J. Gen. Appl. Microbiol., 52, 235–240 (2006) Short Communication Cellular polyamines of lower eukaryotes belonging to the phyla Glauco- phyta, Rhodophyta, Cryptophyta, Haptophyta and Percolozoa Koei Hamana1,* and Masaru Niitsu2 1 Gunma University School of Health Sciences, Gunma 371–8514, Japan 2 Faculty of Pharmaceutical Sciences, Josai University, Saitama 350–0290, Japan (Received February 20, 2006; Accepted June 2, 2006) Key Words—Cryptophyta; Glaucophyta; Haptophyta; Percolozoa; polyamine; Rhodophyta Analysis of cellular polyamine components in the The distribution pattern of three triamines, norspermi- nine lower eukaryotic phyla, the phylum Apicomplexa, dine, homospermidine and spermidine, and two Chlorarachniophyta (Cercozoa), Chlorophyta, Cilio- tetraamines, norspermine and spermine, was almost phora, Dinophyta, Euglenophyta (Euglenozoa), Glau- phylum-specific among the nine phyla; furthermore, a cophyta, Heterokontophyta (Heterokonta) and part of their polyamine components seems to be corre- Rhodophyta, have been studied in order to consider lated to their evolutional endosymbiotic process. the phylogenetic significance of cellular polyamine dis- The remaining three taxa (phyla) among total twelve tributions in early evolution of eukaryotes (Hamana phyla of lower eukaryotes, include the non-photosyn- and Matsuzaki, 1982, 1985; Hamana et al., 1990, thetic phylum Percolozoa without the endosymbiosys 2004a, b). In the nine phyla, the three phototrophic of cyanobacteria and the two photosynthetic phyla phyla Glaucophyta, Rhodophyta and Chlorophyta Cryptophyta and Haptophyta taken secondary en- have plastids by the primary endosymbiosis of a pho- dosymbiotic plastids (Bhattacharya et al., 2004; Cava- totrophic prokaryote, cyanobacterium, and multicellular lier-Smith, 2002, 2003; Falkowski et al., 2004). Cellular species evolved within Rhodophyta and Chlorophyta polyamines in the three phyla were first analyzed in (Bhattacharya et al., 2004; Cavalier-Smith, 1998; the present study. Additional polyamine catalogues of Falkowski et al., 2004; NCBI website, 2006; Rod- some primitive phototrophic members belonging to riguez-Ezpeleta et al., 2005). The other six unicellular Glaucophyta and Rhodophyta were determined. phyla include heterotrophs evolved without the en- Two strains of Glaucophyta, seventeen strains of dosymbiosis of cyanobacteria, phototrophs by sec- Rhodophyta, seven strains of Cryptophyta and eight ondary or tertiary endosymbiotic plastids and non-pho- strains of Haptophyta were supplied from IAM, NIES tosynthetic heterotrophs evolved after the loss of pri- and MBIC, and were cultivated phototrophically in the mary endosymbiotic plastids (Bhattacharya et al., light using the media designed by the culture collec- 2004; Cavalier-Smith, 1998; Falkowski et al., 2004; tions (IAM Catalogue Strains, 2004; Kasai et al., 2004; NCBI website, 2006; Rodriguez-Ezpeleta et al., 2005). MBIC Strain Catalog Algae, 2006). Cyanidioschyzon and Cyamidium species were grown at 37°C. Other * Address reprint requests to: Dr. Koei Hamana, School of red algae and two glaucophytes were grown at 15– Health Sciences, Faculty of Medicine, Gunma University, 25°C. Seven cryptophytes and eight haptophytes were Showa-machi, Maebashi, Gunma 371–8514, Japan. grown at 10–15°C and 15–20°C, respectively. Five E-mail: [email protected] non-photosynthetic species of Percolozoa were 236 HAMANA and NIITSU Vol. 52 Fig. 1. HPLC chromatogram of the whole polyamine extract from Rhodomonas atrorosea NIES-699 (A-a), Por- phyridium sordidum MBIC 10454 (A-b) and Isochrysis galbana MBIC 10554 (A-c), and GC chromatogram of the con- centrated polyamine fraction from Rhodomonas atrorosea NIES-699 (B). Abbreviations for polyamines are shown in Table 1. Printed number on the peaks is elution time or retention time. purchased from ATCC and NIES. Heteramoeba, raphy (GC) was performed on a Shimadzu GC-9A gas Tetramitus and Naegleria species were cultivated at chromatography after the heptafluorobutylation of the 25°C in the dark on the ATCC agar plates (ATCC Bac- concentrated polyamine fraction (Niitsu et al., 1993), teria and Bacteriophages, 1996) spreading with Es- as shown in a typical chromatogram (Fig. 1). cherichia coli IAM 12119 grown in polyamine-free syn- Polyamines were identified by gas chromatography- thetic Eagle MEM medium (Nissui Pharmaceutical Co., mass spectrometry (GC-mass) using a JEOL JMS-700 Tokyo). A species of Percolomonas was grown in URO GC-mass spectrometer (Niitsu et al., 1993). Cellular medium supplemented with wheat grains (Kasai et al., concentrations of polyamines estimated by HPLC are 2004) at 15°C in the dark. shown in Table 1. Living organisms at early stationary stage were har- In euglenophytes and chlorophytes, the photosyn- vested by the centrifugation at 1,500ϫg. In Percolo- thetic cultures in the light and heterotrophic cultures in zoa, trophozoites (amoebae) were harvested and the dark were shown as the same polyamine profiles washed with Artificial seawater SP (Wako Chemicals, (Hamana et al., 2004a, b), suggesting that the two Tokyo) or PBS (Nissui Pharmaceutical Co., Tokyo). growth conditions cannot affect the algal polyamine Packed cells were homogenized in an equal volume of synthesis in the present study. Since the same cold 1 M perchloric acid (HClO4) (Hamana and Matsu- polyamine compositions have been observed in the zaki, 1982, 1985). Polyamines were extracted into axenic and non-axenic (including collected samples 0.5 M HClO4 and analyzed by high-performance liquid from fields) cultures for algae and cyanobacteria be- chromatography (HPLC) on a column of cation-ex- longing to same genus or species (Hamana and Matsu- change resin in a Hitachi L6000 high-speed liquid zaki, 1982; Hamana et al., 2004a, b; Hosoya et al., chromatograph (Hamana et al., 1995), as shown in 2005), non-axenic cultures in the present study were three typical chromatograms (Fig. 1). Gas chromatog- utilized for the determination of major polyamine com- 2006 Polyamines in Glaucophyta, Rhodophyta, Cryptophyta, Haptophyta and Percolozoa 237 Table 1. Cellular polyamine concentrations in lower eukaryotes. Polyamines (mmol/g wet weight) Organism Dap Put Cad NSpd Spd HSpd NSpm Spm Phylum Glaucophyta Cyanophora paradoxa NIES-763 Ϫ 0.80 ϪϪ1.10 ϪϪϪ IAM M-130 (b) 0.02 0.61 0.03 Ϫ 0.63 ϪϪϪ Cyanophora tetracyanea NIES-764 Ϫ 0.02 ϪϪ0.80 ϪϪϪ Glaucocystis nostochinearum IAM M-124 (b) Ϫ 0.96 ϪϪ1.42 ϪϪϪ Phylum Rhodophyta Class Bangiophyceae Order Cyanidiales Cyanidium caldarium (RK-1)IAM R-11* (c) Ϫ 1.28 ϪϪ1.24 ϪϪ0.85 (KS-1) (Nagashima)* (c) Ϫ 0.85 ϪϪ1.11 ϪϪ0.32 Cyanidium sp. MBIC 10236* Ϫ 1.30 ϪϪ0.77 ϪϪ0.20 Cyanidioschyzon melorae NIES-1332* Ϫ 1.57 ϪϪ0.40 ϪϪ0.13 Galdieria sulphuraria IAM M-8* (b) Ϫ 1.72 Ϫ 0.08 3.09 Ϫ 0.59 0.82 Order Porphyridiales Dixoniella grisea MBIC 10460* 0.02 0.35 Ϫ 0.40 0.04 ϪϪ0.02 Porphyridium sordidum MBIC 10454* Ϫ 0.55 ϪϪ0.77 ϪϪ0.18 Porphyridium purpureum IAM R-1* (b) Ϫ 0.67 0.02 Ϫ 0.53 0.04 Ϫ 0.10 IAM R-3* Ϫ 0.60 ϪϪ0.20 ϪϪ0.02 Porphyridium sp. NIES-1032* Ϫ 0.08 ϪϪ0.87 ϪϪ0.08 Rhodella maculata MBIC 10824* Ϫ 0.02 Ϫ 0.02 0.65 Ϫ 0.01 0.01 Rhodella sp. NIES-1036* Ϫ 0.20 Ϫ 0.07 0.35 ϪϪ0.19 Order Stylonematales Rhodosorus sp. MBIC 10854* Ϫ 0.27 Ϫ 0.02 0.15 Ϫ 0.10 0.30 Order Bagniales Porphyra tenera (Yokosuka)* (a) 0.02 0.01 ϪϪ0.01 ϪϪ0.12 Order Erythropeltidales Compsopogn coeruleus NIES-1462* Ϫ 0.03 ϪϪ0.32 0.10 Ϫ 0.02 Compsopogonopsis japonica NIES-1463* Ϫ 0.02 ϪϪ0.30 0.18 Ϫ 0.05 Class Florideophyceae Order Batrachospermales Batrachospermum vagum IAM R-4* Ϫ 0.40 ϪϪ1.15 0.60 Ϫ 0.22 Batrachospermum atrum NIES-1456* Ϫ 0.04 Ϫ 0.20 0.75 0.32 Ϫ 0.02 Batrachospermum helminthosum NIES-1457* Ϫ 0.12 ϪϪ0.60 0.44 Ϫ 0.08 Thorea gaudichaudii NIES-1473* Ϫ 0.02 ϪϪ0.37 0.50 Ϫ 0.22 Thorea okadae NIES-1516* Ϫ 0.02 Ϫ 0.10 0.51 0.45 Ϫ 0.22 Nemalionopsis tortuosa NIES-1467* Ϫ 0.02 ϪϪ0.42 0.30 Ϫ 0.40 Phylum Cryptophyta Chilomonas paramecium NIES-715 Ϫ 0.06 Ϫ 0.08 0.12 ϪϪϪ Chroomonas nordstedtii NIES-706 0.26 0.69 Ϫ 0.02 0.10 ϪϪϪ Cryptomonas ovata NIES-274 Ϫ 0.20 Ϫ 0.35 ϪϪϪϪ Cryptomonas platyuris NIES-276 0.02 0.37 Ϫ 0.35 ϪϪϪϪ Cryptomonas rostratiformis NIES-277 Ϫ 0.20 Ϫ 0.40 ϪϪϪϪ Cryptomonas tetrapyrenoidosa NIES-282 0.02 0.30 Ϫ 0.40 ϪϪϪϪ Rhodomonas atrorosea NIES-699* Ϫ 0.40 Ϫ 0.50 0.08 0.03 0.03 Ϫ Phylum Haptophyta Chrysochromulina hirta NIES-741* Ϫ 0.09 Ϫ 0.05 ϪϪϪϪ Cricosphaera roscoffensis NIES-8 ϪϪϪ0.04 0.07 ϪϪϪ Gephyrocapsa oceanica NIES-353 ϪϪϪϪϪϪϪϪ 238 HAMANA and NIITSU Vol. 52 Table 1. Continued. Polyamines (mmol/g wet weight) Organism Dap Put Cad NSpd Spd HSpd NSpm Spm Hymenomonas coronata NIES-1016* ϪϪϪ0.10 ϪϪ0.14 Ϫ Phaeocystis globosa NIES-388* ϪϪϪϪϪϪϪϪ Prymnesium parvum NIES-1017* Ϫ 0.04 Ϫ 0.05 0.03 Ϫ 0.05 Ϫ Isochrysis galbana MBIC 10554* Ϫ 0.06 Ϫ 0.62 0.21 Ϫ 0.29 Ϫ Pavlova pinguis MBIC 10458* ϪϪϪ0.07 0.52 Ϫ 0.16 0.02 Phylum Percolozoa Class Heterolobosea Heteramoeba clara ATCC 30972* Ϫ 0.20 ϪϪ0.35 ϪϪϪ Naegleria gruberi ATCC 30887* Ϫ 0.35 0.40 Ϫ 0.08 ϪϪϪ Naegleria morganensis ATCC 50351* Ϫ 0.10 0.05 Ϫ 0.17 ϪϪϪ Tetramitus rostratus ATCC 30216* Ϫ 0.05 0.37 Ϫ 0.06 ϪϪϪ Class Percolatea Percolomonas sp. NIES-1441* Ϫ 0.04 0.10 Ϫ 0.26 ϪϪϪ Escherichia coli IAM 12119** 0.45 0.32 0.16 Ϫ 0.01 ϪϪϪ (used as foods for Heterolobosea) Wheat grain*** ϪϪϪϪϪϪϪϪ (used for the medium for Percolatea) Dap, diaminopropane; Put, putrescine; Cad, cadaverine; NSpd, norspermidine; Spd, spermidine; HSpd, homospermidine; NSpm, norspermine; Spm, spermine; Ϫ, not detected (Ͻ0.005); IAM, Institute of Cellular Biosciences, the University of Tokyo, Tokyo, Japan; NIES, National Institute for Environmental Studies, Tsukuba, Japan; ATCC, American Type Culture Collection, Manassas, Virginia, USA; MBIC, the Marine Biotechnology Institute Culture Collection, Kamaishi, Iwate, Japan.