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Marine Biotechnology Research Centre, Korea Ocean Research and Development Institute, Ansan, PO Box 29, 425-600, Republic of Korea
Correspondence
Sang-Jin Kim
s-jkim{at}kordi.re.kr
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ABSTRACT |
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-glucosidase activity. Optimal growth of strain GW14-5T was at pH 7·0 and 37–40 °C and required the presence of 2 % (w/v) NaCl. On the basis of this evidence, strain GW14-5T represents a novel genus and species in the ‘Alphaproteobacteria’ for which the name Kordiimonas gwangyangensis gen. nov., sp. nov. is proposed. The novel order Kordiimonadales is proposed for the distinct phyletic line represented by the genus Kordiimonas. The type strain is GW14-5T (=KCCM 42021T=JCM 12864T).
Published online ahead of print on 13 May 2005 as DOI 10.1099/ijs.0.63684-0.
The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain GW14-5T is AY682384.
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MAIN TEXT |
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). The second edition of Bergey's Manual of Systematic Bacteriology proposed that the class ‘Alphaproteobacteria’ should be divided into six orders: Caulobacterales, ‘Rhizobiales’, ‘Rhodobacterales’, Rhodospirillales, Rickettsiales and ‘Sphingomonadales’ (Garrity & Holt, 2001). In the last few years, 24 new genera, and the novel order ‘Parvularculales’, have been reported in the ‘Alphaproteobacteria’ [Cho & Giovannoni, 2003; refer also to the NCBI taxonomy homepage (http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi) and articles in this journal (http://ijs.sgmjournals.org)]. More than half of these new genera were isolated from marine environments, and most members could survive in oligotrophic environments. In the present study, the taxonomic identification of a novel marine bacterium, strain GW14-5T, belonging to the ‘Alphaproteobacteria’ is described; on the basis of these results, the strain is considered to represent a novel species and genus, Kordiimonas gwangyangensis gen. nov., sp. nov.
Strain GW14-5T was isolated from marine sediments collected in the Gwangyang Bay of the South Sea, Republic of Korea. About 1 g sediment was enriched with 100 p.p.m. each of pyrene and benzo[a]pyrene (BaP) in MM2 liquid medium [18 mM (NH4)2SO4, 1 µM FeSO4.7H2O and 100 µl 1 M KH2PO4/Na2HPO4 buffer solution in 1 litre of aged sea water, pH 7·2] for 2 years (a long-term enrichment method) at a temperature of 10–30 °C. Then, 100 µl slurry was plated onto marine agar 2216 (MA; Difco) after serial dilution. A tiny, translucent creamy white colony with morphology different from that of the other colonies growing on this medium was isolated and designated GW14-5T. Strain GW14-5T was cultivated for 2–3 days at 30 °C on MA for morphological and biochemical characterization.
The degradation rate of BaP by strain GW14-5T was determined by the fluorimetry method (Zhang et al., 2004). Approximately 40 p.p.b. BaP and 1 % 2-hydroxypropyl--cyclodextrin were solubilized in MM2 liquid medium, and strain GW14-5T was inoculated into this mixture. The relative intensity of fluorescence (excitation at 293 nm, emission at 406 nm) was measured after 1, 3, 4 and 10 days incubation. The fluorescence of BaP in the culture medium was significantly decreased during the period of incubation compared with that of the non-inoculated control (Fig. 1). The degradation rates of six different mixed polycyclic aromatic hydrocarbons (PAHs) (fluorene, phenanthrene, anthracene, pyrene, chrysene and BaP) were estimated using the GC method described by Sohn et al. (2004) with 5 p.p.m. of each PAH compound at 30 °C. Based on triplicate samples, the degradation rates of the compounds relative to that of the non-inoculated control were: fluorene, 42·1±19·2 %; phenanthrene, 20·8±16·7 %; anthracene, 13·9±14·3 %; pyrene, 18·1±11·2 %; and BaP, 28·0±4·8 %. Chrysene was not degraded within 1 week of incubation.
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. The Gram reaction was determined using the Bio-Rad Gram-staining kit, and motility was determined with an optical microscope (Zeiss Axioplan) by the hanging drop technique (Skerman, 1967). Cell size and morphology after dehydration were examined with a scanning electron microscope (model S-4300; Hitachi) at 10 kV. Growth temperature was tested over the range 12–48 °C at 4 °C intervals in marine broth 2216 (MB; Difco) in a temperature gradient incubator (TVS126MA; Advantec). Bacterial suspensions for the physiological and biochemical characterizations using an API 20NE test (bioMérieux) and a Microlog GN2 plate (Biolog) were prepared in a 2 % sea salt (Sigma) solution. The physiological, biochemical and morphological characteristics of strain GW14-5T are given below and in Table 1.
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. The dominant fatty acids in strain GW14-5T were i-17 : 1 (46·2 %), i-15 : 0 (15·1 %) and i-17 : 0 (12·6 %). Strain GW14-5T also contained other straight-chain saturated fatty acids (7·3 %), straight-chain unsaturated fatty acids (5·4 %) and a cyclo-fatty acid (2·6 %) (Table 2).
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, using a Symmetry reversed-phase C18 column (Waters). The major respiratory quinone was determined to be MK-5 by HPLC analysis according to the method of Collins (1985).
Genomic DNA extraction, PCR and sequencing of the 16S rRNA gene were conducted using the procedures described in Sohn et al. (2004). Preliminary sequence BLAST analysis and subsequent phylogenetic analyses indicated that strain GW14-5T belonged to the ‘Alphaproteobacteria’ and that its closest recognized relative was Blastochloris sulfoviridis (92·0 % 16S rRNA gene sequence similarity). However, 16S rRNA gene sequence similarity to B. sulfoviridis decreased to 89·3 % after manual alignment and trimming of the sequence; hence this species was excluded from the phylogenetic tree (Fig. 2). Other species belonging to the ‘Rhizobiales’, such as Rhodobium orientis (91·5 %), Methylosinus trichosporium (91·4 %), Blastochloris viridis (90·9 %), Phyllobacterium myrsinacearum (90·8 %) and Parvibaculum lavamentivorans (90·6 %), as well as members of the ‘Rhodobacterales’, such as Paracoccus alkaliphilus (91·0 %) and Amaricoccus macauensis (90·4 %), also showed less than 92 % 16S rRNA gene sequence similarity. Therefore, the 16S rRNA gene sequence of strain GW14-5T was aligned with representative sequences, in the NCBI and RDP databases, of organisms that belong to the seven different orders of the ‘Alphaproteobacteria’ (Cho & Giovannoni, 2003). The program PHYDIT version 3.2 (http://plaza.snu.ac.kr/
jchun/phydit/) was used for this analysis. Phylogenetic distances were calculated from the models of Jukes & Cantor (1969), and the tree was constructed on the basis of the neighbour-joining method (Saitou & Nei, 1987). The resultant phylogenetic tree indicates that strain GW14-5T forms a phyletic line distinct from all seven orders of the ‘Alphaproteobacteria’ (Fig. 2). Furthermore, a specific relationship of the strain to these orders was not seen in the maximum-parsimony analysis. On the basis of the phylogenetic analysis, this novel strain appears to represent a novel order of the ‘Alphaproteobacteria’.
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), indicating that Parvibaculum lavamentivorans should be reclassified as a member of the ‘Parvularculales’ or another new order instead of ‘Rhizobiales’.
Strain GW14-5T was clearly distinct, both phenotypically and ecologically, from other members of the ‘Alphaproteobacteria’. Many strains belonging to the ‘Sphingomonadales’, Rhodospirillales and ‘Rhizobiales’ contain photosynthetic pigments, but strain GW14-5T does not. It does not associate with plant roots, in contrast to Mesorhizobium loti. It has a relatively low DNA G+C ratio similar to Rickettsia prowazekii (Weiss & Moulder, 1984) but does not require a host organism. The major fatty acid of members of the ‘Alphaproteobacteria’, such as Parvularcula bermudensis (Cho & Giovannoni, 2003), Parvibaculum lavamentivorans (Schleheck et al., 2004), Roseobacter litoralis (Labrenz et al., 1999), Phyllobacterium myrsinacearum (Mergaert et al., 2002), Paracoccus haeundensis (Lee et al., 2004) and Novosphingobium pentaromativorans (Sohn et al., 2004), is octadecenoic acid (18 : 1) (Table 2). That of strain GW14-1T is iso-heptadecenoic acid (i-17 : 1).
The results of our morphological, physiological and chemotaxonomic investigations together with the phylogenetic analysis revealed that strain GW14-5T is clearly distinct from other members of the ‘Alphaproteobacteria’. The strain represents a novel species and genus in the ‘Alphaproteobacteria’, for which the name Kordiimonas gwangyangensis gen. nov., sp. nov. is proposed. The new order Kordiimonadales is proposed for the distinct phyletic line represented by the genus Kordiimonas.
Description of Kordiimonadales ord. nov.
Kordiimonadales (Kordii.mo.na.da'les. N.L. fem. n. Kordiimonas type genus of the order; -ales ending to denote an order; N.L. fem. n. Kordiimonadales the order of Kordiimonas).
The description is the same as for the genus Kordiimonas. The type genus is Kordiimonas.
Description of Kordiimonas gen. nov.
Kordiimonas (Kordii.mo'nas. N.L. fem. n. Kordia arbitrary name derived from the abbreviation KORDI, which stands for Korea Ocean Research and Development Institute; L. fem. n. monas a monad, unit; N.L. fem. n. Kordiimonas a micro-organism described by scientists working at KORDI).
Cells are Gram-negative, motile and rod-like in form with a length of 1·3–1·4 µm and width of 0·25 µm. Oxidase and catalase are produced. It is obligately aerobic and weakly halophilic. It has mesophilic characteristics for growth temperature and grows optimally at neutral pH. The dominant fatty acids are branched. The respiratory quinone is MK-5. Phylogenetically, the genus forms the novel order Kordiimonadales of the ‘Alphaproteobacteria’. The type species is Kordiimonas gwangyangensis.
Description of Kordiimonas gwangyangensis sp. nov.
Kordiimonas gwangyangensis (gwang.yang.en'sis. N.L. fem. adj. gwangyangensis from Gwangyang Bay, the geographical origin of the strain).
In addition to the characteristics reported for the genus, the cells form tiny colonies on MA that are circular, translucent, creamy white and grow up to 2–3 mm in diameter after 1 week. Cells are able to grow between 17 and 44 °C and optimally between 37 and 40 °C. No growth is observed below 16 °C or above 48 °C. Growth occurs at pH 6·0–8·5 and optimally at pH 7·0. The optimal salt concentration for growth is 2 %, and the organism has difficulty growing at NaCl concentrations above 4 % or below 0·5 %. It does not reduce nitrate to nitrite. The DNA G+C content is 39·3 mol%. Positive results are obtained for -glucosidase and for the assimilation of glucose, N-acetylglucosamine and maltose. All other tests in API 20NE were negative. Based on the Microlog system, the strain can oxidize 
-cyclodextrin, dextrin, Tweens 40 and 80, N-acetylglucosamine, cellobiose, -D-glucose, maltose, D-trehalose, methyl pyruvate, -hydroxybutyric acid, L-alanyl glycine, L-aspartic acid, L-glutamic acid, glycyl-L-glutamic acid, L-threonine, putrescine and glucose 1-phosphate. The dominant fatty acids are i-17 : 1 (46·2 %), i-15 : 0 (15·1 %) and i-17 : 0 (12·6 %).
The type strain, GW14-5T (=KCCM 42021T=JCM 12864T), was isolated from the sediments of Gwangyang Bay, Republic of Korea.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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TOPABSTRACTMAIN TEXTREFERENCES |
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Cho, J.-C. & Giovannoni, S. J. (2003). Parvularcula bermudensis gen. nov., sp. nov., a marine bacterium that forms a deep branch in the Alphaproteobacteria. Int J Syst Evol Microbiol 53, 1031–1036.
Collins, M. D. (1985). Isoprenoid quinone analysis in bacterial classification and identification. In Chemical Methods in Bacterial Systematics, pp. 267–287. Edited by M. Goodfellow & D. E. Minnikin. London: Academic Press.
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Sohn, J. H., Kwon, K. K., Kang, J.-H., Jung, H.-B. & Kim, S.-J. (2004). Novosphingobium pentaromativorans sp. nov., a high-molecular-mass polycyclic aromatic hydrocarbon-degrading bacterium isolated from estuarine sediment. Int J Syst Evol Microbiol 54, 1483–1487.
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, Uninoculated control; 
, microcosm with strain GW14-5T.