Microbiol. Cult. Coll. June. 2004. p. 3 ― 8 Vol. 20, No. 1 Polyamine Distribution Profiles among Some Members within Delta-and Epsilon-Subclasses of Proteobacteria Koei Hamana1）＊, Tomoko Saito1）, Mami Okada1）, and Masaru Niitsu2） 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）Faculty of Pharmaceutical Sciences, Josai University, Keyakidai 1-chome-1, Sakado, Saitama 350-0295, Japan Cellular polyamines of 18 species（13 genera）belonging to the delta and epsilon subclasses of the class Proteobacteria were analyzed by HPLC and GC. In the delta subclass, the four marine myxobacteria（the order Myxococcales）, Enhygromyxa salina, Haliangium ochroceum, Haliangium tepidum and Plesiocystis pacifica contained spermidine. Fe（III）-reducing two Geobacter species and two Pelobacter species belonging to the order Desulfuromonadales con- tained spermidine. Bdellovibrio bacteriovorus was absent in cellular polyamines. Bacteriovorax starrii contained putrescine and spermidine. Bacteriovorax stolpii contained spermidine and homo- spermidine. Spermidine was the major polyamine in the sulfate-reducing delta proteobacteria belonging to the genera Desulfovibrio, Desulfacinum, Desulfobulbus, Desulfococcus and Desulfurella, and some species of them contained cadaverine. Within the epsilon subclass, three Sulfurospirillum species ubiquitously contained spermidine and one of the three contained sper- midine and cadaverine. Thiomicrospora denitrificans contained cadaverine and spermidine as the major polyamine. These data show that cellular polyamine profiles can be used as a chemotaxonomic marker within delta and epsilon subclasses. Key words: polyamine, spermidine, homospermidine, Proteobacteria The class Proteobacteria is a major taxon of the 18, 26）. Fe（Ⅲ）-reducing members belonging to the gen- domain Bacteria and is phylogenetically divided into the era Pelobacter, Geobacter, Desulfuromonas and alpha, beta, gamma, delta and epsilon subclasses. We Desulfuromusa, form a monophyletic group（the order have reported the cellular polyamine distribution profiles Desulfuromonadales（）2, 3, 4, 20）. Two Pelobacter species, within proteobacteria, as a chemotaxonomic marker（8- previously analyzed, contained spermidine as the major 15）. Within the delta subclass, homospermidine was polyamine（10）. Within the order Campylobacterales of found in some myxobacteria（the order Myxococcales） the epsilon subclass, various species belonging to the （10）comprising the two suborders, Sorangineae and four genera Helicobacter, Wolinella, Campylobacter and Cystobacterineae, and the four families, Myxococcaceae, Arcobacter（2, 6）scarcely contained any polyamines Cystobacteraceae, Polyangiaceae and Archangiaceae（2, but Sulfurospirillum deleyianum contained spermidine （10）. In the present study, polyamines of some new mem- ＊ Correspondence author bers of the delta and epsilon subclasses were analyzed. accepted: June 6, 2004 Newly validated 20 delta and epsilon proteobacteria ― 3 ― Polyamines of Delta and Epsilon Proteobacteria Hamana et al． Table 1. Polyamines of delta and epsilon subclasses of the class Proteobacteria Organism Medium ― Temp.（℃）Polyamine（µ mol/g wet cell） Dap Put Cad Spd HSpd Spm Agm Delta subclass Myxococcales Enhygromyxa salina JCM 11769T JCM 341 ― 30 －－－0.54 － 0.10 － Haliangium ochraceum JCM 11303T JCM 341 ― 30 －－－1.15 － 0.31 0.03 Haliangium tepidum JCM 11304T JCM 341 ― 37 －－－0.70 － 0.15 0.02 Plesiocystis pacifica JCM 11591T JCM 341 ― 30 － 0.10 － 0.65 － 0.02 － Desulfovibrionales Desulfovibrio africanus NCIMB 8401T NCIMB17 ― 37 0.01 0.12 0.02 0.95 － 0.02 0.03 Desulfovibrio desulfuricans IFO 13699 （a）－－－1.16 － 0.01 0.02 subsp. desulfuricans Desulfovibrio desulfuricans NCIMB 8307T NCIMB17 ― 30 － 0.24 1.98 0.40 －－－ subsp. desulfuricans Desulfovibrio desulfuricans NCIMB 9335T NCIMB104 ― 30 － 0.02 0.01 1.11 － 0.05 0.03 subsp. aestuarii Desulfovibrio salexigens NCIMB 8403T NCIMB104 ― 30 － 0.05 0.04 0.65 －－0.20 Desulfovibrio vulgaris NCIMB 9442T NCIMB17 ― 30 － 0.35 0.10 0.50 －－0.10 subsp. oxamicus Desulfovibrio vulgaris NCIMB 8303T NCIMB17 ― 30 － 0.02 0.01 1.50 － 0.10 0.03 subsp. vulgaris Syntrophobacterales Desulfacinum infernum NCIMB 13416T NCIMB109 ― 60 － 0.02 － 0.68 － 0.06 － Desulfobacterales Desulfobulbus propionicus NCIMB 12907T NCIMB17 ― 37 － 0.02 － 0.88 － 0.04 － Desulfococcus multivorans NCIMB 12965T NCIMB295 ― 30 － 0.02 － 0.70 － 0.04 － Desulfuromonadales Pelobacter acidigallici ATCC 49970T （a）－－－1.45 －－0.24 Pelobacter massiliensis ATCC 49973T （a）－－－1.20 －－0.20 Geobacter sulfurreducens ATCC 51573T ATCC1957 ― 30 －－－1.10 －－0.03 Geobacter hydrogenophilus ATCC 51590T ATCC1957 ― 30 －－－0.92 －－0.10 Desulfurellales Desulfurella acetivorans ATCC 51451T ATCC1920 ― 55 －－－0.97 － 0.02 － Bdellovibrionales Bdellovibrio bacteriovorus NCIMB 9529T ATCC137 ― 30 －－－0.05 － 0.10 － 199 ― 30 －－－－－－－ Bdellovibrio bacteriovorus IFO 14261 （a）－2.10 －－－ －－ Incertae sedis Bacteriovorax starrii ATCC 15145T （a）－1.52 0.10 1.77 －－－ （Bdellovibrio starrii） Bacteriovorax stolpii ATCC 27052T （a）－－－0.70 0.90 0.07 － （Bdellovibrio stolpii） Epsilon subclass Campylobacterales Sulfurospirillum barnesii ATCC 700032T ATCC2034 ― 30 －－－1.10 － 0.01 － （“Geospirillum barnesii”） Sulfurospirillum arsenophilum ATCC 700056T ATCC2018 ― 30 0.02 － 0.85 0.10 － 0.03 － （“Geospirillum arsenophillus”） Sulfurospirillum deleyianum ATCC 51133T （a）－－－1.25 －－－ Incertae sedis Thiomicrospira denitrificans ATCC 33889 ATCC1255 ― 26 －－0.40 2.10 －－－ Note: Dap, 1, 3 ― diaminopropane; Put, putrescine; Cad, cadaverine; Spd, spermidine; HSpd, homospermidine; Spm, spermine; Agm, agmatine; NCIMB, The National Collections of Industrial, Food and Marine Bacteria, Aberdeen, Scotland, UK; ATCC, American Type Culture Collection, Manassas, Virginia, USA; IFO, Institute for Fermentation, Osaka, Japan; JCM, Japan Collection of Microorganisms, RIKEN, Saitama, Japan; T, Type strain; ―, not detectable （<0.005）. Quotation marks indicate that the scientific name has not been validly published. Former names are shown in parentheses.（a）was cited from Hamana and Takeuchi（10）. ― 4 ― Microbiol. Cult. Coll. June. 2004. p. 3 ― 8 Vol. 20, No. 1 were grown in the media designated by the culture col- um. A little amount of spermidine and spermine was lections（JCM Catalogue of Strains, 2002 and The incorporated into this organism from the ATCC 137 medi- Catalogue of Strains of NCIMB, 1994 and The Catalogue um containing these amines（Table 1）. The strain IFO of Strains of ATCC, 1996）and in the polyamine-free 199 14261 contained putrescine but not triamines（10）. The medium（Nissui Pharmaceutical Co., Tokyo, Japan）, at polyamine profile of Bdellovibrio bacteriovorus is unique the optimum growth temperature listed in Table 1. The within this subclass. The two Bacteriovorax species dif- cells in the stationary phase were harvested. The whole fer in their polyamine profiles, however, Bacteriovorax polyamine extract in 0.5 M perchloric acid was analyzed starrii contained putrescine and spermidine and by high-performance liquid chromatography（HPLC） Bacteriovorax stolpii contained spermidine and homo- （10）. Gas chromatography（GC）was performed after spermidine（10）. heptafluorobutyrization of the concentrated polyamine The sulfate reducers such as Desulfovibrio, sample（21）. Desulfobulbus, Desulfococcus, Desulfacinum, Desulfobacter, Cellular concentrations of 1, 3-diaminopropane, putre- Desulfobacterium, Desulfomicrobium, Desulfomonas, scine, cadaverine, agmatine, spermidine, homospermidine Desulfomonile, Desulfonema, Desulfosarcina, Desulforhopalus, and spermine are shown in Table 1. Spermine detected as Desulfohalobium, Desulfotulus, Desulforhobdus, a minor component was judged as a contaminant from Desulfuromonas, Syntrophus, and Syntrophobacter phy- the media containing spermine. logenetically spread within the delta subclass（5, 16, 19, 23）. Polyamine analysis of a strain of Desulfovibrio The delta subclass desulfuricans has been reported previously（10）. In the Our previous study showed that several myxobacteria present study, polyamines of Desulfobulbus propionicus, belonging to the genera Myxococcus, Cystobacter, Desulfococcus multivorans, Desulfacinum infernum, and Stigmatella, Melittangium, Polyangium and Archangium seven species of the genus Desulfovibrio, were analyzed. ubiquitously contained homospermidine as the major Cadaverine level differed among the three strains of polyamine（10）. The new marine myxobacteria, Desulfovibrio desulfuricans（Table 1）, correlating to Enhygromyxa salina, Haliangium ochroceum, their phylogenetic heterogeneity（5）. Spermidine was Haliangium tepidum and Plesiocystis pacifica phyloge- the major polyamine in all four Desulfovibrio species and netically located in Sorangineae of Myxococcales（7, 17- all three species belonging to the genera Desulfobulbus 19）contained spermidine as the major polyamine and （the order Desulfobacterales）, Desulfococcus（the order spermine as a minor polyamine, as shown in Table 1. Desulfobacterales）and Desulfacinum（Syntrophobacterales）, Although widespread occurrence of homospermidine which are located in other clusters divergent from the within terrestrial myxobacteria serves as a chemotaxo- Desulfovibrio clade（the order Desulfovibrionales）. nomic marker for Myxococcales, homospermidine was not Desulfurella acetivorans, a thermophilic, acetate-oxidizing found in the four marine slightly halophilic myxobacteria. and sulfur-reducing proteobacterium, contained spermi- The two Geobacter species,