Distribution of polyamines in actinomycetes

FEMS MicrobiologyLetters 41 (1987) 211-215 Published by Elsevier 211 FEM 02717 Distribution of polyamines in actinomycetes K o e i H a m a n a a a n d Shigeru M a t s u z a k i b Received 2 December1986 Accepted 3 December1986 Key words: Actinomycete; Polyamine; Putrescine; Spermidine; Spermine; Cadaverine 1. SUMMARY Polyamines were analyzed in 23 species of actinomycetes including mesophilic, halophilic, alkalophilic and thermophilic strains. Putrescine and spermidine were found to be the predominant polyamines in all the actinomycetes examined. Spermine was also found as a major polyamine in most of the thermophilic actinomycetes, and is the most abundant polyamine in Thermoactinomyces vulgaris. In some of the mesophiles and alkalophiles, the concentration of spermine was much lower than that of other polyamines. Cadaverine was detected in almost all species examined, while agmatine was found only in some. Unusual polyamines, such as norspermidine, homospermidine, norspermine, thermospermine and aminopropylcadaverine were not detected in any of the actinomycetes examined. 2. I N T R O D U C T I O N Polyamines such as putrescine, spermidine and spermine are polycationic compounds which are widely distributed in nature. Several lines of evidence show that these polyamines are essential for growth and the regulation of macromolecular synthesis in almost all organisms [1]. In eubacteria, both putrescine and spermidine are abundantly present [2]. However, the spermine concentration is very low or essentially zero in most of them; it has been detected as a major polyamine only in a limited number of species of microorganisms, for example, Acetobacterium [3], Agrobacterium [4] and thermophilic Bacillus [5,6]. These prokaryotes are thought to have a specific spermine synthase [2,31. Recently several novel polyamines have been identified in Thermus thermophilus [6]. Extreme thermophiles contain more thermine, thermospermine and caldopentamine at high temperatures than at lower temperatures [2,6]. It seemed us of interest to know if grow conditions affect the polyamine composition of actinomycetes. Although several polyamine-containing antibiotics have been found in actinomycetes [7], it is not known which polyamines are present in these organisms. The present study examines the distribution of polyamines in actinomycetes, including mesophilic, halophilic, alkalophilic and thermophilic strains. Correspondence to: Koei Hamana, Collegeof Medical Care and Technology, Gunma University, Maebashi 371, Japan. 0378-1097/87/$03.50 © 1987 Federation of European MicrobiologicalSocieties College of Medical Care and Technology, and b Department of Physiology, Institute of Endocrinology, Gunma University,Japan 212 3. MATERIALS A N D M E T H O D S A 4 3.1. Cultures and growth conditions 3.2. Analysis of polyamines The pellets of organisms were repeatedly washed with distilled water and then homogenized in an equal volume of cold 1.0 M HCIO 4. Polyamines in HC104 extracts were analyzed using high-performance liquid chromatography (HPLC) as described previously [10]. Some samples were analyzed before and after hydrolysis in the presence of 6 M HCI at l l 0 ° C for 24 h or with 2 M Ba(OH)2 at 120°C for 20 h. The identity of polyamines fractionated by column chromatography [10], was confirmed by thin-layer chromatography (TLC) on silica gel plates (Merck) with n-butanol-acetic acid-pyridine-formalin (3 : 3 : 2 : 1, v / v ) [11]. This method separates therm0spermine from spermine. To identify the polyamines, the HPLC analysis was also performed before and after the enzymatic cleavage using certain amine oxidases [12]. The enzymes used were putrescine oxidase purified from Micrococcus (Tokuyama Soda, Fujisawa, Japan) [13], polyamine oxidase from Aspergillus [14] and agmatine oxidase from Penicillium [15] (Amano, Aichi, Japan). .¢ ~ 1 .._ ,,, B 1 I J ~ 1 ~ 14 /5 tJJ O M li ..._.1 ,,, ~ I l 1 I C 2 ~.A 5 iI D I 1 6 I I , 52 1 4 0 J2'0 i 2@ 1 36 44 ELUTIONTIME(min) i Fig. 1. Elution profiles of polyamines of Streptomyces thermodiastaticus (A), T. vulgaris (B), Actinopolyspora halophila (C), and Nocardia autotrophica (D). Polyamines were separated by high-performance ion-exchange liquid chromatography. Elution patterns were followed by o-phthalaldehyde. Peaks (1), putrescine; (2), cadaverine; (3), histamine; (4), spermidine; (5), spermine and (6) agmatine. 4. RESULTS A N D DISCUSSION Typical HPLC profiles of polyamines from actinomycetes are shown in Fig. 1. Putrescine, cadaverine, histamine, spermidine, spermine and agmatine were resolved as 6 distinct peaks. Unusual polyamines such as norspermidine, Most of the actinomycetes were grown in media containing yeast extract, polypeptone, a n d / o r meat extract, as described in the Catalogue of Strains (2nd ed., 1984) of the Japan Collection of Microorganisms (JCM, Wako, Saitama, Japan) or the List of Cultures (7th ed., 1984) of the Institute for Fermentation (IFO, Osaka, Japan). Mesophilic actinomycetes were grown at 28°C. A halophilic strain (Actinopolyspora halophila) was grown at 37°C in the presence of 20% NaC1 [8]. A1kalophilic actinomycetes were cultured at pH 6.5 or 10.0 at 28°C [9]. Thermophilic strains were grown at either 40, 45, or 50 ° C. Eagle's medium, a polyamine-free synthetic medium, was also used. Cultures growing aerobically in shaking liquid media were harvested at 72 h. 213 5 A J e P1 LLI ! I P2 P3 Z LLI U C,O LLI B 0 d LL I,I A J I...U t i t n t I I PI P21~P3 A 0 A 16 I .I p I C I 32 I I | 48 ELUTION TIME (min) Fig. 2. Elution profiles of isolated polyamine fraction of peak 5 of T vulgaris(A) and its oxidation products after incubation with putrescine oxidase at pH 9.0 (B) or polyamine oxidase at pH 7.0 (C). Both peak 5 and authentic spermine were completely cleaved by 1 unit of putrescine oxidase at pH 9.0. The products were identical to those from authentic spermine. The oxidation products of authentic thermospermine are shown as P1, P2, P3, P'I, P'2, and P'3, with arrows. actinomycetes, especially by thermophiles. Spermine probably has some functional role in these thermophiles. In Bacillus, spermine is not detectable in most norspermine, aminopropylcadaverine and homospermidine were also resolved in this separation system, but were not detected in any of these strains. Peaks 1, 2 and 4 disappeared completely and peak 5 only partially after incubation with putrescine oxidase, and peaks 4 and 5 disappeared after incubation with polyamine oxidase. The retention times of peaks 1-5 corresponded to those of putrescine, cadaverine, histamine, spermidine, and spermine, respectively. To confirm their identity, the major polyamines in some strains were fractionated on a large scale and then cleaved enzymatically. With an excess of putrescine oxidase (1 unit per tube) at p H 9, peak 5 disappeared completely. The oxidation products wer (...truncated)