Chromomycin A3 Is an Antibiotic Produced by Streptomyces Griseus

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. As can be seen in Fig . 2, the elution patterns of the acid soluble fractions from the chromomycin A3 treated and control cells were virtuely similar on qualitative basis, comprising fourteen main peaks including nineteen nucleotide components . However, as expected from higher labeling, marked increase of several nucleotide components was evident in the case of the chromomycin As treated cells. As shown in Table II, the amounts of 5'-CMP, 5'-GMP and their di- and triphosphate derivatives increased approxi mately ten-fold on the dry weight basis (mg.) of the bacterial cells as compared with the FIG. 3. The effect of chromomycin A3 on the untreated cells. On the other hand, increase early stage of the incorporation of 2-C14-uracil in the amounts of 5'-UMP, 5'-AMP and their into RNA and 2-C"-leucine into protein fractions. di-nucleotides was found to be in less extent. After preincubation for 10 minutes at 37°C, The Inhibitory Effect of Chromomycin A3 upon 2-C14-uracil (-0-) or 2-C14-leucine (--0--) was the Incorporation of C14-Labeled Amino Acids and incorporated into control and chromomycin A3- C14-Uracil into Protein and RNA-The inhibition treated cells (10 ƒÊg./ml.). One ml. aliquots of of the incorporation of C14-amino acids into the samples were taken after appropriate time intervals. After extraction of the acid soluble proteins by the action of chromomycin As fraction, RNA fraction was extracted by hydrolysis was examined with C14-labeled algal protein with 0.5N KOH for 16 hours at 37°C and hot hydrolysates (specific activity, I mC per mM 5%o TCA-precipitable substances were filtered on carbon) under the standard condition. Table Millipore filter as protein fraction and counted

III showed that the formation of labeled by a gas flow counter.

TABLE III The Effects of Chromomycin A3 on the Incorporation of C14-AminoAcids into the Protein Fraction

The cells were incubated for 60 min. in the basal medium containing casamino acid of 0.1 Mg. /MI. and C14-algal protein hydrolysate, 0.1 p.c/ml. 358 M. KIDA, M. UJIHARA, E. OHMURA and K. KAZIWARA continued for 2 minutes and ceased completely tions of chromomycin A3 from 5 X 10_1 ,ƒÊg. to after 3 minutes, as shown in Fig. 3. From 5•~10-1pg. per ml. For complete inhibition, these results, it can be postulated that 10 hg. per ml. of chromomycin A3 was chromomycin A3 affects first the metabolism necessary as revealed by the curves showing of nucleic acids and then protein synthesis. the relation between the degree of inhibition The Inhibitory Activity of ChromomycinA3 upon and the concentrations of chromomycin A3 in the Incorporationof C'¢-LabeledUracil and Adenine Fig. 4. Further, spesific concen trations into RNA and RNA-Nucleotide Fractions-When required for half inhibition of labeling RNA the bacterial cells were incubated with were found to be 1.8 x l0-1ƒÊg. per ml. for different concentrations of chromomycin A3, adenine and 1.4•~10-1ƒÊg. per ml. for uracil it was found that labeling of RNA with respectively. The difference in the inhibitory radioactive uracil and adenine was concomi concentrations depending upon the two tantly suppressed with increasing concentra- nucleotide bases made it likely to be involved in the metabolic changes of the acid soluble nucleotide pool. In order to substantiate this assumption, RNA fraction was prepared from the cells cultivated in the basal medium containing 0.1 ƒÊg. per ml. of chromomycin A3 for 60 minutes, hydrolyzed with alkali and analysed by column chromatography (12). From analytical results in Table ‡W, it is apparent that the inhibition of labeling AMP and UMP in RNA was much greater in the presence of 0.1 ƒÊg. per ml. of chromomycin A3 than that of GMP and CMP. However, with higher concentration of chromomycin A3 (10 ƒÊg. per FIG. 4. The effect of chromomycin A3 on the ml.), the inhibition of the incorporation incorporation of 8-CI"-adenine and 2-C14-uracil into RNA fraction. occurred equally irrespective of the nucleotide Logarithmically growing cells were incubated bases used. for 60 minutes at 37°C and RNA fraction was The Efect of ChromomycinA3 upon sRNA extracted as described in " EXPERIMENTAL ". and Ribosomal RNA-For further confirmation -0-: 8-C"-adenine , --0--: 2-C14-uracil. of the above results, separation of RNA

TABLE ‡W Inhibition of the Synthesis of Alkaline-Hydrolyzed Nucleotides of RNA by Chromomycin A3.

Incubation for 60 min. at 37•Ž. Effect of Chromomycin As upon Nucleic Acid Metabolism 359 fractions by column chromatography on The uptake of C14-labeled uracil into CMP methylated albumin (9) was carried out for was generally more strongly suppressed by the extract of the cells after incubation with the treatment of the cells with chromomycin C14-labeled adenine and uracil for 60 minutes in the presence of 0.1 ƒÊg. of chromomycin A3. As a control experiment, the extract of the untreated cells was analysed in the same way. The elution patterns was illustrated in Fig. 5. Approximately 1.3 times increase in total amounts of the acid soluble fraction of the chromomycin A3 treated cells was observed for that of the control. Comparison of the specific activities of the nucleic acid compo nents thus resolved was summarized in Table V, indicating that the degree of inhibition is clearly related to the molecular weight of the components in the order of 23S ribosomal RNA>16S RNA>sRNA. Chromomycin A3 also affected the incorporation of labeled precursors into DNA at the same extent as the case of sRNA. To study the base-specific inhibition of incorporation of C14-labeled precursors into RNA, the degree of labeling the four nucleotide constituents of the fractionated RNA compo nents was determined as described previously. FIG. 5. Methylated albumin column chro The results summarized in Table VI showed matography of phenol extracted nucleic acids. that the incorporation of labeled adenine into Incubation for 60 minutes at 37°C with or GMP in 16 S-RNA was more strongly inhibited without the addition of chromomyein A3 (0.1 ƒÊg./ than that into AMP whereas inverse relation- ml.) -!- 0,1). 269, --0-- radioactivity of ship in labeling between GMP and AMP in 8-C14-adenine incorporated per 4 ml. fraction. sRNA was observed. On the other hand, Linear gradient elution with 0.2 M to 1.0M NaCl no significant difference was found between in 0.05 M phosphate buffer pH 6.8. Fraction size, these two nucleotide constituents in 23 S-RNA.The uptake4 ml./ of C14-labeledtube. uracil into CMP was generally more strongly suppressed by the treatment of the cells with chromomycin

TABLE V

The Effect of Chromomycin A3 on the Incroporation of 8-C14-Adenineand 2-C14-Uraeil into RNA Components

Chromomycin A3, 0. 1 ƒÊg./ml. Incubation at 37°C for 60 min. Specific activity, c.p.m./mg. RNA. 360 M. KIDA, M. UJIHARA, E. OHMURA and K. KAZIWARA

TABLE V I

The Effect of Chromomycin A3 on the Incorporation of 8-C14-Adenine and 2-C14-Uracil into the Nucleotides of sRNA, 16S and 23S Ribosomal RNA.

Chromomycin A3, 0.1 ƒÊg./ml. Incubation at 37°C for 60 min. Specific activity, c.p.m./p mole of nucleotide.

A3 than that of UMP. From these results, rather than accelerating secondary breakdown it may be considered that there is no definite of newly synthesized RNA molecules. This relation between the inhibition of RNA was further supported by the evidence that synthesis and the metabolic changes of the although the original content of RNA per acid soluble nucleotides, although some base- cell remained unaltered, a marked increase specific suppression of the formation of in total amounts of the acid soluble fraction RNA-nucleotides was observed as indicated as well as its higher labeling was observed in Table VI. after the treatment of rapidly growing cells with chromomycin A3. Similarly, with chro DISCUSSION momycin A3 treated resting cells, the pool of Inhibitory action of chromomycin A3 the acid soluble nucleotides increased while upon nucleic acid metabolism of tumor cells labeling RNA proceeded normally. has been postulated in recent investigations According to recent studies (13), similar by several workers (3-5, 17). In the present inhibitory activities were attributed to actino studies using the cells of Bacillus subtilis SB-15, mycin D which was shown to inhibit DNA we have found the chromomycin A3 at dependent RNA synthesis. Analogous features suitable concentrations was highly effective in the activities of these two antibiotics are in inhibiting the incorporation of P32; and seen in the following results. That chromo C14-labeled nucleotide precursors into RNA mycin A3 suppresses RNA synthesis prior and that this inhibition for labeling RNA to complete inhibition of protein formation was accompanied by simultaneous increase is apparently compatible with the results in the amounts and radioactivities of the reported by Levinthal et al. (14) that acid soluble nucleotides. These findings led demonstrated the inhibitory action of actino us to the assumption that chromomycin A3 mycin D on de none synthesis of RNA of acts as a specific inhibitor against some Bacillus subtilis. In ultracentrifugal examina metabolic processes involving RNA synthesis, tion, Kersten (15) showed the binding of Effect of Chromomycin A3 upon Nucleic Acid Metabolism 361

chromomycin A3 to DNA from E. coli after Moreover it is interesting to note that an appropriate time of contact. Furthermore, the inhibition of the incorporation of labeled the inhibition of DNA dependent RNA substances into RNA is most remarkable in polymerase by the action of chromomycin ribosomal RNA. Sibatani et al. (19) has A3 has been confirmed by the work of reported a similar result of irradiation of UV Hartmann (16). light on the RNA synthesis in E. coli cells. As observed with actinomycin treated

cells by Schoff1 (17), Usui* found that SUMMARY chromomycin A3 caused not only electronmicroscopic deformation in the nucleolus of 1. Chromomycin A3 affects remarkably the Yoshida sarcoma cells but also inhibited the pathway of RNA synthesis rather than the formation of grains thymidine-H3 incorpo- those of DNA and protein. rated. 2. The antibiotic acts also on the acid However, in contrast to these consistent soluble fraction of the bacteria and causes a results, Kajiro et al. (18) reported that specific quantitative change of the nucleotide chromomycin As was effective in inhibiting components. The amounts of guanine- and the maturation of RNA but not DNA phage. cytosine-nucleotides of treated bacteria in- It was found by Egami* that chromomycin crease more remarkably than those of A3 as well as puromycin were inhibitory adenine- and uracil-nucleotides. against the synthesis of streptolysin S by 3. Chromomycin As inhibits more cell-free system, whereas actinomycin D was strongly the incorporation of radioactive inactive. Thus, these results concerning the precursors into 23S RNA than into low biological activities of chromomycin A3 and molecular weight RNA. actinomycin D were still inconclusive but suggestive that there might be an appreciable The authors wish to thank Dr. S. Tatsuoka, difference in the mode of action between General Manager, Research and Development Division, these two antibiotics. and Dr. K. Kanazawa, Director, Biological Research Since chromomycin A3 was effective in Laboratories, for the continued encouragement. They suppressing the RNA synthesis from acid are also indebted to Prof. J. Kawamata of Osaka soluble nucleotides, resulting in the accumul University for a generous gift of Bacillus subtilis SB-15, ation of cytosine- and guanine-nucleotides, it Dr. K. Mizuno for a pure sample of chromomycin A3, and Dr. Y. Hashimoto's help for the accomplish can be assumed that action of chromomycin ment of the manuscript. A3 is involved in the initial phase of metabolic pathways leading to the synthesis of nucleic acids, possibly the metabolism of the nucleo REFERENCES tides. Moreover, a marked increase in the (1) Tatsuoka, S., Tanaka, K., Miyamoto, M., guanylic and cytidylic contents of the acid Morita, K., Kawamatsu, Y., Nakanishi, K., soluble fraction is indicative of the base Nakadaira, Y., and Bhacca, N.S., Proc. Japan. specific inhibition of RNA-nucleotides in the Acad., 40, 236 (1964) bacterial cells treated with a concentration (2) Kaziwara, K., Watanabe, J., and Usui, T., necessary for half inhibition of labeling RNA. Cancer Chemotherapy Reports, CCNSC No. 13, p. 99 Thus, further extensive studies are (1961) required to elucidate these problems (3) Sato, K., Okamura, N., Utagawa, K., Ito, Y., and Watanabe, M., Sci. Repts. Research Inst. relating to the disturbance of metabolic Tohoku Univ., C9, 224 (1960) regulation of nucleotide level with respect to guanine- and cytosine-nucleotides by (4) Wakisaka, G., Uchino, H., Nakamura, T., Sotobayashi, H., Shirakawa, S., Adachi, A., the action of chromomycin As. and Sakurai, M., Nature, 198, 385 (1963) (5) Yano, M., Kusakari, T., and Miura, Y., J. * Personal communication. Biochem., 53, 6 (1963)* Personal communication. 362 M. KIDA, M. UJIHARA, E. OHMURA and K. KAZIWARA

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