J. Gen. App!. Microbiol., 40, 75-82 (1994) Short Communication POLYAMINE ANALYSIS OF THE GENERA AQUA SPIRILL UM, MAGNETOSPIRILL UM, OCEANOSPIRILL UM AND SPIR ILL UM KOEI HAMANA,* TAKESHI SAKANE,' AND AKIRA YOKOTA' College of Medical Care and Technology, Gunma University, Maebashi 371, Japan 'Institute for Fermentation , Osaka, Yodogawa-ku, Osaka 532, Japan (Received August 10, 1993; Accepted November 26, 1993) On the basis of DNA base composition and limited numbers of physiological characteristics, Gram-negative spirilla belonging to the Proteobacteria are divided into the genera Aquaspirillum, Oceanospirillum, Spirillum, Azospirillum, Herbaspi- rillum, Sulfurospirillum and Rhodospirillum (3,17, 22, 23, 27, 28) . Although DNA- rRNA hybridization studies have revealed that the genus Aquaspirillum is a heterogeneous taxon, no phenotypic information is available to differentiate the various phylogenetic branches of aquaspirilla (20). These organisms distributed in several distinct groups that are intermingled with the members of Proteobacteria belonging to the alpha or beta subclasses (20). Oceanospirillum was created for marine aerobic spirilla requiring seawater for growth and is also a heterogeneous group (19). The genus Spirillum, containing the single species S. volutans, is located in the beta subclass (17,27,28). Recently, Magnetospirillum gryphiswal- dense and M. magnetotacticum (formerly Aquaspirillum magnetotacticum) were reported as the new members of spirilla of the alpha subclass (21). Studies on polyamines have provided valuable chemotaxonomic information on the phylogenetic relationships within Proteobacteria; absence or presence of 2- hydroxyputrescine, spermidine and homospermidine are related to grouping into the alpha (rRNA superfamily IV), beta (rRNA superfamily III) and gamma (rRNA superfamily I + II) subclasses (1,2,4-15). In this study, polyamines of 18 species (20 strains) of Aquaspirillum, 2 species of Magnetospirillum, 11 species (13 strains) of Oceanospirillum and 3 species of Spirillum were analyzed to evaluate taxonomic significance of polyamine pattern as a chemotaxonomic marker. * Address reprint requests to: Dr . Koei Hamana, College of Medical Care and Technology, Gunma University, 3-39-15 Showa-machi, Maebashi 371, Japan. 75 76 HAMANA, SAKANE, and YOKOTA VOL. 40 The organisms were cultured in 199 medium (polyamine-free) (Flow Lab., Irvine, U.K.), pH 7.0, 199S medium (199 medium dissolved in 70% synthetic seawater, pH 7.0), 199SW medium (199 medium dissolved in seawater, pH 7.0), PY medium (1% peptone-0.2% yeast extract-0.1 % MgSO4, pH 7.0) and/or ATY medium (0.1 % sodium acetate-0.05 % sodium thioglycolate-0.0l % yeast extract- 0.01 % NH4Cl-0.01 % MgSO4.7H2O-0.05 % K2HP04 20 [IM ferric citrate, pH 6.9) at 30°C. Some strains could not grow in 199 medium but grew in PY medium or ATY medium. After cultivation for 2 days, the cells were harvested by centrifuga- tion at 5,000 X g for 20 min at 4°C and homogenized in cold 0.5 N HC104 to extract polyamines. The polyamines extracted were analyzed by high-performance liquid chromatography (HPLC) on a column (4 mm I.D. X 50 mm) of cation-exchange resin (Hitachi #2619F) kept at 70°C. The elution system consisted of a linear gradient system which is prepared from two buffer systems of 0.045 M sodium citrate-0.060 M citric acid-0.064 M NaCI and 0.20 M sodium citrate-2.0 M NaCI. Polyamines eluated on the HPLC were detected by heating with o-phthalaldehyde reagent. Before HPLC, acid-hydrolysis, alkaline-hydrolysis or periodate oxidation of the polyamine samples were also performed to identify polyamine peaks (9,12) . The polyamine data obtained from the cells harvested at the stationary phase of growth or logarithmically growing phase are shown in Table 1. Although cellular polyamine contents are affected by medium composition and growth phase, these growth conditions have no influence on the distribution profile of major polyamine components in the mesophilic, neutrophilic and organophilic strains of Proteobacteria tested, as was observed previously (9, 11, 14, 15). The Aquaspirillum species were separated into six groups based on their polyamine patterns; 2-hydroxyputrescine-putrescine type (Aquaspirillum metamor- phum, A, giesbergeri, A, anulus, A. delicatum, A. gracile, A. sinuosum, A. auto- trophicum, A. serpens and A. putridiconchylium ), 2-hydroxyputrescine-putrescine- spermidine type (A. aquaticum and A, psychrophilum ), putrescine-spermidine type (A. peregrinum ), putrescine-spermidine-homospermidine type (A. itersonii ), 2- hydroxyputrescine-putrescine-spermidine-homospermidine type (A. polymorphum ) and diaminopropane-2-hydroxyputrescine-putrescine-cadaverine type (A. dispar). Significant amount of putrescine, cadaverine and spermidine were detected in Spirillum volutans. "S. lunatum" and "S. plemorphum" (which have not been validly published) contained putrescine, cadaverine and spermidine. Magnetospi- rillum gryphiswaldense and MMmagnetotacticum contained putrescine and sper- midine, or putrescine, cadaverine and spermidine, respectively. All Oceanospirillum species contained spermidine as the major polyamine and putrescine as the minor polyamine. The putrescine level of 0. pusillum was relatively higher than that of other Oceanospirillum species. In order to assay amino acid decarboxylation, lysates of the cells harvested at logarithmic growth phase were incubated with the reaction mixtures containing 1 mM amino acids as the substrates (9). Ornithine decarboxylase activity to produce putrescine was detected in all strains used in this study. Lysine decarboxylase 1994 Polyamines in Spirilla 77 Table 1. Cellular concentrations of polyamines in Aquaspirillum, Magnetospirillum, Oceanospirillum and Spirillum. 78 HAMANA,SAKANE, and YOKOTA VOL. 40 Table 1. (continued) 1994 Polyamines in Spirilla 79 activity to form cadaverine was found in A. dispar, S. volutans, "S. plemorphum" and MMmagnetotacticum. Diaminopropane was produced from 2,4-diaminobutyric acid by the incubation of the lysate of A. dispar. Our results show that members of the genus Aquaspirillum are heterogeneous on their polyamine distribution profiles. The presence of 2-hydroxyputrescine was common in the Aquaspirillum species belonging to the beta-1 or beta-2 subgroup (rRNA superfamily III) of Proteobacteria. A. aquaticum and A. psychrophilum are distinguished by the presence of spermidine in addition to putrescine and 2- hydroxyputrescine, suggesting phylogenetic divergence from other 7 species (A. metamorphum, A. giesbergeri, A. anulus, A. delicatum, A. gracile, A. sinuosum, A. autotrophicum) of the beta-1 subgroup within the family Comamonadaceae (20). Synonymity between A. aquaticum and Comamonas terrigena was proposed by DNA-rRNA hybridization (26). It has been proposed that A. serpens, A. putridi- conchylium and A. dispar are located on another divergent cluster (the beta-2 subgroup) by rRNA cistron similarity dendrogram (20). However, the former two were unfortunately indistinguishable in the polyamine profiles from the 9 species belonging to Comamonadaceae. The remaining species A. dispar, containing diaminopropane and cadaverine in addition to 2-hydroxyputrescine and putrescine, is a unique species. A. dispar should be located on a different branch divergent from Comamonadaceae and the group containing A. serpens and A. putridiconchylium within the beta subclass. Alcaligenes, Acidovorax, Variovorax, Xylophilus, Hydroge- nophaga and Comamonas, belonging to the beta subclass, contained 2-hydroxypu- trescine and putrescine as the major polyamines (4,15). 2-Hydroxyputrescine emerged as the ubiquitous component of absolute specificity within the beta subclass. Spermidine appears to be a useful characteristic for the arrangement of the members. For selective identification of some species the presence of diamino- propane or cadaverine could be relevant. A. peregrinum, A, itersonii and A, polymorphum differ in dominant polyamines from the above Aquaspirillum species. These three Aquaspirillum belonging to rRNA superfamily IV (the alpha subclass of Proteobacteria) (20) have a polyamine pattern specific to the alpha subclass, i.e., putrescine-homospermidine type, putres- cine-spermidine type or putrescine-spermidine-homospermidine type. Uniform polyamine pattern with putrescine and spermidine was detected in the members of alpha-3 subgroup and generally the species belonging to the alpha-2 subgroup showed a polyamine pattern with homospermidine as the dominant component (4, 11,15). Azospirillum brasilense andAzz lipoferum (alpha-1), contained putrescine and homospermidine (13). Rhodospirillum rubrum (alpha-1) contained putrescine, sper- midine and homospermidine (6). With respect to the polyamine profile A, itersonii (alpha-1) resembles the Rhodospirillum species rather than the Azospirillum species. A. peregrinum, which is devoid of homospermidine, and A. polymorphum, contain- ing 2-hydroxyputrescine, seem to be located on a cluster divergent from both Azospirillum and Rhodospirillum in the alpha subclass. A, polymorphum is quite unique in containing both 2-hydroxyputrescine and homospermidine. 80 HAMANA, SAKANE, and YOKOTA VoL. 40 Oceanospirillum is referred to rRNA superfamily II in the gamma subclass of Proteobacteria (4,17,19). All Oceanospirillum species tested in this study contained putrescine and spermidine. 0. pusillum belongs to the alpha subclass (rRNA superfamily IV) (19) and can be distinguished by the putrescine level from the typical Oceanospirillum species. The