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1 School of Biological Sciences, Seoul National University, 56-1 Shillim-dong, Kwanak-gu, Seoul 151-742, Republic of Korea
2 Microbiology Lab, Marine Biology Division, Korea Ocean Research and Development Institute, Ansan PO Box 29, Seoul 425-600, Republic of Korea
Correspondence
Jongsik Chun
jchun{at}snu.ac.kr
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ABSTRACT |
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7c and/or iso-C15 : 0 2-OH) and DNA G+C content (38 mol%) of the Antarctic isolate were consistent with those of the genus Flavobacterium. In contrast, several phenotypic characters can be used to differentiate this isolate from other flavobacteria. The polyphasic data presented in this study indicated that this isolate should be classified as a novel species in the genus Flavobacterium. The name Flavobacterium antarcticum sp. nov. is therefore proposed for the Antarctic isolate; the type strain is AT1026T (=IMSNU 14042T=KCTC 12222T=JCM 12383T).
The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain AT1026T is AY581113.
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MAIN TEXT |
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TOPABSTRACTMAIN TEXTREFERENCES |
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). On the basis of polyphasic evidence, the Antarctic strain represents a novel species in the genus Flavobacterium, for which the name Flavobacterium antarcticum sp. nov. is proposed. A soil sample was collected from a penguin habitat near the King Sejong Station on King George Island, Antarctica (62° 14' 01·2'' S 58° 46' 47·4'' W). Isolation was carried out using marine agar 2216 (MA; Difco) at 10 °C following enrichment for 2 days in marine broth 2216 at 4 °C. The isolate was cultured routinely on R2A (Difco) at 15 °C and maintained as a glycerol suspension (20 %, w/v) at –80 °C.
16S rRNA genes were enzymically amplified from a single colony. Primers, PCR conditions and sequencing methods were described elsewhere (Chun & Goodfellow, 1995). The sequence of strain AT1026T was aligned manually with representative sequences of the family Flavobacteriaceae obtained from GenBank. Phylogenetic trees were inferred using the Fitch–Margoliash (Fitch & Margoliash, 1967), maximum-likelihood (Felsenstein, 1993), maximum-parsimony (Fitch, 1972) 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). Resultant tree topologies were evaluated by bootstrap analyses (Felsenstein, 1985) based on 1000 resamplings. Alignment and phylogenetic analyses were carried out using the jPHYDIT program (available at http://chunlab.snu.ac.kr/jphydit) and PAUP 4.0 (Swofford, 1998) as described previously (Chun et al., 2000). An almost complete 16S rRNA gene sequence of strain AT1026T (1406 bp) was obtained. Preliminary sequence comparison with 16S rRNA gene sequences held in GenBank indicated that our isolate was related closely to the genus Flavobacterium. The newly determined sequence was then aligned manually against representatives of Flavobacterium species using bacterial 16S rRNA secondary structure. The regions available for all sequences (positions 46–1477; Escherichia coli numbering system), excluding positions likely to show ambiguous alignment (positions 76–94), were used to construct the phylogenetic trees. Based on 16S rRNA gene sequence similarity, the Antarctic isolate shared 91·3–96·4 % similarity with members of the genus Flavobacterium and the closest bacterial relatives with validly published names were Flavobacterium tegetincola (96·4 %). Flavobacterium flevense (95·9 %), Flavobacterium micromati (95·6 %) and Flavobacterium xinjiangense (95·5 %). Strain AT1026T and F. tegetincola formed a robust (96 % bootstrap value) monophyletic clade (Fig. 1) in all trees inferred in this study, and formed a further monophyletic clade with F. flevense and Flavobacterium johnsoniae in Fitch–Margoliash, maximum-likelihood and maximum-parsimony trees. On the basis of our phylogenetic analysis, it is evident that our isolate is a member of the genus Flavobacterium and represents a novel genomic species.
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) by non-linear regression using the R 1.8.1 package (R Foundation for Statistical Computing, 2003). Square-root growth rate–temperature plots showed that the notional minimum, optimum and maximum growth temperatures were respectively –14·5, 21·2 and 25·1 °C (Fig. 2). When tested on R2A (between 5 and 35 °C at 5 °C intervals), strain AT1026T grew at 25 °C, but not at 30 °C. From the definition of Isaksen & Jørgensen (1996), our Antarctic isolate can be defined as a psychrotolerant bacterium. The minimum doubling time of strain AT1026T was about 4·9 h. Growth at different pH (between pH 4 and 12 with an interval of 1) and NaCl concentrations [between 0 and 7 % (w/v) at 1 % intervals] was determined using sea-salt-free ZoBell's medium. KOH and HCl (both at 6 M) were used to adjust the final pH. Anaerobic and microaerophilic growth was checked under anaerobic (with 4–10 % CO2) and microaerobic (with 5–15 % O2 and 5–12 % CO2) conditions, using GasPak Plus and CampyPak Plus systems (BBL) at 15 °C for up to 1 month. The results of tests for growth conditions are given in the species description.
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). Congo red adsorption was tested by directly flooding colonies on agar plates with 0·01 % aqueous Congo red solution.
Standard physiological and biochemical tests were performed at 15 °C as described previously (Smibert & Krieg, 1994). Hydrolysis of alginate (0·5 %, w/v), casein [50 % skimmed milk (Difco), v/v], carboxymethyl cellulose [0·5 % carboxymethyl cellulose (Sigma), w/v], chitin (0·5 % colloidal chitin, w/v), egg yolk (5 %, w/v), elastin (0·5 %, w/v), starch (0·2 %, w/v), Tween 80 (1 %, v/v) and L-tyrosine (0·5 %, w/v) was tested using R2A as the basal medium. PEK7 agar (Reichenbach, 1991) and DNase test agar (Difco) were respectively used for pectinase and DNase assays. Production of H2S was investigated using triple-sugar iron agar (Difco). Phenylalanine deaminase activity was determined on phenylalanine agar (Smibert & Krieg, 1994; yeast extract, 3 g; L-phenylalanine, 1 g; Na2HPO4, 1 g; NaCl, 5 g; Bacto agar, 12 g; distilled water, 1 l). Alkaline reaction on Christensen's citrate was tested on Christensen citrate agar (Christensen, 1949). Arginine dihydrolase and urease activities were determined using Thornley's semi-solid medium (Thornley, 1960) and Christensen urea agar (Christensen, 1946), respectively. Acid production from carbohydrates was examined for up to 1 month using modified O/F agar plates (Leifson, 1963; casitone, 1·0 g; yeast extract, 0·1 g; ammonium sulfate, 0·5 g; Tris base, 0·5 g; phenol red, 0·01 g; Bacto agar, 15 g; distilled water, 1 l; adjusted to pH 7·0). Nitrate and nitrite reduction, indole production, aesculinase, gelatinase, 
-galactosidase and assimilation of sole carbon sources (glucose, arabinose, mannose, mannitol, N-acetylglucosamine, maltose, gluconate, caprate, adipate, malate, citrate and phenylacetate) were tested using the API 20NE kit (bioMérieux), and other enzymic activities were determined using the API ZYM kit (bioMérieux). The results of morphological, biochemical and physiological tests are given in Table 1 and the species description.
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and analysed by HPLC (Waters) as described by Collins (1985). DNA G+C content was determined by HPLC analysis of deoxyribonucleosides as described by Mesbah et al. (1989), using a reverse-phase column (Supelco). Fatty acid methyl esters analysis was performed by GLC according to the Microbial Identification (MIDI) System using 5-day-old cells. The chemotaxonomic properties of the test strain are consistent with those of the genus Flavobacterium and are given in the species description. The major fatty acid composition of our isolate is similar to those of phylogenetically related species, but differs somewhat from them in quantities (Bernardet et al., 1996; McCammon & Bowman, 2000).
In the phylogenetic trees, our Antarctic isolate clearly belongs to the genus Flavobacterium and forms a distinct phyletic line with low 16S rRNA gene sequence similarity (96·8 %), which indicates that it represents a novel genomic species (Stackebrandt & Goebel, 1994). Moreover, a number of phenotypic characteristics (Table 1
) readily differentiate the Antarctic strain from other related Flavobacterium species. The polyphasic data obtained in this study clearly show that the test strain merits novel species status within the genus Flavobacterium. The name Flavobacterium antarcticum sp. nov. is therefore proposed for strain AT1026T.
Description of Flavobacterium antarcticum sp. nov.
Flavobacterium antarcticum (ant.arc'ti.cum. L. neut. adj. antarcticum southern, and, by extension, pertaining to Antarctica).
Gram-negative, oxidase- and catalase-positive and psychrotolerant. Cells are rod-shaped with rounded ends, approximately 0·5–1·3x0·3–0·4 µm and non-motile. Colonies are convex, translucent, glistening, butyrous, yellow, circular with entire margins and becoming mucoid after prolonged incubation on R2A and AOA. Does not glide or adhere to agar plates. Flexirubin-type pigment is absent. Congo red is not adsorbed. Spores are not formed. Growth occurs on R2A, AOA, MA, NA and TSA, but not on cetrimide or MacConkey agar. Growth is aerobic. Grows weakly under microaerobic conditions (with about 5–15 % O2 and 5–12 % CO2 created by CampyPak Plus system) and poorly under anaerobic conditions (with about 4–10 % CO2 created by GasPak Plus system). Growth occurs at pH 6–10 (optimum pH 7) and 0–4 % NaCl (optimum 0 %). Grows at 5–24·1 °C, with notional minimum, optimum and maximum growth temperatures of –14·5, 21·2 and 25·1 °C. Minimum doubling time is 4·9 h. Decomposes Tween 80, but not alginate, chitin, carboxymethyl cellulose, elastin, starch or tyrosine. Produces brown pigment weakly on tyrosine agar. Positive reaction for arginine dihydrolase. Negative reactions for nitrate reduction, urease, L-phenylalanine deaminase, H2S production, indole production and alkalinization on Christensen's citrate agar. Produces acid from D-glucose and maltose, but not from D-cellobiose, D-fructose, D-mannitol, D-raffinose, D-salicin, D-trehalose, D-xylose, lactose, L-arabinose, L-rhamnose or sucrose. Alkaline phosphatase, esterase lipase (C8), leucine arylamidase, valine arylamidase, acid phosphatase and naphthol-AS-BI-phosphohydrolase are positive; trypsin, 
-glucosidase and N-acetyl--glucosaminidase are weakly positive; esterase (C4), lipase (C14), cystine arylamidase, -chymotrypsin, -galactosidase, -galactosidase, -glucuronidase, -glucosidase, -mannosidase and -fucosidase are negative in API ZYM kits. Cannot assimilate any of the compounds contained in API 20NE kits as sole carbon sources. Other physiological and biochemical characteristics are given in Table 1. Maximum absorption peak of pigment is at 452 nm and the next shoulder peak is at 479 nm. Major isoprenoid quinone is MK-6. Predominant cellular fatty acids are iso-C15 : 1 G (15·3 %; the double bond position is unknown), iso-C15 : 0 (15·8 %) and C16 : 17c and/or iso-C15 : 0 2-OH (10·9 %; the two fatty acids cannot be separated by GLC with the MIDI system). Smaller amounts of iso-C14 : 0 (2·6 %), C14 : 0 (2·1 %), anteiso-C15 : 1 A (3·3 %; double bond position unknown), anteiso-C15 : 0 (6·7 %), C15 : 16c (1·5 %), C15 : 0 (8·2 %), iso-C16 : 1 H (3·2 %; double bond position unknown), iso-C16 : 0 (4·1 %), iso-C15 : 0 3-OH (6·2 %), iso-C17 : 19c (1·7 %), anteiso-C17 : 19c (1·4 %), iso-C16 : 0 3-OH (4·5 %), C16 : 0 3-OH (2·2 %), C18 : 15c (1·0 %) and iso-C17 : 0 3-OH (3·0 %) are also present. DNA G+C content is 38 mol%.
The type strain, AT1026T (=IMSNU 14042T=KCTC 12222T=JCM 12383T), was isolated from a soil sample of a penguin habitat near the King Sejong Station on King George Island, Antarctica.
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ACKNOWLEDGEMENTS |
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Bernardet, J.-F., Nakagawa, Y. & Holmes, B. (2002). Proposed minimal standards for describing new taxa of the family Flavobacteriaceae and emended description of the family. Int J Syst Evol Microbiol 52, 1049–1070.[Abstract]
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r is the square-root of growth rate. The minimum, optimum and maximum temperatures are respectively –14·5, 21·2 and 25·1 °C.