Microb. Resour. Syst. Dec.32（2 ）：1792016 ─ 186, 2016 Vol. 32, No. 2 Additional analysis of cyanobacterial polyamines ─ Distributions of spermidine, homospermidine, spermine, and thermospermine within the phylum Cyanobacteria ─ Koei Hamana1)＊, Takemitsu Furuchi2), Hidenori Hayashi1) and Masaru Niitsu2) 1)Faculty of Engineering, Maebashi Institute of Technology 460-1 Kamisadori-machi, Maebashi, Gunma 371-0816, Japan 2)Faculty of Pharmaceutical Sciences, Josai University, 1-1, Keyakidai, Sakado, Saitama 350-0295, Japan To further catalogue the distribution of cyanobacterial cellular polyamines, we used HPLC and HPGC to newly analyze the acid-extracted polyamines from 14 cyanobacteria. The colony-forming Nostoc verrucosum (“Ashitsuki”) and Nostoc commune (“Ishikurage”), as well as Anabaena species (Nostocales), contained homospermidine. The thermo-halotolerant Spirulina subsalsa var. salina (Spirulinales), as well as freshwater Spirulina strains, contained spermidine. Putrescine, spermidine, and homospermidine were found in freshwater colony-forming Aphanothece sacrum (“Suizenji-nori”), whereas the halotolerant Aphanothece halophytica and Microcystis species (Chroococcales) contained spermidine alone. In addition to putrescine, spermidine, homospermidine and agmatine, thermospermine was found as a major polyamine in haloalkaliphilic Arthrospira platensis (“Spirulina”) (Oscillatoriales). In the Synechococcales, chlorophyll b-containing Prochlorococcus marina contained spermidine, and chlorophyll d-containing Acaryochloris marina contained spermidine and homospermidine. Thermophilic Thermosynechococcus vulcanus and Thermosynechococcus sp. NK55a, as well as Thermosynechococcus elongatus, contained low levels of spermine in addition to homospermidine. The present 14 and previous 126 strains of cyanobacteria were categorized into spermidine-dominant types, homospermidine-dominant types and spermidine-homospermidine-mix types, but their polyamine profiles have not yet been established as chemotaxonomic markers. Key words: ‌cyanobacteria, homospermidine, polyamine, thermospermine Bacterial cellular polyamine distribution profiles 2005). Diaminopropane, putrescine, cadaverine, and are related to the phylogenetically classified loca- agmatine have been found sporadically. Spermine tions of the bacteria, as well as to the growth tem- and thermospermine have been tentatively detected perature, pH, and salt conditions in bacterial growth as minor polyamines in some thermophiles and halo- environments (Hamana, 2002a; Hamana & Hosoya, alkaliphiles (Hamana et al., 2008). The triamine nor- 2006; Hamana & Matsuzaki, 1992; Hamana et al., spermidine, the tetra-amine norspermine, and penta- 2014; Hosoya et al., 2006). In the oxygen-producing amines are widely distributed in algae (Hamana et photosynthetic cyanobacteria, as the origins of the al., 2013, 2016) but have not been found in cyanobac- plastids of photosynthetic eukaryotic algae, it is use- teria. ful to consider the polyamine profiles and the che- Although cyanobacterial classification has under- motaxonomic usage of the polyamine components in gone phylogenetic analyses based on molecular studies of the early evolution of algae and plants. sequence data (Komarek et al., 2014; MCC-NIES, The cellular polyamines of 126 cyanobacterial 2016; NCBI, 2016), the taxonomy of cyanobacterial strains have been roughly separated into spermi- class, order, family, genus and species is conflicting dine-dominant types and homospermidine-dominant (Oren, 2004; Watanabe, 1995). Here, we analyze the types (Hamana et al., 1983, 1988, 2008; Hosoya et al., cellular polyamines of the 14 newly available cyano- bacteria, including thermophilic (thermotolerant), halophilic (halotolerant), haloalkaliphilic (alkaline-halo- ＊Corresponding author philic; alkali- and salt-tolerant), polysaccharide-colony E-mail: [email protected] forming, chlorophyll (Chl) b-containing, and Chl Accepted: July 11, 2016 d-containing strains, in order to add them to cyano- ─ 179 ─ Polyamines of cyanobacteria Hamana et al. bacterial polyamine catalogues. We also re-exam- chromatography (HPLC) of the concentrated poly- ined some of our previous cyanobacterial polyamine amines on a Hitachi L6000 with a column of cation- data by using mass cultivation of cyanobacteria and exchange resin [Hitachi 2619F (=Hitachi 2720); 4 mm new chromatographic techniques. Triamines (sper- I.D.×50 mm] (Hamana, 2002b; Hamana et al., 2008; midine and homospermidine) are produced from the Hosoya et al., 2005), instead of using the old-type of diamine putrescine or the guanidinoamine agmatine, HPLC with a column of Kyowa Seimitsu 62210F and then the tetra-amines (spermine and thermo- (4.8 mm I.D.×80 mm) (Hamana et al., 1983, 1988); spermine) are produced from spermidine. The cellu- samples were then quantified by using post-labeled lar distribution of the triamines and tetra-amines is fluorometry after heating with o-phthalaldehyde. thus focused on the phylum Cyanobacteria. After heptafluorobutyrization of the concentrated Seven unique cyanobacteria supplied by NIES polyamines, we performed high-performance gas (National Institute for Environmental Studies, Japan) chromatography (HPGC) with a SHIMADZU GC-17A and NBRC (Biological Resource Center, National and GC-mass spectrometry (HPGC-MS) on a JEOL Institute of Technology and Evaluation, Japan) were JMS-700 equipped with a long capillary column of cultured phototrophically in the light (10-14 h/24 h) InertCap 1MS (0.32 mm I.D.×30 m, df 0.25 mm) (GL at either 20-25℃(mesophiles), 40-50℃(thermotoler- Sciences, Tokyo, Japan) (Furuchi et al., 2015a, b; ants) or 55-60℃(thermophiles) by using 1-10 L of Niitsu et al., 2014), instead of using standard GC with the liquid media designed by NIES (MCC-NIES, a short packed column of 3% SE-30 Chromosorb 2016) or NBRC (NBRC, 2016). Liquid cultures (1 L) of WHP (Gasukuro Kogyo, Tokyo, Japan) (3 mm I.D.× two cyanobacterial DBT strains were purchased 2.1 m) (Hamana et al., 1983, 1988, 2008; Hosoya et al., from DBT (Department of Biotechnology, Institute 2005; Niitsu et al., 1993). He gas was used as the car- of Environmental Biology Co., Shizuoka, Japan). rier gas and in the flame ionization detector. HPGC Although both axenic and non-axenic (i.e. not proven on a SHIMADZU GC-17A was operated at the col- to be axenic) cyanobacterial strains have been used, umn temperature 120-16℃/min-280℃. HPGC on a disadvantage for the use of non-axenic strains was JEOL JMS-700 was operated at the column tempera- not found in the cyanobacterial polyamine analyses ture 90-16℃/min-280℃ and mass spectra were (Hamana et al., 2008; Hosoya et al., 2005). Two dried obtained by using electron impact mode at an ioniza- powder products (A grade and B grade) of tion energy of 70 eV. The molar concentrations of “Spirulina” (Arthrospira platensis) cultured in open cellular polyamines per gram of wet weight of the culture tanks at the Kumejima Factory of Japan starting pellets, as estimated from the HPLC and Algae, Okinawa, Japan were obtained from Japan HPGC analyses using authentic polyamine stan- Algae Co., Ltd. (Tokyo, Japan). Another dried pow- dards, are shown in Table 1 and are compared der product“Spirulina” cultured in the field pools of among those of neighboring species. Hainan DIC Microalgae Co., Ltd., China was obtained In the order Nostocales, some of the Anabaena spe- from DIC Lifetech Co., Ltd. (Tokyo, Japan). cies (Anabaena circinalis, Anabaena akankoensis, “Suizenji-nori” (Aphanothece sacrum) has been cul- Anabaena affinis, and Anabaena spiroides=Anabaena tured by Endoukanagawa-dou Co. (Asakura, crassa) were reclassified into the new genus Fukuoka), Kisen-dou Co. (Asakura, Fukuoka), Tansei- Dolichospermum (NBCI Taxonomy Browser, 2016). dou Co. (Kashima, Kumamoto), and Green Science A mass culture of the DBT strain of filamentous A. Material Inc. (Mashiki, Kumamoto), in Japan. Dried spiroides, as well as other Anabaena and sheets of“Suizenji-nori” were purchased from Dolichospermum species previously analyzed, con- Endoukanagawa-dou Co. in the present study. tained homospermidine as the major polyamine. Organisms (1-10 g wet weight) harvested at the The aquatic (freshwater) Nostoc verrucosum early stationary phase in our laboratory or dried “Ashitsuki” (Sakamoto et al., 2011) as well as the ter- powders (5-10 g) purchased, were homogenized in restrial Nostoc commune“Ishikurage” (Arima et al., 5% perchloric acid (PCA). The PCA extract was 2012) are unique as colony-forming cyanobacteria passed through a column of a cation-exchange resin with extracellular polysaccharides; they contained (Dowex 50 WX8; 1 cm I.D.×3 cm) and eluted from homospermidine as the major polyamine. All of the the column with 6M HCl to concentrate the poly- Nostoc and Anabaena species analyzed had the same amines. We performed high-performance liquid polyamine profile; however, the taxonomy of the two ─ 180 ─ Microb. Resour. Syst. Dec. 2016 Vol. 32, No. 2 Table 1 Cellular polyamine concentrations of cyanobacteria Polyamines (mmol/g wet weight) Phylum Cyanobacteria Ref. Dap Put Cad Spd HSpd Spm TSpm Agm 3 4 5 34 44 343 334 Order Nostocales Anabaena spiroides DBT − 0.11 − − 1.22 − − − Anabaena spiroides NIES-79 2005 − 0.01 − − 0.35 − − − Anabaena circinalis NIES-41 2005 − 0.20 − − 0.57 − − − Anabaena compacta NEIS-806 2005 − − − − 1.50 − − − Anabaena cylindrica IAM M-1 1983 − 0.01 − 0.01 2.18 − − − Anabaena cylindrica IAM M-253 2005 − − − − 1.55 − − − Anabaena planctonica NIES-814 2005 − − − − 0.85 − − − Anabaena solitaria