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Home , Aquimonas, Asticcacaulis, Azohydromonas, Betaproteobacteria, Deltaproteobacteria, Derxia

Ann Gunma Health Sci 27：1－16，2006 1

Cellular Polyamine Catalogues of the Five Classes of the : Distributions of Homospermidine within the Class , Hydroxyputrescine within the Class , Norspermidine within the Class , and Spermine within the Classes and

Koei HAMANA1)＊, Wakako SATO1), Kanako GOUMA1), Jia YU1), Yurie INO1), Yukiko UMEMURA1), Chiharu MOCHIZUKI1), Kenji TAKATSUKA1), Yoshie KIGURE1), Nagisa TANAKA1), Takashi ITOH2) and Akira YOKOTA3)

(Received September 30, 2006; Accepted December 11, 2006)

Abstract : Cellular polyamines extracted from reclassified or newly validated 47 alphaproteobacteria, 46 betaproteobacteria, 96 gammaproteobacteria, 12 deltaproteobacteria and 10 epsilonproteobacteria were analyzed by high-performance liquid chromatography. Homospermidine was widely distributed within the class Alphaproteobacteria, however, homospermidine-dominant type, spermidine-dominant type and homospermidine/spermidine- dominant type were found and the three triamine profiles were -specific. The all genera belonging to the class Betaproteobacteria, ubiquitously contained putrescine and 2- hydroxyputrescine. Triamines were absent in almost betaproteobacteria. Many genera, including psychrophilic , of the class Gammaproteobacteria, contained putrescine and spermidine as the major polyaminenes. Diaminopropane and norspermidine were selectively distributed in several genera of the class Gammaproteobacteria. Spermidine was the major polyamine in the classes Deltaproteobacteria and Epsilonproteobacteria. Spermine was found in some thermophiles within Betaproteobacteria, Deltaproteobacteria and Epsilonproteobacteria, suggesting that the occurrence of spermine correlate to their thermophily. Additional these polyamine catalogues serve for the classification of the phylum Proteobacteria, as a chemotaxonomic marker.

Key words : Homospermidine, Hydroxyputrescine, Norspermidine, Polyamine, Proteobacteria, Spermidine, Spermine

1)Department of Laboratory Sciences, School of Health Sciences, Faculty of Medicine, Gunma University, 39-15 Showa-machi 3-chome, Maebashi, Gunma 371-8514, Japan 2)Microbe Division, BioResouece Center, RIKEN, Hirosawa 2-1, Wako, Saitama 351-0198, Japan 3)Laboratory of Biosciences, Institute of Molecular and Cellular Biosciences, The University of Tokyo, 1-1 Yayoi 1- chome, Bunkyo-ku, Tokyo 113-0032, Japan ＊Reprint address : Gunma University School of Health Sciences, Maebashi 371-8514, Japan 2

INTRODUCTION within Proteobacteria. Polyamine analyses were The phylum Proteobacteria (formerly the class carried out in the reclassified and 96 newly validated Proteobacteria) of the contains the gammaproteobacteria belonging to eight orders (42 five classes (formerly subclasses) genera) including various marine psychrophiles. Alphaproteobacteria, Betaproteobacteria, Within the class Deltaproteobacteria, some Gammaproteobacteria, Deltaproteobacteria and contained homospermidine, however, it 1, 2) Epsilonproteobacteria . Diamines such as was absent in the other deltaproteobacteria diaminopropane (1,3-diaminopropane), putrescine, containing spermidine6, 12). Spermidine was the major cadaverine and hydroxyputrescine (2- polyamine in the members of the class hydroxyputrescine), triamines such as spermidine, Epsilonproteobacteria, previously analyzed6, 12). To norspermidine, homospermidine, hydroxyspermidine survey spermine distribution in thermophilic 1 (2-hydroxyspermidine) and acetylspermidine (N - proteobacteria, polyamines of 12 newly published acetylspermidine), a triamine, spermine, and a deltaproteobacteria including a novel thermophile and guanidinoamine, agmatine, have been found as their 10 newly published epsilonproteobacteria including cellular polyamine components. Our studies on five novel thermophiles were analyzed. polyamine distribution profiles within proteobacteria already provided valuable chemotaxonomic MATERIALS AND METHODS 3-12) informations on their classifications . Afterward, The proteobacteria were grown at the optimum many new members of proteobacteria have been growth temperature in the media designated by the isolated and validated in Bergey’ Manual of culture collections (listed in Tables 1-4) as shown by 1) Systematic Bacteriology , National Centre for Medium No., Marine broth (MB) (DIFCO Ltd., Detroit, 2) Biotechnology Information (NCBI) and Validation USA), Nutrient broth (NB) (Nissui Pharmaceutical Co. Lists of International Journal of Systematic and Ltd., Tokyo, Japan), NB supplemented with 5% NaCl 13) Evolutionary Microbiology . (NB-NaCl), Trypticase soy broth (TSB) (BBL, Bectone As shown in the previous studies, a novel triamine, Dickinson and Company, Cockeysville, MD, USA), homospermidine, was selectively distributed in the Brain heart infusion broth (BHIB) (Nissui), yeast class Alphaproteobacteria and a group of the class extract-mannitol medium (YM), peptone-yeast extract 6, 9, 11, 14- Gammaproteobacteria within Proteobacteria medium (PY) and PY dissolved in seawater (PYSW). 16) . Homospermidine distribution in the reclassified Polyamine-free synthetic 199 medium (Nissui and 47 newly validated alphaproteobacteria (belonging Pharmaceutical Co. Ltd), 199 medium supplemented to 39 genera), divided into five orders, were with 5% NaCl (199NaCl) and 199 medium dissolved in detarmined to elucidate variations in their polyamine seawater (199SW) were also used. at the profiles, and to evaluate the usefulness of polyamine stationary phase were harvested by centrifugation. pattern as a phenotypic marker. The pellets were homogenized in 0.5 M perchloric A novel hydroxydiamine, hydroxyputrescine, acid (HClO4). Polyamines were extracted into HClO4 6, 8, 14, widespread within the class Betaproteobacteria and analyzed by high-performance liquid 17) . Its distribution was limited in betaproteobacteria chromatography (HPLC) on a Hitachi L6000 High- 20) within Proteobacteria. To determine the occurrence Speed Liquid Chromatograph . Estimated cellular of hydroxypolyamines, cellular polyamines of the concentrations of polyamines are listed in Tables 1, 2, reclassified and 46 newly validated betaproteobacteria 3 and 4. belonging to 21 genera were analyzed. Norspermidine was widespread within the RESULTS AND DISCUSSION gammaproteobacteria belonging to the order Class Alphaproteobacteria (Table 1) 6, 7, 10, 18, Vibrionales of the class Gammaproteobacteria Order 19) . This uncommon triamine was found in this class The wide distribution of homospermidine in this and it has never been detected in other four classes order has been known. Homospermidine was found in 3 Cellular concentrations of polyamines alphaproteobacteria Table 1. 4 5 the newly analyzed three species as profiles of spermidine and homospermidine are well as Sphingosinicella microcystinivorans21), in heterogeneous in the six Brevundimonas species. the present study. The major polyamine was Others spermidine in Sphingomonas azotoformans, Homospermidine was distributed in the newly chungbukense, Sphingobium validated Pleomorphomonas and Kordiimonas amiense, Sphingobium japonicum, Sphingopyxis species, however, their phylogenetic positions within taejonensis and three Novosphingobium species. the class Alphaproteobacteria are unknown. The Sphingomonas soli and two Shinella species major triamine in Terasakiella pusilla was contained spermidine and homospermidine as the spermidine. major polyamines, indicating a unique triamine profile within this order. Other 13 authentic Sphingomonas Class Betaproteobacteria (Table 2) species contained homospermidine. It has been It has been demonstrated that 66 betaproteobacteria, reported that three Sphingobium species, six consisting the six orders , Novosphingobium species and three Sphingopyxis Hydrogenophilales, Methylophilales, Neisseriales, species contained spermidine22). New Erythrobacter and , ubiquitously and Porphyrobacter species contained spermidine contained hydroxyputrescine in addition to alone. These results suggest that the distribution of putrescine6, 8). Hydroxyputrescine has not been homospermidine in this order is genus-specific, detected in almost other four classes of the phylum however, are chemotaxonomically conflicting with the Proteobacteria. Therefore, it has been proposed that classification of some Sphingomonas species. the occurrence of this hydroxydiamine served as a Order chemotaxonomic marker for the class Dinoroseobacter shibae contained spermidine and Betaproteobacteria. homospermidine. Spermidine was the major , , Caldimonas , polyamine in the new alphaproteobacteria located in , , , , the genera, Paracoccus, Pelagibaca, , , , Catallibacterium, Jannaschia, Pseudovibrio, , Hylemonella, Janthinobacter- Roseovarius, Pseudorhodobacter and Yagia species, ium, , Mitsuaria, , , analyzed in the present study. and Wautersia belonging to Order Burkholderiales, and “Pseudomonas Homospermidine and spermidine were found in butanovora” belonging to Rhodocyclales, Azospirillum and Tistrella, whereas spermidine was Hydrogenophilus belonging to Hydrogenophilales, detected in Defluvicoccus, Saccharibacter, Kozakia, and Alysiella, Conchiformibium, Bergeriella, Asaia, Neoasaia, Gluconobacter and Acetobacter Simonsiella and belonging to species, analyzed in the present study. Neisseriales, analyzed in the present study, Order Rhizobiales ubiquitously contained putrescine and Methylocapsa acidiphila, Kaistia adipata and hydroxyputrescine as the major polyamines. Hoeflea phototrophica contained spermidine alone. Hydroxyputrescine was found also in Thiobacter Rhodopseudomonas faecalis contained subterraneus, Sivamonas terrae and “Pseudomonas homospermidine alone. Pseudaminobacter defluvii jianii” classified into this class. Spermidine was and Roseomonas lacus contained spermidine and found in thermophiles and some mesophilic species homospermidine. Three types on triamine profile analyzed in the present study, however, phylogenetic were observed in this order. significance of this triamine was not clear. Order Hydroxyspermidine was produced by Asticcacaulis taihuensis contained spermidine, aminopropyltransfer into hydroxyputrescine, and homospermidine and agmatine. Phenylobacterium coexisted with hydroxyputrescine in the koreense contained spermidine alone. Distribution betaproteobacteria containing spermidine as a major 6 Cellular concentrations of polyamines betaproteobacteria Table 2. 7 8 polyamine. Norspermidine and homospermidine have Pseudoalteromonas phenolica and Psychromonas not been found in the betaproteobacteria analyzed in marina growing at 15℃ is interesting, however, the previous6, 8) and present studies. spermine has been detected ordinarily in thermophilic Spermidine and spermine were detected in a proteobacteria as shown in the present study. moderately thermophilic species of Hydrogenophilus, Spermine was not found in the psychrophilic H. hirschii, growing at 60℃ as a major polyamine, Psychromonas, Shewanella, Colwellia and Moritella suggesting that the occurrence of spermine, a tetra- species growing under 10℃. amine, correlate to its thermophily upper 60℃. On Order Vibrionales the other hand, significant amount of spermine was Norspermidine was found in three new species of not found in slightly thermophilic (thermotolerant) Vibrio and three new species of Photobacterium. Caldimonas manganoxidans growing at 45℃, This triamine had been detected in almost species of 19) Hydrogenophilus thermoluteolus growing at 50℃ these two genera previously analyzed . and Thiobacter subterraneus growing at 55℃. Order Oceanospirillales Uptake of spermine by other mesophilic species from Former Oceanospirillum species have been media is not excluded. devided into the three genera Oceanospirillum, Pseudospirillum and Oceanobacter belonging to the Class Gammaproteobacteria (Table 3) order Oceanospirillales and the genus Marinobacter Order Alteromonadales belonging to the order Alteromonadales1, 2, 13). Norspermidine was detected in two newly analyzed However, the analyzed species belonging to the four species of Psychromonas in the present study and a genera as well as the related genera Marinomonas, Moritella species previously analyzed7). Four types of Halomonas and Chromohalobacter belonging to this polyamine profiles, putrescine alone, putrescine plus order contained putrescine and spermidine as the cadaverine, hydroxyputrescine plus putrescine, and major polyamine. Microbulbifer and Agarivorans putrescine plus spermidine, were found in Shewanella contained cadaverine and spermidine. Occurrence of species including newly analyzed species. Cadaverine cadaverine seems to be species-specific in the genera was the major polyamine in the two new species of Halomonas and Marinomonas. Moritella, however, spermidine had not been found in Order this genus. Two species of Colwellia contained no Almost species of the genera Azotobacter, cellular polyamines. Polyamine disribution profiles Azorhizophilus, Psychrobacter, Moraxella and were varied within the mesophilic/psychrophilic Pseudomonas contained putrescine, cadaverine and marine proteobacteria belonging to the genera spermidine. Two Cellvibrio species contained Psychromonas, Shewanella, Colwellia and Moritella, putrescine alone. A new species as well as previously indicating that these genera are phylogenetically and analyzed species of Acinetobacter contained chemotaxonomically conflicting taxa. diaminopropane alone23). Hydroxyputrescine was detected in several Order Shewanella and a Colwellia species belonging to this Five Thiothrix species of the order Thiotrichales order. The occurrence of this hydroxydiamine related were heterogeneous in their polyamine profiles. to branching point of the two classes Thiomicrospira and Methylophaga had same Betaproteobacteria and Gammaproteobacteria, polyamine profile, putrescine plus spermidine. therefore, serve as a chemotaxonomic marker for Order betaproteobacteria within the phylum Proteobacteria. Among Pseudoxanthomonas, Rhodanobacter and Spermidine was the major polyamine in Alteromonas, Aquimonas species analyzed in the present study, a Idiomarina, Marinobacter, Marinobacterium, species of Pseudoxanthomonas, P. koreensis, lacked Pseudoalteromonas and Ferrimonas species. polyamines. Others contained spermidine as the Pseudidiomarina taiwanensis contained putrescine major polyamine. Chemotaxomomic significance of alone. The occurrence of spermine in the absence of cellular polyamines, found in P. 9 Cellular concentrations of polyamines gammaproteobacteria Table 3. 10 11 12 koreensis as well as two Colwellia species within the sulfate-reducing genus of the order Gammaproteobacteria, is unclear. , D. brasiliensis, D. aerotolerans, Order Chromatiales D. alkalitolerans, D. frigidus and D. ferrireducens, Three Thioalkalivibrio species and Thiovirga as well as other four Desulfovibrio species analyzed sulfuroxydans ubiquitously contained spermidine previously6, 12), contained spermidine. Although alone. Rheinheimera pacifica as well as thermophilic acetivorans (the order Halothiobacillus species contained putrescine and Desulfurellales) and infernum (the spermidine. order ) were poor in spermine, Order Enterobacteriales the major occurrence of spermine was found in a A new Erwinia species analyzed in the present thermophile, ehrlichii, in the study, as well as other four spcies and a related present study. Distribution of cadaverine and Pectobacterium species, contained acetylspermidine agmatine seems to be genus- or species-specific in and diaminopropane. It has been reported that these this class. two polyamines were serectively distributed in the family of this order of the class Class Epsilonproteobacteria (Table 4) Gammaproteobacteria18). However, Aquamonas Hydrogen-oxidizing Hydrogenimonas thermophila, fontana located in this order contained putrescine a novel thermophile growing at 55℃, is located in the and spermidine. order , and contained spermidine Others and spermine as the major polyamines. Mesophilic Salinisphaera species analyzed in the present sulfur-oxidizing autotrophica and study contained putrescine, cadaverine and agmatine. located in the order Campylobacterales, contained spermidine alone. Class Deltaproteobacteria (Table 4) Other various heterotrophic, mesophilic Within the class Deltaproteobacteria, many species epsilonproteobacteria belonging to the families belonging to the heterotrophic order Myxoccocales Campylobacteraceae and Helicobacteraceae of the contained homospermidine, whereas previously order Campylobacterales, contained also spermidine analyzed other deltaproteobacteria belonging to other as the major polyamine6, 12). six orders , Desulfovibrionales, Spermine was found as a major polyamine in two , Syntrophobacterales, thermophilic -ammonifying Desulfurellales and Bdellovibrionales contained species and a thermophilic, acidophilic hydrogen- spermidine as the major polyamine6, 12). In the oxidizing species of the new order present analysis, sulfate-reducing Nautiliales. Mesophilic nitrate-reducing cetonicum belonging to the order Desulfobacterales Nitratifractor salsuginis and mesophilic sulfur- contained cadaverine and homospermidine whereas oxidizing Sulfurovum lithotrophicum and sulfur- other two Desulfobacterium species contained reducing Thioreductor micantisoli contained cadaverine and spermidine. The occurrence of spermidine alone. Thermophilic nitrate-reducing homospermidine in D. cetonicum is the first datum tergarcus contained spermine as the and is unique within this order. main polyamine component. Polyamines of various In the order Desulfuromonadales, thermophilic chemolithoautotrophs belonging to Geothermobacter ehrlichii belonging to the Fe(Ⅲ)- deltaproteobacteria and epsilonproteobacteria have reducing family Geobacteraceae contained spermidine been analyzed. However, polyamine profiles related to and spermine as the major polyamines. On the other their optimum growth temperature rather than their hand, psychrotolerant Geopsychrobacter chemolithoautotrophic type. The occurrence of electrodiphilus belonging to Geobacteraceae as well spermine in the chemolithotrophic , thermophilic as mesophilic and species, deltaproteobacteria growing at 50-60℃ and contained spermidine alone. New species of the epsilonproteobacteria growing at 50-55℃ was 13 Cellular concentrations of polyamines deltaproteobacteria and epsilonproteobacteria Table 4. 14 observed. Norspermidine was the major polyamine in the Photobacterium and Vibrio species analyzed, Polyamine distribution profiles in the phylum belonging to the order Vibrionales, and was detected Proteobacteria as a major polyamine in two species of Psychromonas Homospermidine was distributed as the major and one species of Moritella, within the order polyamine in the alphaproteobacteria belonging to the Alteromonadales. In the order Alteromonadales, the nine genera, Sphingomonas and Sphingosinicella genera Shewanella, Colwellia and Marinobacter within the order Sphingomonadales, Dinoroseobacter were heterogeneous in their hydroxyputrescine, within the order Rhodobacterales, Azospirillum and cadaverine and spermidine levels. Spermidine was Tistrella within the order Rhodospirillales, the major polyamine in the other six orders Rhodopseudomonas and Pseudaminobacter within Oceanospirillales, Pseudomonadales, Thiotrichales, the order Rhizobiales, and Asticcacaulis and Xanthomonadales, Chromatiales and Brevundimonas within the order Caulobacterales. Enterobacteriales. Homospermidine was not found in The three triamine profiles, homospermidine- the all gammaproteobacteria analyzed. dominant type, spermidine-dominant type and Diaminopropane was the main component in homospermidine/spermidine-dominant type, were Acinetobacter of Pseudomonadales23) and a major genus-specific. These results suggest the varieties of polyamine in Erwinia and Pectobacterium of their triamine profiles and the usefulness of triamine Enterobacteriales18). Diaminopropane and pattern as a phenotypic marker in the class norspermidine, produced from diaminopropane by an Alphapoteobacteria. The occurrence of aminopropylation, were selectively detected in some homospermidine instead of spermidine was not genera. These polyamine distribution profiles serve ubiquitous in the genera Sphingomonas and for the chemotaxonomy of gammaproteobacteria, Brevundimonas, indicating that chemotaxonomic however, the profiles are conflicting in some genera. usefulness of polyamine profile is not agree with the Desulfobacterium cetanicum contained criteria of these genera. Spermidine and homospermidine and other deltaproteobacteria, homospermidine are produced by the different , Geothermobacter, Geopsychrobacter pathways, aminopropyl transfer or aminobutyl and Desulfovibrio species, contained spermidine as transfer, however, the two biosynthetic activities the major polyamine. Cadaverine distributed in correlate to their phylogenetic locations at genus level Desulfobacterium, Desulfococcus and Desulfovibrio within the class Alphaproteobacteria. species. Spermidine was found as a major polyamine All betaproteobacteria analyzed ubiquitously in epsilonproteobacteria. Spermine was detected in a contained hydroxyputrescine and putrescine. novel thermophile of Geothermobacter within the Spermidine was sporadicaly distributed in some class Deltaproteobacteria and in the five novel members. Norspermidine and homospermidine were thermophiles belonging to the genera not found. Since hydroxyputrescine was almost Hydrogenimonas, Caminibacter, Lebetimonas and selectively distributed in the class Betaproteobacteria Nitratiruptor of the class Epsilonproteobacteria, including 66 species previously analyzed6, 8), the suggesting that the occurrence of spermine correlate occurrence of hydroxyputrescine in 46 strains also to their thermophily. The widespread distribution of serve as a chemotaxonomic marker for moderate thermophiles containing spermine within betaproteobacteria within the phylum Proteobacteria. the phylum Proteobacteria is suggested. Additional Putrescine is produced by ornithine decarboxylation data on cellular polyamine catalogues, shown in the and then hydroxylated. When spermidine was present study, serve as a chemotaxonomy for further synthesized from putrescine, hydroxyspermidine was classification of the phylum Proteobacteria. also produced from hydroxyputrescine. In addition to spermidine, spermine was dominant in a thermophilic Acknowledgements. The authors are indebted to Hydrogenophilus species within this class. ATCC, IAM (Dr. R. Yoguchi), JCM, MBIC, NBRC (Dr. 15

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