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1 Korea Research Institute of Bioscience and Biotechnology (KRIBB), 52 Oeundong, Yusong, Daejeon 305-333, Republic of Korea
2 Department of Microbiology and Microbial Engineering, School of Life Sciences, Fudan University, Shanghai 200433, China
3 proBionic Corp., KRIBB, 52 Oeundong, Yusong, Daejeon 305-333, Republic of Korea
4 Department of Applied Microbiology, Yeungnam University, Kyeongsan-si, Kyeongbuk 712-749, Republic of Korea
Correspondence
Yong-Ha Park
peter{at}yumail.ac.kr
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ABSTRACT |
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7c as the dominant cellular fatty acid. Strain Iso 3T grew at NaCl concentrations of 1–10 % and temperatures of 4–30 °C. The optimal growth temperature was 20 °C. Analysis of the 16S rRNA gene sequence revealed that this strain is affiliated with a subcluster of the Alphaproteobacteria. However, strain Iso 3T generated metabolic energy by sulfide oxidation. The 16S rRNA gene sequence similarity between strain Iso 3T and the type strain of the most closely related species, Sulfitobacter pontiacus, was 97.7 %. DNA–DNA relatedness between strain Iso 3T and Sulfitobacter pontiacus DSM 10014T was 24.1 %. On the basis of phenotypic properties and phylogenetic distinctiveness, strain Iso 3T is classified within a novel Sulfitobacter species, for which the name Sulfitobacter litoralis sp. nov. is proposed, with the type strain Iso 3T (=KCTC 12521T=DSM 17584T).
The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain Iso 3T is DQ097527.
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). According to recently reported data, members of the genus Sulfitobacter are ubiquitous marine bacteria (Ivanova et al., 2004). Strains of the genus Sulfitobacter, which is a member of the Roseobacter group, were first isolated in 1995 from the Black Sea (Ivanova et al., 2004; Sorokin, 1995). Members of the Roseobacter group are abundant in the marine environment (Gonzalez et al., 1999), but many isolates are as yet undescribed. In this study, we describe a Sulfitobacter-like strain, designated Iso 3T, which we have characterized by phenotypic, genetic and chemotaxonomic analyses.
Strain Iso 3T was isolated from a water sample from the East Sea in Korea using the dilution plating technique. The strain was grown routinely on marine agar 2216 (MA; Difco) at 20 °C and replated every 2 days. As reference strains, the most closely related type strains by 16S rRNA gene sequence similarity, Sulfitobacter pontiacus DSM 10014T, Sulfitobacter brevis DSM 11443T, Sulfitobacter delicatus ATCC BAA-321T and Sulfitobacter dubius ATCC BAA-320T, were obtained from the DSMZ and the ATCC and grown under the same conditions. The morphology of live cells and the presence of flagella were investigated using light microscopy (Nikon E600) and transmission electron microscopy (TEM). For TEM observations, cells from exponentially grown cultures were negatively stained with 1 % (w/v) phosphotungstic acid. After air drying, the grid was examined using a model H-7600 transmission electron microscope (Hitachi). Growth at various NaCl concentrations, temperatures and pH values was measured in marine broth 2216 (MB; Difco). API 20 NE test strips (bioMérieux) were used to analyse the biochemical and physiological traits of the bacterial strains and standard microbiological methods were used for Gram staining and assessment of motility and enzyme reactions of catalase (with 5 % H2O2) and oxidase. The ability to oxidize sulfite was tested as described by Pukall et al. (1999).
Bacterial strains grown on MA plates at 20 °C for 2 days were used for fatty acid methyl ester (FAME) analysis. FAMEs were extracted and prepared according to the standard protocol of the MIDI/Hewlett Packard Microbial Identification System (Sasser, 1990). Chromosomal DNA was extracted and purified according to the method described by Sambrook et al. (1989). The 16S rRNA gene was amplified by PCR using two universal primers, as described previously (Yoon et al., 1998). Sequencing of the amplified 16S rRNA gene and phylogenetic analysis was performed as described by Yoon et al. (2003). DNA–DNA hybridization was performed fluorometrically by the method of Bae et al. (2005) using Cy5-labelled DNA probes and genome-spotted microarrays. The signal produced by self-hybridization was normalized to 100 % and the relative intensities of genomic DNA of other strains were determined as percentage relatedness. The presence of bacteriochlorophyll a (BChl a) was identified by PCR amplification of the phototrophism-related gene pufM, which encodes the M subunit of the photosynthetic reaction centre and is distributed universally amongst aerobic phototrophic bacteria (Achenbach et al., 2001; Kim et al., 2006).
Physiological and biochemical characteristics and morphological traits of cells and colonies of strain Iso 3T are shown in Table 1 or are given in the species description. Cells of strain Iso 3T were Gram-negative. They occurred singly or in irregular clusters or rosette-like aggregates. Strain Iso 3T grew at 4–30 °C (optimum 20 °C), in media of pH 5.0–10.5 (optimum pH 7.0–8.0), but not below pH 4.0 or above pH 12.0, and showed growth in up to 10 % NaCl (optimum 6.0–7.0 %).
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-chymotrypsin, naphthol-AS-BI-phosphohydrolase, -galactosidase, 
-galactosidase, -glucuronidase, -glucosidase, -glucosidase, N-acetyl--glucosaminidase, -glucosaminidase, -mannosidase and -fucosidase were absent.
The major cellular fatty acids of strain Iso 3T are unsaturated fatty acids such as 18 : 17c (85.6 %) and 11-methyl 18 : 17c (9.1 %) (Table 2). The fatty acid composition was similar to that observed in other members of the genus Sulfitobacter. The presence of 18 : 17c as the dominant fatty acid is representative of several major phyletic groups within the Alphaproteobacteria (Labrenz et al., 2000).
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The 16S rRNA gene sequence of strain Iso 3T determined in this study was 1355 nucleotides in length. Phylogenetic trees based on 16S rRNA gene sequences from members of different genera within the Rhodobacteraceae, including the Sulfitobacter group, showed that Iso 3T falls within the cluster of Sulfitobacter species (Fig. 1). Iso 3T exhibited 16S rRNA gene sequence similarities of 97.7–96.8 % to the type strains of four Sulfitobacter species and 96.3 % to Staleya guttiformis DSM 11458T. DNA–DNA relatedness between Iso 3T and the most closely related type strains, Sulfitobacter pontiacus DSM 10014T, Sulfitobacter brevis DSM 11443T, Sulfitobacter dubius ATCC BAA-320T and Sulfitobacter delicatus ATCC BAA-321T, were respectively 24.1, 5.7, 2.3 and 0.1 %, indicating that strain Iso 3T can be considered a member of a novel taxon. Considering the phenotypic, phylogenetic and genotypic characteristics of the isolate, we concluded that Iso 3T belongs to the genus Sulfitobacter. However, based on the phylogenetic and DNA–DNA hybridization data, we propose that Iso 3T should be the type strain of a novel species of the genus, Sulfitobacter litoralis sp. nov.
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Colonies are uniformly round, smooth and slightly yellowish after incubation for 48 h on MA. Cells are Gram-negative, short rods and are motile by means of polar flagella. Cells occasionally form rosette-like aggregates. Growth occurs at 4–30 °C, with an optimum at 20 °C. Neutrophilic (pH 5.0–9.0; optimum pH 7.0–8.0). Halophilic; NaCl [1.0–10.0 % (w/v); optimum 6.0–7.0 % (w/v)] is required for growth. Not able to reduce nitrate. Glucose, arabinose, mannose, N-acetylglucosamine, maltose, gluconate, caprate, citrate and phenylacetate are not utilized, but mannitol, adipate and malate are utilized. Catalase- (with 5 % H2O2) and oxidase-positive. Oxidation of sulfite is observed. BChl a is not produced. The principal cellular fatty acids are 18 : 17c (85.6 %) and 11-methyl 18 : 17c (9.1 %).
The type strain, Iso 3T (=KCTC 12521T=DSM 17584T), was isolated from a water sample obtained from the East Sea, Korea.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Bae, J. W., Rhee, S. K., Nam, Y. D. & Park, Y. H. (2005). Generation of subspecies level-specific microbial diagnostic microarrays using genes amplified from subtractive suppression hybridization as microarray probes. Nucleic Acids Res 33, e113.
Gonzalez, J. M., Kiene, R. P. & Moran, M. A. (1999). Transformation of sulfur compounds by an abundant lineage of marine bacteria in the alpha-subclass of the class Proteobacteria. Appl Environ Microbiol 65, 3810–3819.
Ivanova, E. P., Gorshkova, N. M., Sawabe, T., Zhukova, N. V., Hayashi, K., Kurilenko, V. V., Alexeeva, Y., Buljan, V., Nicolau, D. V. & other authors (2004). Sulfitobacter delicatus sp. nov. and Sulfitobacter dubius sp. nov., respectively from a starfish (Stellaster equestris) and sea grass (Zostera marina). Int J Syst Evol Microbiol 54, 475–480.
Kim, B.-C., Park, J. R., Bae, J.-W., Rhee, S.-K., Kim, K.-H., Oh, J.-W. & Park, Y.-H. (2006). Stappia marina sp. nov., a marine bacterium isolated from the Yellow Sea. Int J Syst Evol Microbiol 56, 75–79.
Labrenz, M., Tindall, B. J., Lawson, P. A., Collins, M. D., Schumann, P. & Hirsch, P. (2000). Staleya guttiformis gen. nov., sp. nov. and Sulfitobacter brevis sp. nov., -3-Proteobacteria from hypersaline, heliothermal and meromictic antarctic Ekho Lake. Int J Syst Evol Microbiol 50, 303–313.[Abstract]
Pukall, R., Buntefuß, D., Frühling, A., Rohde, M., Kroppenstedt, R. M., Burghardt, J., Lebaron, P., Bernard, L. & Stackebrandt, E. (1999). Sulfitobacter mediterraneus sp. nov., a new sulfite-oxidizing member of the -Proteobacteria. Int J Syst Bacteriol 49, 513–519.
Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989). Molecular Cloning: a Laboratory Manual, 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
Sasser, M. (1990). Identification of bacteria by gas chromatography of cellular fatty acids. Technical Note 101. Newark, DE: MIDI Inc.
Shiba, T. (1991). Roseobacter litoralis gen. nov., sp. nov., and Roseobacter denitrificans sp. nov., aerobic pink-pigmented bacteria which contain bacteriochlorophyll a. Syst Appl Microbiol 14, 140–145.
Sorokin, D. Y. (1995). Sulfitobacter pontiacus gen. nov., sp. nov. – a new heterotrophic bacterium from the Black Sea, specialized on sulfite oxidation. Microbiology (English translation of Mikrobiologiia) 64, 295–305.
Wagner-Döbler, I., Rheims, H., Felske, A., El-Ghezal, A., Flade-Schröder, D., Laatsch, H., Lang, S., Pukall, R. & Tindall, B. J. (2004). Oceanibulbus indolifex gen. nov., sp. nov., a North Sea alphaproteobacterium that produces bioactive metabolites. Int J Syst Evol Microbiol 54, 1177–1184.
Yoon, J. H., Lee, S. T. & Park, Y.-H. (1998). Inter- and intraspecific phylogenetic analysis of the genus Nocardioides and related taxa based on 16S rDNA sequences. Int J Syst Bacteriol 48, 187–194.
Yoon, J. H., Kim, H., Kim, I. G., Kang, K. H. & Park, Y.-H. (2003). Erythrobacter flavus sp. nov., a slight halophile from the East Sea in Korea. Int J Syst Evol Microbiol 53, 1169–1174.
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50 % are shown at nodes. Bar, 0.1 substitutions per nucleotide position.