Chromomycin A3 Is an Antibiotic Produced by Streptomyces Griseus
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The Journal of Biochemistry, Vol. 59, No. 4, 1966 The Effect of Chromomycin A3 upon Nucleic Acid Metabolism of Bacillus subtilis SB-15 By MAKOTO KIDA*, MASAKO UJIHARA**, EINOSUKE OHMURA* and KYO KAZIWARA** (From *.ilicrobiological Research Laboratories and **Biological Research Laboratories, Research and DevelopmentDivision, Takeda Chemical Industries, Ltd., Higashiyodogawa-ku, Osaka) (Received for publication, October 16, 1965) Chromomycin A3 is an antibiotic produced and nucleic acids in growing cells of Bacillus by Streptomyces griseus No. 7 and its chemical subtilis SB-15 in connection with protein characterization has been recently established synthesis. by the exhaustive work of T a t s u o k a and his co-workers (1). Like similar antibiotics, MATERIALS AND METHODS this compound was shown to exhibit a potent Chromomycin A3, Lot No. 103 was a gift of Dr. bacteristatic effect on gram-positive bacteria K. M i z u n o in this laboratories. Bacillus subtilis SB-15 and antitumor activities against several trans was kindly given by Dr. J. Kawamata of Osaka plantable tumors (2). The recent reports University. Leucine-2-C14, 0.05ƒÊc per 0.62 mg., uracil- concerning the mechanism of action provided 2-C14, 2.6 me per mM, adenine-8-C'", 0.9 me per mM evidence that chromomycin A3 might act as and radioactive inorganic phoshate, 1 me per 0.025mg. a metabolic inhibitor for the synthetic path- were purchased from Daiichi Kagaku Co., Tokyo. Unless otherwise noted, bacterial cells were cultivated ways of nucleic acids in the neoplastic cells at 37°C in the basal medium consisting of (NH4)2HPO4, (3-5). Electronmicroscopic observations of 2.5 g. ; KH-P04, 1.5 g. ; NaCl, 5.0 g. ; MgSO4.7H2O, the chromomycin-treated Yoshida sarcoma 0.1 g. ; glucose, 5.0 g. ; casamino acid, 3.0 g. in 1,000 ml. cells revealed that several remarkable of distilled water. morphological changes occurred in both In the incorporation experiments using labeled nucleus and nucleolus***. In the biochemical leucine, uracil and adenine, the concentration of studies, it was found that the incorporation casamino acid was adjusted to 0.1 mg. per ml. When of radioactive inorganic phosphate into RNA the incorporation of P32, into the resting cells was and DNA of ascites sarcoma 180 cells was examined, the cells were incubated in 0.05 M Tris-HC1 significantly inhibited after exposure to buffer of pH 7.1. In order to study the effect of chromomycin A3 while energy producing the antibiotic on the growth of bacteria, overnight culture in 50 ml. of a bouillon medium at 37°C was systems such as respiration and glycolysis remained unaffected (3). Further, similar poured into 250 ml. of the basal medium and incubated for 3 hours. The cells during logarithmic phase were inhibitory actions upon the formation of RNA collected by centrifugation and resuspended in the were demonstrated by tracer experiments medium. Then, the optical density of cell suspension using human bone marrow cells (4) and was adjusted to between 0.3 and 0.4 in the Coleman ascites hepatoma cells of rats (5). However, photometer at 660m,,-,. little is yet known about the metabolic sites Extraction of the cells for the preparation of aci( of nucleic acids synthesis where chromomycin soluble nucleotides, RNA and DNA was carried ou A3 really inhibits. In order to approach these essentially according to the method of Schmidt Tannhauser (6). The nucleotide fraction solubl problems, we have studied in the present investigation the effect of chromomycin A3 in cold 6% perchloric acid and alkaline hydrolysate on the metabolism of acid soluble nucleotides of RNA were first treated with activated charcoal ant then subjected to column chromatographic analysis of *** Usui , T., personal communication. Dowex I (formate form). Subsequent characterizatioi *** U s u i, T., personal communication. 353 354 M. KIDA, M. UJIHARA, E. OHMURA and K. KAZIWARA of each component thus obtained was made by a incubated. The growth rate of the cells was chromatographic method modified by Kobata et al. followed by measuring the changes in the (7). For the isolation of higher molecular nucleic optical densities of cell suspensions at appro acids, the cells collected were extracted with 90% priate time intervals. As shown in Fig. 1, phenol containing 0.2%o sodium dodecyl sulfate (8), comparison of the growth curves in the followed by dialysis against 0.01 M Tris-HC1 buffer containing 5x l0-3 M MgCl.2 and the dialysate was presence of different amounts of the antibiotic separated by chromatography on methylated albumin with those of the control culture indicated column (9). The fractions corresponding to sRNA, 16 S-RNA and 23 S-RNA were hydrolyzed with 0.5 N potassium hydroxide at 37°C for 18 hours. The resulting nucleotide constituents were analysed by a conventional paper electrophoresis. For measuring the incorporation of C'4-labeled amino acids into proteins, the protein fractions were isolated from the cells by means of Schneider' s procedure (10). Protein was determined by the method of Lowry (11). Radioactivity was determined by a Nuclear- Chicago thin window gas flow counter. RESULTS Effect of ChromomycinA3 on the Growth of Bacillus subtilis SB-15-In order to examine the effect of chromomycin A3 upon the growth of the bacteria, the cells collected FIG. 1. The effect of chromomycin A3 on after cultivation for 16 hours at 37°C were the growth of B. subtilis SB-15. resuspended in the basal medium andincubated.Incubation The growth at rate37°C. of the cells was followed by measuring the changes in the optical densities of cell suspensions at appro-priate time intervals. As shown in Fig. 1, comparison of the growth curves in the presence of different amounts of the antibiotic with those of the control culture indicated TABLE I The Effect of Chromomycin A3 on the Incorporation of P323 into Acid Soluble, RNA and DNA Fractions The cells were incubated at 37°C with the basal medium containing 1 ƒÊc per ml. of P321. Effect of Chromomycin A3 upon Nucleic Acid Metabolism 355 that the addition of 1.0 ƒÊg. per ml. of thus obtained were fractionated into the chromomycin A3 to the medium resulted in nucleic acid components as described in complete inhibition of the bacterial growth "MATERIALS AND METHODS ". whereas only slight growth retardation effect In Table I was summarized the radioac was observed with 0.05 jag. per ml. tivity incorporated into each fraction. It is Based on this observation, 10 ƒÊg, per ml. evident that both total radioactivity and of the antibiotic was used in further experi specific activity of the acid soluble fraction ments in order to ensure complete inhibition increased after 20 and 60 minutes. In contrast, of cell suspension of the initial optical density 55% and 72.5% decreases in the specific activi of 0.3-0.4. ties of the RNA fractions were observed as For studying incorporation of P321 into compared with the untreated cells. These the acid soluble nucleotides, RNA and DNA, results led to two possible interpretations ; aliquots of the cells collected during logarithmic secondary degradation of newly-synthesized growth phase were incubated with P32; in the RNA and the inhibition of metabolic processes presence of 10 ƒÊg. per ml. of chromomycin A3. involving RNA synthesis by the action of chro After 20 and 60 minutes' incubation, the cells momycin A3. However, the first possibility were removed and extracted with perchloric could be eliminated since the specific activity of acid and potassium hydroxide. The extracts the acid soluble fraction was markedly higher FIG. 2. Elution pattern of the acid soluble nucleotides from Dowex 1 column chromatography. The acid soluble fraction was applied to a column (0.5•~40cm.) of Dowex 1 X10 (formate form, 200-400 mesh). Concave gradient elution was carried out according to the previously described method (7). The flow rate was about 12 ml. per hour and fractions of 4 ml. per tube were collected at 5°C. 356 M. KIDA, M. UJIHARA, E. OHMURA and K. KAZIWARA ,than that of RNA. Thus, RNA synthesis soluble fraction decreased possibly as a result should be inhibited prior to its degradation. of consecutive degradation of the labeled With the resting cells treated under the nucleotides during incubation with Tris same condition, the formation of labeled RNA buffer. occurred at the same level as the control after These changes in the acid soluble incubation for 20 minutes while the radioac nucleotides pool of both growing and resting tivity of the acid soluble fraction sharply bacterial cells suggest that the effect of increased. Increasing the incubation time to chromomycin A3 might primarily related to 60 minutes, the radioactivity in the acid the metabolism of the acid soluble nucleotides. TABLE II Analysis of the Acid Soluble Nucleotides of Chromomycin A3 Treated and the Control Cells 1) UX was an uracil derivative of the molar ratio of Pi to uracil of 2. 2) AX composed of adenine, 1: ribose, 1: Pi, 4. 3) ( ): total optical density at 260 mƒÊ. Effect of Chromomycin A3 upon Nucleic Acid Metabolism 357 Quantitative Change of the Acid Soluble proteins was inhibited for 80% after 20 Fraction-For quantitative determination of minutes. However, in the experiments of the acid soluble nucleotide components, the short periods of incubation with chromomycin cells were treated with 10 ƒÊg. per ml. of A3, it was found that the incorporation of chromomycin A3 for 60 minutes and extracted labeled uracil into RNA stopped immediately with cold 6% perchloric acid. After treatment whereas labeling proteins with leucine-C14 still with activated charcoal, the extract was subjected to chromatographic analysis on Dowex i column.