Biological Function of Gramicidin: Studies on Gramicidin-Negative Mutants (Peptide Antibiotics/Sporulation/Dipicolinic Acid/Bacillus Brevis) PRANAB K

Proc. NatS. Acad. Sci. USA Vol. 74, No. 2, pp. 780-784, February 1977 Microbiology

Biological function of gramicidin: Studies on gramicidin-negative mutants (peptide antibiotics/sporulation/dipicolinic acid/Bacillus brevis) PRANAB K. MUKHERJEE AND HENRY PAULUS Department of Metabolic Regulation, Boston Biomedical Research Institute, Boston, Massachusetts 02114; and Department of Biological Chemistry, Harvard Medical School, Boston, Massachusetts 02115 Communicated by Bernard D. Davis, October 28,1976

ABSTRACT By the use of a rapid radioautographic EXPERIMENTAL PROCEDURE screening rocedure, two mutants of Bacillus brevis ATCC 8185 that have lost the ability to produce gramicidin have been isolated. These mutants produced normal levels of tyrocidine and Bacterial Strains. Bacillus brevis ATCC 8185, the Dubos sporulated at the same frequency as the parent strain. Their strain, was obtained from the American Type Culture Collec- spores, however, were more heat-sensitive and had a reduced tion. Strain S14 is a streptomycin-resistant derivative of B. brevis 4ipicolinic acid content. Gramicidin-producing revertants oc- ATCC 8185, isolated on a streptomycin-gradient plate without curred at a relatively high frequency among tie survivors of mutagenesis. It grows well at 0.5 mg/ml of streptomycin, but prolonged heat treatment and had also regained the ability to produce heat-resistant spores. A normal spore phenotype could growth is retarded by streptomycin at 1.0 mg/ml. Strain B81 also be restored by the addition of gramicidin to cultures of the is a rifampicin-resistant derivative of strain S14, isolated on mutant strain at the end of exponential growth. On the other rifampicin-gradient plates after mutagenesis of spores with hand, the addition of dipicolinic acid could not cure the spore ethyl methanesulfonate (11). Strain B81 produces no detectable defect. These results provide strong evidence that the inability spores or gramicidin or tyrocidine, but has retained the strep- to produce gramicidin is directly responsible for the observed tomycin-resistant character of its parent. The isolation of spore defects. Indeed, they unambiguously demonstrate a gramicidin-negative mutants and their revertants is described function of a peptide antibiotic in bacterial sporulation. The possibility that this function consists of the regulation of tran- in Results. Al mutant strains were maintained as stab cultures scription during the transition from growth to sporulation is in nutrient agar at room temperature or on nutrient agar slants discussed. with intermittent cloning to screen out revertants. The parent strain was stored at -200 as a suspension of heat-activated The biological function of peptide antibiotics has been the spores in 50% (vol/vol) glycerol. subject of much speculation and debate, the hypotheses pro- Rapid Screening for Gramicidin-Negative Mutants. The posed ranging from antibacterial defense (1, 2) and detoxication method used was based on the replica-printing technique de- of unwanted metabolites (3, 4) to the regulation of bacterial veloped by Raetz (12). Spores of B. brevis S14 were treated with sporulation (5, 6). We favor a regulatory role in sporulation, the mutagen ethyl methanesulfonate by the procedure of Ito which is consistent with the observations that peptide antibiotics and Spizizen (11), washed, and incubated in tryptone broth for are produced.during the transition from vegetative growth to 1 hr at 37'. Samples were then plated on potato dextrose agar sporulation and that they inhibit vegetative growth of the at a dilution to give about 150 colonies per plate. The plates producing organism. More specifically, we have postulated that were incubated at 370 for 36 hr and imprinted upon sterile disks peptide antibiotics regulate RNA synthesis during the early of Whatman no. 42 filter paper. These were then impregnated stages of sporulation by selectively inhibiting the transcription with a solution containing KCI (0.33 M), CaCl2 (1 mM), MgC12 of genes that function only during vegetative growth (6, 7). (2 mM), MnCI2 (2 MM), FeSO4 (1 MAM), glucose (1 g/liter), and This hypothesis has been examined in some detail in Bacillus chloramphenicol (100 mg/liter). After 10 min at 370, the paper brevis ATCC 8185, which produces two kinds of antibiotics: was transferred to a solution containing the same salts as before, gramicidin, a family of pentadecapeptides with blocked amino- as well as 2 mM each of L-alanine, L-leucine, L-valine, and and carboxyl-termini, and tyrocidine, a family of cyclic deca- glycine; 0.1 MuCi of DL-[3-'4C]tryptophan (34 mCi/mmol); and peptides (8). Studies with purified RNA polymerase have re- chloramphenicol (100 mg/liter); and incubated at 370 for 30 vealed that both gramicidin (6, 7, 9) and tyrocidine (6, 9, 10) min. The disks were then immersed in cold 0.3 M trichloro- inhibit transcription at concentrations at which they are pro- acetic acid and, after 30 min at 40, carefully washed with 80 duced early during sporulation. Moreover, gramicidin was ml of cold 0.3 M trichloroacetic acid and then with cold water found to inhibit specifically the interaction of RNA polymerase on a large sintered-glass funnel with light suction. After drying, with DNA (7), consistent with the regulation of promoter rec- the filters were exposed to Kodak RP/M-54 x-ray film for 36 ognition. Nevertheless, experiments of this kind cannot provide hr, the films were developed, and the paper disks were stained conclusive proof for a regulatory function of antibiotics-this with Coomassie Blue to reveal bacterial colonies, as described must come from the study of mutants that have a specific lesion by Raetz (12). After comparing the developed film with the in antibiotic synthesis. stained filter disk, any colonies that were stained but produced In this paper, we describe the isolation and characterization no exposure of the film were retrieved from the original agar of mutants of B. brevis that have lost the ability to produce plates (which had been stored at 40 for the duration of the gramicidin. Our results show that these mutants produce de- procedure) as possible gramicidin-negative mutants for further fective spores, and that this defect can be cured by the addition screening. of gramicidin during the early stages of sporulation. Radiometric Assay for Antibiotic Synthesis. Bacillus brevis 780 Downloaded by guest on September 26, 2021 Microbiology: Mukherjee and Paulus Proc. Natl. Acad. Sci. USA 74 (1977) 781

A S14 Table 1. Incorporation of "4C-labeled amino acids into gramicidin and tyrocidine by Bacillus brevis mutants Relative amino acid incorporation into Strain Gramicidin* Tyrocidinet S14 100 100 B81 0 0 Ml 1 98 Ml M5 M5 1 92 M1R 72 81 M5R 48 56 * Incorporation of [14C]tryptophan (100 33,700 cpm/ml of culture). t Incorporation of [14C]ornithine (100 4140 cpm/ml of culture). B S14 agar) seeded with S. faecalis. After 48 hr at 40 to allow diffusion of the antibiotics, the plates were incubated at 370 for about 10 hr to reveal the zones of growth inhibition. Heat Treatment of Spores. Cultures of B. brevis were grown in the medium of Hanson et al. (13) for 48 hr, diluted 105-fold with distilled water, and heated in a water bath at 800 for ap- propriate periods of time. The samples were quickly chilled in ml W5 ice, diluted as necessary, and plated on Penassay agar to de- termine the frequency of viable spores as colony-forming units. Assay of Dipicolinic Acid. The dipicolinic acid content of spores was measured by the method of Janssen et al. (15). Materials. Rifampicin, chloramphenicol, and L amino acids FIG. 1. Antibiotic production by Bacillus brevis mutants. were purchased from Calbiochem; streptomycin sulfate from Gramicidin and tyrocidine were extracted and assayed microbiolog- Pfizer; ethyl methanesulfonate from Eastman; dipicolinic acid ically as described under Experimental Procedure. (A) Each filter from Aldrich; DL-[3-'4C]tryptophan (34 mCi/mmol) and paper disk was impregnated with 1 ml of total antibiotic extract DL-[5-14C]ornithine (7.7 mCi/mmol) from New England (gramicidin plus tyrocidine). (B) Each filter paper disk was impregnated with 1 ml of antibiotic extract after passage through Dowex-50 Nuclear; and bacteriological culture media from Difco. to remove tyrocidine. was grown to early stationary phase (250 Klett units with a no. RESULTS 42 filter; 5 X 108 cells per ml) in the medium of Hanson et al. Isolation of Gramicidin-Negative Mutants. A total of 9300 (13). Samples (1 ml) were removed, treated with chloram- colonies were screened by our radioautographic procedure for phenicol (100 ,ug) at 370 for 10 min, and then supplemented the ability to produce gramicidin. Of these, 12 colonies ap- either with 2 ,mol of L-phenylalanine and 0.1 ,Ci of DL-[3- peared gramicidin-negative. Preliminary screening revealed 14C]tryptophan (to measure gramicidin synthesis) or with 2 mM all of the strains to be streptomycin-resistant like the parent, but L-tryptophan and 0.1 MCi of DL-[5-14C]ornithine (for tyrocidine 10 were unable to sporulate. Labeling with radioactive pre- synthesis). After 5 min at 370, the reaction was terminated by cursors of gramicidin and tyrocidine showed that the two spo- the addition of 0.6 M trichloroacetic acid (1 ml), followed by rogenic strains were able to incorporate amino acids into tyro- heating at 1000 for 15 min. The precipitate was collected on cidine but not into gramicidin (Table 1). In contrast, the 10 GF/C glass fiber filters and washed with 0.3 M trichloroacetic asporogenic strains were unable to incorporate label into either acid and then with water. The filters were dried and their ra- antibiotic (not shown) and in this respect behaved like dioactivity was determined in a toluene-based scintillation fluid pleiotropic asporogenic mutants isolated by other procedures, with a liquid scintillation spectrometer. such as the rifampicin-resistant strain B81 (Table 1). The results Microbiological Antibiotic Assay. Antibiotics were detected of the labeling experiments were confirmed by microbiological microbiologically using a paper disk plate assay with Strepto- assay of antibiotics produced by the mutant strains. As shown coccus faecalis ATCC 10541 as the indicator organism. Total in Fig. 1, total antibiotic production (gramicidin plus tyrocidine) antibiotic (gramicidin plus tyrocidine) was extracted from 24-hr by strains Ml and M5 was similar to that by the parent, but cultures of B. brevis, grown in the medium of Hanson et al. (13), gramicidin was almost completely absent from the antibiotic by the addition of 1.5 volumes of ethanol. After 12 hr at 20°, mixture produced. The growth rate of strains Ml and M5 in the residue was removed by centrifugation. One portion of the liquid media was the same as that of the parent strain. However, extract was assayed directly for total antibiotic activity, while the colonies of the mutants on solid media differed from those another portion was passed through a small column of Dowex of the parent, being relatively translucent like those of aspo- 50X2 (H+) in ethanol, which selectively removes tyrocidine, rogenic strains. the effluent being used for the assay of gramicidin (14). For Properties of Mutant Spores. Although the gramicidin- antibiotic assay, samples were applied in small portions to filter negative mutants, Ml and M5, seemed to sporulate normally paper disks with intermittent drying, and the disks were then when spores were assayed by heating at 800 for 20 min, pro- placed on agar plates (per liter: 10 g of tryptone, 5 g of yeast longed heating at 80° revealed that the mutant spores were extract, 16 g of Na2HPO4-7H20, 10 g of glucose, and 15 g of inactivated considerably more rapidly than spores of the parent Downloaded by guest on September 26, 2021 782 Microbiology: Mukherjee and Paulus Proc. Natl. Acad. Sci. USA 74 (1977)

Table 2. Heat resistance of spores ofBacillus brevis mutants Table 3. Dipicolinic acid content of spores of Bacillus brevis mutants Viable spores Viable spores Ratio of after 20 min after 3 hr survivors, Viable spores at 800 at 800 3 hr/ Grami- after 20 min Dipicolinic Dipicolinic Strain (spores/nl) (spo6res/nl) 20 min cidin at 800 acid content acid/spore Strain added* (spores/nl) (Mglml) (pg) S14 72 40 0.56 Ml 67 3.2 0.05 S14 - 72 118 1.64 M5 63 1.8 0.03 Ml - 78 22 0.28 M1R 64 31 0.49 M5 - 7.3 26 0.36 M5R 56 22 0.40 S14 + 70 115 1.64 M1 + 72 77 1.07 M5 + 75 84 1.12 strain (Table 2). After 3 hr at 800, 5060% of wild-type spores * Gramicidin (200 jg/ml) was added when the cells had reached a remained viable, compared to only 5-10%6 of the mutant spores turbidity of 250 Klett units. (Fig. 2 and Table 2). When the spores were assayed for dipicolinic acid, the mutant spores were found to have an ap- cultures whose spores had been centrifuged and washed with proximately 5-fold reduced level (Table 3). fresh medium before heat treatment still exhibited the increased Isolation of Revertants. The increased heat sensitivity of heat resistance, whereas spores of untreated cultures, heated mutant spores provided a convenient means for isolating rev- in the presence of 200 ,g/ml of gramicidin, remained heat ertants. Spores from strains Ml and M5 were heated at 800 for sensitive (not shown); 3 hr and plated on potato dextrose agar. When the survivor colonies were examined for gramicidin production by the ra- DISCUSSION dioautographic procedure, a surprisingly large fraction (about 10%) were found to be gramicidin-positive. Two randomly By screening about 10,000 colonies with our radioautographic chosen'gramicidin-producing colonies (MiR and M5R) were prooedure, we succeeded in isolating 12 strains that were unable examined more closely. They had largely regained the ability to produce gramicidin. These fell into two classes: (a) mutants to incorporate ['4C]tryptophan into gramicidin (Table 1) and that could synthesize neither graimicidin nor tyrocidine and the resistance of their spores to inactivation at 800 had been were unable to sporulate, and (b) mutants that were gramici- restored almost to the wild-type level (Table 2). din-negative but produced tyrocidine and were able-to form Effect of Gramicidin Addition. When cultures of strains Ml spores. The first class presumably represents mutants blocked and M5 were treated with gramicidin (200 ,ug/ml) at the end in the early stages of sporulation, analogous to SpoO mutants of exponential growth and then permitted to sporulate, the of B. subtilis (16), which have lost the ability to express most number of spores seen after heating at 800 for 20 min was not sporulation functions, among them antibiotic synthesis. Their affected, but the spores' heat stability, measured by heating at phenotype resembles that of asporogenic rifampicin-resistant 800 for 3 hr, was substantially increased (Table 4), as was their mutants, such as strain B81, which are also unable to form both content of dipicolinic acid (Table 3). Addition of dipicolinic kinds of antibiotic. The second mutant class is normal in spor- acid, however, had no effect on the heat stability of the'mutant ulation frequency and tyrocidine production, but does not spores (Table 4). The time of gramicidin addition was very produce gramicidin. This phenotype is consistent with a specific critical. Maximum heat stability of spores was observed when lesion in gramicidin biosynthesis, even though we have not gramicidin was added just at the end of exponential growth (250 confirmed this-conclusion by assay of the gramicidin biosyn- Klett units); earlier or later addition produced a much smaller thetic enzymes, a task that would be difficult on account of their response (Fig. 3). It should be noted that, in these experiments, instability in ttro (17). The relative frequency of the two the spores were diluted 105-fold with distilled water before mutant classes was found to be 5:1. Since sporulation involves heating, so that the concentration of gramicidin during heat treatment was insignificant. Moreover, gramicidin-treated Table 4. Effect of gramicidin and dipicolinic acid on heat resistance of spores of Bacillus brevis mutants 100 (I) Viable Viable W 80 spores spores Ratio of 0 .60 after 20 after 3 hr survivors min at 80° at 80° at 3 hr (spores/ (spores/ and at (>40 Strain Additions* nl) nl) 20 min S14 None 70 39 0.56 z w20 Gramicidin 71 41 0.58 Dipicolinate 72 39 0.59 C.) Ml None 69 3.8 0.06 Gramicidin 71 20 0.28 IC0 Dipicolinate 72 6.2 0.09 1 2 3 AT M5 None 70 4.4 0.06 TIME 80- (hours) Gramicidin 69 21 0.30 FIG. 2. Heat resistance of spores of Bacillus brevis mutants. Dipicolinate 72 5.3 0.07 Spores were heated at 80° for the times indicated and their viability * was assayed as described under Experimental Procedure. (r-rn), Gramicidin (200 ug/ml). or dipicolinic acid (200 Ag/ml) was added strain S14; (A --- A), strain Ml; (0 -), strain M5. at 250 Klett units. Downloaded by guest on September 26, 2021 Microbiology: Mukherjee and Paulus Proc. Natl. Acad. Sci. USA 74 (1977) 783 explanation was suggested by earlier studies with other Bacillus ...... 400 species, which showed that mutants unable to synthesize dip- 300 E icolinic acid could not form heat-resistant spores (20, 21). In- z co 0 200 deed, upon analyzing spores of our mutant strains, we found .Pz/1.1"', e co .c 0.3 r cc that the gramicidin-negative mutants had a much lower dip- -,, 0 0 I icolinic acid content than the parent strain. Moreover, the ad- J 60 100 I 04= I dition of which restored almost normal 0.3 J U5 gramicidin, heat resis- J JIJIJI tance to the mutant spores, caused a corresponding increase in 0.2 I 40 i 50 0 i w a: i a cc their dipicolinic acid content. In contrast, the addition of dip- 0 icolinic acid could not restore a normal spore phenotype. This 20 20 failure could be due to the inability to utilize exogenous dip- co C: 5I icolinic acid, as suggested by the fact that only a small increase

2 4 6 8 (hr) in spore dipicolinate was observed in these experiments (results TIME OF GRAMICIDIN ADDMON not shown). On the other hand, it is possible that the inability to synthesize dipicolinic acid is only one of several defects that FIG. 3. Effect of gramicidin addition on the heat resistance of have resulted from the absence of gramicidin. This conclusion spores of Bacillus brevis strain M5. Gramicidin (200 ,g in 4 ,ul of ethanol) was added to 1 ml portions of a culture of B. brevis strain M5 is supported by the observation that the gramicidin-negative at various times during the growth cycle. Incubation was continued mutants also have greatly reduced levels of an intracellular for 48 hr and the heat stability of the spores was measured. (.... ), serine proteinase (unpublished results). Growth curve during gramicidin addition; (0-0), viable spores after We must therefore examine more closely the mode of action 20 min at 800; (0-0), ratio of survivors after heating for 3 hr and for of gramicidin in bacterial sporulation. In order for the normal 20 min. spore phenotype to be restored, gramicidin must be added to the mutant strains within a relatively short time period. This about 40 operons (18), i.e., perhaps 80 to 120 genes, while critical period corresponds to the transition from vegetative gramicidin synthesis must involve at least 15 enzymes (19), this growth to stationary phase, when gramicidin synthesis would ratio would be more or less expected if we are dealing with ordinarily occur in the parent strain, and before the time when single mutational events. We do not know whether the observed dipicolinic acid is produced (6). It is at this time of the growth mutant frequency has been affected by the treatment of spores cycle that alterations in the apparent template specificity of with ethyl methanesulfonate, for we have not done the laborious RNA polymerase are first observed (22). As mentioned in the control experiment without mutagen. However, considering introduction, we had found earlier that gramicidin can mod- the large number of genes involved in sporulation and grami- ulate the activity and specificity of purified RNA polymerase cidin synthesis, the frequency of our mutants is not much (6, 7), and we postulated that this might be a reflection of the greater than what would be expected for spontaneous events. biological function of the antibiotic. More specifically, we Because, even under highly mutagenic conditions, ethyl proposed that the peptide antibiotic might function to turn off methanesulfonate gives rise to but few double mutants (11), we the transcription of vegetative genes that are not essential for can be fairly confident that we are dealing with single muta- sporulation (6). According to this view, the absence of grami- tional alterations. cidin should not actually prevent sporulation but should lead The observation that the spores produced by the second class to an imbalance in transcription during the transition from of gramicidin-negative mutants are relatively heat-sensitive vegetative growth to sporulation. This is consistent with our and have a reduced content of dipicolinic acid is of great in- observation that the absence of gramicidin still permits the terest because it suggests that gramicidin is essential for normal formation of spores, albeit defective ones. On the other hand, spore formation. An alternate interpretation of these results, the possibility that gramicidin acts by other mechanisms, for that the spore defect and inability to produce gramicidin are example by affecting the cation permeability of membranes the results of two unrelated genetic lesions, seems unlikely for (5, 23), cannot be excluded at this time. several reasons. First, as already discussed, the relative fre- It may be of interest to compare the mutants described in this quency of the two classes of gramicidin-negative mutants is paper with other antibiotic-negative mutants isolated from consistent with a single mutational event. Second, two inde- various Bacillus species. Most of those were also unable to pendently isolated gramicidin-negative mutants have the same sporulate (24-27), but because the biochemical nature of their spore phenotype. This would be quite a coincidence if both defect was not defined, the possibility could not be eliminated were double mutants. Third, the frequency of gramicidin- that their lesion was in a pleiotropic regulatory element that producing survivors after prolonged heat treatment of mutant affected both antibiotic synthesis and sporulation. Conse- spores was quite high (about 10%), and two randomly chosen quently, even though the existence of such mutants suggested gramicidin producers had also regained heat resistance of some connection between antibiotic production and sporulation, spores. This is strong evidence that both properties were af- it could not establish a causal relationship between these pro- fected by a single mutation, but does not eliminate the possi- cesses. In spite of this uncertainty, one set of mutants of this type bility of a polar or regulatory mutation that affected the ex- is of special interest in relation to our work. These mutants of pression of two genes. Fourth, the addition of gramicidin to the B. subtilis, isolated in the laboratory of Bose (27, 28) on the basis mutant strains during the early stages of sporulation restored of their inability to produce mycobacillin, were at first thought the heat-resistant character and normal dipicolinic acid content to sporulate normally, but upon further examination proved of the spores. This observation alone proves conclusively that to have abnormally heat-sensitive spores (29), a phenotype the inability to produce gramicidin is directly responsible for reminescent of our gramicidin-negative mutants. Unfortu- the spore defect. It therefore unambiguously demonstrates a nately, the response of these strains to the addition of myco- function of a peptide antibiotic in bacterial sporulation. bacillin has not been tested, and consequently the possibility What is the basis of the increased heat sensitivity of mutant of a polar or regulatory mutation that affects both mycobacillin spores that results from the absence of gramicidin? A possible synthesis and sporulation cannot be excluded. Some of these Downloaded by guest on September 26, 2021 784 Microbiology: Mukherjee and Paulus Proc. Natl. Acad. Sci. USA .74 (1977) ambiguities can be avoided by the use of mutants with a specific 6. Sarkar, N. & Paulus, H. (1972) Nature New Blol. 239, 228- defect in antibiotic synthesis. For example, Kurahashi and co- 230. workers (30) have isolated five mutants of B. brevi ATCC 9999 7. Paulus, H. & Sarkar, N. (1976) in Molecular Mechanisms in the that were unable to synthesize gramicidin S. a- cyclic deca- Control of Gene Expression, eds. Nierlich, D. P., Rutter. W. J. & Fox, C. F. (Academic Press, New York), pp. 177-194. peptide related to tyrocidine, because one of the biosynthetic 8. Hotchkiss, R. D. (1944) Ado. Enzymol. 4,153-199. enzymes was defective. Four of these mutants sporulated at a 9. Ristow, H., Schazschneider, B., Bauer, K. & Kleinkauf, H. (1975) greatly reduced frequency, suggesting an involvement of Blochim. Biophys. Acta 390,246-252. gramicidin S in sporulation. However, because studies on rev- 10. Schazschneider, B., Ristow, H. & Kleinkauf, H. (1974) Nature ertants or on the effect of gramicidin addition have not been 249,757-759. carried out, the possibility remains that these results could also 11. Ito, J. & Spizizen, J. (1971) Mutat. Res. 13,93-96. be due to a double or polar mutation. Moreover, another 12. Raeft, C. R. H. (1975) Proc. Natl. Acad. Sci. USA 72, 2274- gramicidin-S-negative mutant, although biochemically not 2278. characterized, produced a high level of spores. These were not 13. Hanson, R. S., Blicharska, J. & Szulxnajster, J. (1964) Biochem. studied further, but in the light of our results, the examination Blophys. Res. Commun. 17,1-7. The a 14. Sarges, R. & Witkop, B. (1965) J. Am. Chem. Soc. 87, 2011- of their heat stability would be of interest. properties of 2020. mutant strain of B. licheniformis that lacks one of the enzymes 15. Janwen, F. W., Lund, A. J. &-Anderson, L. E. (1958) Science 127, of bacitracin synthesis (31, 32) are also problematical. This strain 26-27. appears to sporulate at the same frequency as the parent, but 16. Young, F. E. & Wilson, G. A. (1972) in Spores V, eds. Halvorson, neither the heat stability of the spores nor their dipicolinic acid H. O., Hanson, R. & Campbell, L. L. (American Society for Mi- content has been examined. Moreover, the production of a low crobiology, Washington, D.C.), pp. 77-106. level of bacitracin or of an altered antibiotic could escape de- 17. Bauer, K., Roskoski, R., Kleinkauf, H. & Lipmann, F. (1972) tection by the relatively insensitive microbiological and enzy- Biochemistry 11,3266-3271 mic assays. These cases illustrate how difficult it is to define 18. Hranueli, D., Piggot, P. J. & Mandelstam, J. (1974) J. Bacterlol. between antibiotics and spor- 119,684-690. clearly the relationship peptide 19. Lipmann, F. (1973) Acc. Chem. Res. 6,361-67. ulation. It was only through the good fortune that the sporula- 20. Fukuda, A. & Gilvarg, C. (1968) J. Biol. Chem. 243, 3871- tion defect of gramicidin-negative mutants can be phenotyp- 3876. ically cured by the addition of gramicidin that we could un- 21. Halvorson, H. 0. & Swanson, A. (1969) in Spores IV, ed. ambiguously demonstrate a function of this antibiotic in spore Campbell, L. L. (American Society for Microbiology, Bethesda, formation. Md.), pp. 121-132. 22. L6sick, R. & Sonenshein, A. L. (1969) Nature 224,357. This research was sortedhy Grant GM-23149 from the National 23. Hodgson, B. (1970) J. Theor. Blol. 30,111-119. Institute of General MedHclSciences. H.P. was the recipient of the 24. Paulus, H. (1967) in Antibiotics, eds. Gottlieb, D. & Shaw, P. O. Career Development Award 1-K3-GM-9848 from the U.S. Public (Springer, Berlin), Vol. 2, pp. 254-267. Health Service, and P.K.M. is the recipient of a research fellowship 25. Schmitt, R. & Freese E. (1968) J. Bacteriol. 96,1255-1265. from The Medical Foundation, Inc., Boston, Mass. 26. Schaeffer, P. (1969) Bacteriol. Rev. 33,48-71. 27. Ray, B. & Bose, S. K. (1971) J. Gen. Appl. Microblol. 17,491- 498. 1. Krasil'nikov, N. A. (1958) Soil Microorganisms and Higher 28. Ray, B. & Bose, S. K. (1974) Acta Microblol. Pol. Ser. A 6, Plants (U.S.S.R. Acad. Sci., Moscow). 101-109. 2. Pollock, M. R. (1971) Proc. R. Sock London Ser. B 179, 385- 29. Ray, B. & Bose, S. K. (1974) Folia Microblol. (Prague) 19, 401. 203-208. 3. Weinberg, E. D. (1971) Perspect. Biol. Med. 14,565-577. 30. Kambe, M., Imae, Y. & Kurahashi, K. (1974) J. Blochem. 75, 4. Dhar, M. M. & Khan, A. W. (1971) Nature 233,182-184. 481-493. 5. Sadoff, H. L. (1972) in Spores V, eds. Halvorson, H. O., Hanson, 31. Haavik, H. I. & Thomassen, S. (1973) J. Gen. Microblol. 76, R. & Campbell, L. L. (American Society for Microbiology, 451-454. Washington, D.C.), pp. 157-166. 32. Haavik, H. I. & Froyshov, 0. (1975) Nature 254,79-82. Downloaded by guest on September 26, 2021