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Home , Desemzia incerta, Holophagales, Methanocella paludicola, Methanolobus profundi, Thermoleophilum, Thermus

Journal of Japanese Society for (2009) Vol.8 (2)

Journal of Japanese Society for Extremophiles (2009) Vol.8, 59-68 ORIGINAL PAPER

Hamana Ka, b, Hayashi Ha, b, Niitsu Mc and Itoh Tb

Polyamine analysis of thermophilic, acidophilic, alkaliphilic and radio-tolerant belonging to the domain Bacteria and , and extreme belonging to the domain -Polyamine catalogues of bacterial (eubacterial) and archaeal (archaebacterial) extremophiles IV-

aFaculty of Engineering, Maebashi Institute of Technology, Maebashi, Gunma 371-0816, Japan. bJapan Collection of Microorganisms, RIKEN, BioResource Center, Wako, Saitama 351-0198, Japan. cFaculty 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: August 3, 2009 /Revised: September 3, 2009 /Accepted: September 7, 2009

Abstract Cellular polyamines of 25 newly validated but not in hyperthermophilic as well as in thermophilic, acidophilic, alkaliphilic or radio-resistant the mesophilic genera, Methanobacterium, Methano- bacteria (eubacteria) and 35 methanogenic, thermophilic saeta, , and Methanofollis. or extremely halophilic archaea (archaebacteria) were Some methanogenic mesophiles contained spermine. analyzed by HPLC and GC. In the domain Bacteria, Long and branched polyamines were not found in 23 4 quaternary branched penta-amines, N -bis(aminopropyl) extreme halophiles and halalkaliphiles belonging to the 4 norspermidine and/or N -bis (aminopropyl)spermidine, order Halobacteriales. were detected in the extreme thermophiles, Hydrogenovirga and Thermodesulfidibacter of the Key words:archaea, archaebacteria, extreme , phylum and Thermus of the phylum , polyamine, Thermus-Deinococcus. A liner penta-amine, homo­ caldopentamine, was found in moderately thermophilic Introduction Thermodesulfovibrio. Spermine, a tetra-amine, was found in the moderate thermophiles, Meiothermus, Analyses ofcellular polyamine components have already Exilispira, Desulfurispora, Desulfotomaculum, provided valuable chemotaxonomic information as to the Caloramator and Calditerrivibrio but not in slightly classification of the domain Bacteria (Eubacteria) and the thermophilc Thiofava and mesophilic radio-resistant domain Archaea (Archaebacteria) as reported in Deinococcus. An acidophilic slight thermophile, Polyamine catalogues of bacterial (eubacterial) and Acidimicrobium, contained only cadaverine as a novel archaeal (archaebacterial) extremophiles I, II and III in polyamine profile. The occurrence of spermine in this journal15-17). Although the cellular concentration mesophilic Acanthopleuribacter belonging to the phylum levels of polyamines should be changed based on their was a unique profile. Alkaliphilic culture medium composition and growth phase, it has Alkalibacterium were devoid of cellular been suggested that polyamine distribution profiles in polyamines. In the phylum of the eubacteria and archaebacteria are related to their domain Archaea, the branched penta-amines were optimum growth pH, temperature and salt concentration distributed in the extremely thermophilic genera, as well as their phylogenetically classified locations. Methanocaldococcus, Archaeoglobus and Thermococcus Ionic interaction of polycationic polyamines to nucleic

59 Journal of Japanese Society for Extremophiles (2009) Vol.8 (2) acids and other acidic components in the cells is Materials and Methods important for their growth. Various , extreme thermophiles and Newly validatedeubacteria and archaebacteria were moderate thermophiles are distributed as diverse supplied from JCM (Japan Collection of Microorganisms, eubacterial phyla or orders3,4). The cellular occurrence RIKEN, Wako, Saitama, Japan) and NBRC (Biological oflinear penta- and hexa-amines, tertiary branched Resource Center, National Institute of Technology and tetra-amines and quaternary branched penta-amines in Evaluation, Kisarazu, Chiba, Japan) and cultivated in a hyper-/extreme thermophiles shows novel polyamine liquid media or on the agar media as designated by the synthetic abilities possibly associated with their culture collections under aerobic or anaerobic conditions. thermophily14,17,24). A tetra-amine, spermine, was Medium pH and culture temperature used for optimum detected as a major polyamine in various moderate growth are given in Tables 1 and 2. The organisms in thermophiles and thermoacidophiles14,17,24). However the the stationary phase were harvested. The pellets of degree of thermophily is estimated roughly and cannot organisms were washed with an NaCl solution (same salt be defined exactly. Further polyamine analyses of new concentration as the culture medium) and then members of eubacterial thermophiles are expected. On homogenized in equal volumes of cold 1M perchloric the other hand, polyamines of several acidophilic, acid (PCA)(HClO4). The 0.5 M (5 %) PCA extract was alkaliphilic and radio-tolerant eubacterial mesophiles subjected to a Dowex 50W column to concentrate have been demonstrated5,6,25);however, unique polyamine polyamines7,21). The concentrated polyamine fractions distribution profile has not been presented. were analyzed by high-performance liquid An archaebacterial phylum, Euryarchaeota, chromatography (HPLC) on a column of cation- encompassed diverse lineages, including: six orders of exchange resin in a Hitachi L6000 high-speed liquid methanogens, two orders of thermophiles, one order of chromatograph developed in our laboratory7,21). , and one order of extreme halophiles1,2). Polyamines were detected with the o-phthalaldehyde Another phylum,Crenarchaeotawas comprised of four reagent.The polyamine fraction was further purified on a thermophilic orders1,2). Long linear and/or branched column of Whatman CM237,21). Gas chromatography polyamines and a guanidinoamine (as a polyamine (GC) was performed on a Shimadzu GC-9A gas component), agmatine, found in the thermophiles grown chromatograph after heptafluorobutyrization of the in high thermal environment that seem to be important in purified polyamine samples21,26). Polyamines were stabilizingthe cellular nucleic acid structure as a major identified by gas chromatography-mass spectrometry function of the high basic polyamines14,15,16,23). These (GC-mass) using a JEOL JMS-700 mass polyamine syntheses were salt dependent in the spectrometer21,26). Molar concentrations of cellular thermophiles. Archaebacterial extreme halophiles grown polyamines per gram of wet cell pellet (μmol-nmol/g wet in high salt media showed low cellular concentrations of wt. cell), estimated from the HPLC and GC analyses, are spermidine, spermine and/or agmatine. The polyamine shown in Tables 1 and 2. levels varied in haloalkaliphilic species within the extreme halophiles15,17). Results and Discussion Additional distribution catalogues of long linear polyamines, branched polyamines and agmatine in 60 Domain Bacteria (Eubacteria) newly validated extremophiles distributed in the two Phylum Aquificae prokaryotic domains (25 eubacteria and 35 Extremely thermophilic Hydrogenivirga okinawensis3) euryarchaebacteria, respectively) were presented here to grown at 75 °C, as well as the Hydrogenivirga highlight their significance for extremophily. In the caldilitoris previously analyzed17), belonging to the first present study, cellular polyamine analyses of several family Aquificaceae of the order Aquificales (phylum eubacterial thermophiles, acidophiles and , Aquificae), contained N4-bis(aminopropyl)norspermidine and radio-tolerant eubacteria, and archaebacterial (3(3)(3)3, abbreviation in the tables) as a major methanogens, thermophiles and extreme halophiles were polyamine (Table 1). Extremely thermophilic Aquifex, included to search for unique polyamine profiles in the Hydrogenobacter, Hydrogenobaculum and Thermocrinis extremophiles. species of the family also contained N4-bis (aminopropyl)norspermidine as a major polyamine17,24). Another branched penta-amine, N4-bis(aminopropyl)

spermidine (3(3)(3)4), was detected as a minor 60 Journal of Japanese Society for Extremophiles (2009) Vol.8 (2) polyamine in some strains of these species. A newly found in the strains HB8 (type strain) and HB27 but not validated extreme thermophile, Thermosulfidibacter detected in the strain TMY. Differences in the takaii3) grown at 70 °C, contained the two tertiary distribution of penta-amines were observed among branched penta-amines, N4-bis(aminopropyl) Thermus species and T. thermophiles strains grown at the norspermidine and N4-bis(aminopropyl)spermidine, as same growth temperature. shown in the present study (Table 1). The taxonomic Penta-amines have not been detected in the four location of the thermophile within the order of the Meiothermus species, M. chliarophilus, M. ruber, M. Aquificales has not been made clear. The genera silvanus and M. taiwanensis grown at 55-60°C 19,24). A Balnearium, Thermovibrio and Desulfuro- bacterium of new species of Meiothermus, M. timidus, grown at 60 °C, the second family Desulfurobacteriaceae, contained contained spermidine, homospermidine (44) and N4-bis(aminopropyl) spermidine17,24). The genera spermine (343) as its major polyamines (Table 1). Hydrogenothermus, Persephonella and Sulfuri- Thermospermine (334), an isomer of spermine, was hydrogenibium of the third family Hydrogenothermaceae detected as a minor polyamine component and contained N4-bis(aminopropyl)norspermidine17,24). penta-amines were not found in the moderate Distribution of the two quaternary branched thermophile. penta-amines seems to be family-specificwithin the order Aquificales (phylum Aquificae). Phylum Deferribacters It has been shown that moderately thermophilic Phylum Deinococcus-Thermus Deferribacter species belonging to the order The gram-positive mesophilic order Deinococcales Deferribacterales, D. abysai and D. desulfuricans grown (family, Deinococcaceae) is radiation-resistant3,4). It is at 60-62 °C, contained cadaverine (5), spermidine and speculated that polyamines protect DNA structure from spermine25). On the other hand, moderately thermophilic radiation damage by its ionic binding to DNA in the Calditerrivibrio nitroreducens3) grown at 60 °C, extremophiles. It has been reported that spermidine contained spermidine as their major polyamine, as shown (34) was detected ubiquitously as the major cellular in the present study (Table 1). Spermine and agmatine polyamine in the five Deinococcus species; D. were detected as minor polyamines; however, the two proteolyticus, D. radiodurans, D. radiophiles, D. grandis, amines seem to correlate with moderate thermophily in and D. radiopugnans5). In the present study, five new this organism. Deinococcus species analyzed also contained a common triamine, spermidine, as their major polyamine (Table 1). Phylum Putrescine (4) and agmatine (Agm) levels were varied in The occurrence of norspermidine (33), norspermine the Deinococcus species. Cellular concentrations of the (333), spermine, and homocaldopentamine (3334), in polyamines were normal. These results suggest that addition to spermidine, has been reported previously in cellular polyamine components do not relate directly to moderately thermophilic Thermodesulfovibrio their radio-tolerancy. However, these polyamines are yellowstonii grown at 60 °C 19). The newly validated widely distributed in various radio-sensitive usual four species (five strains) of the eubacteria. Thermodesulfovibrio3) analyzed in the present study, Cellular polyamine analyses of many species of the contained ubiquitously spermidine, spermine and a linear extremely thermophilic genus Thermus belonging to the penta-amine (Table 1). Identification of homo- order (family Thermaceae) have been caldopentamine and/or thermopentamine (3343) was reported12,13,17,19,20,24). Linear penta-amines were incomplete; however, the occurrence of linear ubiquitously distributed within the genus. However, penta-amine(s) and the absence of branched penta­ quaternary branched penta-amines were found to be amines in the moderate thermophiles belonging to the limited in the strains belonging to T. thermophiles and T. order Nitrospirales of the phylum proved interesting. filiformis, grown at 70-75 °C. In the present study, a new strain, TMY of T. thermophiles3) isolated in Japan, Phylum and grown at 75 °C, contained a linear penta-amine, Slightly thermophilic Thiofaba tepidiphila3), grown at caldopentamine (3333) and a quaternary branched 45 °C, belonging to the order Chromatiales of the class penta-amine, N4-bis (aminopropyl)norspermidine (Table Gammaproteobacteria, contained putrescine, spermidine 1). Homocaldopentamine (3334), thermopentamine and homospermidine and lacked tetra-amines (Table 1). (3343) and N4-bis (aminopropyl)spermidine have been Tetra-amines have not been detected in the 61 Journal of Japanese Society for Extremophiles (2009) Vol.8 (2) othermesophilic gammaproteobacteria such as Thiovirga, Clostridium species previously analyzed, belonging to Halothiobacillus, Thioalkalivibrio and Rheinheimers the order Clostridiales of this phylum, ubiquitously located in the order22). A tetra-amine, spermine, has contained a tetra-amine, spermine, as a major polyamine been found as a major polyamine in many moderately but did not produce penta-amines10,17,24). thermophilic proteobacteria grown at 50-55 °C within the five classes, Alphaproteobacteria, Betaproteo- Phylum Acidobacteria bacteria, Gammaproteobacteria, Deltaproteobacteria, It is well known that acidophilic Acidobacterium and Epsilonproteobacteria of the phylum Proteo- capsulatum belonging to the order Acidobacteriales and bacteria22). On the other hand, a branched penta-amine, a Fe(III)-reducing bacterium, N4-bis (aminopropyl)spermidine, has been detected in belonging to the order Holophagales, contained only moderately thermophilic Desulfothermus species, grown homospermidine 25). Cellular polyamines of a novel at 55-60 °C, belonging to the class Delta- marine bacterium, Acanthopleuribacter pedis, belonging proteobacteria17). It was the first report on the to the new order Acanthopleuribacterales of this occurrence of branched penta-amine in moderately phylum3) were analyzed in the present study (Table 1). thermophilic eubacteria. This organism grown at 25 °C in the three neutral pH media, marine broth, synthetic 199 seawater and Phylum synthetic F12 seawater (F12-SW) contained at relatively A novel, slightly/moderately thermophilic eubacterium, similar levels spermidine and spermine as its major Exilispira thermophila, grown at 50 °C, belonging to the polyamines. The culture harvested from F12-SW rich order Spirochaetales, which was recently validated3), in arginine, contained agmatine as a minor polyamine, contained spermidine and spermine as its major suggesting that the organism produces arginine polyamines, as shown in the present study (Table1). decarboxylase activity. The high spermine (a tetra-amine) level found in the mesophilic Phylum Acanthopleuribacter species phylogenetically located in Cellular polyamines have not been detected in the two this phylum is very unique in the cellular polyamine species of alkaliphilic Alkalibacterium, belonging to the distribution profiles found in various mesophilic family of the order Lactobacillales of eubacteria. However, the occurrence of the tetra-amine this phylum grown in organic media used for their has been almost limited to moderate or extreme speedy growth25). The other two Alkalibacterium thermophiles within the domain Bacteria. species, A. iburiens and A. indicireducens, grown at pH 10 and analyzed here, were absent in cellular polyamine Phylum components (Table 1). The absence of cellular Slightly thermophilic, acidophilic Acidimicrobium polyamines in the alkaliphiles seems to be related to their ferrooxidans3) grown at 45 °C and a pH of 2.0, contained alkaliphily,but the correlation between the two is not only a diamine, cadaverine (Table 1). Putrescine, clear. Although most of the eubacteria of the family spermidine and spermine were not detected in the Carnobacteriaceae, grown in mesophilic and neutral pH slightly thermophilic belonging to the order growth conditions, contained a significant amount of Acidimicrobiales of this phylum, while the three putrescine and/or spermidine, Alkalibacterium polyamines showed wide distributions in various olivoapovliticus, grown at pH 7.2 and incerta, moderately thermophilic actinobacteria6). This is the grown at pH 7.4, lacked polyamnes25). Spermidine was first report on the occurrence of cadaverine alone within detected as the major polyamine in Marinilactibacillus the phylum Actinobacteria. Furthermore, this is also species grown at an alkaline pH of 8.525). the first report within eubacteria and archaebacteria, Moderate thermophiles, Desulfurispora thermophila, which is interesting due to the phylogenetic and Desulfotomaculum alcoholivorax, Desulfotomaculum chemotaxonomic significance of the cellular polyamine hydrothermale, and Caloramator australicus grown at profile. Acidophily did not affect to long polyamine 55-60 °C, contained spermidine and spermine (Table1). distribution, as found in acidophilic Acidobacterium Other moderately thermophilic two Pelotomaculum, species (grown at pH 4.0) of the phylum Acidobacteria seven Desulfotomaculum, a Caloramator and four (Table 1).

62 Journal of Japanese Society for Extremophiles (2009) Vol.8 (2)

Table 1.Cellular concentrations of polyamines of themophilic, acidophilic, alkaliphilic and radio-resistant bacteria (eubacteria)

4, putrescine; 5, cadaverine; 33, norspermidine; 34, spermidine; 44, homospermidine; 333, norspermine; 3(3)3, N4-aminopropylnorspermidine; 3(3)4, N4-aminopropylnorspermine; 343, spermine; 334, thermospermine; 344, aminopropylhomospermidine; 3333, caldopentamine; 3343, thermopentamine; 3334, homocaldopentamine; 3(3)(3)4, N4-bis(aminopropyl)spermidine; 3(3)(3)3, N4-bis(aminopropyl)norspermidine; 33333, caldohexamine; Agm, agmatine; *, not calculated; -, not detected (<0.005); T, type strain; ℃, culture temperature; ATCC, American Type Culture Collection, Manassas, Virginia, USA; JCM, Japan Collection of Microorganisms, RIKEN, Wako, Saitama, Japan; NBRC, Biological Resource Center, National Institute of Technology and Evaluation, Kisarazu, Chiba, Japan;NCIMB, the National Collection of Industrial, Food and Marine Bacteria, Aberdeen, Scotland, UK. (a), (b) and (c) were cited from 16), 25) and 19), respectively.

63 Journal of Japanese Society for Extremophiles (2009) Vol.8 (2)

Domain Archaea (Archaebacteria) 60 °C belonging to the same order15), the occurrence of Phylum Euryarchaeota the branched penta-amine in Methanocaldococcus seems Six methanogenic orders to correlate to their extreme thermophily. Archaebacterial methanogens are phylogenetically It has been reported that the hyperthermophilic divided into six orders belonging to the Methanopyrus kandleri strain AV19 (type strain) of the phylumEuryarchaeota of the domain Archaea1,2). In the order Methanopyrales contained a significant amount of order Methanobacteriales, the five Methanobacterium agmatine, suggesting that the occurrence of agmatine species analyzed showed conflicting results in regards to correlates to its thermophily13). Another strain, strain the occurrence of spermidine or homospermidine as their 116, recently classified as a strain of M. kandleri, major polyamines (Table 2). Moderately thermophilic contained spermidine as its major polyamine, as reported Methanothermobacter and Methanothermus species here (Table 2). In spite of the common absence of long belonging to this order, contained spermine as previously polyamines, the difference in polyamine profile between reported whereas spermine has not been detected in the the two methanogenic hyperthermophiles, grown at mesophilic Methanobacterium as well as mesophilic 95-100 °C is mysterious. However, long linear and/or Methanobrevibacter or Methanosphaera15,16,27). branched polyamines have been detected in other Methanocella paludiocola located in the new sixth extremely thermophilic or hyper-thermophilic eubacteria methanogenic order Methanocellales1) contained homo- and archaebacteria. spermidine as its major polyamine (Table 2). This is the first polyamine analysis on the new genus of the new Order Archaeoglobales order. Three extremely thermophilic Archaeoglobus species, Three different polyamine profiles were found in the including a new species, A. infectus1) grown at 70 °C and three species of Methanosaeta belonging to the order analyzed here, and the previously analyzed A. flugidus (Table 2), suggesting that they should and A. profundus grown at 85 °C 11), ubiquitously be belong to a different genera. It has been reported contained N4-bis (aminopropyl)spermidine and N4-bis that four Methanosarcina species ubiquitously contained (aminopropyl)norspermidine (Table 2). The occurrence homospermidine as their major polyamine. Methano- of the two branched penta-amines as the major lobus profundi belonging to the order contained polyamines in the genus of the order Archaeoglobales is putrescine, homospermidine and agmatine (Table 2). unique among the domain Archaea. The synthetic Distribution profiles of spermidine and homospermidine ability of the two triamines, norspermidine and were heterogeneous and conflicted with the order. spermidine, increases during the occurrence of the two Methanofollis ethanolicus contained putrescine, branched penta-amines in these extreme thermophiles. cadaverine and homospermidine, as shown in the present Relatively lower polyamine levels in A. infectus might be study (Table 2). In the order Methanomicrobiales, due to the low growth temperature of 70 °C. polyamine distribution profiles varied within six other genera, Methanolinea (putrescine, spermidine and Order Thermococcales spermine), Methanomicrobium (spermidine), Methano- N4-bis (aminopropyl)spermidine and agmatine found as culleus (spermidine and spermine), Methanogenium the major polyamine in a new Thermococcus species, T. (putrescine), Methanospirillum (putrescine and sper- onnurineus1), grown at 80 °C (Table 2), as well as other midine) and Methanocalculus (putrescine, spermidine extremely thermophilic Thermococcus, Pyrococcus and and homospermidine) as analyzed in previous Palaeococcus species belonging to the order 16,17,27) studies . Thermococcales, have been reported in previous Now, two extremely thermophilic Methano- studies15,16,18,23). In addition, temperature (70-85 °C) caldococcus species belonging to the order Methano- and salt (0.15-2.5 % NaCl) dependent synthesis of the coccales, M. fervens and M. infernus, grown at 85 °C, branched penta-amine and agmatine in these extreme 4 contained N -bis (aminopropyl)spermidine as a major thermophiles was reported on previously16). The polyamine (Table 2). This branched penta-amine has polyamine level decreased under the growth conditions also been found in three other Methanocaldococcus at 70 °C or in 0.15 % NaCl16). T. onnurineus grown at 15) species previously analyzed . Since 80 °C in the presence of 2.5 % NaCl contained a higher 4 N -bis(aminopropyl)spermidine has not been detected in agmatine concentration among the 22 Thermococcus mesophilic Methanococcus grown at 37 °C and species previously analyzed. moderately thermophilic Methanothermococcus grown at 64 Journal of Japanese Society for Extremophiles (2009) Vol.8 (2)

In the order Thermales, extremely thermophilic Thermus Order Halobacteriales as well as Vulcanithermus and Marinithermus contained Polyamines of 117 strains of mesophilic, extremely several linear and branched penta-amines and halophilic archaebacteria (informally halobacteria), hexa-amines. Moderately thermophilic Oceamithermus belonging to the order Halobacteriales of the phylum and Meiothermus contained a tetra-amine, spermine, and Euryarchaeota have been analyzed8,9,15,27). lacked penta-amines. Other various moderately or Twenty-three newly published species (type strain), slightly thermophilic eubacteria contained only spermine. including the five new genera Haladaptatus, Halopiger, Additional polyamine catalogues of new thermophilic Haloplanus, Haloquadratum, and Halovivax grown at members should be presented to define their association 30-37 °Cin the organic media containing 20 % NaCl, with thermophily. Acidophilic, alkaliphilic and were analyzed in the present study. A trace amount of radio-tolerant eubacterial-mesophiles contained diamines spermidine and/or spermine was detected in 20 strains and triamines, however, some lacked them. Further (Table 2). Cellular polyamine could not be detected in polyamine analyses of mesophilic extremophiles are also some halobacteria as shown in this study and previous useful as a chemotaxonomic marker, furthermore, as an studies8,9,15,27). Agmatine has been sporadically extremophilic property. distributed within halobacteria grown at pH factors Methanogenic euryarchaebacteria are distributed into between 7.0-9.08,9,15,27); however, it was detected in two six orders and order-specific their polyamine profiles strains in our present report (Table 2). A halalkaliphile, have been proposed. Moderately thermophilic Natronorubrum sulfidifaciens, grown at a pH of 9.5, Methanothermobacter and Methanothermococcus, and contained a higher agmatine level than other extremely thermophilic Methanothermus contained halalkaliphiles grown at pH 8.5-9.0 8,9,15,21). The spermine. Extremely thermophilic Methanocaldo- cellular accumulation of agmatine is possible in coccus and Methanotorris contained a quaternary halalkaliphiles. On the other hand, a high spermine branched penta-amine. The absence of the tetra-and level was sporadically found in halobacteria8,9,15,21). penta-amines in hyperthermophilic Methanopyrus Polyamines of halobacteria were detected at a nmol/g containing spermidine or agmatine is a unique polyamine wet weight of high salt-containing cell pellet, while other profile. New methanogens available in the future various eubacteria and archaebacteria were present at a should be analyzed to evaluate chemotaxonomic level of μmol/g wet weight. Since it is known that significance of cellular polyamine type. In major parts of intracellular polyamines are released into euryarchaebacteria, the branched penta-amines were the culture medium in high salt growth conditions,poor ubiquitously distributed within the thermophilic orders cellular polyamine levels correlate to less binding to Archaeoglobales and Thermococcales but not in the intracellular nucleic acid and protein components. acidophilic (mesophilic/moderately thermophilic) order Thermoplasmatales. The branched penta-amines have Conclusion not been detected in the crenarchaebacterial, In eubacteria, quaternary branched penta-amines, N4-bis thermophilic orders Caldisphaerales, Desulfurococcales (aminopropyl)norspermidine and N4-bis(aminopropyl) and Thermoproteales, and thermoacidophilic order spermidine, were distributed in thehyper-/extreme Sulfolobales, in which spermine and linear penta-amines thermophiles belonging to the order Aquificales in have been detected. Low levels of spermidine, addition to the modelately thermophilic Desulfothermus spermine and agmatine were ubiquitously found in of the class Deltaproteobacteria and other many various euryarchaebacterial extreme halophiles extreme/moderate thermophiles belonging to the orders belonging to the order Halobacteriales. Analysis of Thermoanaerobacteriales, Clostridiales and Bacillales. intracellular accumulation and extracellular secretion of A linear penta-amine has been found moderately polyamines in the extreme halophiles under high thermophilic Thermodesulfovibrio belonging to the order temperature and alkaline pH growth conditions are Nitrospirales and the extreme thermophiles belonging to planning. the orders Thermotogales and Thermodesulfobacteriales.

65 Journal of Japanese Society for Extremophiles (2009) Vol.8 (2) Table 2.Cellular concentrations of polyamines of methanogenic, thermophilic and extremely halophilic archaea (archaebacteria) Culture Polyamines (μmol/g wet wt. cell): 343/ Phylum Euryarchaeota ℃ pH 4 5 34 44 343 3(3)4 3(3)4 3(3)(3)4 Agm Order Methanobacteriales Methanobacterium formicicum JCM 10132T (a) 37 7.0 0.01 - 0.10 - 0.45 - 0.45 - - Methanobacterium sp. JCM 10400 (a) 37 7.4 0.09 - 2.30 ------Methanobacterium bryantii NBRC 104951T 37 7.4 - - - 0.20 0.60 - 0.60 - - Methanobacterium ivanovii NBRC 104952T 37 7.0 - - 0.10 - 0.02 - 0.02 - - Methanobacterium sp. NBRC 105039 37 7.2 0.02 0.03 0.25 - 0.50 - 0.50 - - Order Methanocellales Methanocella paludicola JCM 13418T 37 7.0 0.35 - - 1.40 - - - - - Order Methanosarcinales Methanosaeta thermophila NBRC 101360T (b) 55 7.0 - - 1.50 - - - - 0.05 - Methanosaeta concilii JCM 10134T (c) 37 7.0 - - - 0.50 - - - - - Methanosaeta harundinacea NBRC 104789T 37 7.0 - - 0.24 0.26 0.40 - 0.40 - - Methanolobus profundi NBRC 104158T 30 7.0 0.90 - - 0.72 - - - - 0.25 Order Methanopyrales Methanopyrus kandleri AV19 JCM 9639T (a) 95 6.5 - - 0.04 - 0.02 - 0.02 - 0.22 Methanopyrus kandleri 116 JCM 15049 100 6.5 0.20 - 0.55 - 0.02 - 0.02 - - Order Methanococcales Methanocaldococcus fervens JCM 15782T 85 6.0 0.18 - 0.68 - * * 0.76 0.90 0.04 Methanocaldococcus infernus JCM 15783T 85 6.0 0.09 - 0.16 - * * 0.36 0.80 0.21 Order Methanomicrobiales Methanofollis ethanolicus JCM 15103T 37 6.5 0.68 0.70 - 0.30 - - - - - Polyamines (μmol/g wet wt. cell): 333/ Order Archaeoglobales 33 34 343 3(3)4 3(3)3 3(3)(3)4 3(3)(3)3 Archaeoglobus fulgidus JCM 9528T (d) 85 6.9 - 0.56 2.28 0.20 - 1.44 0.24 Archaeoglobus profundus JCM 9629T (d) 85 6.9 - 0.60 2.10 - - 0.57 1.47 Archaeoglobus infectus NBRC 100649T 70 6.5 0.03 0.15 0.02 - 0.35 0.45 0.09 Polyamines (μmol/g wet wt. cell): 343/ 4 34 343 3(3)4 3(3)4 3(3)(3)4 Agm Order Thermococcales Thermococcus onnurineus JCM 13517T 80 7.0 0.10 0.90 * * 0.82 0.46 0.55 Polyamines (nmol/g wet wt. cell): Order Halobacteriales 4 5 34 343 Agm Halalkalicoccus jeotgali JCM 14584T 37 7.4 - - - 0.05 - Haladaptatus paucihalophilus JCM 13897T 30 7.0 - - - 0.04 - Haloarcula amylolytica JCM 13557T 37 7.0 - - 0.04 0.07 - piscisalsi JCM 14661T 37 7.0 - - - 0.03 - Halobacterium noricense JCM 15102T 37 7.0 - - - 0.04 - Halococcus qingdaonensis JCM 13587T 37 7.0 - - - - - Halococcus thailandensis JCM 13552T 37 7.0 - - 0.02 0.10 - Haloferax elongans JCM 14791T 37 7.4 - - - 0.04 - Haloferax larsenii JCM 13917T 37 7.0 - - - 0.04 - Haloferax mucosum JCM 14792T 37 7.0 - - 0.01 0.12 - Haloferax prahovense JCM 13924T 37 7.0 - - - - - Halopiger xanaduensis JCM 14033T 37 8.0 - - - 0.06 - Haloplanus natans JCM 14081T 37 7.0 - - 0.15 - - Haloquadratum walsbyi JCM 12705T 37 7.5 - - - - - Halorubrum arcis JCM 13916T 37 7.0 - - - 0.05 - Halorubrum eijinorense JCM 14265T 37 7.0 - - - 0.05 - Halorubrum kocurii JCM 14978T 37 7.0 - - 0.10 0.82 - Halorubrum litoreum JCM 13561T 37 7.0 - - - 0.10 - Halovivax asiaticus JCM 14624T 37 7.4 - - - 0.10 0.15 Halovivax ruber JCM 13892T 37 7.0 - - - 0.02 - Natrinema gari JCM 14663T 37 7.0 - - - 0.01 - Natronococcus jeotgali JCM 14583T 37 7.4 - - - 0.08 - Natronorubrum sulfidifaciens JCM 14089T 37 9.5 - - 0.17 0.03 0.50 4, putrescine; 5, cadaverine; 33, norspermidine; 34, spermidine; 44, homospermidine; 333, norspermine; 3(3)3, 4 4 N -aminopropylnorspermidine; 3(3)4, N -aminopropylspermidine; 343, spermine; 3(3)(3)3, N4-bis(aminopropyl)norspermidine; 3(3)(3)4, N4-bis(aminopropyl)spermidine; Agm, agmatine; *, not calculated; -, not

detected (<0.005); T, type strain; ℃, culture temperature; JCM, Japan Collection of Microorganisms, RIKEN, Wako, Saitama, Japan; NBRC, Biological Resource Center, National Institute of Technology and Evaluation, Kisarazu, Chiba, Japan. (a), (b), (c) and (d) were cited from 27), 15), 16) and 11), respectively.

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Acknowledgments belonging to Rhodothermus, Thermus and We would like to thank JCM and NBRC (Dr. K. Mori Thermonema. J. Gen. Appl. Microbiol. 38: 575-584. and Dr. Y. Uchino) for their generous donation of 13) Hamana, K., Hamana, H., Shinozawa, T., Niitsu, M., eubacterial and archaebacterial strains. Samejima, K., and Itoh, T. 1999. Polyamines of the thermophilic eubacteria belonging to the genera References Aquifex, Thermodesulfobacterium, Thermus and 1) Archaea, The NCBI Entrez website 2009. Meiothermus, and the thermophilic archaebacteria National Center for Information. belonging to the genera Sulfurisphaera, http://www.ncbi.nlm.nih.gov/Taxonomy/ Sulfophobococcus, Stetteria, Thermocladium, 2) Archaea, Bergey’s Manual of Systematic Bacteriology, Pyrococcus, Thermococcus, Methanopyrus and 2 nd ed. Vol. 1. D. R. Boone and R. W. Castenholz eds. Methanothermus. Microbios 97: 117-130. Springer, 2001. 14) Hamana, K. and Hosoya, R. 2006. Polyamines of 3) Bacteria, The NCBI Entrez Taxonomy website 2009. thermophilic Eubacteria and Archaebacteria. National Center for Biotechnology Information. Chemistry and Biology 44: 320-330 ( in Japanese ). http://www.ncbi.nlm.nih.gov/Taxonomy/ 15) Hamana, K., Hosoya, R., and Itoh, T. 2007. 4) Bacteria, Bergey’s Manual of Systematic Bacteriology, Polyamine analysis of methanogens, thermophiles and 2 nd ed. Vol. 1. D. R. Boone and R. W. Castenholz eds. extreme halophiles belonging to the domain Archaea. J. Springer, 2001. Jpn. Soc. Extremophiles 6: 25-31. 5) Hamana, K. 1994. Polyamine distribution patterns in 16) Hamana, K., Hosoya, R., Niitsu, M., Hayashi, H., aerobic gram-positive cocci and some radio-resistant and Itoh, T. 2008. Temperature and salt dependent bacteria. J. Gen. Appl. Microbiol. 40: 181-195. long linear and branched polyamine syntheses in the 6) Hamana, K. 1999. Polyamine distribution catalogues thermophiles belonging to the domain Archaea. J. Jpn. of clostridia, acetogenic anaerobes, actinobacteria, Soc. Extremophiles 7: 21-27. , heliobacteria and haloanaerobes within 17) Hamana, K., Hosoya, R., Yokota, A., Niitsu, M., gram-positive eubacteria -Distribution of spermine and Hayashi, H., and Itoh, T. 2008. Distribution of long agmatine in thermophiles and halophiles-. Microbiol. linear and branched polyamines in the thermophil Cult. Coll. 15: 9-28. belonging to the domain Bacteria. J. Jpn. Soc. 7) Hamana, K. 2002. Extraction and HPLC analysis of Extremophiles 7: 10-20. bacterial polyamines. Ann. Gunma Health Sci. 23: 18) Hamana, K. and Itoh, T. 2001. Polyamines of the 149-153 ( in Japanese ). hyperthermophilic archaebacteria belonging to the 8) Hamana, K., Hamana, H., and Itoh, T. 1995. genera Thermococcus and Methanothermus and two Ubiquitous occurrence of agmatine as the major new genera Caldivirga and Palaeococcus. Microbios polyamine within extremely halophilic archaebacteria. 104: 105-114. J. Gen. Appl. Microbiol. 41: 153-158. 19) Hamana, K., Niitsu, M., Samejima, K., Itoh, T., 9) Hamana, K., Hamana, H., and Itoh, T. 1998. Hamana, H., and Shinozawa, T. 1998. Polyamines of Polyamine analysis of extremely halophilic the thermophilic eubacteria belonging to the genera archaebacteria. Ann. Gunma Health Sci. 19: 1-4. Thermotoga, Thermodesulfovibrio, Thermoleophilum, 10) Hamana, K., Hamana, H., Niitsu, M., and Samejima, Thermus, Rhodothermus and Meiothermus, and the K. 1996. Polyamines of thermophilic Gram-positive thermophilic archaebacteria belonging to the genera anaerobes belonging to the genera Caldicellulosiruptor, Aeropyrum, Picrophilus, Methanobacterium and Caloramator, Clostridium, Coprothermobacter, Methanococcus. Microbios 94: 7-21. Moorella,Thermoanaerobacter and 20) Hamana, K., Niitsu, M., Samejima, K., and Thermoanaerobacterium. Microbios 85: 213-222. Matsuzaki, S. 1991. Polyamine distribution in 11) Hamana, K., Hamana, H., Niitsu, M., Samejima, K., thermophilic eubacteria belonging to Thermus and and Itoh, T. 1996. Polyamines of hyperthermophilic Acidothermus. J. Biochem. 109: 444-449. archaebacteria, Archaeoglobus, Thermococcus, Pyro- 21) Hamana, K., Otsuka, E., Eguchi, F., and Niitsu, M. baculum and Sulfolobus. Microbios 87: 69-76. 2005. Occurrence of homospermidine and canaval- 12) Hamana, K., Hamana, H., Niitsu, M., Samejima, K., mine as a major polyamine in mushrooms. Mushroom and Mtsuzaki, S. 1992. Distriution of unusual long and Sci. Biotech. 13: 95-102. branched polyamines in thermophilic eubacteria 22) Hamana, K., Sato, W., Gouma, K., Yu, J., Ino, Y., Umemura, Y., Mochizuki, C., Takatsuka, K., Kigure, 67 Journal of Japanese Society for Extremophiles (2009) Vol.8 (2)

Y., Tanaka, N., Itoh, T., and Yokota, A. 2006. Cellular 25) Hosoya, R., Yokoyama, Y., Hamana, K., and Itoh, T. polyamine catalogues of the five classes of the phylum 2006. Polyamine analysis within the eubacterial Proteobacteria. Ann. Gunma Health Sci. 27: 1-16. thirteen phyla Acidobacteria, Actinobacteria, Chlorobi, 23) Hamana, K., Tanaka, T., Hosoya, R., Niitsu, M., and , Chrisiogenetes, Deferribacteres, Itoh, T. 2003. Cellular polyamines of the acidophilic, , Firmicutes, , thermophilic and thermoacidophilic archaebacteria, , Nitrospirae, , and Acidilobus, Ferroplasma, Pyrobaculum, Pyrococcus, . Microbiol. Cult. Coll. 22: 21-33. Staphylothermus, Thermococcus, Thermodiscus and 26) Niitsu, M., Samejima, K., Matsuzaki, S., and Vulcanisaeta. J. Gen. Appl. Microbiol. 49: 287-293. Hamana, K. 1993. Systematic analysis of naturally 24) Hosoya, R., Hamana, K., Niitsu, M., and Itoh, T. occurring linear and branched polyamines by gas 2004. Polyamine analysis for chemotaxonomy of chromatography-mass spectrometry. J. Chromatogr. thermophilic eubacteria: Polyamine distribution 641: 115-123. profiles within the orders Aquificales, 27) Tanaka, T., Hamana, K., and Itoh, T. 2002. Thermotogales, Thermodesulfobacteriales, Thermales, Polyamine analysis of extremely halophilic Thermoanaerobacteriales, Clostridiales and Bacillales. archaebacteria and methanogenic archaebacteria. Ann. J. Gen. Appl. Microbiol. 50: 271-287. Gunma Health Sci. 23: 137-143.

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