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1 Marine Works Japan Ltd, Kamariyahigashi, 2-16-32 4F, Kanazawa-ku, Yokohama 236-0042, Japan
2 Department of Environmental Sciences, Faculty of Science, Shinshu University, Asahi, 3-1-1, Matsumoto 390-8621, Japan
3 Department of Microbiology and Immunology, Graduate School of Medicine, Tohoku University, Seiryo-machi, 2-1, Aoba-ku, Sendai 980-8575, Japan
4 Hitachi Plant Engineering & Construction Co., Ltd, Matsudo 271-0064, Japan
5 Department of Laboratory Sciences, School of Health Sciences, Faculty of Medicine, Gunma University, Showa-machi 3chome, 39-15, Maebashi 371-8514, Japan
6 Department of Ecological Engineering, Toyohashi University of Technology, Toyohashi 441-8580, Japan
7 Department of Biology and Geosciences, Faculty of Science, Shizuoka University, Oya, 836, Shizuoka 422-8529, Japan
Correspondence
Tomoko Maruyama
maruyamat{at}mwj.co.jp
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7c (36–41 %) and 16 : 17c (33–36 %). Hydroxy fatty acids were mainly 2-OH 14 : 0 (11–13 %), and 3-OH fatty acids were absent. Glycosphingolipids were detected. Ubiquinone-10 and homospermidine were present as the major quinine and polyamine, respectively. Thus, it is proposed that the three strains represent a new genus and species of the family Sphingomonadaceae with the name Sphingosinicella microcystinivorans gen. nov., sp. nov. The type strain is Y2T (=KCTC 12019T=JCM 13185T).
The GenBank/EMBL/DDBJ/accession numbers for the 16S rRNA gene sequences of strains Y2T, MDB2 and MDB3 are AB084247, AB219940 and AB219941.
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; Kuiper-Goldman et al., 1999). A microcystin-degrading bacterium designated strain Y2T was isolated using diluted nutrient agar (Nissui Pharmaceutical) from a eutrophic lake, Lake Suwa, Japan, during the blooming period of toxic Microcystis (Park et al., 2001). This strain was able to degrade microcystin-RR, -YR and -LR and its isomer 6(Z)-Adda microcystin-LR and to grow in inorganic media containing microcystin as the sole carbon source as well as in diluted nutrient broth (Park et al., 2001). A phylogenetic analysis of strain Y2T based on 16S rRNA gene sequences revealed that it represents a deeply branching lineage within the cluster of the sphingomonads, including the genera Blastomonas, Novosphingobium, Sphingobium, Sphingopyxis and Sphingomonas (Park et al., 2001). Later, we isolated two other strains (MDB2 and MDB3) of microcystin-degrading bacteria from the Tenryu River in Japan. These strains were phylogenetically similar to strain Y2T. In the present study, we describe the taxonomic properties of these three strains of microcystin degraders and propose to classify them in a novel genus and species.
General cell morphology, Gram reaction, spore formation and motility by means of flagella were studied under an Olympus light microscope (U-LH 1000) by NCIMB Japan (Shizuoka, Japan). Colony shape was observed after the cells were incubated at 30 °C for 48 h on nutrient agar (Oxoid). Biochemical tests were performed by NCIMB Japan using an API 20NE kit according to the manufacturer's instructions (API bioMérieux) and by conventional tests for activity of catalase and oxidase, gas/acid production from glucose and oxidation/fermentation from glucose, as described previously (Barrow & Feltham, 1993). Analysis of cellular fatty acids was performed by NCIMB Japan using the Sherlock Microbial Identification system (version 5.0; MIDI Inc.) according to the manufacturer's instructions. Cellular fatty acids were extracted from cells grown on trypticase soy (SCD) agar (Becton Dickinson) at 30 °C for 24 h and analysed as methyl esters. Glycosphingolipids were analysed by TLC as described previously (Takeuchi et al., 2001). Respiratory quinone profiles were studied as described previously (Hiraishi et al., 1996; Iwasaki & Hiraishi, 1998). Polyamines were analysed as previously reported (Hamana & Takeuchi, 1998; Hamana et al., 2003). Genomic DNA was extracted and purified by the phenol extraction method as described previously (Saito & Miura, 1963) and DNA base composition was determined by the HPLC method of Katayama-Fujimura et al. (1984). After genomic DNA was prepared by the PrepMan method (Applied Biosystems), 16S rRNA genes were amplified by PCR and sequenced with a MicroSeq Full 16S rDNA Bacterial Sequencing kit (Applied Biosystems) by NCIMB Japan. Sequence similarities were studied using the BLAST program (Altschul et al., 1997). Related sequences including type strains of established genera of the family Sphingomonadaceae were obtained from GenBank/EMBL/DDBJ. Multiple alignments of sequence data, calculation of evolutionary distances and construction of a neighbour-joining phylogenetic tree (Saitou & Nei, 1987) were performed with the CLUSTAL W program (Thompson et al., 1994) using bootstrap values based on 1000 replications.
Strains Y2T, MDB2 and MDB3 were Gram-negative, non-spore-forming rods measuring 0·6–0·7 µm in width and 0·8–1·0 µm in length. Cells were motile by means of single polar flagella. All three strains formed yellow colonies on nutrient agar (Oxoid) after 48 h incubation at 30 °C. The temperature range for growth was 10–37 °C and the optimum temperature was 30 °C. No growth occurred at 45 °C. The pH range for growth was 7–9. The strains were strictly aerobic and chemo-organotrophic. They exhibited positive reactions for oxidase and catalase but negative reactions in the oxidation/fermentation test and gas/acid production test with glucose. Other physiological and biochemical characteristics of strains Y2T, MDB2 and MDB3 were compared with those of type strains of the phylogenetically related genera Sphingomonas, Sphingobium, Novosphingobium and Sphingopyxis (Table 1). In assimilation tests using 12 carbon sources, strains Y2T, MDB2 and MDB3 were shown to assimilate citrate only. The strains did not assimilate glucose, L-arabinose, D-mannose, N-acetyl-D-glucosamine, maltose, gluconate, n-caproate, adipate, DL-malic acid or phenylacetate. Strains Y2T, MDB2 and MDB3 exhibited negative reactions for all other phenotypic tests: nitrate reduction, 
-galactosidase, aesculin hydrolysis, urease, gelatin hydrolysis, indole production, glucose fermentation and arginine dihydrolase. A negative reaction for nitrate reduction, which was proposed as a phenotypic marker to distinguish the four genera of the family Sphingomonadaceae (Takeuchi et al., 2001), was characteristic of Sphingobium, Novosphingobium and some species of Sphingomonas.
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, the major fatty acids of the three strains were 18 : 17c (36–41 %) and 16 : 17c (33–36 %). Minor fatty acids were 16 : 0 (7–8 %), 16 : 15c (3 %) and 14 : 0 (1–2 %). The main component of the hydroxy fatty acids was 2-OH 14 : 0 (11–13 %), and 3-OH fatty acids were absent. Analysis of lipid extracts by TLC revealed the presence of glycosphingolipids in all three strains. The major respiratory quinone was Q-10. The polyamine detected was homospermidine [1·5 µmol (g wet cells)–1], as reported for the genus Sphingomonas. The DNA G+C content of the three strains ranged from 63·6 to 63·7 mol%.
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), B9 (99·3 %; AB159609; Harada et al., 2004) and IC075 (99·3 %; AB196249; Inoue et al., 2005). Strains 7CY and B9 were also microcystin-degrading bacteria, isolated independently from Lake Suwa. The microcystin-degrading processes of strains 7CY and B9 were quite similar to that of strain Y2T, as several common degradation products were detected (Park et al., 2001; Harada et al., 2004; Ishii et al., 2004). Saito et al. (2003) reported that strain Y2T possessed a gene, mlrA, that encodes a hydrolytic enzyme to open the cyclic peptide of microcystins (Bourne et al., 2001). These findings suggest that strains Y2T, 7CY and B9 are highly similar. Strain IC075 was able to degrade carbazole, which is an aromatic compound similar in structure to dioxins. Although there has been no report of the ability of strain IC075 to degrade microcystin, the high 16S rRNA gene sequence similarity between strains Y2T and IC075 implies that the latter strain may be able to degrade microcystin. A phylogenetic tree based on 16S rRNA gene sequences revealed that strains Y2T, MDB2 and MDB3 formed a distinct clade together with strains 7CY, B9 and IC075 within the family Sphingomonadaceae (Kosako et al., 2000). However, this clade was separate from any of the established genera of this family, in particular Sphingomonas, Sphingobium, Novosphingobium and Sphingopyxis (Fig. 1); strains Y2T, MDB2 and MDB3 showed 16S rRNA gene sequence similarity of 90·9–94·4 % to the type strains of the respective type species.
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), we aligned the sequences of strains Y2T, MDB2, MDB3, 7CY, B9 and IC075. Nucleotide signatures specific to the 16S rRNAs of strains Y2T, MDB2 and MDB3 were the same as those of the genus Sphingomonas sensu stricto reported by Takeuchi et al. (2001), i.e. C : G at position 52 : 359, G at position 134, G at position 593, G : C at position 987 : 1218 and U : G at position 990 : 1215 (Escherichia coli numbering; Brosius et al., 1978). The same nucleotide signatures were found in the other microcystin degraders strains 7CY (Ishii et al., 2004) and B9 (Harada et al., 2004) and the carbazole-utilizing strain IC075 (Inoue et al., 2005).
As described above, the phylogenetic data demonstrate clearly that strains Y2T, MDB2 and MDB3 are members of the family Sphingomonadaceae. However, since strains Y2T, MDB2 and MDB3 form a distinct phylogenetic cluster within this family, it is difficult to allocate them to any of the previously described genera (Fig. 1
). 16S rRNA gene sequence similarities between strains Y2T, MDB2, MDB3 and the type strains of species of established genera were low, ranging from 90·9 to 94·9 %. Takeuchi et al. (2001) reported that the genera of the family Sphingomonadaceae were separated at approximately <95 % 16S rRNA gene sequence similarity. Chemotaxonomic and phenotypic data support the conclusion that these strains are members of the family Sphingomonadaceae (Tables 1 and 2). Glycosphingolipids and ubiquinone-10 were present. Strains Y2T, MDB2 and MDB3 contained 18 : 17c and 16 : 17c as the dominant fatty acids and 2-OH 14 : 0 as the major hydroxy fatty acid (Takeuchi et al., 1993, 2001; Kämpfer et al., 1997; Tiirola et al., 2005) and 3-OH fatty acids were absent (Takeuchi et al., 1993) (Table 2). The polyamine of the microcystin-degrading strains was homospermidine, as was the case for the genus Sphingomonas sensu stricto, whereas all other genera noted above contained spermidine (Takeuchi et al., 2001; Hamana et al., 2003). The ability to reduce nitrate was absent from our strains as well as from Sphingobium and Sphingopyxis strains.
By a combination of a number of chemotaxonomic and phenotypic characteristics listed above (see Tables 1 and 2), together with phylogenetic information of the formation of a distinct clade within the family Sphingomonadaceae and low 16S rRNA gene sequence similarity (<95 %) to related genera, it is most appropriate to conclude that these novel microcystin-degrading strains should be classified in a novel genus and species of the family Sphingomonadaceae. The name Sphingosinicella microcystinivorans gen. nov., sp. nov. is proposed for the three strains.
Description of Sphingosinicella gen. nov.
Sphingosinicella (Sphin.go.si'ni.cel'la. N.L. n. sphingosinum sphingosine; L. fem. n. cella a store-room and in biology a cell; N.L. fem. n. Sphingosinicella sphingosine-containing cell).
Cells are Gram-negative, non-spore-forming rods, motile by means of polar flagella. Colonies are yellow. Strictly aerobic and chemo-organotrophic. Catalase- and oxidase-positive. Nitrate is not reduced to nitrite. The major fatty acids are 18 : 17c and 16 : 17c. 2-Hydroxy fatty acids are present, with 2-OH 14 : 0 predominating. 3-Hydroxy fatty acids are absent. Glycosphingolipids are produced. Respiratory quinone is predominantly Q-10. Homospermidine is the major polyamine component, as for the genus Sphingomonas. Placed phylogenetically in the family Sphingomonadaceae. The characteristic 16S rRNA signatures are the same as for the genus Sphingomonas: 52 : 359 (C : G), 134 (G), 593 (G), 987 : 1218 (G : C) and 990 : 1215 (U : G). The type species is Sphingosinicella microcystinivorans.
Description of Sphingosinicella microcystinivorans sp. nov.
Sphingosinicella microcystinivorans (mi.cro.cys'ti.ni.vo'rans. N.L. n. microcystinum microcystin; L. part. adj. vorans devouring; N.L. part. adj. microcystinivorans microcystin-degrading).
Shows the following properties in addition to those given in the genus description. Cells are 0·3–0·7x0·6–1·0 µm. Citrate only is assimilated. Negative reactions are observed for hydrolysis of aesculin, gelatin and urease, activity of -galactosidase, indole production, glucose fermentation, arginine dihydrolase and assimilation of glucose, L-arabinose, D-mannose, N-acetyl-D-glucosamine, maltose, gluconate, n-caproate, adipate, DL-malic acid and phenylacetate. Major fatty acids are 18 : 17c (33–36 %) and 16 : 17c (36–41 %); 16 : 0 (7–8 %), 16 : 15c (3 %) and 14 : 0 (1–2 %) are produced as minor components. Major 2-hydroxy fatty acid is 2-OH 14 : 0 (11–13 %); 2-OH 16 : 0 (1 %) is produced as a minor component. Polyamine is homospermidine [1·5 µmol (g wet cells)–1]. The DNA G+C content is 63·6–63·7 mol%.
The type strain, strain Y2T (=KCTC 12019T=JCM 13185T), was isolated from a toxic Microcystis blooming lake, Lake Suwa, Japan. Strains MDB2 and MDB3, isolated from the Tenryu River, Japan, are reference strains.
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