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School of Biological Sciences and Institute of Microbiology, Seoul National University, 56-1 Shillim-dong, Kwanak-gu, Seoul 151-742, Republic of Korea
Correspondence
Jongsik Chun
jchun{at}snu.ac.kr
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). On the basis of the polyphasic evidence presented, the Antarctic strains are considered to represent two distinct novel species in the genus Flavobacterium. Strains AT1042T and AT1048T were isolated from soil samples collected on the Weaver Peninsula (62° 14' 07.8'' S 58° 46' 33.3'' W) and in a penguin habitat near the King Sejong Station (62° 12' 17.6'' S 58° 47' 40.2'' W), respectively, on King George Island, Antarctica. 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 isolates were cultured routinely on R2A (Difco) at 15 °C and maintained as a glycerol suspension (20 %, w/v, in distilled water) at –80 °C.
The 16S rRNA gene was enzymically amplified from a single colony. Primers, PCR conditions and sequencing methods were as described by Chun & Goodfellow (1995). The sequences of strains AT1042T and AT1048T were 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, 1971) 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 (Jeon et al., 2005) and PAUP 4.0 (Swofford, 1998) as described by Chun et al. (2000) and Kim et al. (2005). Almost complete 16S rRNA gene sequences of strains AT1042T (1402 bp) and AT1048T (1395 bp) were obtained. Preliminary sequence comparison with 16S rRNA gene sequences held in GenBank indicated that the new isolates were related closely to the genus Flavobacterium. The newly determined sequences were then aligned manually against representatives of Flavobacterium species using bacterial 16S rRNA gene secondary structure. The regions available for all sequences (positions 48–1468; Escherichia coli numbering system), excluding positions likely to show ambiguous alignment (positions 76–94), were used to generate phylogenetic trees. 16S rRNA gene sequence similarity between strains AT1042T and AT1048T was 98.9 %, and the two Antarctic isolates were consistently recovered as a monophyletic clade (100 % bootstrap support) within the genus Flavobacterium in all tree-making methods used in this study. Strain AT1042T showed highest 16S rRNA gene sequence similarity to Flavobacterium flevense ATCC 27944T (96.9 %), followed by Flavobacterium psychrolimnae LMG 22018T (96.7 %), Flavobacterium limicola ST-82T (96.6 %) and Flavobacterium saccharophilum NCIMB 2072T (96.5 %). Strain AT1048T showed highest 16S rRNA gene sequence similarity with F. psychrolimnae LMG 22018T (97.0 %), followed by F. flevense ATCC 27944T (96.9 %), F. saccharophilum NCIMB 2072T (96.7 %) and F. limicola ST-82T (96.6 %). In the phylogenetic trees generated in this study, strains AT1042T and AT1048T represented a distinct phyletic line corresponding to novel species in the genus Flavobacterium (Fig. 1).
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) as these isolates shared high 16S rRNA gene similarity (98.9 %), corresponding to 16 nt differences. The two isolates shared a low DNA–DNA relatedness value of 34 %, which is well below the threshold (70 %) recommended for determining bacterial species (Wayne et al., 1987). It is clear from DNA–DNA hybridization experiments that strains AT1042T and AT1048T belong to separate genomic species.
Chemotaxonomic characteristics were determined from cultures grown at 15 °C on R2A. Menaquinone was isolated from 7-day-old cells according to the method of Minnikin et al. (1984) 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), by using a reversed-phase column (Supelco). Fatty acid methyl ester analysis was performed by GLC according to the Microbial Identification (MIDI) System using 5-day-old cells. The chemotaxonomic properties of the strains were consistent with those of the genus Flavobacterium and are given in the species descriptions below. The major fatty acids of the new isolates (Table 1) were similar to those of phylogenetically related species, although in slightly different proportions (Bernardet et al., 1996; Tamaki et al., 2003; Van Trappen et al., 2005).
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) by non-linear regressions using the R 1.8.1 statistical package (R Foundation for Statistical Computing, 2003). Square-root growth rate – temperature plots showed that the notional minimum, optimum and maximum growth temperatures were –29.7, 15.3 and 19.9 °C, respectively, for strain AT1042T and –29.7, 14.3 and 18.9 °C, respectively, for strain AT1048T (Fig. 2). When tested on R2A (between 5 and 25 °C at 5 °C intervals), both strains failed to grow at 25 °C. Following the definition of Isaksen & Jørgensen (1996), the Antarctic isolates can be defined as psychrophiles. The minimum doubling times of strains AT1042T and AT1048T were about 2.9 and 6.8 h, respectively. Growth at different pH (between pH 4 and 12 at intervals of 1 pH unit) and NaCl concentrations [between 0 and 7 % (w/v) at intervals of 1 %] was determined using sea-salt-free ZoBell's medium. KOH and HCl (both at 6 M) were used to adjust the final pH. Growth was also assessed 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. Detailed results of these growth experiments are given in the species descriptions.
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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 by Smibert & Krieg (1994). Hydrolysis of alginate (0.5 %, w/v), casein [50 % skimmed milk (Difco), v/v], carboxymethylcellulose (CM-cellulose) [0.5 % CM-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, 1992) and DNase test agar (Difco) were used for pectinase and DNase assays, respectively. Production of H2S was investigated using triple-sugar iron agar (Difco). Phenylalanine deaminase activity was determined on phenylalanine agar (Smibert & Krieg, 1994; 3 g yeast extract, 1 g L-phenylalanine, 1 g Na2HPO4, 5 g NaCl, 12 g Bacto agar, per litre of distilled water). 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; 1.0 g casitone, 0.1 g yeast extract, 0.5 g ammonium sulfate, 0.5 g Tris base, 0.01 g phenol red, 15 g Bacto agar, per litre of distilled water, adjusted to pH 7.0). Nitrate and nitrite reduction, production of indole, aesculinase, gelatinase and 
-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 enzyme activities were determined using the API ZYM kit (bioMérieux). The results of morphological, biochemical and physiological tests are given in Table 2 and in the species descriptions.
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-galactosidase, urease, arginine dihydrolase and 
-fucosidase and acid production from lactose, D-trehalose, sucrose and D-cellobiose. Therefore, we conclude that strains AT1042T and AT1048T should be assigned to the genus Flavobacterium as the type strains of two separate novel species.
Description of Flavobacterium weaverense sp. nov.
Flavobacterium weaverense (wea.ver.en'se. N.L. neut. adj. weaverense pertaining to the Weaver Peninsula, the geographical origin of the type strain).
Gram-negative, oxidase- and catalase-positive and psychrophilic. Cells are non-motile long rods during the exponential phase (approx. 1.6–12.5x0.3–0.5 µm) and become short rods during the stationary phase (approx. 0.7–1.5x0.3–0.4 µm). Colonies are convex, translucent, glistening, viscid, yellow, circular with entire margins and become mucoid after prolonged incubation on R2A or AOA. Does not glide or adhere to agar plates. Flexirubin-type pigments are 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. Grows well under aerobic conditions, weakly under microaerobic conditions (with 5–15 % O2 and 5–12 % CO2, created by use of the CampyPack Plus system) and poorly under anaerobic conditions (with 4–10 % CO2, created by use of the GasPack Plus system). Growth occurs at pH 6–12 (optimum pH 6–7) and with 0–3 % NaCl (optimum 1 %). Grows at 5.0–19.9 °C with notional minimum, optimum and maximum growth temperatures of –29.7, 15.3 and 19.9 °C, respectively. Minimum doubling time is 2.9 h. Decomposes starch and Tween 80, but not alginate, chitin or elastin. Positive reaction for urease. Negative reactions for L-phenylalanine deaminase, indole production and alkalinization on Christensen's citrate agar. Test for arginine dihydrolase is positive on Thornley's semi-solid medium, but negative with the API 20NE kit. 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. In API ZYM kits, alkaline phosphatase, esterase lipase (C8), leucine arylamidase, valine arylamidase, acid phosphatase, naphthol-AS-BI-phosphohydrolase, -glucosidase, N-acetyl--glucosaminidase and -fucosidase activities are present; weak cystine arylamidase and trypsin activities are present; and esterase (C4), lipase (C14), -chymotrypsin, -galactosidase, -galactosidase, -glucuronidase, -glucosidase and -mannosidase activities are absent. Assimilates glucose, mannose, N-acetylglucosamine and maltose as sole carbon sources in API 20NE kits, but not arabinose, mannitol, gluconate, caprate, adipate, malate, citrate or phenylacetate. Other physiological and biochemical characteristics are given in Table 2
. Maximum absorption peak of pigment is at 451 nm and the next shoulder is at 479 nm. Major isoprenoid quinone is MK-6. Predominant cellular fatty acids are C15 : 1
6c, iso-C16 : 1 H and iso-C16 : 0 3-OH; the full fatty acid profile is given in Table 1. DNA G+C content of the type strain is 37 mol%.
The type strain, AT1042T (=IMSNU 14048T=KCTC 12223T=JCM 12384T), was isolated from a soil sample from the Weaver Peninsula, King George Island, Antarctica.
Description of Flavobacterium segetis sp. nov.
Flavobacterium segetis (se.ge'tis. L. gen. n. segetis of the soil).
Gram-negative, oxidase- and catalase-positive and psychrophilic. Cells are rod-shaped with rounded ends, approximately 1.1–2.3x0.2–0.3 µm and non-motile. Colonies are convex, translucent, glistening, butyrous, orange, circular with entire margins and become mucoid after prolonged incubation on R2A or AOA. Does not glide or adhere to agar plates. Flexirubin-type pigments are 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. Grows well under aerobic conditions, weakly under microaerobic conditions (with 5–15 % O2 and 5–12 % CO2 created by use of the CampyPack Plus system) and poorly under anaerobic conditions (with 4–10 % CO2 created by use of the GasPack Plus system). Growth occurs at pH 6–11 (optimum pH 7) and with 0–3 % NaCl (optimum 0 %). Grows at 5.0–21.8 °C with notional minimum, optimum and maximum growth temperatures of –29.7, 14.3 and 18.9 °C, respectively. Minimum doubling time is 6.8 h. Decomposes starch and Tween 80, but not alginate, chitin or elastin. Negative reactions for arginine dihydrolase, urease, L-phenylalanine deaminase, indole production and alkalinization on Christensen's citrate agar. Produces acid from D-cellobiose, D-glucose, D-trehalose, maltose and sucrose, but not from D-fructose, D-mannitol, D-raffinose, D-salicin, D-xylose, L-arabinose or L-rhamnose. Produces acid from lactose after prolonged incubation (4 weeks). In API ZYM kits, alkaline phosphatase, leucine arylamidase, valine arylamidase, acid phosphatase, naphthol-AS-BI-phosphohydrolase, -glucosidase and N-acetyl--glucosaminidase activities are present; weak esterase lipase (C8), cystine arylamidase, trypsin and -glucosidase activities are present; and esterase (C4), lipase (C14), -chymotrypsin, -galactosidase, -galactosidase, -glucuronidase, -mannosidase and -fucosidase activities are absent. Assimilates glucose, mannose, N-acetylglucosamine (weakly) and maltose as sole carbon sources in API 20NE kits, but not arabinose, mannitol, gluconate, caprate, adipate, malate, citrate or phenylacetate. Other physiological and biochemical characteristics are given in Table 2. Maximum absorption peak of pigment is at 472 nm and the next shoulder is at 452 nm. Major isoprenoid quinone is MK-6. Predominant cellular fatty acids are iso-C16 : 0 3-OH and summed feature 3 (containing C16 : 17c and/or iso-C15 : 0 2-OH); the full fatty acid profile is given in Table 1. DNA G+C content of the type strain is 35 mol%.
The type strain, AT1048T (=IMSNU 14050T=KCTC 12224T=JCM 12385T), 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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r is the square-root of growth rate.