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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 iso- lated. 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 cul- ture). 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 impreg- nated 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.

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