchungen zur Wirkungsweise von Megacin Cx auf Bacillus megaterium KM. Z. Allg. Mikrobiol. 10:373-382. 8. Durner, K., and F. Mach. 1966. Physiologische Untersuchungen eines Bacteriocines aus Bacillus megaterium 337. Zentralbl. Bakteriol. Parasitenkd. Infektionskr. Hyg. Abt. II Orig. 120:565-575.
PRODUCTION OF MEGACINS C AND CX
37
9. Fredericq, P. 1958. Colicins and colicinogenic factors. Symp. Soc. Exp. Biol. 12:104-122. 10. Hale, E. M., and R. D. Hinsdill. 1975. Biological activity of staphylococcin 462: bacteriocin from Staphylococcus aureus. Antimicrob. Agents Chemother. 7:7481. 11. Henneberry, R. C., and B. C. Carlton. 1973. Characterization of the polydisperse closed circular deoxyribonucleic acid molecules of Bacillus megaterium. J. Bacteriol. 114:625-631. 12. Holland, I. B. 1961. The purification and properties of megacin, a bacteriocin from Bacillus megaterium. Biochem. J. 78:641-648. 13. Holland, I. B. 1963. Effect of a bacteriocin preparation (Megacin C) on DNA synthesis in Bacillus megaterium. Biochem. Biophys. Res. Commun. 13:246-250. 14. Holland, I. B. 1965. A bacteriocin specifically affecting DNA synthesis inBacillus megaterium. J. Mol. Biol. 12:429-438. 15. Holland, I. B., and C. F. Roberts. 1964. Properties of a new bacteriocin formed by Bacillus megaterium. J. Gen. Microbiol. 35:271-285. 16. Ivanovics, G., and L. Alfoldi. 1954. A new antibacterial principle: megacine. Nature (London) 174:465. 17. Jetten, A. M., and G. D. Vogels. 1973. Characterization and extrachromosomal control of bacteriocin production in Staphylococcus aureus. Antimicrob. Agents Chemother. 4:49-57., 18. Kawakami, M., and 0. E. Landman. 1966. Retention of episomes during protoplasting and during propagation in the L state. J. Bacteriol. 92:398-404. 19. May, J. W., R. H. Houghton, and C. J. Perret. 1964. The effect of growth at elevated temperatures on some heritable properties of Staphylococcus aureus. J. Gen. Microbiol. 37:157-169. 20. Mayr-Harting, A., A. J. Hedges, and R. C. W. Berkeley. 1972. Methods for studying bacteriocins, p. 315422. In J. R. Norris and D. W. Ribbons (ed.), Methods in microbiology, vol. 7A. Academic Press Inc., London. 21. Miles, A. A., and S. S. Misra. 1938. The estimation of the bactericidal powers of the blood. J. Hyg. 38:732748. 22. Nomura, M. 1967. Colicins and related bacteriocins. Annu. Rev. Microbiol. 21:257-284. 23. Novick, R. P. 1969. Extrachromosomal inheritance in bacteria. Bacteriol. Rev. 33:210-263. 24. Warren, R., M. Rogolsky, B. B. Wiley, and L. A. Glasgow. 1974. Effect of ethidium bromide on elimination of exfoliative toxin and bacteriocin production in Staphylococcus aureus. J. Bacteriol. 118:980-985.
Vol. 11, No. 1 Printed in U.S.A.
Production of Megacins C and Cx: Presumptive Evidence of Extrachromosomal Control HELEN D. DONOGHUE' Bacteriology Department, The Medical School, Bristol University, Bristol BS8 lTD, England
Received for publication 3 June 1976
Exposure of growing cultures of Bacillus megaterium C4A- to ethidium bromide or an elevated growth temperature was found to eliminate megacin C production. Ethidium bromide resulted in a cure rate of up to 13%. Growth at 430C gave a cure rate of up to 99%. Megacin C production was lost spontaneously at a rate of 4% or less. There was a greater rate of spontaneous loss of megacin Cx production by B. megaterium 337, up to 14%. Growth at 430C resulted in a cure rate of up to 24% in this organism. Reversion to the Meg+ state by cured clones has never been demonstrated. These observations support the hypothesis that production of megacins C and Cx is plasmid mediated. Meg- clones adsorbed more megacin than either parent strain and were more susceptible to megacin action.
Megacins are bacteriocins produced by strains of Bacillus megaterium. "Megacine" was first described by Ivanovics and Alfoldi (16). Later it was realized that the term covered a number of different agents. Megacin C appears to be similar in its effect to certain colicins (22), acting on deoxyribonucleic acid (DNA) synthesis in susceptible organisms (1315). Megacin Cx, first thought to be similar to megacin C (6, 8), is now known to inhibit protein synthesis in susceptible bacteria (7). Little is known of the genetic regulation of bacteriocin production by gram-positive organisms, although some staphylococcins are apparently plasmid mediated and can be cured by an elevated growth temperature or by ethidium bromide (3, 4, 17, 24). Kawakami and Landman (18) noted that megacin C production could not be cured by acriflavine treatment, yet Donoghue (5) observed that production of megacins C and Cx could be eliminated by incubating Meg+ cultures at 4300. This paper examines the effect of chemical curing agents or an elevated initial incubation temperature on the production of megacin C or Cx by two strains of B. megaterium. (This paper is taken in part from a Ph.D. thesis submitted by H.D.D. to Bristol University.) MATERIALS AND METHODS
netics, University of Leicester, England. B. megaterium 337 (MegCx+) was obtained from G. Ivinovics, Institute of Microbiology, University Medical School, Szeged, Hungary. B. megaterium 162u is a streptomycin-resistant indicator strain that was originally isolated from soil. Media. Oxoid blood agar base no. 2 (4%, wt/vol; NA) was used for routine subculture and counts of bacteria. The liquid medium used was peptone-water, which contained 1% (wt/vol) each of peptone and tryptone (Difco) and 0.05% (wt/vol) NaCl. Soft agar used for overlayering plates contained 1.6% (wt/vol) Difco nutrient broth, 0.5% (wt/vol) NaCl, and 0.7% (wt/vol) agar. Peptone-glucose broth (12) was used for megacin production and broth cultures. Megacin. Megacins C and Cx were produced according to the method described by Donoghue (5). Crude culture supernatants were used as a source of both megacins. Demonstration of megacin production. Cultures were diluted in saline, and 0.1 ml of appropriate dilutions was spread on NA plates. After overnight incubation at 370C, plates showing well-distributed colonies were replicated onto NA plates seeded with 162u (0.1 ml of an overnight 162u peptone-water culture per 4 ml of soft agar). The replicated plates were kept at 5°C overmight and then incubated at 37°C for 8 to 12 h. Meg- colonies were easily identified by the absence of an inhibition zone (Fig. 1). Elimination by chemical agents. A growing C4Aculture in peptone-glucose broth was diluted 1:10 and 1:100 in warm broth, and an aqueous acriflavine solution added to give a final concentration of 10.0, 1.0, or 0.1 pg/ml. The organisms were incubated at 370C overnight, together with control cultures without acriflavine. To examine the effect of culture age, C4A- was inoculated into peptone-glucose broth and samples were taken at various times after incubation. The optical density of samples was adjusted with warm broth to give a reading of 0.06 to 0.07
Organisms. B. megaterium C4A- (MegA-C+) was obtained from I. B. Holland, Department of GeX Present address: School of Medical Sciences, University of Bradford, Bradford, West Yorkshire BD7 lDP, England. 34
35
PRODUCTION OF MEGACINS C AND CX
VOL. 11, 1977
(Unicam SP600, 610 nm). Volumes (10 ml) of diluted sample were mixed with various concentrations of aqueous acriflavine or ethidium bromide solutions. These and control cultures were incubated overnight at 37°C and then tested for megacin production. Effect of temperature. Two peptone-water cultures of the test organism, inoculated from the same colony, were set up. One was incubated overnight at 37°C and the other at 43°C. Samples were then tested for megacin production. All subsequent incubation was at 37C. Adsorption of megacin and susceptibility to megacin action. The organisms to be used for megacin adsorption were grown as a confluent layer on the surface of a large (304.8 mM2 or 12 by 12 inch) NA plate. After overnight incubation, they were scraped off the agar, suspended in saline, and washed twice with saline. A sample from each cell suspension was counted by the Miles and Misra method (21), using triplicate plates. Equal volumes of sterile megacin solution and either undiluted or diluted cell suspen-
FIG. 1. Demonstration of megacin production by replica plating on to agar seeded with the indicator strain B. megaterium 162u. (a) Meg+ colony; (b) Meg' colony; (c) Meg- colony.
sion were mixed and left for 1 to 2 h at 5°C to allow adsorption to occur. The bacterial cells were then spun down, resuspended in saline, and counted. The supernatant was assayed for residual megacin by a punch-hole assay (20). The percentage of megacin adsorbed was calculated, and when different strains were compared, the figures were corrected to allow for differences in cell numbers.
RESULTS
A summary of the results is shown in Table 1. Chemical curing agents. Acriflavine, at a concentration of 0.1, 1.0, or 10.0 ,ug/ml, had little curing effect on C4A- cultures containing 1.3 x 106, 2.5 x 107, or 2.8 x 108 viable organisms per ml. A concentration of 10 ug of acriflavine per ml was bactericidal except in the undiluted C4A- culture. Acriflavine had no curing effect on C4A- cells obtained from 1- to 5-h-old cultures. In a similar experiment, there was an increase in the proportion of Meg- colonies when 1 x 10-5 M ethidium bromide was added to a 3-h (exponential phase) culture of C4A-. The cure rate was 13% (15/116), compared with 2% (4/196) in the control. Effect of high temperature. An initial incubation temperature of 43°C was highly effective in eliminating megacin C production. A maximal cure rate of 99% (110/111) could be obtained by this procedure. Using data from several experiments, an average cure rate of 85% (468/ 550) was realized after initial growth at 43°C, compared with a rate of 1.7% (17/975) in the controls. Using strain 337, there was a slight increase in cure rate of megacin Cx production. In one experiment a rate of 24% (44/184) was noted, compared with 2% (4/198) in the corresponding control. Average rates, however, were 10.6% (125/1,174) at 430C and 6.3% (54/854) at 370C. Spontaneous loss of megacin Cx production. Because MegCx- clones appeared to arise so readily, individual MegCx+ colonies were examined to see whether they too contained
TABLE 1. Comparison of the effect of various curing agents on megacin production Treatment
Ethidium bromide (1 x 10-5 M) Control
Acriflavine (1.0 ,ug/ml) Control
Growth at 43°C Control a NT, Not tested.
Megacin C Megacin Cx No. of colonies cured/ Maximum No. of colonies cured/ Maximum no. no. tested no.tested percent cured percent cured
15/116
4/196
12.9 2.0
NT NT
NT NT
3/111
0/89
2.7 0
NT NT
NT NT
110/111 2/152
99.1 1.3
44/184 4/196
23.9 2.0
36 DONOGHUE MegCx- cells. Both MegCx+ colonies and those colonies surrounded by a hazy zone, termed MegCx+, contained a significant proportion of viable Meg- cells, 13.5% (8/133) and 9.8% (19/ 193), respectively. Reversion from Meg- to Meg+. No Megclone has yet been shown to have reverted to the Meg+ state. From the data currently available, if reversion does occur it must be at a rate less than 0.22% (0/456) for MegCx and 0.073% (0/1,371) for MegC. Activity spectrum of Meg+ and Meg- organisms. Using an overlay technique, both Meg+ strains were apparently nonsusceptible to megacins C and Cx. Meg- clones were susceptible to both megacins. This was the only difference in activity spectrum observed. In broth cultures, Meg+ strains could be shown to be megacin susceptible when high multiplicities of megacin were used. A typical result as shown in Fig. 2. Adsorption of megacin. Meg- cultures adsorbed more megacin than Meg+ cells (Fig. 2). In this example, cells of the indicator organism 162u adsorbed about five times more megacin
00
1.0. megacin
100
100.0
miltiplicity
FIG. 2. Adsorption of megacin Cx to MegCx+ and MegCx- cells of B. megaterium and their subsequent survival. Symbols: (-) Survival curve after an "infinite" time for megacin adsorption. Log,, survivor ratio plotted against megacin multiplicity. (A) Adsorption curve. Log,. percent megacin adsorbed per cell plotted against megacin multiplicity.
ANTIMICROB. AGENTS CHEMOTHER.
than did MegCx- cells of strain 337. The Megcells adsorbed approximately 50 times more megacin Cx than did the MegCx+ cells. DISCUSSION An elevated incubation temperature has been shown to eliminate megacin C production highly effectively from B. megaterium C4A-. Ethidium bromide has a lesser effect. These observations, when compared with staphylococcal studies (1, 3, 17, 19, 24), strongly suggest that the determinant for megacin C production is situated on a plasmid. Failure to eliminate megacin C production with acriflavine is not unexpected. Curing of plasmids is highly unpredictable, and agents active against one organism may have no effect even on another strain of the same species (3, 10). Too little is known of the genetics ofB. megaterium to enable plasmid linkage to be established by the other criteria suggested by Novick (23). It is known that a high proportion of the DNA in B. megaterium is in the form ofheterogeneous circular DNA segments (2, 11). Although an additional element could not be distinguished in one Meg+ strain examined, the existence of a megacin plasmid has not been disproved. The megacin Cx determinant is probably also plasmid bome, but since its high rate of spontaneous loss masks the effect of possible curing agents, B. megaterium 337 cultures were not tested with chemical curing agents. The demonstration of mixed clones of strain 337 is rather surprising, since it would be expected that a Meg- cell arising in a Meg+ colony would be killed immediately by the megacin being produced by adjacent cells. Possibly most of this megacin is adsorbed to the cell walls of the producing organisms or rapidly diffiuses away into the surrounding agar. If Meg- organisms are being killed, their proportions as reported in this paper may be underrepresented. Previous work (5) has demonstrated the presence of megacin on the surface of Meg+ cells. This may explain the low levels of megacin adsorption found in Meg+ cultures. However, the difference in megacin susceptibility between Meg+ and Meg- cultures of the same bacterial strain suggests that, as with other bacteriocins (9), there may be an immunity substance present in the cytoplasm of Meg+ organisms that can be overwhelmed by high megacin levels. B. megaterium is a particularly suitable organism for future investigations along these lines since its cell wall can be completely removed by treatment with lysozyme. Kawakami
VOL. 11, 1977
and Landman (18) have already shown that the capacity of C4A- to produce megacin C is not lost when cultures are converted into protoplasts. The study of megacin production and its action on protoplasts should, therefore, prove most rewarding. ACKNOWLEDGMENTS The advice and helpful discussions of the late Anna Mayr-Harting, formerly of this Department, are gratefully acknowledged. Vilma Stanisich is thanked for her constructive criticism of the manuscript. Most of the work was carried out while I was in receipt of an M.R.C. scholarship for training in research methods. LITERATURE CITED 1. Bouanchaud, D. H., M. R. Scavizzi, and Y. A. Chabbert. 1968. Elimination by ethidium bromide of antibiotic resistance in enterobacteria and staphylococci. J. Gen. Microbiol. 54:417-425. 2. Carlton, B. C., and D. R. Helinski. 1969. Heterogeneous circular DNA elements in vegetative cultures ofBacillus megaterium. Proc. Natl. Acad. Sci. U.S.A. 64:592-599. 3. Dajani, A. S., and Z. Taube. 1974. Plasmid-mediated production of staphylococcin in bacteriophage type 71 Staphylococcus aureus. Antimicrob. Agents Chemother. 5:594-598. 4. Dastidar, S. G., S. Mitra, S. N. Sarkar, and A. N. Chakrabarty. 1974. Transformation with bacteriocin factors in staphylococci. J. Gen. Microbiol. 84:245252. 5. Donoghue, H. D. 1972. Properties and comparative starch-gel electrophoresis of megacins from several Bacillus megaterium strains. J. Gen. Microbiol. 72:473-483. 6. Durner, K. 1970. Anreicherung, Reinigung und Charakterisierung eines Bacteriocins ausBacillus megaterium 337. Z. Allg. Mikrobiol. 10:93-102. 7. Durner, K. 1970. Untersuchungen zur Wirkungsweise von Megacin Cx auf Bacillus megaterium KM. Z. Allg. Mikrobiol. 10:373-382. 8. Durner, K., and F. Mach. 1966. Physiologische Untersuchungen eines Bacteriocines aus Bacillus megaterium 337. Zentralbl. Bakteriol. Parasitenkd. Infektionskr. Hyg. Abt. II Orig. 120:565-575.
PRODUCTION OF MEGACINS C AND CX
37
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