J. Gen. Appl. Microbiol., 41, 153-158 (1995)

Short Communication

UBIQUITOUS OCCURRENCE OF AGMATINE AS THE MAJOR POLYAMINE WITHIN EXTREMELY HALOPHILIC ARCHAEBACTERIA

KOEI HAMANA,* HIROSHI HAMANA,' ANDTAKASHI IT0H2

College of Medical Care and Technology, Gunma University, Maebashi 371, Japan 'Faculty of Engineering, Gunma University, Kiryu 376, Japan 2Japan Collection of , Institute of Physical and Chemical Research (RIKEN), Wako 351-01, Japan

(Received May 12, 1994; Accepted January 11, 1995)

Previous studies have indicated that bacterial polyamine patterns may be useful for chemotaxonomic evaluations (6). In the course of investigations on the possible existence of unusual polyamines in extremely halophilic archaebacteria, we detected agmatine as the major polyamine and arginine decarboxylase (ADC) activity which produces agmatine from arginine in the cells of some strains of and Halococcus (5, 8). These extreme halophiles were devoid of any other polyamines and of lysine decarboxylase (LDC) and ornithine decarboxylase (ODC) activities which produce cadaverine and putrescine, respectively (5, 8). Extremely halophilic, nonmethanogenic archaebacteria are comprised of essentially two groups: extreme halophiles that grow at neutrality include the genera Halo- bacterium, Haloarcula, Haloferax and Halococcus and extremely haloalkaliphilic isolates that grow only at high pH include the genera and (3,10,13). In the present study, polyamines of 41 strains of the published of extremely halophilic, nonmethanogenic archaebacteria listed in Table 1 were analyzed to evaluate polyamine type as a chemotaxonomic marker. Organisms belonging to the genera Halobacterium, Haloarcula, Haloferax and Halococcus were grown in the synthetic Onishi-McCance-Gibbons medium (OMG medium), pH 6.2 (11). Natronobacterium pharaonis was grown in OMG medium adjusted pH at 9.0. The medium contains 15 amino acids including arginine and lysine, AMP, UMP, trisodium citrate, glycerol, various inorganic salts, 2% MgSO4 7H20 and 25% NaCI but not ornithine and polyamines. Three other species of Natronobacterium and Natronococcus occultus which did not grow in

* Address reprint requests to: Dr . Koei Hamana, College of Medical Care and Technology, Gunma University, 3-39-15 Showa-machi, Maebashi 371, Japan.

153 154 HAMANA, HAMANA, and ITOH VOL. 41 1995 Polyamines of Extremely Halophilic Archaebacteria 155 156 HAMANA, HAMANA, and ITOH VOL. 41

OMG medium were cultivated in JCM No. 166 medium (corresponding to ATCC medium 1394), pH 8.5, containing casamino acids (Difco Laboratories, Detroit, MI, U.S.A.) or JCM No. 167 medium (corresponding to ATCC medium 1590), pH 9.0, containing casamino acids and yeast extract (Difco Laboratories). After incubation for 5-7 days at 37°C in liquid media or on solid media (agar slant), the cells were harvested. The polyamines extracted from the cells with cold 0.5 M perchloric acid were analyzed by high-performance liquid chromatography (HPLC) on a column of cation-exchange resin, as described previously (7). A main peak corresponding to agmatine was detected on the HPLC chromat- ograms of the perchloric acid extracts from the extremely halophilic archaebacteria grown in polyamine-free 0MG medium (Fig. 1). The peak was stable for the acid hydrolysis with 6 M HCl at 110°C for 24 h but degradated by the alkaline hydrolysis with 1 M NaOH at 110°C for 24 h. After the alkaline hydrolysis putrescine was detected in the samples. These results indicated that the alkaline-labile peak was assigned to a guanidinoamine, agmatine. The cellular agmatine levels were about 1-10 nmol/g wet cell pellet (Table 1). The haloalkaliphiles cultivated in either alkaline liquid media or on alkaline solid slant media were poor in agmatine, suggesting poor production of agmatine or leakage of agmatine into the media under these growth conditions. Putrescine was detected as a major polyamine in H. saccharovorum JCM 8865 and H. lacusprofundi JCM 8891 (Table 1). Putrescine levels in the two organisms were not increased by the supplement of ornithine into OMG medium, indicating the absence of ODC activity. Putrescine was sporadi- cally detected as a minor component in some other halophiles grown in OMG

Fig. 1. Typical HPLC chromatogram of polyamines of Haloarcula hispanica JCM 8911 grown in 0MG medium (A), Halobacterium saccharovorum JCM 8865 grown in 0MG medium (B), Natronobacterium pharaonis JCM 8858 grown in 0MG medium (C) and Natronobacterium gregoryi JCM 8860 grown in JCM No. 167 medium (D). Abbreviations for polyamines are shown in Table 1. 1995 Polyamines of Extremely Halophilic Archaebacteria 157 medium. Since it is known that putrescine can be formed by enzymatic hydrolysis of agmatine in various eubacteria (6), agmatine may possibly be converted to putrescine in these organisms. Other polyamines were not detected ( < 0.05 nmol/g wet cell) in the extreme halophiles grown in the polyamine-free synthetic OMG medium. Putrescine, spermidine and spermine detected in the haloalkaliphiles grown in JCM No. 166 or No. 167 medium were probably incorporated from yeast extract and casamino acids containing putrescine, cadaverine, spermidine and spermine (Table 1). Since No. 166 and No. 167 media were poor in agmatine, endogeneous agmatine synthesis was suggested in addition to the uptake of agma- tine from the media. We have already shown the occurrence of agmatine as the major polyamine in haloneutrophilic Halobacterium salinarium ATCC 2934 (H. halobium R1), H. cutirubrum NRC 34001, and four strains of Halococcus morrhuae (NRC 147072, 16006, 16012, 16018) grown in OMG medium or OMG medium supplemented with yeast extract (8). Excretion of agmatine into the media and uptake of other polyamines into the cells from the latter medium containing polyamines were observed (8). Lack of detectable polyamines or very low concentrations of polyamines in some extremely halophilic archaebacteria cultivated in the media containing polyamines have been reported (1, 2, 9). However, agmatine was not analyzed, and furthermore, uptake of polyamines from the media could not be excluded in these reports. The present study shows synthesis of agmatine as the major polyamine and absence of other polyamines except for the sporadical occurrence of putrescine in various extremely halophilic archaebacteria belonging to the six published genera. This polyamine profile is unique and has never been found in the other members of archaebacteria (i.e. methanogens, extreme thermo- philes and acidothermophiles) and eubacteria including halophiles (6, 9,12). Wide- spread distribution of agmatine as the major polyamine in extremely halophilic, nonmethanogenic archaebacteria serve for the chemotaxonomic evaluation within archaebacteria. The absence of polyamines in Methanobacterium and Methano- brevibacter species which are included in the family of methanogenic archaebacte- ria, Methanobacteriaceae, was reported (9,12). Agmatine was not analyzed in the reports. The other methanogenic archaebacteria belonging to other families and various thermophilic archaebacteria contained at least one of triamines, tetraamines and pentaamines at ~imol/g wet cell level (4, 9). Ubiquitous absence of these long polyamines in the family including the six genera of non- methanogenic extreme halophiles and the family Methanobacteriaceae support the phylogenetically short-distant locations of the two families in archaebacteria (14).

We are indebted to IAM Culture Collection, Center for Cellular and Molecular Research, Institute of Molecular and Cellular Biosciences, The University of Tokyo for supplying of bacterial strains. 158 HAMANA, HAMANA, and ITOH VOL. 41

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