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1 Korea Research Institute of Bioscience and Biotechnology (KRIBB), PO Box 115, Yusong, Taejon, Korea
2 Department of Microbiology, College of Medicine, Chungang University, 221 Heukseok-dong, Seoul, Korea
3 Health Technology Planning and Evaluation Board, 57-1 Noryangjin-dong, Seoul, Korea
Correspondence
Jung-Hoon Yoon
jhyoon{at}kribb.re.kr
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ABSTRACT |
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The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain JG-241T is AY928901.
Levels of DNA–DNA relatedness between strain JG-241T and the type strains of six recognized Nesterenkonia species are shown in a supplementary table available in IJSEM Online.
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TOPABSTRACTMAIN TEXTREFERENCES |
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) as Nesterenkonia halobia (Stackebrandt et al., 1995). Subsequently, five more Nesterenkonia species, Nesterenkonia lacusekhoensis (Collins et al., 2002), Nesterenkonia halotolerans and Nesterenkonia xinjiangensis (Li et al., 2004) and Nesterenkonia sandarakina and Nesterenkonia lutea (Li et al., 2005) have been described. Phylogenetic analyses based on 16S rRNA gene sequences showed that the genus Nesterenkonia belongs to the family Micrococcaceae of the order Actinomycetales (Stackebrandt et al., 1995, 1997). In the study presented here, we report on the taxonomic characterization of a slightly halophilic, vivid-yellow- or orange–yellow-pigmented Nesterenkonia-like bacterial strain, JG-241T, which was isolated from jeotgal, a traditional Korean fermented seafood.
Strain JG-241T was isolated by the usual dilution plating technique on marine agar 2216 (MA; Difco) at 30 °C. The type strains of six Nesterenkonia species were used as reference strains. N. halobia DSM 20541T, N. lacusekhoensis DSM 12544T, N. halotolerans DSM 15474T and N. xinjiangensis DSM 15475T were obtained from the Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ), Braunschweig, Germany. N. sandarakina KCTC 19011T and N. lutea KCTC 19013T were obtained from the Korean Collection for Type Cultures, Taejon, Korea. The cell morphology was examined by light microscopy (E600; Nikon) and transmission electron microscopy. The presence of flagella was examined by transmission electron microscopy using cells from exponentially growing cultures. Colony morphology and colour were examined by using colonies grown on MA and on solid PYGV medium (Staley, 1968). The Gram reaction was determined by using the bioMérieux Gram stain kit according to the manufacturer's instructions. Growth at various NaCl concentrations was investigated in marine broth 2216 (MB; Difco) and trypticase soy broth (Difco). Growth in the absence of NaCl was investigated in trypticase soy broth prepared according to the formula of the Difco medium except that no NaCl was used. Growth at various temperatures (4–40 °C) was measured on MA. The pH range for growth was determined in MB adjusted to various pH values (pH 4.5–9.5, with increments of 0.5 pH units) prior to sterilization by the addition of HCl and Na2CO3. Growth under anaerobic conditions was determined after incubation in an anaerobic chamber on MA and on MA supplemented with nitrate, both of which had been prepared anaerobically using a nitrogen atmosphere. Catalase and oxidase activities and the hydrolysis of casein, starch and Tweens 20, 40, 60 and 80 were determined as described by Cowan & Steel (1965). The hydrolysis of hypoxanthine, tyrosine and xanthine was investigated on MA at the substrate concentrations described by Cowan & Steel (1965). Nitrate reduction and the hydrolysis of aesculin, gelatin and urea were studied as described previously (Lanyi, 1987) with the modification that artificial seawater was used for preparation of media. The artificial seawater contained the following (l–1 distilled water): 23.6 g NaCl, 0.64 g KCl, 4.53 g MgCl2.6H2O, 5.94 g MgSO4.7H2O and 1.3 g CaCl2.2H2O (Bruns et al., 2001). H2S production was tested as described previously (Bruns et al., 2001). Enzyme activity was determined by using the API ZYM system (bioMérieux). Acid production from carbohydrates was determined as described by Leifson (1963). The utilization of substrates as sole carbon and energy sources was tested according to the method of Baumann & Baumann (1981), using supplementation with 2 % (v/v) Hutner's mineral base (Cohen-Bazire et al., 1957), 1 % (v/v) vitamin solution (Staley, 1968) and 0.01 % (w/v) yeast extract (Difco). Susceptibility to antibiotics was tested on MA plates, using antibiotic discs containing the following: polymyxin B, 100 U; streptomycin, 50 µg; penicillin G, 20 U; chloramphenicol, 100 µg; ampicillin, 10 µg; cephalothin, 30 µg; gentamicin, 30 µg; novobiocin, 5 µg; tetracycline, 30 µg; kanamycin, 30 µg; lincomycin, 15 µg; oleandomycin, 15 µg; neomycin, 30 µg; and carbenicillin, 100 µg. Other physiological and biochemical tests were performed with the API 20E system (bioMérieux).
Cell mass for DNA extraction and for analyses of the cell wall, polar lipids and menaquinones was obtained by cultivation in MB at 30 °C. Chromosomal DNA was isolated and purified according to the method described previously (Yoon et al., 1996), with the exception that RNase T1 was used in combination with RNase A. The 16S rRNA gene sequence was amplified by means of a PCR using two universal primers as described previously (Yoon et al., 1998). Sequencing of the 16S rRNA gene and phylogenetic analysis were performed as described by Yoon et al. (2003). The DNA G+C content was determined by the method of Tamaoka & Komagata (1984), with the modification that the DNA was hydrolysed and the resultant nucleotides were analysed by reverse-phase HPLC. Preparation of cell-wall peptidoglycan was carried out using the method described by Schleifer & Kandler (1972), and the peptidoglycan amino acids were determined using an automated amino acid analyser (model L-8500A; Hitachi). Preparation of cell-wall peptidoglycan and determination of the peptidoglycan structure was also performed at the DSMZ by using modified versions of the methods of Rhuland et al. (1955), Schleifer & Kandler (1972), Schleifer (1985) and MacKenzie (1987). Menaquinones were extracted according to the method of Komagata & Suzuki (1987) and analysed using reversed-phase HPLC and a YMC ODS-A (250x4.6 mm) column. For fatty acid methyl ester analysis, cell mass of strain JG-241T and reference strains was harvested from MA plates after cultivation for 5 days at 30 °C. The fatty acid methyl esters were extracted and prepared according to the standard protocol of the MIDI/Hewlett Packard Microbial Identification System (Sasser, 1990). Polar lipids were extracted according to the procedures described by Minnikin et al. (1984) and were identified by two-dimensional TLC followed by spraying with appropriate detection reagents (Minnikin et al., 1984; Komagata & Suzuki, 1987). DNA–DNA hybridization was performed fluorometrically by the method of Ezaki et al. (1989), using photobiotin-labelled DNA probes and microdilution wells: five replications were performed for each sample. The highest and lowest values obtained in each sample were excluded and the means of the remaining three values were quoted as the DNA–DNA relatedness values. Repetitive extragenic palindromic DNA-PCR (rep-PCR) genomic fingerprinting using REP, BOX and (GTG)5 PCR primers was performed as described by Rademaker et al. (1998). Computer-assisted analysis of the genomic fingerprints was performed by using the software GelCompar II, version 1.5 (Applied Maths). Similarity among patterns was calculated on the basis of Pearson's similarity coefficient, and a dendrogram was constructed using the UPGMA algorithm (Sneath & Sokal, 1973).
Morphological, cultural, physiological and biochemical properties of strain JG-241T are shown in Table 1 or are given in the species description (see below). The almost-complete 16S rRNA gene sequence of strain JG-241T determined in this study comprised 1480 nt, representing approximately 96 % of the Escherichia coli 16S rRNA gene sequence. Comparative 16S rRNA gene sequence analyses revealed that strain JG-241T is phylogenetically most closely related to Nesterenkonia species, showing highest sequence similarity (99.7 %) with N. sandarakina YIM 70009T (Fig. 1). The 16S rRNA gene sequence similarity values between strain JG-241T and the type strains of the other Nesterenkonia species were 96.7 % (N. halobia and N. lacusekhoensis), 97.0 % (N. xinjiangensis) and 99.3 % (N. halotolerans and N. lutea). Sequence similarities with respect to other species used in the phylogenetic analysis were below 95.6 % (Fig. 1).
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peptidoglycan type based on L-lys–gly–D-Asp (described by Schleifer & Kandler, 1972). The predominant menaquinones found in strain JG-241T were MK-7 (59.8 %), MK-8 (20.3 %), MK-9 (11.0 %) and MK-6 (8.8 %). The cellular fatty acid profile showed the presence of large amounts of straight and branched fatty acids; the major fatty acids were anteiso-C15 : 0 and anteiso-C17 : 0 (Table 2). This fatty acid profile was generally similar to those of Nesterenkonia species analysed in this study (Table 2). For some fatty acids, the proportions present in Nesterenkonia species differed between this study and the studies of Li et al. (2004, 2005), perhaps because of differences in cultivation conditions. The major polar lipids detected in strain JG-241T were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylinositol and an unidentified glycolipid. This polar lipid profile was similar to those of Nesterenkonia species, although it differed from those of N. lacusekhoensis and N. xinjiangensis in that phosphatidylcholine was absent (Table 1
). The DNA G+C content of strain JG-241T was 68.0 mol%.
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). The DNA–DNA relatedness data were sufficient to identify strain JG-241T as representing a genomic species that is distinct from recognized Nesterenkonia species (Wayne et al., 1987). Mean levels of DNA–DNA relatedness between strain JG-241T and the type strains of six recognized Nesterenkonia species were in the range 11–53 % (see Supplementary Table S1 in IJSEM Online). There were differences between strain JG-241T and recognized Nesterenkonia species in several phenotypic, particularly chemical, properties, including the presence of MK-9, differences in the content of some fatty acids (particularly anteiso-C17 : 0), NaCl requirements and some physiological and biochemical properties (Table 1). Therefore, on the basis of the data presented above, strain JG-241T should be placed within the genus Nesterenkonia as a member of a novel species, for which the name Nesterenkonia jeotgali sp. nov. is proposed.
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Cells are cocci (diameter 0.5–0.8 µm) on MA at 30 °C; some cells are oval in the very early growth phase. Colonies are smooth, circular, slightly convex or raised, glistening and 1.0–2.0 mm in diameter after 5 days cultivation at 30 °C on MA. Colonies on MA are vivid yellow in colour at 30 °C, but orange–yellow in colour at 25 °C. Colonies on solid PYGV medium are light yellow in colour at 25 and 30 °C. Optimal growth temperature is 25–30 °C; growth occurs at 4 and 36 °C, but not at 37 °C. Optimal pH for growth is 7.5–8.5; growth occurs at pH 6.0, but not at pH 5.5. Optimal growth occurs in the presence of 2–5 % (w/v) NaCl; growth occurs in the presence of 0–16 % NaCl (w/v). Growth does not occur under anaerobic conditions on MA or on MA supplemented with nitrate. Tyrosine is hydrolysed, but aesculin, casein, hypoxanthine, xanthine and Tweens 20, 40 and 60 are not hydrolysed. Methyl red reaction is positive. In assays with the API ZYM system, alkaline phosphatase, esterase (C4), esterase lipase (C8) and leucine arylamidase are present, but lipase (C14), valine arylamidase, cystine arylamidase, trypsin, -chymotrypsin, acid phosphatase, naphthol-AS-BI-phosphohydrolase, N-acetyl-
-glucosaminidase, -mannosidase, -fucosidase, arginine dihydrolase, ornithine decarboxylase and tryptophan deaminase are absent. D-Galactose, acetate, citrate, succinate, L-malate and pyruvate are utilized. Benzoate, salicin, formate and L-glutamate are not utilized. Acid is produced from L-arabinose, D-cellobiose, D-glucose, maltose, D-mannose, D-melezitose, D-ribose and sucrose, but not from D-fructose, melibiose, D-raffinose, L-rhamnose, myo-inositol or D-sorbitol. Susceptible to penicillin G, chloramphenicol, ampicillin, cephalothin, novobiocin and carbenicillin, but not to polymyxin B, gentamicin, kanamycin or neomycin. The cell-wall peptidoglycan type is L-lys-gly–D-Asp. The predominant menaquinones are MK-7, MK-8 and MK-9. The major fatty acids are anteiso-C15 : 0 and anteiso-C17 : 0. The major polar lipids are diphosphatidylglycerol, phosphatidylglycerol, phosphatidylinositol and an unidentified glycolipid. The DNA G+C content is 68.0 mol% (determined by HPLC). Other phenotypic properties are given in Table 1.
The type strain, JG-241T (=KCTC 19053T=JCM 12610T), was isolated from jeotgal, a traditional Korean fermented seafood.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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TOPABSTRACTMAIN TEXTREFERENCES |
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Bruns, A., Rohde, M. & Berthe-Corti, L. (2001). Muricauda ruestringensis gen. nov., sp. nov., a facultatively anaerobic, appendaged bacterium from German North Sea intertidal sediment. Int J Syst Evol Microbiol 51, 1997–2006.[Abstract]
Cohen-Bazire, G., Sistrom, W. R. & Stanier, R. Y. (1957). Kinetic studies of pigment synthesis by nonsulfur purple bacteria. J Cell Comp Physiol 49, 25–68.
Collins, M. D., Lawson, P. A., Labrenz, M., Tindall, B. J., Weiss, N. & Hirsch, P. (2002). Nesterenkonia lacusekhoensis sp. nov., isolated from hypersaline Ekho Lake, East Antarctica, and emended description of the genus Nesterenkonia. Int J Syst Evol Microbiol 52, 1145–1150.[Abstract]
Cowan, S. T. & Steel, K. J. (1965). Manual for the Identification of Medical Bacteria. London: Cambridge University Press.
Ezaki, T., Hashimoto, Y. & Yabuuchi, E. (1989). Fluorometric deoxyribonucleic acid-deoxyribonucleic acid hybridization in microdilution wells as an alternative to membrane filter hybridization in which radioisotopes are used to determine genetic relatedness among bacterial strains. Int J Syst Bacteriol 39, 224–229.
Komagata, K. & Suzuki, K. (1987). Lipids and cell-wall analysis in bacterial systematics. Methods Microbiol 19, 161–207.
Lanyi, B. (1987). Classical and rapid identification methods for medically important bacteria. Methods Microbiol 19, 1–67.
Leifson, E. (1963). Determination of carbohydrate metabolism of marine bacteria. J Bacteriol 85, 1183–1184.
Li, W.-J., Chen, H.-H., Zhang, Y.-Q., Schumann, P., Stackebrandt, E., Xu, L.-H. & Jiang, C.-L. (2004). Nesterenkonia halotolerans sp. nov. and Nesterenkonia xinjiangensis sp. nov., actinobacteria from saline soils in the west of China. Int J Syst Evol Microbiol 54, 837–841.
Li, W.-J., Chen, H.-H., Kim, C.-J., Zhang, Y.-Q., Park, D.-J., Lee, J.-C., Xu, L.-H. & Jiang, C.-L. (2005). Nesterenkonia sandarakina sp. nov. and Nesterenkonia lutea sp. nov., novel actinobacteria, and emended description of the genus Nesterenkonia. Int J Syst Evol Microbiol 55, 463–466.
MacKenzie, S. L. (1987). Gas chromatographic analysis of amino acids as the N-heptafluorobutyryl isobutyl esters. J Assoc Off Anal Chem 70, 151–160.[Medline]
Minnikin, D. E., O'Donnell, A. G., Goodfellow, M., Alderson, G., Athalye, M., Schaal, A. & Parlett, J. H. (1984). An integrated procedure for the extraction of bacterial isoprenoid quinones and polar lipids. J Microbiol Methods 2, 233–241.[CrossRef]
Onishi, H. & Kamekura, M. (1972). Micrococcus halobius sp. n. Int J Syst Bacteriol 22, 233–236.
Rademaker, J. L. W., Louws, F. J. & de Bruijn, F. J. (1998). Characterization of the diversity of ecologically important microbes by rep-PCR genomic fingerprinting. In Molecular Microbial Ecology Manual, supplement 3, pp. 3.4.3.1–3.4.3.26. Edited by A. D. L. Akkermans, J. D. van Elsas & F. J. de Bruijn. Dordrecht: Kluwer.
Rhuland, L. E., Work, E., Denman, R. F. & Hoare, D. S. (1955). The behaviour of the isomers of ,
-diaminopimelic acid on paper chromatograms. J Am Chem Soc 77, 4844–4846.[CrossRef]
Sasser, M. (1990). Identification of bacteria by gas chromatography of cellular fatty acids. Technical Note 101. Newark, DE: MIDI.
Schleifer, K. H. (1985). Analysis of the chemical composition and primary structure of murein. Methods Microbiol 18, 123–156.
Schleifer, K. H. & Kandler, O. (1972). Peptidoglycan types of bacterial cell walls and their taxonomic implications. Bacteriol Rev 36, 407–477.
Sneath, P. H. A. & Sokal, R. R. (1973). Numerical Taxonomy. San Francisco: Freeman.
Stackebrandt, E., Koch, C., Gvozdiak, O. & Schumann, P. (1995). Taxonomic dissection of the genus Micrococcus: Kocuria gen. nov., Nesterenkonia gen. nov., Kytococcus gen. nov., Dermacoccus gen. nov., and Micrococcus Cohn 1872 gen. emend. Int J Syst Bacteriol 45, 682–692.
Stackebrandt, E., Rainey, F. A. & Ward-Rainey, N. L. (1997). Proposal for a new hierarchic classification system, Actinobacteria classis nov. Int J Syst Bacteriol 47, 479–491.
Staley, J. T. (1968). Prosthecomicrobium and Ancalomicrobium: new prosthecate freshwater bacteria. J Bacteriol 95, 1921–1942.
Tamaoka, J. & Komagata, K. (1984). Determination of DNA base composition by reverse-phase high-performance liquid chromatography. FEMS Microbiol Lett 25, 125–128.
Wayne, L. G., Brenner, D. J., Colwell, R. R. & 9 other authors (1987). International Committee on Systematic Bacteriology. Report of the ad hoc committee on reconciliation of approaches to bacterial systematics. Int J Syst Bacteriol 37, 463–464.
Yoon, J.-H., Kim, H., Kim, S.-B., Kim, H.-J., Kim, W. Y., Lee, S. T., Goodfellow, M. & Park, Y.-H. (1996). Identification of Saccharomonospora strains by the use of genomic DNA fragments and rRNA gene probes. Int J Syst Bacteriol 46, 502–505.
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 rRNA gene 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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