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Polyamine Analysis of Thermophilic, Acidophilic, Alkaliphilic and Radio

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Polyamine Analysis of Thermophilic, Acidophilic, Alkaliphilic and Radio Journal of Japanese Society for Extremophiles (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 bacteria belonging to the domain Bacteria and methanogens, thermophiles and extreme halophiles belonging to the domain Archaea -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 Methanopyrus as well as in thermophilic, acidophilic, alkaliphilic or radio-resistant the mesophilic genera, Methanobacterium, Methano- bacteria (eubacteria) and 35 methanogenic, thermophilic saeta, Methanolobus, Methanocella 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 halophile, phylum Aquificae and Thermus of the phylum methanogen, polyamine, thermophile 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 Acidobacteria was a unique profile. Alkaliphilic culture medium composition and growth phase, it has Alkalibacterium species were devoid of cellular been suggested that polyamine distribution profiles in polyamines. In the phylum Euryarchaeota 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 hyperthermophiles, 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). acidophiles, 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 alkaliphiles, 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
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