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Phylogenetic analysis of the genus Aquaspirillum based on 16S rRNA gene sequences
FEMS Microbiology Letters 212 (2002) 165^169
www.fems-microbiology.org
Phylogenetic analysis of the genus Aquaspirillum based on 16S rRNA gene sequences Linxian Ding , Akira Yokota Laboratory of Bioresources, Institute of Molecular and Cellular Biosciences, The University of Tokyo, Yayoi, Bunkyo-ku, Tokyo 113-0032 Japan Received 19 February 2002 ; accepted 6 April 2002 First published online 31 May 2002
Abstract Phylogenetic analysis of 15 species of the genus Aquaspirillum based on 16S rRNA gene (rDNA) sequences indicated that the genus Aquaspirillum is phylogenetically heterogeneous and the species could be divided into four groups as follows: Aquaspirillum serpens, the type species of this genus, A. dispar and A. putridiconchylium are situated in the family Neisseriaceae; members of the second group, A. gracile, A. delicatum, A. anulus, A. giesbergeri, A. sinuosum, A. metamorphum and A. psychrophilum, are included in the family Comamonadaceae; the two members of the third group, A. arcticum and A. autotrophicum, are included in the family Oxalobacteriaceae; and members of the fourth group, A. polymorphum, A. peregrinum, and A. itersonii, are included in the alpha-subdivision of Proteobacteria. Thus, phylogenetic studies indicated that all the species excepting A. serpens, the type species, should be transferred to distinct genera. 2 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : 16S rDNA; Phylogeny; Aquaspirillum
1. Introduction The genus Aquaspirillum was established in 1973 by Hylemon et al., in which 13 species were described [1]. After that, Terasaki reported two subspecies, Aquaspirillum itersonii subsp. nipponicum and Aquaspirillum peregrinum subsp. integrum [2,3]. Kumar et al. described Aquaspirillum bengal [4], but that species has been transferred to A. serpens by Boivin et al. on the basis of DNA^DNA hybridization [5]. Strength et al. reported Aquaspirillum fasciculus [6], which was lately transferred to the genus Prolinoborus as P. fasciculus based on DNA^rRNA hybridization studies [7]. Aragno et al. [8] isolated a new hydrogen-oxidizing species, Aquaspirillum autotrophicum. Schleifer et al. established the genus Magnetospirillum, and they proposed to transfer Aquaspirillum magnetotacticum to this genus as Magnetospirillum magnetotacticum [9,10]. Willems et al. had changed Aquaspirillum aquaticum to Comamonas terrigena [11,12]. Butler et al. described a new psychrophilic bacterium Aquaspirillum arcticum [13].
Therefore, the genus Aquaspirillum includes 15 species at present. Most of the strains in this genus were isolated from fresh water (pond water, stream water, or Antarctic mosses), and all the species are aerobic and helical shaped and do not grow in the presence of 3% NaCl [14,15]. DNA^rRNA hybridization studies [7,16], chemotaxonomic investigation of the genus based on cellular fatty acids [17], respiratory quinone composition and G+C contents of DNA [17], polyamine analysis [18], and 16S rDNA sequencing [19,20] indicate that the genus Aquaspirillum is quite heterogeneous and is composed of many distinct groups intermingled phylogenetically with the related Gram-negative bacteria. In this study, we investigated the phylogenetic position of the species of the genus Aquaspirillum based on 16S rDNA sequences.
2. Materials and methods 2.1. Bacteria strains and cultivation
* Corresponding author. Tel. : +81 (3) 5841 7828; Fax : +81 (3) 5841 8490. E-mail address : [email protected] (L. Ding).
We used nine species of the genus Aquaspirillum in this study. A. serpens IAM (IAM Culture Collection, Institute of Molecular and Cellular Biosciences, The University of
0378-1097 / 02 / $22.00 2 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. PII : S 0 3 7 8 - 1 0 9 7 ( 0 2 ) 0 0 7 4 7 - 4
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Tokyo, Japan) 13944T , A. autotrophicum IAM 14942T , and A. dispar IAM 14943T were grown in medium B-33 [21](peptone 10.0 g, succinic acid 1.0 g, (NH4 )2 SO4 1.0 g, MgSO4 W7H2 O 1.0 g, FeCl3 W6H2 O 2.0 mg, MnSO4 W5H2 O 2.0 mg, distilled water 1.0 l, pH 6.8); A. anulus IAM 14948T , A. delicatum IAM 14955T , A. giesbergeri IAM 14949T , A. itersonii subsp. nipponicum IAM 14945T , A. peregrinum subsp. integrum IAM 14946T , and A. putridiconchylium IAM 14964T were grown in medium B-104 [21] (Polypepton 10.0 g, yeast extract 2.0 g, MgSO4 W7H2 O 1.0 g, distilled water 1.0 l, pH 7.0); A. arcticum IAM 14963T was grown in medium B-52 [21] (trypticase soy agar 40.0 g (BBL 11042), distilled water 1.0 l, pH 7.0), and A. polymorphum IAM 14441T was grown in medium B-66 [21] (peptone 10.0 g, yeast extract 5.0 g, liver, infusion 25.0 g, glucose 3.0 g, glycerol 15.0 g, NaCl 3.0 g, distilled water 1.0 l, pH 7.0). Bacterial strains were grown aerobically at 30‡C except for A. arcticum, which was grown at 4 ‡C. 2.2. Polymerase chain reaction (PCR) ampli¢cation of 16S rDNA and sequencing PCR for the ampli¢cation of 16S rDNA was carried out by using cell lysate extracted from organisms [22]. We used universal primers 8F (5P-AGAGTTTGATCCTGGCTCAG-3P) and 1510R (5P-GGCTACCTTGTTACGA-3P) for PCR ampli¢cation of 16S rDNA. The conditions for PCR ampli¢cation were 1 min at 94‡C and then 30 cycles of 1 min at 94‡C, 1 min at 55‡C, and 1.5 min at 72‡C, followed by a ¢nal step for 2 min at 72‡C. The PCR products were puri¢ed by using the GFXtm PCR DNA and Gel Band Puri¢cation Kit (Amersham Pharmacia Biotech, Inc., NJ, USA). 16S rDNA sequencing primers 8F, 520F, 926F, 350R, 700R, 1110R, and 1510R were used in the sequencing reactions. The 16S rDNA sequencing of all strains was performed as described previously [23]. 2.3. Phylogenetic analysis Seventy-four species of 16S rRNA (rDNA) sequences obtained from a DNA databank were aligned using CLUSTAL W, version 1.74 [24]. Nucleotide substitution rates (Knuc values) were calculated, and the phylogenetic tree was constructed by using the neighbor-joining algorithms [25]. The nucleotide sequence data of A. serpens IAM 13944T (AB074518), A. dispar IAM 14943T (AB074526), A. arcticum IAM 14963T (AB074523), A. autotrophicum IAM 14942T (AB074524), A. peregrinum subsp. integrum IAM 14946T (AB074521), A. itersonii subsp. nipponicum IAM 14945T (AB074520), A giesbergeri
IAM 14949T (AB074522), A. anulus IAM 14948T (AB074527), and A. putridiconchylium IAM 14964T (AB076000) reported in this paper will appear in the DDBJ nucleotide sequence databases with the accession numbers listed. The nucleotide sequence of Escherichia coli from Brosius et al. [26] was used as the outgroup. Similarity values of 16S rDNA sequences were calculated by the use of PAUP*, Version 4.068. [27].
3. Results and discussion We ampli¢ed 16S rDNA from nine species of the genus Aquaspirillum and determined their nucleotide sequences. The base arrangement length ampli¢ed from 16S rDNA fragments ranged from 1380 to 1503. A phylogenetic tree based upon nucleotide substitution values is depicted in Fig. 1. The 16S rDNA similarity values are shown in Table 1. Fig. 1 showed that 15 species (four subspecies) of genus Aquaspirillum have been divided into four groups. A. serpens, the type species of this genus, is situated in the family Neisseriaceae. Group I, A. serpens and A. putridiconchylium, originates from one branch, but the sequence similarity was only 93.0%. A. dispar was close to the genus Microvirgula aerodenitri¢cans with the sequence similarity of 98.8%. A. arcticum and A. autotrophicum in the second group (group II) belonged to the family Oxalobacteriaceae. A. arcticum formed a cluster with Duganella zoogloeoides and Janthinobacterium, and the sequence similarities among them were 96.9^97.2%. A. autotrophicum was close to Herbaspirillum frisingense, where the sequence similarity was 96.6%. As members of group III, A. delicatum, A. giesbergeri, A. anulus, A. sinuosum, A. metamorphum, A. psychrophilum, and A. gracile belonged to the family Comamonadaceae, and three species, A. anulus, A. sinuosum, and A. giesbergeri formed a cluster. The 16S rDNA sequence similarities among these were 98.3^98.7%. Two species, A. psychrophilum and A. metamorphum, formed a cluster, and the sequence similarity between them was 95.1%. A. gracile formed a cluster with the genus Delftia, but the sequence similarity between them was 93.8.%. A. delicatum stands alone. Groups I, II, and III belong to the beta-subdivision of Proteobacteria. As the fourth group, the three species A. polymorphum, A. peregrinum, and A. itersonii were included in the alpha-subdivision of Proteobacteria. Two species (four subspecies), A. peregrinum subsp. peregrinum, A. peregrinum subsp. integrum, A. itersonii subsp. nipponicum, and A. itersonii subsp. itersonii, were very close to each other and originates from one branch. The sequence similarities
C Fig. 1. Phylogenetic tree displaying the relationships among species of the genus Aquaspirillum and the members of the families Neisseriaceae, Oxalobacteriaceae, Comamonadaceae and alpha-subdivision of Proteobacteria. Scale bar represents 1 nucleotide substitution per 100 nucleotides. Bootstrap values of 1000 resamplings are shown at the branch points.
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Table 1 Similarity matrix based on 16S rDNA comparisons
between subspecies of each species were 97.2 and 97.8%, respectively, but those between the species was about 90%. A. polymorphum was close to Magnetospirillum gryphiswaldence by the sequence similarity of 95.9%. As was indicated in Table 1, all the species of the genus were diverged from the type species A. serpens the sequence similarities among them were 81.5^93.0%. As indicated in the phylogenetic tree and similarity matrix, the genus Aquaspirillum was found to be quite heterogeneous. These results were in accordance with those obtained by Pot et al. [7], Wen et al. [19], and Kawasaki et al. [20]. Based on the 16S rDNA sequences, placement of three species, A. itersonii, A. peregrinum, and A. polymorphum, in the alpha-subdivision of Proteobacteria has already been reported [20]. Based on DNA^rRNA hybridization, Pot et al. showed that the three species A. serpens, A. putridiconchylium, and A. dispar belonged to rRNA superfamily III [7], and, in this study, we found that these three species belong to the family Neisseriaceae (group I). Wen et al. determined the 16S rDNA sequences for ¢ve species, A. delicatum, A. sinuosum, A. metamorphum, A. psychrophilum, and A. gracile, and found that they belong to the family Comamonadaceae [19], but we found that seven species, including those ¢ve and two others, A. giesbergeri and A. annulus, belong to the family Comamonadaceae. We have shown here the phylogenetic positions of the 15 species of genus Aquaspirillum, and examined that the
genus is a quite heterogeneous. Final re-arrangement of the genus should await further studies, including characterization of phenotype, comparative chemotaxonomy, and DNA^DNA hybridization.
References [1] Hylemon, P.B., Wells Jr., J.S., Krieg, N.R. and Jannasch, H.W. (1973) The genus Spirillum : taxonomic study. Int. J. Syst. Bacteriol. 23, 340^380. [2] Terasaki, Y. (1973) Studies on the genus Spirillum Ehrenberg. II. Comments on type and reference strains of Spirillum and description of new species and new subspecies. Bull. Suzugamine Women’s Coll. Nat. Sci. 17, 1^71. [3] Terasaki, Y. (1979) Transfer of ¢ve species and two subspecies of Spirillum to other genera (Aquaspirillum and Oceanospirillum), with emended descriptions of the species and subspecies. Int. J. Syst. Bacteriol. 29, 130^144. [4] Kumar, R., Banerjee, A.K., Brwdre, J.H., McElroy, L.J. and Krieg, N.R. (1974) Isolation, characterization, and taxonomy of Aquaspirillum bengal sp. nov.. Int. J. Syst. Bacteriol. 24, 453^458. [5] Boivin, M.F., Morris, V.L., Lee-Chan, E.C.M. and Murray, R.G.E. (1985) Deoxyribonucleic acid relatedness between selected members of the genus Hylemon, Wells, Krieg, and Jannasch 1973 emended to include Aquaspirillum bengal as subjective synonym. Int. J. Syst. Bacteriol. 35, 512^517. [6] Strength, W.J., Isani, B., Linn, D.M., Williams, F.D., Vandermolen, G.E., Laughon, B.E. and Krieg, N.R. (1976) Isolation and characterization of Aquaspirillum fasciculus sp. nov., a rod-shaped nitrogen¢xing bacterium having unusual £agella. Int. J. Syst. Bacteriol. 26, 253^268.
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L. Ding, A. Yokota / FEMS Microbiology Letters 212 (2002) 165^169 [7] Pot, B., Willems, A., Gillis, M. and Deler, J. (1992) Intra-and intergeneric relationships of the genus Aquaspirillum: Prolinobors, a new genus for Aquaspirillum fasciulus, with the species Prolinoborus fasciculus comb. nov. Int. J. Syst. Bacteriol. 42, 44^57. [8] Aragno, M. and Schlegel, H.G. (1978) Aquaspirillum autotrophicum, a new species of hydrogen-oxidizing, facultatively atotrophic bacteria. Int. J. Syst. Bacteriol. 28, 112^116. [9] Schleifer, K.H., Schu«ler, D., Spring, S., Weizenegger, M., Amann, R., Ludwig, W. and Ko«hler, M. (1991) The genus Magnetospirillum gen. nov. Description of Magnetospirillum gryphiswaldens sp. nov. and transfer of Aquaspirillum magnetotacticum to Maganetospirillum maganetotacticum comb. nov. Syst. Appl. Microbiol. 14, 379^385. [10] Eden, P., Schmidt, T.M., Blakemore, R.P. and Pace, N.R. (1991) Phylogenetic analysis of Aquaspirillum magnetotacticum using polymerase chain reaction-ampli¢ed 16S rRNA-speci¢c DNA. Int. J. Syst. Bacteriol. 41, 324^325. [11] Willems, A., Pot, B., Falsen, E., Vandamme, P., Gillis, M., Kersters, K. and De Ley, J. (1991) Polyphasic taxonomic study of the emended genus Comamonas: relationship to Aquaspirillum aquaticum, E. Falsen group 10, and other clinical isolates. Int. J. Syst. Bacteriol. 41, 427^444. [12] Willems, A., De Ley, J., Gillis, M. and Kersters, K. (1991) Comamonadaceae, a new family encompassing the acidovorans rRNA complex, including Varivorax paradoxus gen. nov., comb. nov., for Alcaligenes paradoxus (Davis, 1969). Int. J. Syst. Bacteriol. 41, 445^ 450. [13] Butler, B.J., McCallum, K.L. and Inniss, W.E. (1989) Characterization of Aquaspirillum arcticum sp. nov., a new psychrophilic bacterium. Syst. Appl. Microbiol. 12, 263^266. [14] Krieg, N.R. (1984) Genus Aquaspirillum Hylemon, Wells, Krieg and Jannasch 1973, 361AL . In: Bergey’s Manual of Systematic Bacteriology (Krieg, N.R., Holt, J.G., Eds.) Vol. 1, pp. 72^90. Williams and Wilkins, Baltimore, MD. [15] Pot, B. et al. (1992) The genus Aquaspirillum. In: The Prokaryotes, 2nd edn. (Ballows, A., Tru«per, H.G.M., Dwonkin, Hander, W., Schleifer, K.H., Eds.), Vol. 3, pp. 2569^2582. Springer-Verlag, New York, NY. [16] De Ley, J. and De Smedt, J. (1975) Improvements of the membrane
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¢lter method for DNA :rRNA hybridization. Antonie van Leeuwenhoek J. Microbiol. Serol. 41, 287^307. Sakane, T. and Yokota, A. (1994) Chemotaxonomic inverstigation of heterotrophic, aerobic and microaerophilic spirilla, the genera Aquaspirillum, Magnetospirillum and Oceanospirillum. Syst. Appl. Microbiol. 17, 128^134. Hamana, K. et al. (1994) Polyamine analysis of the genus Aquaspirillum Magnetospirillum, Oceanospirillum and Spirillum. J. Gen. Appl. Microbiol. 40, 75^82. Wen, A., Fegan, M., Hayward, C., Chakraborty, S. and Sly, L.I. (1999) Phylogenetic relationships among members of the Comamonadaceae, and description of Delftia acidovorans (den Dooren de Jong 1926 and Tamaoka et al., 1987) gen. nov., comb. nov. Int. J. Syst. Bacteriol. 49, 567^576. Kawasaki, H. (1997) Phylogenetic relationships of the helical-shaped bacteria in the alpha- Proteobacteria inferred from 16S rDNA sequences. J. Gen. Appl. Microbiol. 43, 89^95. IAM Catalogue of Strains (1998) pp. 277^285. The Microbiology Research Foundation, Tokyo, Japan. Hiraishi, A. (1992) Direct automated sequencing of 16S rDNA ampli¢ed by polymerase chain reaction from bacterial cultures without DNA puri¢cation. Lett. Appl. Microbiol. 15, 210^213. Uchino, Y., Yokota, A. and Sugiyama, J. (1997) Phylogenetic position of the marine subdivision of Agrobacterium species based on 16S rRNA sequence analysis. J. Gen. Appl. Microbiol. 43, 243^247. Thompsom, J.D., Higgins, D.G. and Gibson, T.J. (1994) CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position speci¢c gap penalties and weight matrix choice. Nucleic Acids Res. 22, 4673^4680. Saitou, N. and Nei, M. (1987) The neighbor-joining methods: A new method for reconstructing phylogenetic trees. Mol. Biol. Evol. 4, 406^425. Brosius, J., Dull, T.J., Sleeter, D.D. and Noller, H.F. (1981) Gene organization and primary structure of a ribosomal RNA operon from Escherichia coli. Mol. Biol. 148, 107^127. Swo¡ord, D.L. (1998) PAUP*. Phylogenetic Analysis Using Parsimony (*and other methouds) Version 4. Sinauer Associates, Sunderland, MA.
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Phylogenetic analysis of the genus Aquaspirillum based on 16S rRNA gene sequences Linxian Ding , Akira Yokota Laboratory of Bioresources, Institute of Molecular and Cellular Biosciences, The University of Tokyo, Yayoi, Bunkyo-ku, Tokyo 113-0032 Japan Received 19 February 2002 ; accepted 6 April 2002 First published online 31 May 2002
Abstract Phylogenetic analysis of 15 species of the genus Aquaspirillum based on 16S rRNA gene (rDNA) sequences indicated that the genus Aquaspirillum is phylogenetically heterogeneous and the species could be divided into four groups as follows: Aquaspirillum serpens, the type species of this genus, A. dispar and A. putridiconchylium are situated in the family Neisseriaceae; members of the second group, A. gracile, A. delicatum, A. anulus, A. giesbergeri, A. sinuosum, A. metamorphum and A. psychrophilum, are included in the family Comamonadaceae; the two members of the third group, A. arcticum and A. autotrophicum, are included in the family Oxalobacteriaceae; and members of the fourth group, A. polymorphum, A. peregrinum, and A. itersonii, are included in the alpha-subdivision of Proteobacteria. Thus, phylogenetic studies indicated that all the species excepting A. serpens, the type species, should be transferred to distinct genera. 2 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : 16S rDNA; Phylogeny; Aquaspirillum
1. Introduction The genus Aquaspirillum was established in 1973 by Hylemon et al., in which 13 species were described [1]. After that, Terasaki reported two subspecies, Aquaspirillum itersonii subsp. nipponicum and Aquaspirillum peregrinum subsp. integrum [2,3]. Kumar et al. described Aquaspirillum bengal [4], but that species has been transferred to A. serpens by Boivin et al. on the basis of DNA^DNA hybridization [5]. Strength et al. reported Aquaspirillum fasciculus [6], which was lately transferred to the genus Prolinoborus as P. fasciculus based on DNA^rRNA hybridization studies [7]. Aragno et al. [8] isolated a new hydrogen-oxidizing species, Aquaspirillum autotrophicum. Schleifer et al. established the genus Magnetospirillum, and they proposed to transfer Aquaspirillum magnetotacticum to this genus as Magnetospirillum magnetotacticum [9,10]. Willems et al. had changed Aquaspirillum aquaticum to Comamonas terrigena [11,12]. Butler et al. described a new psychrophilic bacterium Aquaspirillum arcticum [13].
Therefore, the genus Aquaspirillum includes 15 species at present. Most of the strains in this genus were isolated from fresh water (pond water, stream water, or Antarctic mosses), and all the species are aerobic and helical shaped and do not grow in the presence of 3% NaCl [14,15]. DNA^rRNA hybridization studies [7,16], chemotaxonomic investigation of the genus based on cellular fatty acids [17], respiratory quinone composition and G+C contents of DNA [17], polyamine analysis [18], and 16S rDNA sequencing [19,20] indicate that the genus Aquaspirillum is quite heterogeneous and is composed of many distinct groups intermingled phylogenetically with the related Gram-negative bacteria. In this study, we investigated the phylogenetic position of the species of the genus Aquaspirillum based on 16S rDNA sequences.
2. Materials and methods 2.1. Bacteria strains and cultivation
* Corresponding author. Tel. : +81 (3) 5841 7828; Fax : +81 (3) 5841 8490. E-mail address : [email protected] (L. Ding).
We used nine species of the genus Aquaspirillum in this study. A. serpens IAM (IAM Culture Collection, Institute of Molecular and Cellular Biosciences, The University of
0378-1097 / 02 / $22.00 2 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. PII : S 0 3 7 8 - 1 0 9 7 ( 0 2 ) 0 0 7 4 7 - 4
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Tokyo, Japan) 13944T , A. autotrophicum IAM 14942T , and A. dispar IAM 14943T were grown in medium B-33 [21](peptone 10.0 g, succinic acid 1.0 g, (NH4 )2 SO4 1.0 g, MgSO4 W7H2 O 1.0 g, FeCl3 W6H2 O 2.0 mg, MnSO4 W5H2 O 2.0 mg, distilled water 1.0 l, pH 6.8); A. anulus IAM 14948T , A. delicatum IAM 14955T , A. giesbergeri IAM 14949T , A. itersonii subsp. nipponicum IAM 14945T , A. peregrinum subsp. integrum IAM 14946T , and A. putridiconchylium IAM 14964T were grown in medium B-104 [21] (Polypepton 10.0 g, yeast extract 2.0 g, MgSO4 W7H2 O 1.0 g, distilled water 1.0 l, pH 7.0); A. arcticum IAM 14963T was grown in medium B-52 [21] (trypticase soy agar 40.0 g (BBL 11042), distilled water 1.0 l, pH 7.0), and A. polymorphum IAM 14441T was grown in medium B-66 [21] (peptone 10.0 g, yeast extract 5.0 g, liver, infusion 25.0 g, glucose 3.0 g, glycerol 15.0 g, NaCl 3.0 g, distilled water 1.0 l, pH 7.0). Bacterial strains were grown aerobically at 30‡C except for A. arcticum, which was grown at 4 ‡C. 2.2. Polymerase chain reaction (PCR) ampli¢cation of 16S rDNA and sequencing PCR for the ampli¢cation of 16S rDNA was carried out by using cell lysate extracted from organisms [22]. We used universal primers 8F (5P-AGAGTTTGATCCTGGCTCAG-3P) and 1510R (5P-GGCTACCTTGTTACGA-3P) for PCR ampli¢cation of 16S rDNA. The conditions for PCR ampli¢cation were 1 min at 94‡C and then 30 cycles of 1 min at 94‡C, 1 min at 55‡C, and 1.5 min at 72‡C, followed by a ¢nal step for 2 min at 72‡C. The PCR products were puri¢ed by using the GFXtm PCR DNA and Gel Band Puri¢cation Kit (Amersham Pharmacia Biotech, Inc., NJ, USA). 16S rDNA sequencing primers 8F, 520F, 926F, 350R, 700R, 1110R, and 1510R were used in the sequencing reactions. The 16S rDNA sequencing of all strains was performed as described previously [23]. 2.3. Phylogenetic analysis Seventy-four species of 16S rRNA (rDNA) sequences obtained from a DNA databank were aligned using CLUSTAL W, version 1.74 [24]. Nucleotide substitution rates (Knuc values) were calculated, and the phylogenetic tree was constructed by using the neighbor-joining algorithms [25]. The nucleotide sequence data of A. serpens IAM 13944T (AB074518), A. dispar IAM 14943T (AB074526), A. arcticum IAM 14963T (AB074523), A. autotrophicum IAM 14942T (AB074524), A. peregrinum subsp. integrum IAM 14946T (AB074521), A. itersonii subsp. nipponicum IAM 14945T (AB074520), A giesbergeri
IAM 14949T (AB074522), A. anulus IAM 14948T (AB074527), and A. putridiconchylium IAM 14964T (AB076000) reported in this paper will appear in the DDBJ nucleotide sequence databases with the accession numbers listed. The nucleotide sequence of Escherichia coli from Brosius et al. [26] was used as the outgroup. Similarity values of 16S rDNA sequences were calculated by the use of PAUP*, Version 4.068. [27].
3. Results and discussion We ampli¢ed 16S rDNA from nine species of the genus Aquaspirillum and determined their nucleotide sequences. The base arrangement length ampli¢ed from 16S rDNA fragments ranged from 1380 to 1503. A phylogenetic tree based upon nucleotide substitution values is depicted in Fig. 1. The 16S rDNA similarity values are shown in Table 1. Fig. 1 showed that 15 species (four subspecies) of genus Aquaspirillum have been divided into four groups. A. serpens, the type species of this genus, is situated in the family Neisseriaceae. Group I, A. serpens and A. putridiconchylium, originates from one branch, but the sequence similarity was only 93.0%. A. dispar was close to the genus Microvirgula aerodenitri¢cans with the sequence similarity of 98.8%. A. arcticum and A. autotrophicum in the second group (group II) belonged to the family Oxalobacteriaceae. A. arcticum formed a cluster with Duganella zoogloeoides and Janthinobacterium, and the sequence similarities among them were 96.9^97.2%. A. autotrophicum was close to Herbaspirillum frisingense, where the sequence similarity was 96.6%. As members of group III, A. delicatum, A. giesbergeri, A. anulus, A. sinuosum, A. metamorphum, A. psychrophilum, and A. gracile belonged to the family Comamonadaceae, and three species, A. anulus, A. sinuosum, and A. giesbergeri formed a cluster. The 16S rDNA sequence similarities among these were 98.3^98.7%. Two species, A. psychrophilum and A. metamorphum, formed a cluster, and the sequence similarity between them was 95.1%. A. gracile formed a cluster with the genus Delftia, but the sequence similarity between them was 93.8.%. A. delicatum stands alone. Groups I, II, and III belong to the beta-subdivision of Proteobacteria. As the fourth group, the three species A. polymorphum, A. peregrinum, and A. itersonii were included in the alpha-subdivision of Proteobacteria. Two species (four subspecies), A. peregrinum subsp. peregrinum, A. peregrinum subsp. integrum, A. itersonii subsp. nipponicum, and A. itersonii subsp. itersonii, were very close to each other and originates from one branch. The sequence similarities
C Fig. 1. Phylogenetic tree displaying the relationships among species of the genus Aquaspirillum and the members of the families Neisseriaceae, Oxalobacteriaceae, Comamonadaceae and alpha-subdivision of Proteobacteria. Scale bar represents 1 nucleotide substitution per 100 nucleotides. Bootstrap values of 1000 resamplings are shown at the branch points.
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Table 1 Similarity matrix based on 16S rDNA comparisons
between subspecies of each species were 97.2 and 97.8%, respectively, but those between the species was about 90%. A. polymorphum was close to Magnetospirillum gryphiswaldence by the sequence similarity of 95.9%. As was indicated in Table 1, all the species of the genus were diverged from the type species A. serpens the sequence similarities among them were 81.5^93.0%. As indicated in the phylogenetic tree and similarity matrix, the genus Aquaspirillum was found to be quite heterogeneous. These results were in accordance with those obtained by Pot et al. [7], Wen et al. [19], and Kawasaki et al. [20]. Based on the 16S rDNA sequences, placement of three species, A. itersonii, A. peregrinum, and A. polymorphum, in the alpha-subdivision of Proteobacteria has already been reported [20]. Based on DNA^rRNA hybridization, Pot et al. showed that the three species A. serpens, A. putridiconchylium, and A. dispar belonged to rRNA superfamily III [7], and, in this study, we found that these three species belong to the family Neisseriaceae (group I). Wen et al. determined the 16S rDNA sequences for ¢ve species, A. delicatum, A. sinuosum, A. metamorphum, A. psychrophilum, and A. gracile, and found that they belong to the family Comamonadaceae [19], but we found that seven species, including those ¢ve and two others, A. giesbergeri and A. annulus, belong to the family Comamonadaceae. We have shown here the phylogenetic positions of the 15 species of genus Aquaspirillum, and examined that the
genus is a quite heterogeneous. Final re-arrangement of the genus should await further studies, including characterization of phenotype, comparative chemotaxonomy, and DNA^DNA hybridization.
References [1] Hylemon, P.B., Wells Jr., J.S., Krieg, N.R. and Jannasch, H.W. (1973) The genus Spirillum : taxonomic study. Int. J. Syst. Bacteriol. 23, 340^380. [2] Terasaki, Y. (1973) Studies on the genus Spirillum Ehrenberg. II. Comments on type and reference strains of Spirillum and description of new species and new subspecies. Bull. Suzugamine Women’s Coll. Nat. Sci. 17, 1^71. [3] Terasaki, Y. (1979) Transfer of ¢ve species and two subspecies of Spirillum to other genera (Aquaspirillum and Oceanospirillum), with emended descriptions of the species and subspecies. Int. J. Syst. Bacteriol. 29, 130^144. [4] Kumar, R., Banerjee, A.K., Brwdre, J.H., McElroy, L.J. and Krieg, N.R. (1974) Isolation, characterization, and taxonomy of Aquaspirillum bengal sp. nov.. Int. J. Syst. Bacteriol. 24, 453^458. [5] Boivin, M.F., Morris, V.L., Lee-Chan, E.C.M. and Murray, R.G.E. (1985) Deoxyribonucleic acid relatedness between selected members of the genus Hylemon, Wells, Krieg, and Jannasch 1973 emended to include Aquaspirillum bengal as subjective synonym. Int. J. Syst. Bacteriol. 35, 512^517. [6] Strength, W.J., Isani, B., Linn, D.M., Williams, F.D., Vandermolen, G.E., Laughon, B.E. and Krieg, N.R. (1976) Isolation and characterization of Aquaspirillum fasciculus sp. nov., a rod-shaped nitrogen¢xing bacterium having unusual £agella. Int. J. Syst. Bacteriol. 26, 253^268.
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