Polyamine Analysis for Chemotaxonomy of Thermophilic Eubacteria: Polyamine Distribution Profiles Within the Orders Aquificales, Thermotogales, Thermodesulfobacteriales, Thermales

J. Gen. Appl. Microbiol., 50, 271–287 (2004)

Full Paper

Polyamine analysis for chemotaxonomy of thermophilic eubacteria: Polyamine distribution proﬁles within the orders Aquiﬁcales, Thermotogales, Thermodesulfobacteriales, Thermales, Thermoanaerobacteriales, Clostridiales and Bacillales

Ryuichi Hosoya,1 Koei Hamana,1,* Masaru Niitsu,2 and Takashi Itoh3

1 Gunma University School of Health Sciences, Maebashi, Gunma 371–8514, Japan 2 Faculty of Pharmaceutical Sciences, Josai University, Sakado, Saitama 350–0290, Japan 3 Japan Collection of Microorganisms, RIKEN, Wako, Saitama 351–0198, Japan

(Received May 31, 2004; Accepted October 1, 2004)

Cellular polyamines of 45 thermophilic and 8 related mesophilic eubacteria were investigated by HPLC and GC analyses for the thermophilic and chemotaxonomic significance of polyamine distribution profiles. Spermidine and a quaternary branched penta-amine, N4-bis(aminopropyl)norspermidine, were the major polyamine in Thermocrinis, Hydrogenobacter, Hy- drogenobaculum, Aquifex, Persephonella, Sulfurihydrogenibium, Hydrogenothermus, Balnear- ium and Thermovibrio, located in the order Aquificales. Thermodesulfobacterium and Thermo- desulfatator belonging to the order Thermodesulfobacteriales contained another quaternary penta-amine, N4-bis(aminopropyl)spermidine. In the order Thermotogales, Thermotoga contained spermidine, norspermidine, caldopentamine and homocaldopentamine. The latter two linear penta-amines were not found in Marinitoga and Petrotoga. In the order Thermales, Thermus and Marinithermus contained homospermidine, norspermine and the linear penta-amines. Meiother- mus lacked penta-amines. Vulcanithermus contained linear penta-amines and hexa-amines but not homospermidine. Oceanithermus contained spermine alone. Within the order Thermoanaer- obacteriales, the two quaternary branched penta-amines were found in Thermanaeromonas and Thermoanaerobacter. Caldanaerobacter contained N4-bis(aminopropyl)spermidine. Thermoanaer- obacterium lacked penta-amines. Thermaerobacter of the order Clostridiales contained N4-bis(aminopropyl)spermidine and agmatine. Thermosyntropha, Thermanaerovibrio, Thermo- brachium (the order Clostridiales), Sulfobacillus, Alicyclobacillus, Anoxybacillus, Ureibacillus, Thermicanus (the order Bacillales), Desulfotomaculum, Desulfitobacterium and Pelotomaculum (the family Peptococcaceae) ubiquitously contained spermine. Some thermophiles of Bacillales added linear and branched penta-amines.

Key Words——Alicyclobacillaceae; Aquiﬁcales; polyamine; Thermales; Thermoanaerobacteriales; Thermodesulfobacteriales; thermophile; Thermotogales

Introduction

Aliphatic cellular polyamines, linear diamines, tri- * Address reprint requests to: Dr. Koei Hamana, School of Health Sciences, Faculty of Medicine, Gunma University, amines, tetra-amines, penta-amines and hexa-amines, Showa-machi, Maebashi, Gunma 371–8514, Japan. and quaternary branched penta-amines, were found in E-mail: [email protected] eubacteria (domain Bacteria) and archaebacteria (do- 272 HOSOYA et al. Vol. 50 main Archaea), and are important to stabilize cellular et al., 1998; Fardeau et al., 1995; Imachi et al., 2002; nucleic acid structure as a major function (Hamana, Qiu et al., 2003; Robertson et al., 2001), and the alka- 2002; Hamana and Matsuzaki, 1992). The chemotaxo- liphilic clostridia, Alkaliphilus (Cao et al., 2003; Takai nomic and phylogenetic significance of the distribution et al., 2001b) were available in the present study. of long and branched polyamines, possibly associated Polyamines of thermophilic Anoxybacillus and Ure- with their thermophily, has been proposed (Hamana ibacillus (Belduz et al., 2003; Fortina et al., 2001; and Itoh, 2001; Hamana and Kato, 2000; Hamana Yumoto et al., 2004), as well as thermoacidophilic et al., 1994, 1995, 1996a, b, 1998, 1999, 2001, 2003). Sulfobacillus and Alicyclobacillus (Dufresne et al., The three deeply branching lineages of eubacteria, 1996; Goto et al., 2002a, b, 2003; Matsubara et al., the orders Aquificales, Thermotogales and Thermo- 2002; Tsuruoka et al., 2003), were also determined. desulfobacteriales, are phylogenetically located in Distribution catalogues of long and branched early eubacterial evolution and comprise entirely polyamines in the 53 newly validated species were thermophiles. We have attempted to analyze presented here to elucidate their chemotaxonomic sig- the polyamines of hydrogen-oxidizing Hydrogenother- nificance. Their distribution profiles, at different culture mus, Hydrogenobacter (Stohr et al., 2001; Takai temperatures under acidic, neutral and alkaline condi- et al., 2001a), Persephonella, Sulfurihydrogenibium, tions, were also described. Thermocrinis and Balnearium, and nitrate-reducing Thermovibrio (Eder and Huber, 2002; Huber et al., Materials and Methods 1998, 2002; Nakagawa et al., 2003; Takai et al., 2003a, b), belonging to Aquificales. Polyamines of new The culture media designated by the culture collec- members of Thermotoga, Petrotoga and Marinitoga tions were used according to the Catalogue of Strains (Alain et al., 2002; Balk et al., 2002; L’Haridon et al., (2004) of ATCC, JCM, NBRC and NCIMB. The 2002; Takahata et al., 2001) of Thermotogales, and medium pH and culture temperature used for optimum thermophilic sulfate-reducing, Thermodesulfatator growth are given in Table 1. Thermocrinis, Perse- belonging to the order Thermodesulfobacteriales phonella, Sulfurihydrogenibium, Balnearium and Ther-

(Moussard et al., 2004), were analyzed. movibrio were grown under N2-O2-H2 or H2-CO2. Hy- The thermophilic order Thermales currently com- drogenothermus was aerobically grown. Marinitoga, prises Thermus, Meiothermus, Marinithermus and Petrotoga and Thermotoga were cultivated under N2 or

Oceanithermus (Miroshnichenko et al., 2003a; Sako N2-CO2. Thermodesulfatator was grown under H2-CO2. et al., 2003). A novel member, Vulcanithermus, was Thermus, Marinithermus and Meiothermus were isolated as the ﬁfth genus (Miroshnichenko et al., aerobically grown whereas Vulcanithermus and

2003b). Thus, polyamine proﬁles of new species of the Oceanithermus were grown under a N2 atmosphere. ﬁve genera were determined, in relation to their growth Thermanaeromonas, Thermoanaerobacterium, Cal- temperature and their phylogenetic distance. danaerobacter, Gelria, Thermosyntropha, Ther- Thermophiles are distributed also in the Gram-posi- manaerovibrio, Thermobrachium, Aminobacterium and tives of the orders Thermoanaerobacteriales, Clostridi- Aminomonas were grown anaerobically. Themaer- ales and Bacillales. Polyamines of new species of obacter, Alkaliphilus, Desulfotomaculum, Desulfo- Thermaerobacter (Nunoura et al., 2002; Spanevello sporosinus, Pelotomaculum and Sporotomaculum et al., 2002), Caldanaerobacter (Fardeau et al., 2000, were grown under N2-CO2. Sulfobacillus, Alicyclobacil- 2004; Xue et al., 2001), Thermanaeromonas (Mori lus, Anoxybacillus and Ureibacillus were grown aerobi- et al., 2002) and Thermoanaerobacterium (Cann et al., cally and Thermicanus anaerobically. 2001) were determined. The thermophilic anaerobes, The organisms at stationary phase were harvested

Gelria (Plugge et al., 2002), Thermanaerovibrio and homogenized in equal volumes of cold 1 M HClO4.

(Baena et al., 1999b), Thermosyntropha (Svetlitshnyi The whole HClO4 extract was analyzed by high-perfor- et al., 1996), Thermicanus (Gossner et al., 1999) mance liquid chromatography (HPLC) on a column of and Thermobrachium (Engle et al., 1996), and the cation-exchange resin in a Hitachi L6000 high- mesophiles, Aminobacterium and Aminomonas speed liquid chromatograph (Hamana et al., 1998). (Baena et al., 1999a, 2000), and several anaerobes Polyamines were detected with the o-phthalaldehyde located in the Desulfotomaculum lineage I (Brauman reagent. The whole extract was subjected to a Dowex 2004 Polyamines of thermophilic eubacteria 273 3(3)(3)3 3(3)(3)4 343/ 343/ mol/g wet wt. cell) m 333/ 343/ 334/ ——————0.60— ——————0.80— Polyamines ( 30 08 0. 0. —— .04 ——— —0 — 5.5 5.5 °C pH 3 4 5 33 34 333 3(3)3 334 3(3)3 3(3)4 3(3)4 80 7.0 —75 0.02 7.0 — 0.01 — 0.10 0.56 —70 *62 0.06 7.0 1.20 0.02 7.5 —65 — 0.02 — * 7.0 0.40 0.10 0.0175 * 0.20 * —75 — 0.03 —75 0.03 0.02 — * 1.50 6.0 * — 0.18 — 0.02 — 0.40 0.02 0.30 0.02 * — — — — — 2.40 * * * — — 65 — 0.10 — 6.0 —55 — — 0.08 * —55 7.0 0.15 — — 0.01 7.0 1.60 — 0.0265 1.30 — — 0.90 0.35 — *60 6.9 0.57 0.25 0.15 — 3.1070 6.5 — — — — — — 6.5 — 0.10 0.03 1.20 — * 0.02 0.05 — 0.05 0.04 — 1.15 0.80 — 0.10 — 0.40 — 1.85 — — 0.60 0.04 — 0.79 0.60 — 0.30 — — 0.80 — — — 0.90 0.25 — 0.02 — — — — 0.10 0.10 0.03 0.20 — — — 0.05 — — — — — — — — — — 0.02 °C pH 3 4 5 33 34 44 333 334 3333 3343 3334 33333 (c) 75 7.0 —(c) 70(c) 0.01 65 7.0 — 3.0 — 0.60 — — 0.50 — — — — — * — 1.50 0.06 0.08 — — — 0.03 * * * 0.03 0.05 — 2.10 0.02 — — * — 2.60 0.01 * 1.12 0.02 (b) 85 6.5 — — — 0.02 — — — — 0.02 —(d) 55 — 7.2 1.60 —(a) — 65 0.57 8.0 0.16 — 0.02(a) 0.55 0.08 80 — — 6.5 0.02 0.02 — 0.02 0.17 0.36 — — — 0.20 — 0.15 — — — 0.66 0.04 0.40 — 0.20 — 1.25 — — — — 0.59 T T T T T T T T T T T T T T T able 1. Cellular concentration of polyamines thermophilic eubacteria. T T T T T TCC BAA-301 TCC 51224 TCC 51869 JCM 12110 JCM 11663 JCM 11234 A JCM 8795 JCM 9492 JCM 10560 JCM 11468 JCM 10974 JCM 11386 JCM 8158 JCM 7687 JCM 11477 JCM 11970 A JCM 11235 A JCM 10099 JCM 11233 Hydrogenothermaceae Desulfurobacteriaceae Aquificaceae Thermotogaceae Thermotogales hydrogenophilus subterraneum Aquificales Thermovibrio ammonificans Persephonella hydrogeniphila Petrotoga olearia Thermotoga lettingae Hydrogenobaculum acidophilum Aquifex pyrophilus Hydrogenobacter subterraneus Thermovibrio ruber Hydrogenothermus marinus Balnearium lithotrophicum Thermocrinis albus Hydrogenobacter Hydrogenobacter thermophilus Sulfurihydrogenibium Petrotoga miotherma Petrotoga sibirica Thermotoga elfii Thermotoga maritima Marinitoga piezophila Family Family Family Family Order Order 274 HOSOYA et al. Vol. 50 33333 33334 3(3)(3)3 343/ mol/g wet wt. cell) m Polyamines ( 3(3)3/ 343/ 3343/ able 1. (Continued.) T 80 6.580 — 6.5 0.25 — 0.05 0.19 0.80 1.20 0.10 0.65 — 1.40 1.30 — 0.02 0.01 1.52 0.01 0.02 0.04 — 0.02 0.01 70 — 6.7 0.02 — 0.10 — 0.90 — 0.01 — — — — — 0.80 °C pH 3 4 5 33°C 34 pH 44 3 333 4 334 3333 33 3343 3334 34 33333 333 343 334 3333 3343 3334 3(3)4 3(3)(3)4 (f) 70 7.0 — 0.27 — 1.05 1.45 — 0.02 0.40 — — — — (a) 80 6.5 0.20(d) 0.15 70(d) 0.02 70 7.0 1.35 0.02 7.0 1.15 0.14 0.04 0.10 0.02 — 0.05 0.85 0.15 1.42 0.60(b) 0.14 1.40 70 — 0.16 — 7.5(b) 0.05 0.12 65 — 0.02 0.22 0.15 7.4 0.02 0.10 0.45 0.02 — 0.12 0.02 — 0.02 0.18 — — 0.05 0.02 — 0.08 0.77 — 0.05 — 1.05 — 0.04 0.20 — 0.50 — — 0.02 0.03 0.08 1.00 1.50 T T T T T T T T T 70 7.6 — 0.10 0.01 0.25 1.00 0.32 — *70 0.10 7.6 0.30 * — 0.15 0.05 0.01 0.02 0.10 0.01 1.00 0.02 0.38 * — — * 0.05 — 0.10 * — 0.05 0.02 0.02 0.02 * — — — °C pH 3 4 33 34 44 333 3(3)3 333 343 334 334 344 3333 3343 3334 3334 TCC 4964 TCC 35602 TCC 33708 TCC 49049 A NCIMB 12254 JCM 11887 JCM 10881 A JCM 10495 A JCM 10882 A (a) 70 7.6(b) 70 — 0.26 7.6(a) 0.14 70 — 0.68 1.38 0.10 7.6 1.06 0.10 0.04 — 0.75 0.20 1.30 0.09 0.80 *(b) 0.88 1.04 — 70 0.16 0.21 0.02 8.2 0.80 * 0.01 * * 0.02 0.20 0.05 2.50 0.10 0.05 0.18 0.41 0.16 0.42 * 0.10 0.60 * 0.10 0.20 — 0.02 * 0.09 0.02 0.21 * 0.01 0.05 — 0.05 0.20 * 1.40 * — — * 0.05 — 0.06 — — 0.06 0.04 0.05 0.05 — * — — — T T T T T T TCC 43280 TCC 700962 TCC 700961 TCC 25104 A NCIMB 12676 A A A NCIMB 13400 Thermodesulfobacteriaceae Thermaceae brockianus Thermodesulfobacteriales Thermales commune thermophilum aquaticus filiformis oshimai antranikianii igniterrae Thermotoga naphthophila Fervidobacterium islandicum Thermodesulfobacterium Thermodesulfatator indicus Thermotoga petrophila Fervidobacterium nodosum Thermotoga neapolitana Thermosipho japonicus Thermodesulfobacterium Thermus Thermus Thermus Thermus Thermus Thermus Family Family Order Order 2004 Polyamines of thermophilic eubacteria 275 33333 33334 3(3)(3)3 mol/g wet wt. cell) m 343/ 3334/ 3(3)4/ Polyamines ( 3(3)3/ 343/ 3343/ able 1. (Continued.) T 50 7.260 7.270 — 0.05 7.2 — 0.10 0.10 0.20 — 0.10 1.25 0.30 — * 2.10 0.10 * 0.25 1.40 * * * 0.15 0.40 * * * 0.80 * * * 0.15 — 0.30 0.05 * — — 0.09 0.05 * 0.05 * * * * * — — 0.02 — — — — — — — — 0.02 °C pH 3 4 33 34 44 333 3(3)3 333 343 334 334 344 3333 3343 3334 3334 60 7.670 — 0.12 7.0 —60 — 0.0268 0.10 7.3 0.85 0.15 0.9570 0.10 7.3 — 0.75 — 0.90 6.5 — — 0.23 0.12 0.02 — 0.05 — * 0.02 — * 0.24 — 0.05 — 0.02 * — 0.02 0.05 — 0.50 0.15 — — — — — 1.00 0.03 — 0.10 — — — 1.50 0.40 — — * * * * * — — — — — — 0.10 0.10 — — — * 0.20 — — — * — — * 0.02 — * 0.04 — 0.01 — — — — — °C pH 3 4 5 33 34 44 333 343 334 334 344 444 3333 3334 3343 3343 33333 33334 (a) 65 7.2(a) 70 — 0.15 7.2 — — 0.24 0.51 1.70 0.14 0.30 0.14 0.95 — 0.53 0.04 * * 0.22 1.50 0.19 1.00 * * 0.45 0.10 0.05 0.02 0.11 0.03 0.06 0.04 * * — 0.04 0.04 — 0.02 — (b) 55 8.2(a) 0.02 60 0.10 7.2 —(b) 0.01 55 0.04 — 8.2 — 1.54 0.80 0.01 0.01 0.02 0.15 0.12 0.15 1.81 — 0.02 — — * 0.02 0.72 0.01 0.02 1.14 * — — 0.07 — 0.05 0.01 — * — — 0.01 0.01 * — — * — — — * — — — — — — — T T T T T T T T T T TCC 51532 TCC 34958 TCC BAA-399 TCC 27634 A A NCIMB 13440 A JCM 11576 NBRC 100410 JCM 11956 NCIMB 13439 A JCM 10941 chiarophilus ruber silvanus taiwanensis hydrothermalis scotoductus thermophilus mediatlanticus thermophilus ulcanithermus profundus Thermus Thermus Thermus Meiothermus Meiothermus Meiothermus Marinithermus Oceanithermus V Meiothermus 276 HOSOYA et al. Vol. 50 3(3)4 3343 3(3)(3)4 3(3)(3)3 3(3)(3)43 mol/g wet wt. cell) m 343/ 334/ Polyamines ( able 1. (Continued.) T 65 7.575 0.80 7.5 0.75 0.15 — 0.05 0.01 1.15 0.51 0.01 — — — 0.90 * * 0.55 0.10 0.01 * — — 0.05 1.20 * — — 0.17 — — — 70 6.5 — 0.02 0.01 0.10 — 0.15 * 0.85 0.10 — —65 7.5 — 0.15 0.2555 0.20 7.5 — 0.16 0.65 0.90 0.69 0.05 —65 — 6.8 1.40 — — — 0.10 1.62 — — * 0.56 0.50 * — * — — *65 0.46 * 6.855 — 7.0 * — — * 0.10 — 0.24 * — — — — 0.45 — 0.06 — — * 0.15 — — — — — 0.35 — — — * * — * 0.85 — — — — * — — — — — — — — — °C pH 4 5 33 34 44 3(3)3 3(3)4 343 334 344 (e) 60 6.5 0.68 0.14 — 1.30 — — * 0.50 — — — — — — — (e) 60 7.2 0.95(e) 60 — 6.5 0.35 — 0.10(d) 2.80 — 65(d) 7.2 — 60 0.45 0.01 7.1 0.03 — — 0.61(e) — 0.02 60 7.2 — — * 0.50 0.12 0.08 1.25 — 0.45 * — — — 0.80 — 1.48 — — — — * — — * 2.15 — — — — 2.75 — * —(e) — — 60 0.04 2.15 7.0 — — — 0.40(e) — 0.01 — 0.10 50 — — 7.0 0.40 — — — 1.35(e) 0.16 — 0.02 60 1.64 — — 7.3 — — — — 0.64 0.05 1.49 — — — — — — 1.22 * — — — 0.20 * — — — 0.35 — * — — 2.45 — — — — — — — — — — — — — — — — — T T T T T T T T T T T T T T T TCC BAA-17 TCC BAA-262 TCC 53556 TCC 35045 TCC BAA-225 TCC 7956 TCC 33743 TCC BAA-16 TCC 33265 TCC 49915 TCC 33488 TCC 31550 JCM 11007 A JCM 11376 A A IAM 13577 A A A A A A A A A tengcongensis subterraneus Thermoanaerobacteriaceae subsp. acetoethylicus thermocopriae thermohydrosulfuricus saccharolyticum thermosaccharolyticum thermosulfurigenes toyohensis subsp. polysaccharolyticum ethanolicus Thermoanaerobacteriales Caldanaerobacter subterraneus Thermoanaerobacterium Gelria glutamica Thermoanaerobacter brockii Thermoanaerobacter Caldanaerobacter subterraneus Thermoanaerobacterium Thermoanaerobacterium Thermoanaerobacterium zeae Thermoanaerobacter Thermoanaerobacter Thermoanaerobacterium Thermoanaerobacter kivui Thermoanaerobacter Thermanaeromonas Family Order 2004 Polyamines of thermophilic eubacteria 277 343/ 334/ mol/g wet wt. cell) m Polyamines ( able 1. (Continued.) T 707060 7.055 7.3 9.0 —37 7.1 — 0.05 0.0137 0.02 7.2 — 0.02 —60 7.2 0.10 0.55 0.5037 — — 0.45 —37 8.0 — * 11.5 0.50 0.70 — 9.50 0.12 7.5 — — 0.90 * 0.10 0.40 * — — 0.60 — — — — 1.10 * * 0.15 — — 0.45 0.30 — —35 — 0.35 * 0.45 — — 1.15 * 2.5 0.55 * — — 1.15 0.09 0.85 — 1.40 — 0.15 — 0.12 — — — *55 0.75 *45 0.30 — — — — 55 4.5 0.01 * —60 4.0 — —50 — 4.0 — * — —35 4.0 — — * 0.5045 3.0 — — — 0.30 — 55 4.0 — 0.50 0.45 4.0 — — 0.50 — 0.03 0.40 — 4.0 — 0.70 1.04 0.02 — — 0.60 — 1.20 — — — 0.04 1.20 — — 0.22 — 1.00 — — 0.18 — — — 0.24 — — — — — — — — — — 0.05 — — — — — — — — — — — — — — — — — — 0.15 — — — — — — — — — — — 0.03 — — — — 0.02 °C pH 4 5 33 34 343 334 3(3)4 3(3)4°C 3(3)(3)4 Agm pH 4 34 343 3343 33343 33433 3(3)4 3(3)(3)4 3(3)(3)43 Agm (f) 70 7.6 — 0.04 0.16 0.60 * *(g) * 45(g) 0.75(g) 50 2.0 0.60 65(g) 0.70 4.0 — 50 4.0 1.24 0.03 4.0 1.00 — 0.50 0.04 0.31 — — 1.40 — 1.60 — — 0.56 — — — — — — — — — — — — — — — — — 0.34 — — 0.05 0.02 — 0.40 T T T T T T T T T T T T T T T T T TCC BAA-7 TCC 700318 TCC BAA-137 TCC 700317 TCC 49978 TCC BAA-6 TCC 49025 TCC 700253 TCC 51911 IAM 14934IFO 15310 55 4.5 — 0.09 0.70 — — — — — — 0.02 A A A A A A JCM 10712 JCM 11672 A IAM 14935 JCM 10246 A JCM 11223 A JCM 5260 IAM 14883 Clostridiaceae Syntrophomonadaceae Alicyclobacillaceae acidaminovorans Bacillales Clostridiales Alicyclobacillus cycloheptanicus Thermobrachium celere Thermaerobacter subterraneus Thermosyntropha lipolytica Thermanaerovibrio Aminomonas paucivorans Alkaliphilus transvaalensis Alkaliphilus crotonatoxidans Alicyclobacillus acidoterretris Alicyclobacillus acidiphilus Thermaerobacter marianensis Sulfobacillus acidophilus Thermaerobacter nagasakiensis Sulfobacillus disulfidooxidans Alicyclobacillus acidocaldarius Alicyclobacillus herbarius Aminobacterium mobile Family Family Family Order Order 278 HOSOYA et al. Vol. 50 mol/g wet wt. cell) m Polyamines ( 50 7.4 — — 0.22 0.18 — °C pH 4 5 34 343 Agm (g)(g)(g) 37(g) 55(g) 37 7.2 60 7.5 35 7.5 — 7.0 0.02 0.10 6.5 — 0.04 — — 0.35 — 1.20 0.25 0.77 0.05 0.20 2.05 — — — 0.45 0.45 0.57 0.02 — 0.02 — able 1. (Continued.) T 554560 4.555 4.0 4.0 — 4.5 — 0.83 — 0.7555 — 0.06 0.80 0.15 1.04 — 7.0 0.15 — 0.75 0.10 — — — 1.20 — — — 0.10 — —60 — — — — —50 7.3 — — — — —60 7.5 — — — — — — 0.35 0.03 7.3 — — — 0.90 0.90 0.09 — — 0.08 0.70 — — 0.20 1.60 0.04 — — 0.20 — — — — — — — — — — — — — — — — 0.05 — 0.04 °C pH 4 34 343 3343 33343 33433 3(3)4 3(3)(3)4 3(3)(3)43 Agm T T T T T T (h)(h) 75 50 7.0 5.0(h) —(g) 0.06 60(h) 0.60 60 1.90(h) 70 7.3 1.50(h) 1.78 65 7.0 0.90 65 7.0 — — 7.5 — 0.01 1.57 0.10 7.3 — 0.01 0.74 0.76 — 0.96 0.04 0.15 1.60 0.93 — 0.45 — 0.14 0.20 0.02 — 2.50 3.25 — — — — — — — — — — — — — — — — 0.02 — — — 0.01 0.02 — — — — 0.03 0.01 0.04 — — — 0.01 0.01 0.02 — 0.05 0.04 T T T TCC 700041 (g) 35 7.3 — — 0.74 0.14 — TCC 51507 TCC 7946TCC 23193 TCC 49756 (g) 55 7.5 0.04 0.04 0.74 0.10 0.52 TCC 49208 T T T A A IFO 13698 A A A NCIMB 13375 A T T T T T T T TCC 43741 TCC 700890 TCC 49805 TCC 43125 IAM 11062 IFO 15311 NCIMB 13819 JCM 11817 A IAM 14988 IFO 15132 A A A IFO 15316 IFO 15317 (h) 65 7.3 — 1.15 0.50 — — — — — — 0.02 (Aero) NCIMB 13933 (Aero) JCM 12111 (Anaero) 60 7.3 — 0.50 1.05 — — — — — — — (Anaero) 50 7.5 — 0.72 0.60 0.04 — — — — — 0.10 lineage I Bacillaceae Paenibacillaceae Peptococcaceae voinovskiensis Geobacillus stearothermophilus Anoxybacillus Ureibacillus thermosphaericus Bacillus smithii Alicyclobacillus sendaiensis Bacillus schlegelii Alicyclobacillus pomorum Thermicanus aegyptius Bacillus tusciae Bacillus thermocloacae Saccharococcus thermophilus Geobacillus thermocatenulatus Anoxybacillus flavithermus Anoxybacillus gonensis Desulfotomaculum nigrificans Desulfotomaculum ruminis Desulfotomaculum thermobenzoicum Desulfotomaculum thermosapovorans Desulfotomaculum acetoxidans Desulfitobacterium dehalogenans Family Family Family Desulfotomaculum 2004 Polyamines of thermophilic eubacteria 279 homocaldohexamine; 33343, mol/g wet wt. cell) -bis(aminopropyl)norspermidine; 4 m N ana et al.(1996a); (e) Hamana al. spermine; 334, thermospermine; 344, Collections of Industrial, Food and Marine croorganisms, RIKEN, Wako, Saitama, Japan; croorganisms, RIKEN, Wako, Polyamines ( -aminopropylspermidine; 3(3)(3)3, 4 N -bis(aminopropyl)spermine; Agm, agmatine; (Aero), aerobically grown; (Anaero), 4 3055 7.43037 7.0 — 7.6 0.05 6.5 — — — — 0.40 0.05 — 0.10 — 0.50 0.65 — 0.35 — 0.15 0.45 — — °C pH 4 5 34 343 Agm N (g) 37 7.5 0.20 — 0.24 0.06 0.02 able 1. (Continued.) T , type strain; ATCC, American Type Culture Collection, Manassas, Virginia, USA; IAM, IAM Culture Culture Collection, Manassas, Virginia, American Type , type strain; ATCC, T T T T -aminopropylnorspermidine; 3(3)4, T T 4 N -aminopropylspermine; 3(3)(3)43, 4 TCC 23598 (g) 30 7.5 — — 0.97 0.30 — TCC 43876 TCC 700645 N A JCM 11495 NCIMB 13706 A A JCM 10971 0.005); *, data not separated; -bis(aminopropyl)spermidine; 3(3)43, 4 Thermoanaerobacteriaceae Lachnospiraceae N Desulfosporosinus orientis Sporotomaculum syntrophicum Desulfosporosinus meridiei Acetitomaculum ruminis Sporotomaculum hydroxybenzoicum Pelotomaculum thermopropionicus Family Family 3, diaminopropane; 4, putrescine; 5, cadaverine; 33, norspermidine; 34, spermidine; 44, homospermidine; 333, norspermine; 343, (1996c); (f) Hamana et al. (2001); (g) (1999); (h) (1993). °C, culture temperature. Collection, Institute of Molecular and Cellular Biosciences, the University of Tokyo, Tokyo, Japan; JCM, Japan Collection of Mi Tokyo, Collection, Institute of Molecular and Cellular Biosciences, the University Tokyo, and Evaluation, Kisarazu, Chiba, Japan; NCIMB, the National National Institute of Technology NBRC, Biological Resourse Center, Bacteria, Aberdeen, Scotland, UK. (a) Cited from Hamana et al.(1998); (b) al.(1999); (c) al.(1995); (d) Ham anaerobically grown; —, not detected ( 3(3)(3)4, aminopropylhomospermidine; 3333, caldopentamine; 3334, homocaldopentamine; 3343, thermopentamine; 33333, caldohexamine; 33334, thermohexamine; 33433, homothermohexamine; 3(3)3, 280 HOSOYA et al. Vol. 50

50W column to concentrate long and branched [3343] and homocaldopentamine [3334] were sepa- polyamines (Hamana et al., 1998). Gas chromatogra- rately eluted in the HPLC and GC. Typical HPLC chro- phy (GC) was performed after heptafluorobutyrization matograms of the acid extracted polyamines are of the concentrated polyamine samples (Niitsu et al., shown in Fig. 1. Some GC chromatograms of the con- 1993). Polyamines were identified by gas chromatog- centrated polyamine samples are shown in Fig. 2. raphy-mass spectrometry (GC-mass) (Niitsu et al., Polyamine peaks were further identified by GC-mass. 1993). During GC analysis, a quaternary penta-amine, N4- bis(aminopropyl)norspermidine [3(3)(3)3], was con- Results and Discussion verted to a tertiary tetra-amine, [3(3)3], and another quaternary penta-amine, N4-bis(aminopropyl) spermi- HPLC analysis of polyamines has been developed dine [3(3)(3)4] was converted to two tertiary tetra- in our laboratory and resolution of many polyamines amines, [3(3)3] and [3(3)4] (Hamana et al. 1992). Cel- was improved (Hamana and Itoh, 2001; Hamana et al., lular concentrations of polyamines estimated from the 2001). Norspermine [333] and N4-aminopropylnorsper- HPLC and GC analyses are shown in Table 1. A list of midine [3(3)3] were co-eluted in the HPLC. Separation trivial names, systematic names and chemical formu- of thermospermine [334], spermine [343] and N4- lae of polyamines has been recorded elsewhere aminopropylspermidine [3(3)4] was difficult by HPLC. (Hamana, 2002; Hamana and Matsuzaki, 1992; When a concentrated polyamine sample is subjected Hamana et al., 1994). Polyamine profiles within each to GC analysis, they are identified. Thermopentamine order and lineage are followed.

Fig. 1. HPLC analysis of polyamines extracted from (a) Sulfurihydrogenibium subterraneum JCM 11477; (b) Hy- drogenobacter subterraneus JCM 10560; (c) Hydrogenothermus marinus JCM 10974; (d) Thermotoga petrophila JCM 10881; (e) Thermanaeromonas toyohensis JCM 11376; (f) Caldanaerobacter subterraneus subsp. tengcongensis JCM 11007 (grown at 75°C). Abbreviations for polyamines are given in Table 1. 2004 Polyamines of thermophilic eubacteria 281

Aquificales The genera Aquifex, Thermocrinis, Hydrogenobacter and Hydrogenobaculum phylogenetically belong to the family Aquificaceae of the order Aquificales. Perse- phonella, Hydrogenothermus and Sulfurihydro- genibium are located in the family Hydrogenotherma- ceae of the order (Eder and Huder, 2002; Takai et al., 2003a). Thermovibrio and Balnearium together with Desulfurobacterium, represent a tentative family Desulfurobacteriaceae of the order (Huber et al., 2002; Takai et al., 2003b). We reported the occurrence of a quaternary branched penta-amine, N4-bis(aminopropyl)norspermidine, as the main polyamine of a species of Aquifex, a species of Hydrogenobaculum and two Hydrogenobacter species (Hamana et al., 1995, 1999). The eight new members of Aquificales ubiquitously contained N4-bis(aminopropyl)norspermidine as the major polyamine. The ubiquitous and abundant occurrence of N4-bis(aminopropyl)norspermidine within the moderate-, extreme- and hyper-thermophiles, ranging from 62 to 85°C as the optimum growth temperature, char- acterizes the order Aquificales.

Thermotogales Several thermophiles belonging to Thermotogales, previously analyzed, contained liniar triamines, tetra- amines, penta-amines and a hexa-amine (Hamana et al., 1998, 2001). Among the Thermotoga species examined, Thermotoga naphthophila and Thermotoga petrophila growing at 80°C contained norspermidine, norspermine, caldopentamine and thermopentamine, in addition to spermidine and spermine (and/or thermospermine). On the other hand, Thermotoga littingae growing at 65°C was devoid of norspermine, caldopentamine and thermopentamine. Existence of Fig. 2. GC analysis of the concentrated long polyamine homospermidine in T. littingae was confirmed by the fractions from (a-1) Thermus igniterrae ATCC 700962; (a-2) GC analysis. Such difference in the polyamine profile Meiothermus taiwanensis ATCC BAA-399; (a-3) Vulcanithermus of T. littingae, as well as that of Thermotoga elfii, might mediatlanticus JCM 11956; (a-4) Thermanaeromonas toyohen- reflect their distinct phylogenetic position and growth sis JCM 11376; (a-5) Caldanaerobacter subterraneus subsp. temperature from other Thermotoga species. When tengcongensis JCM 11007 (grown at 75°C); (b-1) Thermus Thermotoga maritina was cultivated at lower tempera- antranikianii ATCC 700961. tures, penta-amine and hexa-amine were decreased. GC-column oven temperature was raised from 120 to 280°C Therefore, the difference in the long polyamine compo- in (a) and from 200 to 280°C in (b) (Niitsu et al., 1993). Abbrevi- ations for polyamines are given in Table 1. Retention times sition may be attributed to the difference of growth (min) are printed on each peak. temperature. Likewise, Marinitoga piezophila, Petro- toga olearia and Petrotoga sibirica growing at 55–65°C showed polyamine profiles lacking panta-amines and hexa-amines. The level of norspermidine was low in all 282 HOSOYA et al. Vol. 50 the Petrotoga species and not significantly affected by A marine species, Marinithermus hydrothermalis the growth temperatures in Thermotoga maritina. The growing at 70°C, contained norspermidine, spermi- low norspermidine level in the genus Petrotoga and dine, homospermidine, spermine (and/or thermosper- T. elfii may represent a chemotaxonomic feature. mine) and homocaldopentamine (and/or thermopenta- Within other members of the order Thermotogales mine). Two hexa-amines were detected but not homo- (the family Thermotogaceae), linear penta-amines spermidine in Vulcanithermus mediatlanticus growing have been detected in Fervidobacterium islandicum at 70°C. Oceanithermus profundus growing at 60– growing at 70°C, but not in Fervidobacterium nodosum 68°C contained spermine alone as the major and Thermosipho japonicus growing at 70°C (Hamana polyamine and lacked norspermidine and homosper- et al., 1996a, 2001). midine as well as long and branched polyamines. Therefore, some genus-specific polyamine profiles Thermodesulfobacteriales among the five genera of the Thermacea were ob- The occurrence of a quaternary branched panta- served. amine, N4-bis(aminopropyl)spermidine, in the two The family Thermaceae (the order Thermales) be- species of Thermodesulfobacterium growing at longs to the phylum Deinococcus-Thermus, however, 65–70°C, have been reported in our previous reports mesophilic Deinococcus species belonging to another (Hamana et al., 1996a, 1999). A new member of the family Deinococcaceae (the order Deinococcales) order Thermodesulfobacteriales (the family Thermo- lacked norspermidine, homospermidine, and long and desulfobacteriaceae), Thermodesulfatator indicus, grow- branched polyamines (Hamana, 1994). ing at 70°C, contained the branched penta-amine indi- cating the ubiquitous occurrence of the branched Thermoanaerobacteriales panta-amine in the two extremely-thermophilic, sulfate- Two quaternary penta-amines, N4-bis(amino- reducing genera of this order. propyl)spermidine and N4-bis(aminopropyl)norspermidine, were found as major polyamines in extremely Thermales thermophilic Thermanaeromonas toyohensis growing Six Thermus species previously analyzed contained at 70°C. In addition, a novel tertiary branched penta- many triamines, tetra-amines and penta-amines amine, N4-aminopropylspermine, was detected by GC (Hamana et al., 1991, 1998, 1999), as listed in Table 1. analysis of the concentrated polyamine fraction from A branched tetra-amine, N4-aminopropylnorspermi- this organism. This tertiary penta-amine is produced dine, and a branched penta-amine, N4-bis(amino- from a quaternary hexa-amine, N4-bis(aminopropyl)norspermidine, have been detected in two of propyl)spermine, during GC analysis (Hanama et al., the six species. In the two new members of extremely 1992, 1993). It is strongly suggested that the quater- thermophilic Thermus growing at 70°C, almost the nary branched hexa-amine exists in T. toyohensis. same polyamine components were identified; however, Recently, Caldanaerobacter subterraneus subsp. hexa-amines and branched polyamines were not subterraneus (formerly Thermoanaerobacter subterra- detected. Penta-amines increased at higher growth neus) and Caldanaerobacter subterraneus subsp. temperature of T. thermophilus. A new species of the tengcongensis (formerly Thermoanaerobacter teng- genus Meiothermus, M. taiwanensis had a polyamine congensis) were phylogenetically separated from other pattern similar to that of the three Meiothermus Thermoanaerobacter species (Fardeau et al., 2004). species previously reported (Hamana et al., 1991, N4-bis(aminopropyl)spermidine was found in the two 1998, 1999). Spermidine, homospermidine and sper- Caldanaerobacter species, as a major polyamine, as mine were found as the major polyamine and norsper- well as Thermoanaerobacter kivui and Thermoanaer- midine and norspermine were minor in the moderately obacter thermocopriae, previously reported (1996c). thermophilic genus Meiothermus growing at 55–60°C. Extremely thermophilic C. subterraneus subsp. teng- Although levels of norspermine were affected by congensis growing at 75°C contained norspermidine, growth temperature in T. thermophilus, a significant homospermidine, aminopropylhomospermidine and amount of norspermine as well as norspermidine was thermopentamine as a minor polyamine component. A found in the cells grown at the lower temperature minor occurrence of thermospermine was found by 50°C. GC-mass analysis. When C. subterraneus subsp. 2004 Polyamines of thermophilic eubacteria 283 tengcongensis was cultivated at lower temperatures, (Hamana et al., 1996c). The genus Alkaliphilus con- 55°C and 65°C, it still had the branched penta-amine tained alkaliphilic and non-alkaliphilic species, and in decreased amounts. Moderate thermophilic Ther- alkaliphily did not affect the polyamine composition in moanaerobacterium species and Gelria glutamica, the two species. growing at 55–65°C, contained putrescine, spermidine and spermine and lacked long and branched Bacillales polyamines (Table 1). In Caprothermobacter and The family Alicyclobacillaceae of the order Bacillales Moorella species growing at 60°C, N4-bis(amino- includes mesophilic, acidophiles and moderately propyl)spermidine was found in some species (or slightly) thermophilic acidophiles (Dufresne et al., (Hamana et al., 1996c). Thus, the distribution profiles 1996; Goto et al., 2002a, b, 2003; Matsubara et al., of long and branched polyamines were variable within 2002; Tsuruoka et al., 2003). When two Sulfobacillus the order Thermoanaerobacteriales. species were cultivated at optimum growth temperature, spermine level was higher at higher tempera- Clostridiales tures. In the three Alicyclobacillus species, spermine Strictly-aerobic, thermophilic Thermaerobacter mari- level did not change significantly in temperature anensis contained a quaternary branched penta- ranges between the optimal and 15°C below. Never- amine, N4-bis(aminopropyl)spermidine, and agmatine theless, the levels of spermine among the different (Hamana et al., 2001). Two new species of Thermaer- species seemed to be variable in accordance with their obacter, T. nagasakiensis and T. subterraneus, also optimal growth temperatures. A acidic pH range from contained N4-bis(aminopropyl)spermidine and agma- 3.0 to 4.0 in Alicyclobacillus species did not change tine. These extreme thermophiles, growing at 70°C, significantly in their polyamine profiles. Considering show same polyamine profiles. that neutrophilic, moderate thermophiles of the families Moderately thermophilic anaerobes, belonging to the Bacillaceae and Thermoactinomycetaceae also con- genera Thermosyntropha and Thermanaerovibrio, tained spermine (Hamana, 1999; Hamana et al., growing at 55–60°C, contained spermidine and sper- 1993), it seems that acidophily does not affect the mine, but lacked long and branched polyamines. Two polyamine composition and the occurrence of sper- mesophiles, Aminobacterium and Aminomonas, were mine correlates to moderate thermophily. This family rich in putrescine and spermidine and poor in sper- also contains thermoacidophilic Bacillus tusciae (grow- mine. These findings suggest that the occurrence of ing at 50°C) containing spermidine and spermine and long and branched polyamines are coupled with extremely thermophilic Bacillus schlegelii (growing extreme thermophily within Syntrophomonadaceae, a at 75°C) containing linear penta- and hexa-amines family of the order Clostridiales. Thermophilic Caldicel- and a branched penta-amine (Hamana et al., 1993) lulosiruptor species (growing at 70°C) of this family (Table 1). contained also linear and branched penta-amines In the family Bacillaceae, the occurrence of a qua- (Hamana et al., 2001). ternary branched hexa-amine, N4-bis(aminopropyl)- Moderately thermophilic Clostridium species con- spermine (detected as N4-aminopropylspermine in GC tained spermine as a major polyamine and mesophilic analysis), in addition to spermidine and spermine, was Clostridium species lacked a significant amount of reported in thermophilic Saccharococcus, Geobacillus spermine, as reported in our previous studies (Nazina et al., 2001) and Bacillus species growing at (Hamana, 1999; Hamana et al., 1996c). Two 60–75°C (Hamana et al., 1993). New thermophilic mesophilic anaerobes, Alkaliphilus transvaalensis and Anoxybacillus and Ureibacillus species (growing at Alkaliphilus crotonatoxidans, growing at 37°C, and a 50–65°C) (Belduz et al., 2003; Fortina et al., 2001; moderately thermophilic anaerobe, Thermobrachium Yumoto et al., 2004), analyzed here, contained spermi- celere, growing at 60°C, are located in the family dine and spermine as the major polyamine. Ther- Clostridiaceae. The former two strains contained sper- mopentamine and agmatine were detected in Anoxy- midine as the major polyamine and the latter strain bacillus voinovskiensis. Some polyamine profiles were contained spermine as a major polyamine. Calorama- observed in the both aerobically and anaerobically tor fervidus of this family, growing at 70°C, also con- grown Anoxybacillus species (Table 1). The branched tained spermine but not long branched polyamine hexa-amine was abundant in the extreme ther- 284 HOSOYA et al. Vol. 50 mophiles containing high spermine levels within Bacil- rence of the branched polyamines or the linear long laceae and Thermoanaerobacteriaceae. polyamines seems to be coupled with their ther- A moderately thermophilic acetogen growing at mophily, and serve for a chemotaxonomy. 55°C, Thermicanus aegyptius, belongs to the family Extremely thermophilic Thermus contained long Paenibacillaceae and is related to the other three fami- linear and branched polyamines whereas other ex- lies of this order (Gossner et al., 1999). This organism treme thermophiles, Marinithermus and Vulcanither- contained spermidine and spermine. In Paenibacil- mus, contained long linear polyamines. Moderately- laceae, it is known that mesophilic Paenibacillus slightly thermophilic Meiothermus and Oceanithermus and Brevibacillus species lack spermine whereas lacked them. The presence or absence of homosper- mesophilic Aneurinibacillus species contain a small midine, norspermidine and its derivatives differed in amount of spermine (Hamana, 1999). the five genera; however, they serve as a chemotaxonomic marker within the Thermales. Linear long Desulfotomaculum lineage I polyamines and branched polyamines were simultane- Sulfate-reducing Desulfotomaculum and Desulfo- ously found in some extreme thermophiles belonging sporosinus, sulfite-reducing Desulfitobacterium, propi- to Thermoanaerobacteriales, Clostridiales and Bacil- onate-oxidizing Pelotomaculum and benzoate-degrad- lales located in Gram-positives. Various moderate ing Sporotomaculum, located in the family Peptococ- thermophiles of this taxa contained spermidine and caceae of the order Clostridiales or the family Ther- spermine alone. moanaerobacteriaceae of the order Thermoanaer- It is suggested that the synthetic abilities of long obacteriales, are moderate thermophiles growing at linear polyamines and branched polyamines were sep- 50–60°C or mesophiles growing at 37–30°C (Brauman arately developed in early evolved thermophilic eubac- et al., 1998; Fardeau et al., 1995; Imachi et al., 2002; teria and the two polyamine synthetic pathways were Qiu et al., 2003; Robertson et al., 2001). The mixed during later eubacterial evolution. One of them polyamine pattern of an acetogenic anaerobe, Acetito- seems to be essential for extremely thermophilic eu- maculum ruminis, belonging to the family Lach- bacteria. Norspermidine- and homospermidine-synthe- nospiraceae of the order Clostridiales (Hamana, ses might be phylogenetically developed or elimitated 1999), was cited in Table 1. Although putrescine, sper- in eubacteria independently of their thermophily. midine and/or spermine were found as the major polyamines in the Desulfotomaculum lineage I, sper- Acknowledgments mine was relatively abundant in the moderate ther- We are indebted to JCM, IAM, NBRC, ATCC and NCIMB for mophiles as suggested in our previous report supplying bacterial strains, and M. S. Yoko Yokoyama and M. S. (Hamana, 1999). Some of the thermophiles contained Takehiko Tanaka, Gunma University School of Health Sciences agmatine, suggesting that the cellular occurrence of (Maebashi, Japan) for cultivation of some bacteria and Ms. Satoko Tsuzuki, JCM, RIKEN (Saitama, Japan) for technical as- spermine and agmatine may correlate to their moder- sistance. ate thermophily, as found in the thermophiles of Clostridiales and Bacillales. References

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