46(3):762. 1983,Appl. Environ. Microbiol.  Alírio De Carvalho and Rubens Molinari   Streptomyces aureofaciens Substances Secreted by Nucleic Acid-Related Low-Molecular-Weight Identification of http://aem.asm.org/content/46/3/762 found at: Updated information and services can be These include: CONTENT ALERTS more» alerts (when new articles cite this article), Receive: RSS Feeds, eTOCs, free email http://journals.asm.org/site/misc/reprints.xhtml Information about commercial reprint orders: http://journals.asm.org/site/subscriptions/To subscribe to to another ASM Journal go to: on A pril 8, 2014 by U N E S P - U niversidade E stadual P aulista http://aem .asm .org/ D ow nloaded from on A pril 8, 2014 by U N E S P - U niversidade E stadual P aulista http://aem .asm .org/ D ow nloaded from http://aem.asm.org/content/46/3/762 http://aem.asm.org/cgi/alerts http://aem.asm.org/cgi/alerts http://journals.asm.org/site/misc/reprints.xhtml http://journals.asm.org/site/subscriptions/ http://aem.asm.org/ http://aem.asm.org/ http://aem.asm.org/ http://aem.asm.org/ Vol. 46, No. 3APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Sept. 1983, p. 762-764 0099-2240/83/090762-03$02.00/0 Copyright 0 1983, American Society for Microbiology Identification of Low-Molecular-Weight Nucleic Acid-Related Substances Secreted by Streptomyces aureofaciens ALIRIO DE CARVALHO* AND RUBENS MOLINARI Instituto de Quimica da UNESP, Araraquara, 14800, Sdo Paulo, Brazil Received 12 October 1982/Accepted 8 March 1983 Streptomyces aureofaciens growth in chemically defined medium is actively associated with the secretion of low-molecular-weight nucleic acid-related sub- stances and is linked to low availability of phosphate. Thirteen pure compounds were isolated, of which seven were identified. A previous paper (1) described the develop- ment of a chemically defined medium for the growth of a nonantibiotic producer Streptomy- ces aureofaciens strain associated with the active secretion of a complex mixture of low- molecular-weight nucleic acid-related sub- stances. This note describes the fractionation of the mixture with the purification of 13 sub- stances and identification of 7 compounds as purine and pyrimidine bases, nucleosides, and orotic acid. Fractionation and purification of the ferment- ed deproteinized filtrate was achieved by classi- cal ion-exchange column chromatography (Fig. 1). The identification of the pure compounds was made by classical paper chromatography (2, 6) and UV spectrophotometry (1), with 76 natu- ral and nonnatural purine and pyrimidine bases and derivatives serving as reference substances for standard and differential hydrolysis (3, 5). Figure 1 shows the main fractionation and purification of the crude fermented S. aureofa- ciens broth (1) and shows the partial identifica- tion steps for the pure compounds. Tables 1 and 2 show the well-defined and purified compounds present in the main anionic and cationic fractions of the crude fermented broth. From eight anionic components, only component no. 2 could be identified with one of the 76 standard reference substances. It seems to be identical to orotic acid. The other seven substances could not be identified with any of the reference substances. However, some char- acteristics are as follows. (i) They do not contain phosphorus. (ii) They probably do not contain either D-ribose or D-deoxyribose. (iii) Com- pounds 1, 10, 13, and 14 are easily hydrolyzable, suggesting a purine-like derivative structure. (iv) Compounds 3, 4, and 6 behave as pyrimidine- like derivatives or as simple nitrogen bases. Components 4 and 6 are so similar that they can be taken as the same compound appearing in different fractions. All six well-defined cationic components could be identified, despite their low concentra- tions in the deproteinized filtrate (Table 2). In the previous paper (1), the S. aureofaciens low-molecular-weight substances, related to nu- cleic acid, were presumed to be secondary metabolites actively secreted by the microorgan- ism in all growth phases as a response to low phosphate level, available in the chemically de- TABLE 1. Anionic fraction components Fraction Compound. Rf Avg spectral Concn in Fraction no. ~~~~~~maxima (nm) by Identification or characteristics broth filtrateSn S2 S, and S2 (mg/liter) All 1 0.51 0.53 240; 288 Purine-like derivative All 2 0.60 0.53 280 Orotic acid 11.0 A21 3 0.60 0.61 260 Simple nitrogen base or py- rimidine-like derivative A21 4 0.72 0.77 250; 295 Simple nitrogen base or py- rimidine-like derivative A31 6 0.74 0.75 250; 295 Probably compound 4 A43 10 0.71 0.76 248; 298 Purine-like derivative A51 13 0.66 0.74 248; 290 Purine-like derivative A52 14 0.68 0.65 268 Purine-like derivative Sl, Paper chromatography system 1 (6); S2, system 2 (2). 762 on A pril 8, 2014 by U N E S P - U niversidade E stadual P aulista http://aem .asm .org/ D ow nloaded from http://aem.asm.org/ http://aem.asm.org/ VOL. 46, 1983 NOTES 763 FERMENTED BROTH TCA EPROTZED CO CENTRAED FLTRATE DOWEX- I CHROMATOGRAHY lONIC CM" FRAT1O (AO) 5 ANONC FRTON s DOWEX- 5O CHROMATOGRAPHY DOWEX -I RECHROMATOGRAPIC| FRACTIONS PURIFICATION NON - POLAR 7 CATIONIC FRACrON (C.) FRACTIONS 18 ANIONIC RJIFED FRACrTONS DOWEX-50 RECHROMATOeRAPHY FRACTIONS PURFICATION I 12 CATIONIC PURIFIED FRACTIONS PAPER CHROMATOGRAPHIC ANALYSIS (SI AND S2 SYSTEMS) 122 Rf DISTINCT SPOTS LOCALIZATION UV SPECTRAL ANALYSIS OF THE 22 COtNENTS COMRISON OF THE 22 SPECTRA AND Rf PAIRS WITH 76 KNOWN PURINE AND PYRIMINE DERN 7 UNKNOWN B LOW - CONCENTRATION AND 7 IDENTIFI ENTS SPECTRAL INAC.RQ E COMPONENTSM NENTS HYDROLYTICAL AND PAPER ORarlC ACID RROMATOGRAPHIC ANALYSI ADE T CYTIDINE 4 UNKNOWN P| 3 UNKNOWN NITROGEN INOSINE DERI BASES OR PYRMIDINE CYTOSINERINEDERIV DERIVATIVES CTSN SNANE ANALYTICAL COMPARISON I-METHYL I 6 UNIDENTIFIED DISTINCT COMONENTS (4PURINE DERIVATI AND 2 BASES OR PYRIEMNE DERIVATIVES) QINnFICAT1NI FIG. 1. Purification and identification scheme. on A pril 8, 2014 by U N E S P - U niversidade E stadual P aulista http://aem .asm .org/ D ow nloaded from http://aem.asm.org/ http://aem.asm.org/ APPL. ENVIRON. MICROBIOL. TABLE 2. Cationic fraction components FracnCompound Rf Avg spectral Compound Concn in Fraction no. S, S2 maxima (nm) identification (mg/liter)no. ~~SI by SIand S2 mAtr C12 15 0.29 0.30 264 Adenine 0.2 C21 16 0.49 0.54 277 Cytidine 0.3 C31 17 0.34 0.36 247 Inosine 0.5 C41 18 0.48 0.45 275 Cytosine 1.9 C53 20 0.26 0.18 249 Guanine 0.1 C62 22 0.35 0.43 260 1-Methyladenine 0.1 a S1, Paper chromatography system 1 (6); S2, system 2 (2). fined medium, specifically developed for such a purpose. On the other hand, Simuth and Zelinka (4) have proposed RNA degradation by active RNase as their explanation for the accumulation of hypoxanthine, cytosine, and guanosine by chlortetracycline-producing S. aureofaciens in- dustrial strains. RNA degradation by active ri- bonuclease as the explanation for their observa- tion. The absence of phosphorus in all of our identified and unidentified well-separated com- pounds could be explained either as a conse- quence of nucleotide enzymatic degradation, as supposed by Simuth and Zelinka (4), or by the lack of enough phosphate for normal nucleotide synthesis, as we have presumed (1). The latter hypothesis fit well our observation that orotic acid was the most abundant identified substance of the studied complex mixture, since it is very difficult to think of orotic acid as an RNA degradation product. Another corroborative ob- servation is the absence of uracil and uracil derivatives in all of the fractions studied. LITERATURE CITED 1. Carvalho, A., and R. Molinari. 1976. Production of nucleic acid-related substances by Streptomyces aureofaciens. Rev. Bras. Technol. 7:289-296. 2. Kirby, K. S. 1955. Some new solvent systems for the paper chromatography of nucleic acid degradation products. Bio- chim. Biophys. Acta 18:575-576. 3. Marshak, A., and H. J. Vogel. 1951. Microdetermination of purines and pyrimidines in biological materials. J. Biol. Chem. 189:597-605. 4. Simuth, J., and J. Zelinka. 1970. Nucleic acid degradation products of Streptomyces aureofaciens. J. Antibiot. 23:242-249. 5. Vischer, E., and E. Chargaff. 1948. The composition of the pentose nucleic acids of yeast and pancreas. J. Biol. Chem. 176:715-734. 6. Wyatt, G. R. 1951. The purine and pyrimidine composition of deoxypentose nucleic acid. Biochem. J. 48:584-590. 764 NOTES on A pril 8, 2014 by U N E S P - U niversidade E stadual P aulista http://aem .asm .org/ D ow nloaded from http://aem.asm.org/ http://aem.asm.org/