International Journal of Systematic and Evolutionary Microbiology (2001), 51, 661–666 Printed in Great Britain Hahella chejuensis gen. nov., sp. nov., an extracellular-polysaccharide-producing marine bacterium Hong Kum Lee,1 Jongsik Chun,2 Eun Young Moon,2 Sung-Hwan Ko,1 Deuk-Soo Lee,1 Hyun Sang Lee1 and Kyung Sook Bae2 Author for correspondence: Kyung Sook Bae. Tel: j82 42 860 4610. Fax: j82 42 860 4677. e-mail: ksbae!mail.kribb.re.kr 1 Microbiology Laboratory, A bacterial strain, designated 96CJ10356T, which produced abundant Korea Ocean R & D extracellular polysaccharides and red pigment was isolated from marine Institute, Ansan PO Box 29, Seoul 425-600, Republic of sediment collected from Marado, Cheju Island, Republic of Korea. The organism Korea is Gram-negative, aerobic, rod-shaped and motile. Growth was not observed in 2 Korean Collection for Type the absence of NaCl, and was optimal at an NaCl concentration of 2%. The Cultures, Korea Research strain contained oxidase and catalase, and was able to hydrolyse aesculin and Institute of Bioscience and gelatin. The major cellular fatty acids were saturated or monounsaturated Biotechnology, PO Box 115, Yusong, Taejon straight-chain fatty acids. An almost complete 16S rDNA sequence of the test 305-600, Republic of Korea strain was determined. Phylogenetic analysis based on the neighbour-joining and Fitch–Margoliash methods indicated that the organism formed a distinct phyletic line within the γ Proteobacteria. This relationship was also supported by sequence comparison, as no valid bacterial species showed more than 90% sequence homology with the isolate. It is clear from polyphasic evidence that the isolate merits the status of genus in the γ subclass of the Proteobacteria, and the name Hahella chejuensis gen. nov., sp. nov. is proposed for the marine isolate 96CJ10356T (l KCTC 2396T l IMSNU 11157T). Keywords: Hahella chejuensis gen. nov., polyphasic taxonomy, 16S rDNA sequencing INTRODUCTION Island, Republic of Korea. Our preliminary study indicated that EPSs produced by the isolate are of high ' Microbial extracellular polysaccharides (EPSs) have molecular mass (over 2i10 Da), consist of galactose, been used in a wide variety of industrial applications glucose, xylose and ribose, and have good potential as such as emulsification, gel formation, absorption, film an emulsifying agent (Ko et al., 2000). In the present formation and anticancer treatment (Fu & Tseng, study, we report the polyphasic characterization of 1990; Irene et al., 1990; Martins et al., 1990; Low et this isolate, and propose that it belongs to a novel al., 1998). They have been found in various micro- species, which is named Hahella chejuensis gen. nov., organisms including Alteromonas, Cyanothece, sp. nov. Pseudomonas, Vibrio and Zoogloea (Ikeda et al., 1982; Rodrigues & Bhosle, 1991; Matsuda & METHODS Worawattanamateekul, 1993; Philippis et al., 1993; Raguenes et al., 1996). Microbial EPSs have an Bacterial strains. A marine sediment sample was collected advantage over polysaccharides from other sources, as from Marado, Cheju Island, Republic of Korea, at a depth they can be produced in large quantity using a of 1 m. The sample was diluted with saline (0n85% NaCl), spread onto ZoBell’s medium (5 g peptone, 1 g yeast extract, relatively simple purification process. A bacterial strain 0n01 g FePO%, 15 g agar, 250 ml distilled water, 750 ml aged producing a large amount of EPS was isolated from a seawater), and incubated at 20 mC. A bacterial strain, marine sediment sample collected from Marado, Cheju designated 96CJ10356T, was isolated and shown to produce ................................................................................................................................................. a large quantity of EPS. The strain was maintained as a Abbreviation: EPS, extracellular polysaccharide. glycerol suspension (20%, w\v) at k80 mC. The GenBank accession number for the 16S rDNA sequence of Hahella Morphology. The test strain was grown on basal medium chejuensis KCTC 2396T is AF195410. (BM; 10 g tryptone, 20 g sucrose, 4 g MgSO%, 7 g CaCl#, 01421 # 2001 IUMS 661 H. K. Lee and others 0n07 g KH#PO%,0n08 g K#HPO%, 5 mg FeCl$, 1 mg MnCl#, package (Felsenstein, 1993) was used for all analyses. 1mgNa#MoO%, 1 mg ZnCl#, 10 g NaCl, 1 l distilled water) The resultant neighbour-joining tree topology was evaluated for 3 d at 37 mC. Morphology was observed by a differential by bootstrap analyses (Felsenstein, 1985) based on 1000 interference microscope (Nikon), and Phillips model SEM resamplings. 515 scanning and CM 20 transmission electron microscopes. Nucleotide sequence accession numbers. The GenBank Negative staining was performed with an aqueous solution accession numbers of the published 16S rDNA sequences (2%, w\v) of phosphotungstic acid adjusted to pH 7, as used in this study are Y12579 (Alcanivorax borkumensis described previously (Robinson et al., 1987). DSM 11573T), AF001375 (Colwellia psychroerythraea T Phenotypic tests. The basal medium (BM), incubated at ATCC 27364 ), X67023 (Halomonas elongata ATCC T 37 mC, was used for phenotypic tests, unless otherwise stated. 33173 ), X67022 (Marinobacter hydrocarbonoclasticus The motility of young cells was examined using wet mounts. ATCC 49840T), U58339 (Marinobacterium georgiense DSM Growth under anaerobic conditions was determined by 11526T), AB006769 (Marinospirillum minutulum ATCC T T testing the growth of the strain on BM agar at 30 mCinan 19193 ), U58338 (Microbulbifer hydrolyticus DSM 11525 ), anaerobic chamber (5% CO ,7%H,88%N; Forma AF053734 (Neptunomonas naphthovorans NAG-2N-126T), # # # T Scientific). Catalase production was assayed by using 0n3% M22365 (Oceanospirillum linum ATCC 11336 ), X06684 hydrogen peroxide with a colony taken from BM agar (Pseudomonas aeruginosa DSM 50071T) and D14555 (Zymo- plates. Oxidase activity was determined by the method of bacter palmae IAM 14233T). The sequence of Marinomonas Kovacs (1956). The growth temperature was tested over the communis is in RDP as Mrm. commu2. range of 5–50 mC using BM broth. The requirement of NaCl for growth was tested using YMG broth (10 g glucose, 5 g RESULTS AND DISCUSSION peptone, 3 g yeast extract, 3 g malt extract, 1 l distilled water) supplemented with concentrations of NaCl in the Phenotypic and chemotaxonomic characterization range of 0–20%. The pH range for growth was determined using BM broth adjusted to different pH values over the The strain, isolated from marine sediment, was Gram- range of 3–11. negative, rod-shaped, and catalase- and oxidase-posi- Biochemical tests were done using API 20E and 20NE strips tive. It grew equally well under both aerobic and (bioMe! rieux) following the manufacturer’s instructions. anaerobic conditions. The colony was visible after 2 d Utilization of carbohydrates as sole carbon source was incubation on BM agar at 37 mC. The colour of examined using modified BM that was prepared by replacing colonies changed from pale orange to pinkish red, and tryptone with 0n5% (w\v) NH%Cl, and sucrose with 1% (w\v) carbohydrates. For acid production tests, bromo- " thymol blue was added to a final concentration of 0n05gl− . Pigment. Strain 96CJ10356T was grown in 100 ml BM broth (a) at 30 mC for 7 d. Biomass was removed by centrifugation at 10000 g for 20 min, following the addition of 0n1 g di- atomaceous earth (Sigma). The pigments were extracted from the supernatant using 2 vols methanol and chloroform, and analysed using a scanning UV\Visible spectrophoto- meter (Pharmacia Ultrospec 2000). Cellular fatty acid analysis. Fatty acid methyl esters were prepared from biomass that was scraped from marine TSA [Tryptic Soy Agar (Difco), 50% aged seawater] incubated at 30 mC for 3 d. Cellular fatty acids of the test strain were analysed as methyl esters by GC according to the instructions of the Microbial Identification System (MIDI). Determination of DNA base composition. DNA was prepared according to Chun & Goodfellow (1995). The mol% GjC (b) content of the resultant DNA preparations was determined using the thermal denaturation method (Mandel & Marmur, 1968). 16S rDNA analysis. The primary structure of 16S rDNA was determined as described earlier (Chun & Goodfellow, 1995). The resultant sequence of strain 96CJ10356T was manually aligned with representative sequences of the γ Proteobacteria obtained from the Ribosomal Database Project (Maidak et al., 1997) and GenBank databases, using known 16S rRNA secondary structure information (Gutell, 1994). Phylogen- etic trees were inferred by using the Fitch–Margoliash (Fitch & Margoliash, 1967) and neighbour-joining (Saitou & Nei, 1987) methods. Evolutionary distance matrices for the neighbour-joining and Fitch–Margoliash methods were generated according to the model of Jukes & Cantor (1969). ................................................................................................................................................. The trees were rooted using Rhizobium leguminosarum Fig. 1. Scanning electron micrographs of strain 96CJ10356T : (a) (GenBank accession no. D14513) as an outgroup. The young (1 d) and (b) old (7 d) cultures. Bar, 10 µm. 662 International Journal of Systematic and Evolutionary Microbiology 51 Hahella chejuensis gen. nov., sp. nov. Table 1 Phenotypic properties of isolate 96CJ10356T Table 2 Cellular fatty acid profile of isolate 96CJ10356T ................................................................................................................................................. ,