INTRODUCTION
Studies on cellular polyamine distribution profiles have already provided valuable chemo¬ taxonomic information on the classification of the domain Bacteria (Eubacteria) (Hamana, 2002a; Hamana & Matsuzaki, 1992). Polyamine analysis has been established in the systematics of the phyla Proteobacteria (Hamana et al. , 2000a, 2000b, 2001, 2003a, 2003b, 2004; Hamana & Takeuchi, 1998) and Bacteroidetes (Hamana & Matsuzaki, 1990; Hamana & Nakagawa, 2001a, 2001b; Hosoya & Hamana, 2004), and eubacterial Gram-positives (Hamana, 1994, 1999b, 1999c; Hamana & Satake, 1995; Hamana et al., 2002) and thermophiles (Hamana et al., 1996, 1997, 1998; Hosoya et al., 2004). The significance of this approach for the recently validated eight phyla Acidobacteria, Chloroflexi, Nitrospirae, Chlorobi, Chrysiogenetes, Deferribacteres, Fusobacteria and Gemmatimonadetes, phylogenetically deeply branch- ing in Eubacteria (Boone & Castenholz, 2001), was investigated in the present study. Polyamines of some novel species belonging to the two large Gram-positive groups, the two phyla Firmicutes and Actinobacteria (Boone & Castenholz, 2001), have not been clarified. Polyamine data in the related minor three phyla Fibrobacteres, Planctomycetes and Verrucomicrobia, tentatively published in a local report (Hamana, 1999a), were cited in the pres¬ ent study. Polyamine distributions within 51 newly described (mostly validated) strains of these taxa (total 13 phyla) were analyzed to fill up polyamine distribution catalogues in numerous so far not inves- tigated eubacterial taxa of different lines of descent. Polyamine profiles in thermophiles, psychrophiles, halophiles, acidophiles and alkaliphiles found within the 13 phyla were also analyzed.
MATERIALS AND METHODS
Fifty-one newly available strains were aerobically or anaerobically grown at the optimum growth tem¬ perature in the medium No. designed by the culture collections (ATCC Bacteria Collection 2005, IAM Catalogue of Strains 2004, JCM Catalogue of Strains 2005, and NCIMB Catalogue of Strains 2002), and the media NB, Nutrient Broth (Nissui); NB-U, Nutrient Broth plus 2% urea; MB, Marine Broth 2216 (DIFCO); TSB, Trypticase Soy Broth (BBL); GAM, GAM Broth (Nissui); BA, Blood Agar Medium con- taining heart extract and peptone (Eiken Chemical); 199, polyamine-free synthetic 199 medium, ordinarily used for tissue culture (Nissui); YTA, Yeast extract/ Tryptone/Sodium acetate and PYE, Peptone/Yeast extract. Exponentially growing cells or living cells in the early stationary growth phase (200-1000 mg wet weight) were harvested by centrifugation from 200-1000 ml liquid cultures or 2-4 agar plates. The pellets of organisms were homogenized in 0.5 M perchloric acid (PCA, HClO ). Polyamines were extracted into 0.5 M PCA and analyzed by high¬ performance liquid chromatography (HPLC) on a L6000 High-Speed Liquid Chromatograph (Hitachi) (Hamana, 2002a, 2002b; Hamana et al ., 1998). When no major polyamine (detected at the level of 2.00-0.10 μmol/g wet weight cells) was found and polyamines were nondetectable (<0.005 μmol/g wet weight cells) in the PCA extracts, cell residues after the PCA extraction were hydrolyzed with 6 M HCl at 110℃, for 20 h. By HPLC of the hydrolyzates to confirm complete polyamine extraction into 0.5M PCA, the two nondetectable polyamine data could be expressed as that the organism (grown under the used condition) lacks cellular polyamines. It is well known that the composition of medium, culture temperature and growth stage can influence cellu- lar polyamine content, but cannot change the major polyamine profile. Therefore, the present investiga- tion focused on identifying major cellular polyamine components.
RESULTS AND DISCUSSION
Acidobacteria
A novel Fe (III)-reducing bacterium, Geothrix fermentans (Coates et al., 1999), newly analyzed in the present study, contained homospermidine as the major polyamine (Table 1). This organism as well as Acidobacterium and Holophaga species belongs to the phylum Acidobacteria (Coates et al., 1999). Two acidophilic Acidobacterium capsulatum strains also contained homospermidine alone as reported previ¬ ously (Hamana, 1999a), suggesting a wide distribu- tion of this triamine within this phylum.
Chloroflexi
Two Chloroflexus species and two Herpetosiphon species belonging to the class Chloroflexi contained homospermidine as the major polyamine, as previ¬ ously reported (Hamana et al., 1997). In this study, three newly reported species were subjected to polyamine analysis: one was a slightly thermophilic photosynthetic bacterium, Roseiflexus castenholzii, growing at 50℃ , belonging to the class Chloroflexi (Hanada et al., 2002) and the other two were filamentous slight-thermophiles growing at 55℃, Anaerolinea thermophila and Caldilinea aerophila belonging to the class Anaerolineae (Sekiguchi et al., 2003). The major polyamine of R. castenholzii was homospermidine, suggesting the ubiquitous occurrence of homospermidine within the class Chloroflexi (Table 1). Although tetra-amines were detected as minor polyamines in the thermophilic Chloroflexus species, tetra-amines were not found in this slight-thermophile growing at 55℃. In the class Anaerolineae , the major polyamine of A. thermophila was norspermidine, however, its pro¬ file was similar to that of an extreme thermophile, Thermomicrobium roseum containing norspermine, caldopentamine and caldohexamine of the class Thermomicrobia (Table 1). On the other hand, C. aerophila as well as five slight-thermophiles of the class Chloroflexi contained homospermidine. In A . thermophila and C. aerophila, the major polyamine type did not change in the cultures harvested at dif- ferent growth stages (Table 1). Slightly thermophilic Sphaerobacter thermophilus growing at 55℃ (Demharter et al., 1989; Hugenholtz & Stackebrandt, 2004) as a single species of the
' ' t exposerring  Allee  composerring odesulfovibrio yellowstor Microbiol. Cult. Coll. June 2006 Vol. 22,  No. 1 order Sphaerobacterales of the class Thermomicrobia lacked polyamines when the organism was grown in both organic and synthetic media (Hamana, 1999c). The one species, Thermomicrobium roseum belonging to the order Thermomicrobiales of the class Thermomicrobia, was validated in this phylum (Yakimov et al., 2003). This extreme thermophile, growing at 70℃, contained three penta-amines and a hexa-amine synthesized from spermine, thermo¬ spermine and/or norspermine (Hamana et al , 1998). Homospermidine and branched polyamine were not detected. Two different polyamine patterns observed in the phylum Chloroflexi suggest heterogeneous poly- amine distribution profiles within this phylum.
Nitrospirae
The phylum Nitrospirae phylogenetically compris- es Nitrospira, Leptospirillum, Magnetobacterium and Thermodesulfovibrio species (Boone & Castenholz, 2001; Garrity & Holt, 2001b; Hippe, 2000). We reported that the major polyamine of Leptospillum ferrooxidans was spermidine and extremely (mod- erately) thermophilic Thermodesulfovibrio yellow- stonii, growing at 60℃ , contained norspermidine, norspermine, spermine and homocaldopentamine in addition to spermidine (Hamana et al., 1997, 1998). Nitrospira marina, newly analyzed in the present study, contained spermidine alone (Table 1). The two mesophiles, acidophilic L. ferrooxidans and neu- trophilic N. marina, have same polyamine profiles.
Chlorobi
The major polyamine of two photosynthetic Chlorobium species, slightly thermophilic C. tepidum (growing at 45℃) and mesophilic C. linicola belong- ing to the phylum Chlorobi, was homospermidine (Hamana et al., 1997). Two strains of C. linicola, avail- able in the present study, contained also homosper¬ midine as the major polyamine (Table 2). Since other novel genera belonging to this phylum were not available, the chemotaxonomic significance of the occurrence of homospermidine is unclear.
Chrysiogenetes
The phylum Chrysiogenetes is currently represent- ed by a single species, which was reportedly distinct from members of other phyla (Boone & Castenholz, 2001; Garrity & Holt, 2001a). Chrysiogenes arsena- tis is mesophilic, exhibiting anaerobic respiration in which arsenate serves as the electron acceptor (Macy et al., 1996). This organism contained spermi¬ dine alone as the major polyamine, as shown in the present study (Table 2).
Deferribacteres
Chemoorganotrophic heterotrophs, Deferribacter, Flexistipes, Geovibrio and Denitrovibrio species in this phylum, respire anaerobically with terminal electron acceptors including sulfur, nitrate, Fe(III) and arsenate (Boone & Castenholz, 2001; Garrity & Holt, 2001c; Miroshnichenko et al. , 2003; Takai et al., 2003). The two moderately thermophilic Deferribacter species, D. desulfuricans and D. abyssi, growing at 60-62℃, available in the present study, contained cadaverine, spermidine and spermine (Table 2).
Fibrobacteres
Fibrobacter species only comprise the phylum Fibrobacteres (Boone & Castenholz, 2001). It has been reported previously that three Fibrobacter spe- cies contained spermidine as the major polyamine (Hamana, 1999a). These results were shown again in the present study that compared polyamine pat¬ terns among some related phyla (Table 2).
Fusobacteria
The genera Fusobacterium, Ilybacter, Leptotrichia, Propionigenium, Sebaldella and Streptobacillus spe- cies comprise the phylum Fusobacteria (Boone & Castenholz, 2001). In the present study, polyamines of seven Fusobacterium species available, includ¬ ing newly analyzed F. equinum (Dorsch et al., 2001), were described. Spermidine and spermine were detected in all the mesophilic species (Table 2). Putrescine and/or agmatine were found in three species, however, the significance of the diamine and guanidinoamine distribution is unclear.
Gemmatimonadetes
Gemmatimonas aurantiaca was isolated as a representative of the new eubacterial phylum, Gemmatimonadetes (Zhang et al., 2003). This organ- ism contained homospermidine alone (Table 2). The occurrence homospermidine instead of spermidine in this deeply branching phylum as well as the other early branching different lines of descent, Acidobacteria, Chloroflexi and Chlorobi, is phyloge- netically interesting.

Planctomycetes
The phylum Planctomycetes includes the gen- era  Planctomyces, Gemmata, Isosphaera, Pirellula, Blastopirellula and Rhodopirellula (Boone & Castenholz, 2001; Schlesner et al., 2004; Ward et al., 2000). Homospermidine was found in Rhodopirellula species, analyzed here, in addition to the previ- ously analyzed four genera, as shown in Table 2. Spermine was not detected in Isosphaera pallida growing at 45℃ as well as other mesophilic species. Among them, Planctomyces limnophilus contained spermidine instead of homospermidine. The previ- ous results were cited from our report (Hamana, 1999a), however, similar polyamine data were report- ed simultaneously elsewhere (Griepenburd et al., 1999).
Verrucomicrobia
The phylum Verrucomicrobia includes the genera Prosthecobacter and Verrucomicrobium (Boone & Castenholz, 2001; Ward et al., 2000) and their four species contained spermidine alone, as previously reported (Hamana, 1999a) (Table 2).
Firmicutes
Polyamine analyses of two novel anoxygenic phototrophs and two novel halophilic anaerobes located in the large phylum Firmicutes were added in the present study. The family Heliobacteriaceae located in the order Clostridiales of this phylum comprising the genera Heliobacterium, Heliobacillus, Heliophilum and Heliorestis, is a group of anoxygen- ic phototrophs (Boone & Castenholz, 2001; Madigan, 2001). Three heliobacteria analyzed previously con- tained low levels of cadaverine, spermidine, sperm- ine and agmatine (Hamana, 1999c). Uptake of these polyamines from the media was not excluded. In the present study, slightly thermophilic Heliobacterium modesticaldum and mesophilic Heliophilum fascia- tum were analyzed. Polyamine profiles were identi- cal within the five heliobacteria, however, slightly thermophilic H.modesticaldum, growing at 50℃, was rich in a tetra-amine, spermine (Table 3). In the family Clostridiaceae of this order, thermo¬ philes often show a tendency for tetra- and penta- amines instead of di- and tri-amines (Hamana, 1999c; Hamana et al , 1996; Hosoya et al , 2004). Recently, two newly reported halophilic anaerobes Halanaerobium fermentans and Selenihalanaerobacter shriftii belonging to the order Halanaerobiales of this phylum were published (Blum et al., 2001; Boone & Castenholz, 2001; Kobayashi et al., 2000; Oren, 2000). The two species growing at 10% concentra- tion of NaCl as well as eight other halophilic anaer- obes (Hamana, 1999c), ubiquitously contained spermi¬ dine and spermine (Table 3). In the previous study on aerobic Gram-positive cocci located in the order Bacillales of this phylum, the presence or absence of spermidine was conflict- ing (Hamana, 1994). Polyamines of new members belonging to two families of this order (Boone & Castenholz, 2001) were analyzed. All species of the genera Planococcus (Nakagawa et al., 1996; Yoon et al., 2003), Sporosarcina (except for S. pasteurii) (Yoon et al., 2001b). Planomicrobium (Dai et al., 2005: Yoon et al., 2001a), Filibacter (Clausen et al., 1985) and Kurthia (Boone & Castenholz, 2001) of the fam- ily Planococcaceae contained spermidine as the major polyamine (Table 3). Caryophanon species of the family Caryphanaceae (Farrow et al., 1994) contained spermidine (Table 3). Alkalibacterium, Carnobacterium, Desemzia and Marinilactibacillus species are members of the family Carnobacteriaceae of the order Lactobacillales (Ishikawa et al., 2003; Ntougias & Russell, 2001; Toffin et al., 2005; Yumoto et al., 2004). Two Carnobacterium species and two Marinilactibacillus species contained spermidine as the major polyamine. Polyamines were not detected in a Desemzia species and two Alkalibacterium spe- cies grown in organic media used for their good growth. Further polyamine analysis of these taxa is needed to evaluate chemotaxonomic usefulness.
Actinobacteria
We reported the absence of appreciable amounts of polyamines in slightly thermophilic Rubrobacter radiotolerans growing at 60℃ (Hamana, 1994). In the present study, significant amounts of polyamines were not detected in the moderately thermophilic Rubrobacter xylanophilus growing at 60℃ (Ferreira et al., 1999) and mesophilic Conexibacter woesei (Monciardini et al. , 2003) of the order Rubrobacterales (the subclass Rubrobacteridae) (Boone & Castenholz, 2001) (Table 4). On the other hand, extremely ther- mophilic Thermoleophilum species, growing at 70℃, phylogenetically located in the order, contained nor- spermidine, spermidine and quaternary branched penta-amines (Hamana et al , 1998). Atopobium, Collinsella, Coriobacterium, Eggerthella, Olsenella and Slackia comprise the order Coribacteriales (the subclass Coribacteridae ) of the class Actinobacteria (Boone & Castenholz, 2001; Dewhirst et al., 2001; Kageyama et al., 1999; Wade et al ., 1999). Three Collinsella species, Coriobacterium glomerans, Slackia exigua, Eggerthella lenta and Olsenella uli analyzed in the present study, as well as Atopobium rimae previously reported (Hamana & Satake, 1995), lacked appreciable amounts of cellular polyamines (Table 4). Slightly (moderately)-thermophilic (growing at 60℃ ), acidophilic Acidothermus cellulolyticus, grown in both organic and synthetic media, belonging to the order Actinomycetales (Boone & Castenholz,
Polyamines of eubacterial thirteen phyla Hosoya et al. 2001; Demharter et al., 1989) lacked polyamines (Hamana, 1999c), as shown as a reference datum (Table 4). Polyamines were not detected in vari¬ ous Gram-positives belonging to the two orders Actinomycetales and Bifidobacteriales of this phylum including many slight- or moderate-thermophiles growing at 45-60℃ (Hamana, 1999c). On the other hand, thermophilic Thermoactinomyces species belonging to the order Bacillales of the phylum Firmicutes contained spermidine and spermine as the major polyamines (Hamana, 1999c).
Polyamine profiles of the thermophiles.
psychrophiles, halophiles, acidophiles
and alkaliphiles in the 13 phyla
Homospermidine or norspermidine was the major polyamine in the early deeply branched five phyla Acidobacteria, Chloroflexi, Chlorobi, Gemmatimonadetes and Planctomycetes. Mesophilic species, slightly thermophilic species and acidophilic species in each phyla (or classes) show the same tri¬ amine profile, indicating that slight thermophily and acidophily do not affect polyamine distributions. Thermophilic Thermomicrobium species within the class Thermomicrobia in which slightly alkali- philic Sphaerobacter was isolated, and thermophilic Thermodesulfovibrio species within the phylum Nitrospira in which acidophilic Leptospirillum was isolated, contained norspermine, penta-amines and a hexa-amine. These results suggested that the occurrence of these long polyamines coincided with their extreme/moderate thermophily and had no direct correlation to their optimum alkaline or acidic growth conditions. Within the order Rubrobacterales, extremely ther- mophilic Thermoleophilum species contained qua¬ ternary branched penta-amines whereas polyamines were not detected in slight/moderate thermophiles of Rubrobacter and a mesophile, Conexibacter woesei. Polyamines were not detected in slightly-thermo¬ philic, slightly-acidophilic Actinothermus of the order Actinomycetales as well as mesophilic, neutrophilic species of the order Coriobacteriales. Halophilic anaerobes in the order Clostridiales and halococi in the order Bacillales had no unique polyamine profiles within the orders, suggesting that their halophily does not affect their polyamine pro- files. Within the family Carnobacteriaceae in the order Lactobacillales, polyamines were not detected in the two alkaliphiles, Alkalibacterium olivoapovliti- cus and A. psychrotolerans. The absence of major polyamines was also found in a mesophilic species, Desemzia incerta , in this order. Alkaliphilic, psy¬ chrotolerant Marinilactibacillus psychrotolerans as well as neutrophilic, mesophilic M. piezotolerans contained spermidine alone. Although psychrophilic or psychrotolerant species were found in the orders Bacillales and Lactobacillales, they were similar to the mesophilic species in the two orders, in their polyamine profiles. The occurrence of a tetra-amine, spermine, seems to be correlated to moderate thermophily in many eubacterial thermophiles. Penta-amines were selec- tively produced in extreme thermophiles. Slight- thermophily as well as psychrophily, halophily, acidophily and alkaliphily within the 13 phyla did not correlate directly to their cellular polyamine components as a phylum-, class-, order-, family- or genus-specific chemotaxonomic marker.
ACKNOWLEDGEMENTS
The authors are indebted to the culture collec¬ tions JCM, IAM, NCIMB and ATCC for supplying bacterial strains.