Journal of Japanese Society for Extremophiles (2010) Vol.9 (2) Journal of Japanese Society for Extremophiles (2010) Vol. 9 (2), 75-77 ORIGINAL PAPER a a b Hamana K , Hayashi H and Niitsu M NOTE 4 A tertiary-branched tetra-amine, N -aminopropylspermidine is a major cellular polyamine in an anaerobic thermophile, Caldisericum exile belonging to a new bacterial phylum, Caldiserica a Faculty of Engineering, Maebashi Institute of Technology, Maebashi, Gunma 371-0816, Japan. b Faculty of Pharmaceutical Sciences, Josai University, Sakado, Saitama 350-0290, Japan. Corresponding author: Koei Hamana, [email protected] Phone: +81-27-234-4611, Fax: +81-27-234-4611 Received: November 17, 2010 / Revised: December 8, 2010 /Accepted: December 8, 2010 Abstract Acid-extractable cellular polyamines of Anaerobic, moderately thermophilic, filamentous, thermophilic Caldisericum exile belonging to a new thiosulfate-reducing Caldisericum exile was isolated bacterial phylum, Caldiserica were analyzed by HPLC from a terrestrial hot spring in Japan for the first and GC. The coexistence of an unusual tertiary cultivated representative of the candidate phylum OP5 4 brancehed tetra-amine, N -aminopropylspermidine with and located in the newly validated bacterial phylum 19,20) spermine, a linear tetra-amine, as the major polyamines Caldiserica (order Caldisericales) . The in addition to putrescine and spermidine, is first reported temperature range for growth is 55-70°C, with the 20) in the moderate thermophile isolated from a terrestrial optimum growth at 65°C . The optimum growth 20) hot spring in Japan. Linear and branched penta-amines occurs at pH 6.5 and with the absence of NaCl . T were not detected. The novel cellular polyamine profile Caldisericum exile NBRC 104410 was provided by found in the moderate thermophile has never been NBRC (Biological Resource Center, National Institute observed within various bacteria and archaea previously for Technology and Evaluation, Kisarazu, Chiba, Japan) analyzed. and cultivated anaerobically under a N2/CO2 (80/20) at 65°C in a NBRC No.1039 medium, pH 6.520). Cells in Key words: Caldisericum, polyamine, tertiary branched the stationary phase were harvested and homogenized in tetra-amine, thermophile equal volumes of cold 1.0M perchloric acid (PCA). Cell residues after the PCA extraction were hydrolyzed The chemotaxonomic and phylogenetic significance of with 6M HCl, at 110°C, for 20h. The PCA-extract and the cellular distribution of quaternary branched HCl-hydrolysate were subjected to a Dowex 50W penta-amines, N4-bis(aminopropyl)norspermidine and column to concentrate polyamines1,15). The concent- N4-bis(aminopropyl)spermidine within thermophilic rated polyamines were analyzed by high-performance bacteria belonging to the domain Bacteria and liquid chromatography (HPLC) on a Hitachi L6000 thermophilic archaea belonging to the domain Archaea high-speed liquid chromatograph using a column of 6-10) 1,15) has been proposed . The occurrence of the cation-exchange resin . The concentrated polyamine high-basic quaternary branched penta-amines is possibly fraction was further purified on a column of Whatman 15) associated with their thermophily to stabilize cellular CM23 . Gas chromatography (GC) was performed on nucleic acids and other acidic cellular components under a Shimadzu GC-9A gas chromatograph after 7,23) high thermal environments . On the other hand, heptafluorobutyrization of the purified polyamine 4 12,22) tertiary branched tetra-amines, N -aminopropylnor­ samples . Polyamines were identified by gas 4 spermidine and/or N -aminopropylspermidine have been chromatography-mass spectrometry (GC-Mass) using a 12,22) detected as a minor polyamine component selectively in JEOL JMS-700 mass spectrometer . the bacterial and archaeal thermophiles containing the HPLC chromatogram of the concentrated acid- quaternary branched penta-amines as a major polyamine. extractable polyamine fraction from C. exile showed the In our course of the studies on cellular polyamine occurrence of putrescine (4; abbreviation for the number analyses of newly validated bacterial and archaeal of methylene CH2 between N), spermidine (34) and a thermophiles, a novel polyamine profile was found in a tetra-amine peak (Fig. 1A). Spermine (343) and new member of thermophilic bacteria, Caldisericum N4-aminopropylspermidine (3(3)4) were eluted in the exile, in which an unusual tertiary branched tetra-amine, tetra-amine peak by HPLC. When the purified N4-aminopropylspermidine was detected as a major polyamine fraction was analyzed in GC, the two polyamine in the absence of quaternary branched tetra-amines were detected (Fig. 1B) and identified by penta-amines. GC-Mass. Roughly estimated cellular concentrations ( μ mol/g wet weight of cells) of putrescine (4), 75 Journal of Japanese Society for Extremophiles (2010) Vol.9 (2) spermidine (34), spermine (343) and N4-aminopropyl­ growth conditions are being planned, its unique spermidine (3(3)4) were 0.33, 0.52. 0.30 and 0.60, polyamine profile suggests the phylogenetic uniqueness respectively. Norspermidine (33), norspermine (333), of the polyamine synthetic ability of C. exile located in N4 -aminopropylnorspermidine (3(3)3) and thermo- the new phylum, Caldiserica. spermine (334) were not found. Branched penta- amines, N4-bis(aminopropyl)norspermidine (3(3)(3)3), N4-bis(aminopropyl)spermidine (3(3)(3)4) and N4-aminopropylspermine (3(3)43), and linear penta-amines were not detected. Polyamines were not detected in the HCl-hydrolysate of the cell residues, indicating that the cellular polyamines of C. exile were extracted completely by PCA extraction. Many extreme-/hyper-thermophiles belonging to the bacterial orders Aquificales (of the phylum Aquificae), Thermales (of the phylum Deinococcus-Thermus), Thermodesulfobacteriales (of the phylum Thermo- desulfobacteria), Thermoanaerobacteriales and Clostridiales (of the phylum Firmicutes), Sphingobacteriales (of the phylum Bacteroidetes) and Rubrobacterales (of the phylum Actinobacteria) 2,3,5,6,10,11,13,17), and archaeal orders Methanococcales, Archaeoglobales and Thermococcales (of the phylum Euryarchaeota)4,6,8,9,16), grown at 70-100°C, contained quaternary branched penta-amines as a major polyamine and tertiary branched tetra-amines as minor polyamine components. Some extreme-/hyper-thermophiles belonging to the bacterial orders Thermotogales (of the phylum Thermotogae) and Nitrospirales (of the phylum Nitrospirae)6,10,17) and archaeal orders Sulfolobales and 8,9) Fig. 1. HPLC chromatogram (A) and GC chromatogram Desulfurococcales (of the phylum Crenarchaeota) (B) of the acid-extracted polyamines from Caldisericum contained linear tetra-amines and penta-amines and exile NBRC 104410T grown at 65 ℃ . The arrows lacked branched tetra-amines and penta-amines. indicate the elution position or retention position of 334, Although many moderate thermophiles grown at 3(3)3, 3(3)(3)4 and 3(3)(3)3, respectively. Long linear 55-65℃ were widespread within various bacterial and and branched polyamines were concentrated in the archaeal phyla, they contained a linear tetra-amine, purified polyamine sample for the GC. Abbreviations spermine (343) alone, as the major common tetra-amine 2,5,6,10,13,17,18) for polyamines: 4, putrescine; 34, spermidine; 343, and lacked penta-amines . 4 4 spermine; 334, thermospermine; 3(3)3, N -aminopropyl The coexistence of N -aminopropylspermidine (3(3)4) 4 norspermidine; 3(3)4, N -aminopropylspermidine; with spermine (343) as the major polyamines in the 4 4 3(3)(3)3, N -bis(aminopropyl)norspermidine; 3(3)(3)4, absence of N -bis(aminopropyl)spermidine (3(3)(3)4), N4-bis(aminopropyl)spermidine. was first found in the moderate thermophile, C. exile grown at 55-70°C with optimum growth at 65°C, as shown in the present study. The occurrence of the two Acknowledgment tetra-amines is possibly associated with its moderate We would like to thank NBRC (Dr. K. Mori) for thermophily. However, this novel and simple cellular supplying Caldisericum exile. polyamine profile has never been known in archaea as well as other bacteria except C. exile. A tertiary 4 branchd penta-amine, N -aminopropylspermine (3(3)43) References has been found in extremely/moderately thermophilic 1) Hamana, K. 2002. Extraction and HPLC analysis of Bacillus, Geobacillus, Saccharococcus, Caldicellulo- bacterial polyamines. Ann. Gunma Health Sci. siruptor and Thermoanaeromonas species grown at 23: 149-158 (in Japanese). 55-70°C 14,17) and extremely thermophilic 21) 2) Hamana, K., Hamana, H., Niitsu, M., and Samejima, “ Calditerricola ” species grown at 75-80°C K. 1996. Polyamines of thermophilic belonging to the bacterial phylum Firmicutes but not Gram-positive anaerobes belonging to the genera detected in C. exile, as shown in the present study. In 4 Caldicellulosiruptor, Caloramator, Clostridium, contrast, significant amount of N -aminopropyl­ Coprothermobacter, Moorella, spermidine (3(3)4) was not found in the former six Thermoanaerobacter and Thermoanaerobacterium. thermophiles. However the degree of thermophily in Microbios 85: 213-222. bacterial and archaeal thermophiles is estimated roughly 3) Hamana, K., Hamana, H., Niitsu, M., Samejima, K., and cannot be defined exactly. Although analyses of and Itoh, T. 1996. Distribution of long and branched the cellular polyamine levels of C. exile under different polyamines in thermophilic eubacteria and 76 Journal of Japanese Society for Extremophiles (2010) Vol.9 (2) hyperthermophilic archaebacteria. Microbios 85: Thermaerobacter,