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1 Division of Applied Life Science, EB-NCRC, PMBBRC, Gyeongsang National University, Jinju 660-701, Republic of Korea
2 Division of Environmental Science and Ecological Engineering, Korea University, Seoul 136-701, Republic of Korea
Correspondence
Che Ok Jeon
cojeon{at}gsnu.ac.kr
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ABSTRACT |
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7c and/or iso-C15 : 0 2-OH), C16 : 0, C18 : 17c and C18 : 17c 11-methyl. The strain contained phosphatidylethanolamine and diphosphatidylglycerol as polar lipids. The G+C content of the genomic DNA was 63.3 mol%. The major quinone was Q-8. Phylogenetic analysis of 16S rRNA gene sequences showed that strain EMB325T formed a phyletic lineage with members of the genus Simplicispira and was most closely related to Simplicispira psychrophila LMG 5408T and Simplicispira metamorpha DSM 1837T with similarities of 98.1 and 97.9 %, respectively. Levels of DNA–DNA relatedness between strain EMB325T and S. psychrophila LMG 5408T and S. metamorpha DSM 1837T were 28 and 23 %, respectively. On the basis of chemotaxonomic data and molecular characteristics, strain EMB325T is considered to represent a novel species within the genus Simplicispira, for which the name Simplicispira limi sp. nov. is proposed. The type strain is EMB325T (=KCTC 12608T=DSM 17964T).
The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain EMB325T is DQ372987.
A transmission electron micrograph of cells of strain EMB325T is available as supplementary material in IJSEM Online.
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) and here report the taxonomic characterization of a novel species of the genus Simplicispira. The genus Simplicispira belongs to the family Comamonadaceae and was described by Grabovich et al. (2006). At the time of writing the genus comprised just two recognized species, Simplicispira psychrophila (formerly Aquaspirillum psychrophilum) and Simplicispira metamorpha (formerly Aquaspirillum metamorphum).
Strain EMB325T was isolated from activated sludge that performed EBPR in a laboratory-scale sequencing batch reactor (SBR). Sodium acetate was supplied as the sole carbon source and the operation of the SBR has been described by Jeon et al. (2003). A sludge sample was diluted serially with 1 % (w/v) saline solution and spread on R2A agar (Difco) and incubated at 20 °C for 5 days. Subcultivation was on R2A agar at 30 °C for 3 days. Gram staining was determined using a bioMérieux Gram stain kit according to the manufacturer's instructions. Cell morphology and motility were studied by using phase-contrast microscopy and transmission electron microscopy (JEM-1010; JEOL) as described by Jeon et al. (2005). Growth was tested at different temperatures (4–55 °C) on TSB agar and at different pH values in R2A broth. Media with different pH values were prepared using appropriate biological buffers: Na2HPO4/NaH2PO4 buffer, Na2CO3/NaHCO3 buffer and Na2HPO4/NaOH buffer were used for pH values below 8.0, pH values of 8.0–10.0 and pH 11.0, respectively (Gomori, 1955). Oxidase activity was tested by oxidation of 1 % (w/v) tetramethyl-p-phenylenediamine (Merck). Catalase activity was determined by production of oxygen bubbles in 3 % (v/v) aqueous hydrogen peroxide solution. Hydrolysis of compounds was determined on R2A agar after 7 days incubation according to the methods described by Lanyi (1987) and Gerhardt et al. (1994). Nitrate reduction was performed according to the method of Lanyi (1987). Acid production from carbohydrates was tested as described by Leifson (1963). Oxidation of various substrates, and additional enzyme activities and biochemical features were determined by using the Biolog GN2 MicroPlate assay (Biolog) and API kits (API ZYM and API 20E; bioMérieux) at 30 °C as recommended by the manufacturers, respectively. Strain EMB325T on R2A agar formed white, glistening, translucent, slightly sticky, slightly raised circular colonies with lobate margins when grown at 30 °C for 3 days. Growth was observed at temperatures of 10–40 °C, with an optimum growth temperature of 30 °C. The strain grew in the pH range 6.0–9.0, with an optimum pH for growth of 7.0–8.0. Cells were motile rods (width of 0.7–0.8 µm and length of 1.5–1.7 µm), with a polar flagellum visible at different stages of growth (see Supplementary Fig. S1 in IJSEM Online). By contrast, cells of S. psychrophila and S. metamorpha are spirilla with bipolar flagella. Different cell morphologies and flagella types have been reported for different members of the same genus. For example, cells of Polaromonas naphthalenivorans CJ2T are cocci that lack flagella, but other Polaromonas species and related genera have rod-like cells with flagella (Jeon et al., 2004). Cells of strain EMB325T were Gram-negative, oxidase-positive and catalase-positive. The isolate utilized citrate and reduced nitrate to nitrite. The isolate was negative for production of indole, H2S and acetoin (API 20E). Anaerobic growth was not observed on R2A agar for 7 days at 30 °C but was observed on R2A agar containing 10 mM nitrate.
Analysis of fatty acid methyl esters was performed according to the instructions of the Microbial Identification System (MIDI; Microbial ID). Analyses of polar lipids and isoprenoid quinones were carried out using the methods described by Komagata & Suzuki (1987). The DNA G+C content of strain EMB325T was determined by using an HPLC system fitted with a reversed-phase column (GROM-SIL 100 ODS-2FE; GROM) according to the method of Tamaoka & Komagata (1984). The major respiratory lipoquinone of strain EMB325T was ubiquinone-8 (Q-8). The cellular membrane of the strain contained summed feature 3 (C16 : 17c and/or iso-C15 : 0 2-OH; 47.0 %), C16 : 0 (22.0 %), C18 : 17c (11.9 %), C18 : 17c 11-methyl (7.1 %), C10 : 0 3-OH (3.8 %), C12 : 0 (3.5 %), C14 : 0 (1.9 %), summed feature 7 (an unknown fatty acid of ECL 18.846, C19 : 0 cyclo 10c and/or C19 : 16c; 1.1 %), C8 : 0 3-OH (0.9 %) and C15 : 0 (0.9 %). Polar lipids were dominated by phosphatidylethanolamine; diphosphatidylglycerol was present as a minor component. The G+C content of the genomic DNA of strain EMB325T was 63.3 mol%. Other phenotypic features of strain EMB325T are given in the species description below and are summarized and compared with those of the type strains of closely related species in Table 1. Some of these characteristics, such as cell morphology, flagella type and major cellular fatty acids, allow the differentiation of strain EMB325T from closely related species (Hylemon et al., 1973; Willems et al., 1990; Khan & Hiraishi, 2002; Mechichi et al., 2003; Grabovich et al., 2006).
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). The PCR product was cloned using a TOPO cloning kit (Invitrogen) and sequenced. The resultant 16S rRNA gene sequence (1455 nt) of strain EMB325T was compared with available 16S rRNA gene sequences from GenBank by using the BLAST program (http://www.ncbi.nlm.nih.gov/blast/) to determine the approximate phylogenetic affiliation, and was then aligned with sequences of closely related taxa by using the CLUSTAL W software (Thompson et al., 1994). Phylogenetic trees were constructed using three different methods, neighbour-joining, maximum-likelihood and maximum-parsimony algorithms available in the PHYLIP software, version 3.6 (Felsenstein, 2002). Sequence similarity values were computed using Similarity Matrix version 1.1 (Ribosomal Database Project II; http://35.8.164.52/html/; Cole et al., 2003). A bootstrap analysis was performed according to the algorithm of the Kimura two-parameter model (Kimura, 1980) of the neighbour-joining method in the PHYLIP package. DNA–DNA hybridization experiments were carried out to evaluate levels of relatedness between strain EMB325T and S. psychrophila LMG 5408T and S. metamorpha DSM 1837T. Extracted genomic DNA samples for slot hybridization were fragmented with restriction enzyme HaeIII, and random primed DNA labelling with digoxigenin-dUTP and hybridizations were performed using a digoxigenin High Prime DNA labelling kit (Roche Applied Science) according to the manufacturer's instructions and standard procedures (Sambrook & Russell, 2001; Lim et al., 2005). Phylogenetic analysis using the 16S rRNA gene sequence of strain EMB325T showed that it formed a phylogenetic lineage with S. psychrophila LMG 5408T and S. metamorpha DSM 1837T (Fig. 1) with similarities of 98.1 and 97.9 %, respectively. The topologies of phylogenetic trees built using the maximum-likelihood and maximum-parsimony algorithms also supported the conclusion that strain EMB325T represents a member of the genus Simplicispira (data not shown). However, levels of DNA–DNA relatedness between strain EMB325T and S. psychrophila LMG 5408T and S. metamorpha DSM 1837T were 28±3.5 and 23±2.8 %, respectively (mean±SD of three determinations). The physiological, biochemical and molecular properties of strain EMB325T suggest that it represents a novel species of the genus Simplicispira, for which the name Simplicispira limi sp. nov. is proposed.
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Cells form white, glistening, translucent, slightly sticky, raised circular colonies with lobate margins on R2A agar. Growth occurs at 10–40 °C (optimum at 30 °C). Cells are Gram-negative rods, 0.7–0.8 µm wide and 1.5–1.7 µm long (at 25 °C on R2A agar). Motile by means of a polar flagellum. Nitrate is reduced to nitrite. Catalase- and oxidase-positive. Urea, Tween 80, tyrosine and aesculin are hydrolysed. Casein, Tween 20, hypoxanthine, starch and gelatin are not hydrolysed. Acids are produced from D-raffinose, D-lactose, D-fructose and D-mannitol, but not from myo-inositol, L-arabinose, D-glucose, sorbitol, sucrose, rhamnose, amygdalin, melibiose, D-galactose, D-mannose, arbutin or salicin. Produces alkaline phosphatase, leucine arylamidase, valine arylamidase, trypsin, naphthol-AS-BI-phosphohydrolase and N-acetyl-
-glucosaminidase, but not 
-galactosidase, -glucuronidase, -glucosidase, -mannosidase or -fucosidase. Weak enzymic activity is observed for esterase (C4), esterase lipase (C8), lipase (C14), cystine arylamidase, -chymotrypsin, acid phosphatase, -galactosidase and -glucosidase. Utilizes Tweens 40 and 80, pyruvic acid methyl ester, succinic acid monomethyl ester, -hydroxybutyric acid, -hydroxybutyric acid, -ketovaleric acid, DL-lactic acid, succinamic acid, L-proline and 2,3-butanediol (Biolog GN2). Other organic substrates included in Biolog GN2 microplates are not utilized. Contains a large amount of phosphatidylethanolamine and a small amount of diphosphatidylglycerol as polar lipids. The major isoprenoid quinone is ubiquinone-8 (Q-8). The major fatty acids are summed feature 3 (C16 : 17c and/or iso-C15 : 0 2-OH), C16 : 0, C18 : 17c and C18 : 17c 11-methyl. The DNA G+C content is 63.3 mol% (HPLC).
The type strain, EMB325T (=KCTC 12608T=DSM 17964T), was isolated from sludge performing EBPR in a laboratory-scale SBR.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Felsenstein, J. (2002). PHYLIP (phylogeny inference package), version 3.6a. Distributed by the author. Department of Genome Sciences, University of Washington, Seattle, USA.
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Lu, S., Park, M., Ro, H.-S., Lee, D. S., Park, W. & Jeon, C. O. (2006). Analysis of microbial communities using culture-dependent and culture-independent approaches in an anaerobic/aerobic SBR reactor. J Microbiol 44, 155–161.[Medline]
Mechichi, T., Stackebrandt, E. & Fuchs, G. (2003). Alicycliphilus denitrificans gen. nov., sp. nov., a cyclohexanol-degrading, nitrate-reducing -proteobacterium. Int J Syst Evol Microbiol 53, 147–152.
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