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1 Division of Biology and Ocean Sciences, Inha University, Yonghyun-Dong, Incheon 402-751, Republic of Korea
2 Polar BioCenter, Korea Polar Research Institute, KOPRI, Songdo Techno Park, Incheon 406-840, Republic of Korea
Correspondence
Jang-Cheon Cho
chojc{at}inha.ac.kr
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ABSTRACT |
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), a member the family Flavobacteriaceae (Bernardet et al., 2002; Bernardet & Nakagawa, 2006; Reichenbach, 1989), currently contains one species, Ulvibacter litoralis, isolated from the green alga Ulva fenestrata. In this study, a dark yellow-pigmented bacterial strain, designated IMCC3101T, was isolated from Antarctic coastal seawater and subjected to a taxonomic study according to the minimal standards for the description of new taxa in the family Flavobacteriaceae (Bernardet et al., 2002). On the basis of phenotypic characteristics and phylogenetic data, strain IMCC3101T represents a novel species of the genus Ulvibacter. Strain IMCC3101T was isolated from a seawater sample collected from the coast of King George Island, Weaver Peninsula, Antarctica (6 ° 14' S 5 ° 47' E), using a standard dilution-plating method on marine agar 2216 (MA; Difco) at 20 °C. After the optimum growth temperature of strain IMCC3101T had been determined, cultures were maintained routinely on MA at 25 °C and preserved as a suspension in marine broth (MB; Difco)/glycerol (9 : 1, v/v) at –75 °C.
The methods used for DNA extraction, PCR amplification and sequencing of the 16S rRNA gene were as described in a previous study (Cho & Giovannoni, 2003). The resulting almost-complete 16S rRNA gene sequence (1461 bp) of strain IMCC3101T was aligned with those of its nearest neighbours by using the ARB software package (Ludwig et al., 2004), and levels of 16S rRNA gene sequence similarity were calculated by using the ARB software on the basis of this alignment. An unambiguous segment of 1121 nt, determined from the alignment of the 16S rRNA gene sequences of 16 members of the phylum Bacteroidetes, was used for phylogenetic analyses in PAUP* 4.0 beta 10 (Swofford, 2002). Phylogenetic trees were generated by using the neighbour-joining algorithm (Saitou & Nei, 1987) with the Jukes–Cantor distance model (Jukes & Cantor, 1969) and using the maximum-parsimony (Fitch, 1971) and maximum-likelihood (Felsenstein, 1981) algorithms. The robustness of the neighbour-joining and maximum-likelihood phylogenetic trees was confirmed with bootstrap analyses based on 1000 and 100 resamplings of the sequences, respectively. Preliminary sequence comparisons with 16S rRNA gene sequences deposited in GenBank (Altschul et al., 1997), at the Ribosomal Database Project (RDP-II; Cole et al., 2005) and on the EzTaxon server (http://www.eztaxon.org) indicated that strain IMCC3101T was related closely to the genus Ulvibacter in the family Flavobacteriaceae. The strain showed the highest 16S rRNA gene sequence similarity to U. litoralis KMM 3912T (96.6 %), but showed <93.5 % sequence similarity to members of other genera in the family. In all of the phylogenetic trees generated in this study (Fig. 1), strain IMCC3101T, U. litoralis KMM 3912T and an uncultured coastal bacterium, 2D9 (GenBank accession no. AY274841; Kirchman et al., 2003), formed a robust monophyletic clade. This monophyletic clade was clearly separated from other genera of the family Flavobacteriaceae with high levels of bootstrap support, indicating that strain IMCC3101T represents a novel species within the genus Ulvibacter.
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. Cellular pigments were extracted with acetone/methanol (1 : 1, v/v) from a 4 day culture on MA at 25 °C and their absorption spectra were determined by using a scanning UV/visible spectrophotometer (Optizen 2120UV; Mechasis). The presence of flexirubin-type pigments was investigated by using the bathochromatic shift test with a 20 % (w/v) KOH solution (Bernardet et al., 2002; McCammon & Bowman, 2000). The growth-temperature range and optimum were tested at 3–42 °C on MA. The pH range and optimum for growth were tested on MA adjusted to pH values from 4.0 to 12.0. The NaCl concentrations and optimum for growth were determined in NaCl-free artificial seawater medium (Choo et al., 2007) supplemented with 5.0 g peptone, 1.0 g yeast extract and various concentrations of NaCl (0–15 %, w/v). Catalase and oxidase tests were performed according to standard methods (Smibert & Krieg, 1994). Other biochemical tests and carbon-source oxidation tests were carried out using API 20NE and API ZYM strips (bioMérieux) and in GN2 microplates (Biolog), according to the manufacturers' instructions except that the strips were inoculated with bacterial suspensions in artificial seawater medium and incubated at 25 °C for 3 days. Degradation of macromolecules was tested by incubating strain IMCC3101T at 25 °C for 3 weeks on MA containing macromolecules. The following macromolecules were tested: starch (0.2 %, w/v), casein (10 % skimmed milk, w/v), elastin (0.5 %, w/v), chitin (0.5 %, w/v), agar (1.5 %, w/v) and CM-cellulose (0.2 %, w/v). Hydrolysis was revealed by the formation of clear zones around the colonies either immediately or after flooding of the plate with the appropriate staining solution (Teather & Wood, 1982). Susceptibility to 10 different antimicrobial agents (Choo et al., 2007) was determined by using the disc diffusion method (Jorgensen et al., 1999) on MA incubated for 4 days at 25 °C. The DNA G+C content of strain IMCC3101T was determined by using HPLC according to Mesbah et al. (1989), with a Discovery C18 column (5 µm, 15 cmx4.6 mm; Supelco). Cellular fatty acid methyl esters, extracted and prepared from cultures grown on MA at 25 °C for 4 days, were analysed by the Korean Culture Center of Microorganisms (KCCM, Seoul, Republic of Korea), using the MIDI system. The quinone content was also analysed by the KCCM, using reversed-phase HPLC analysis (Komagata & Suzuki, 1987).
Phenotypic characteristics of strain IMCC3101T are listed in the species description and in Table 1. In summary, cells of strain IMCC3101T were Gram-negative, chemoheterotrophic, obligately aerobic, non-motile, straight rods that produced flexirubin-type pigments. As shown in Table 1, strain IMCC3101T shared a number of characteristics with U. litoralis, including the production of flexirubin-type pigments, a similar DNA G+C content and a similar fatty acid profile. However, there were several phenotypic differences with respect to U. litoralis, e.g. cell and colony morphology, temperature range for growth, gliding motility and the proportions of iso-C15 : 0, iso-C15 : 1 and iso-C16 : 0 (Table 1). These differences, together with the level of 16S rRNA gene sequence similarity (<97 %), support the description of strain IMCC3101T as representing a novel species of the genus Ulvibacter, for which the name Ulvibacter antarcticus sp. nov. is proposed.
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Cells are Gram-negative, chemoheterotrophic, obligately aerobic, straight rods devoid of flagellar and gliding motility. Cells are 0.5–2.3 µm in length and 0.4–0.7 µm in diameter. Colonies grown on MA for 4 days are 0.2–0.4 mm in diameter, circular, shiny with entire edges, viscous and dark yellow in colour. After 3 weeks incubation, colonies reach up to 2 mm in diameter. Growth occurs at 3–25 °C (optimum, 25 °C), at pH 6–10 (optimum, pH 7) and at 1–3 % NaCl (optimum, 2 %). Catalase- and oxidase-positive. Peaks in absorption spectra for the cellular pigments are observed at 397, 450 (major peak) and 470 nm. Flexirubin-type pigments are produced. Starch is degraded, but casein, agar, elastin, CM-cellulose and chitin are not. In API 20NE strips, gelatin hydrolysis is positive, but nitrate reduction, hydrolysis of aesculin and urea, β-galactosidase and arginine dihydrolase activities, indole production and acid production from glucose are negative. In the API ZYM system, alkaline phosphatase, esterase (C4), esterase lipase (C8), leucine arylamidase, valine arylamidase, cystine arylamidase, acid phosphatase and naphthol-AS-BI-phosphohydrolase activities are present, but lipase (C14), trypsin, 
-chymotrypsin, -galactosidase, β-galactosidase, β-glucuronidase, -glucosidase, β-glucosidase, N-acetyl-β-glucosaminidase, -mannosidase and -fucosidase activities are absent. The following carbon substrates are assimilated (Biolog GN2 microplates): -cyclodextrin, glycogen, N-acetyl-D-galactosamine, D-arabitol, D-mannitol, pyruvic acid methyl ester, succinic acid monomethyl ester, D-galacturonic acid, -ketoglutaric acid, -ketovaleric acid, bromosuccinic acid, L-alaninamide, L-alanine, L-alanylglycine, L-asparagine, L-aspartic acid, L-glutamic acid, glycyl L-aspartic acid, glycyl L-glutamic acid, L-leucine, L-ornithine, L-proline, L-serine, L-threonine and urocanic acid. None of the other carbon substrates in Biolog GN2 microplates is assimilated. Susceptible to chloramphenicol (25 µg), erythromycin (15 µg), penicillin G (10 µg), rifampicin (50 µg) and tetracycline (30 µg), but resistant to ampicillin (10 µg), gentamicin (10 µg), kanamycin (30 µg), streptomycin (10 µg) and vancomycin (30 µg). Cellular fatty acids amounting to 
1 % of the total fatty acid content are as follows: iso-C17 : 0 3-OH (19.4 %), iso-C15 : 0 (15.3 %), iso-C16 : 0 (11.0 %), iso-C16 : 0 3-OH (9.2 %), summed feature 3 (comprising C16 : 1
7c and/or iso-C15 : 0 2-OH; 5.9 %), iso-C16 : 1 (5.4 %), C17 : 0 2-OH (4.7 %), iso-C15 : 0 3-OH (4.1 %), iso-C15 : 1 (3.9 %), C17 : 16c (3.4 %), iso-C17 : 19c (2.2 %), C15 : 0 2-OH (2.1 %), iso-C14 : 0 3-OH (1.8 %), iso-C14 : 0 (1.7 %), an unidentified fatty acid with an equivalent chain length of 13.565 (1.6 %), anteiso-C15 : 0 (1.3 %) and C15 : 0 (1.2 %). Traces (<1 %) of the following fatty acids are also present: anteiso-C17 : 19c, C17 : 0 3-OH, C19 : 0, C17 : 18c, C16 : 0, iso-C13 : 0, iso-C12 : 0, C10 : 0 3-OH, iso-C17 : 0, anteiso-C15 : 1, C15 : 16c, C12 : 0 3-OH and anteiso-C17 : 0. The major respiratory quinone is MK-6. The DNA G+C content is 37.0 mol%.
The type strain, IMCC3101T (=KCCM 42686T=NBRC 102682T), was isolated from surface seawater from Maxwell Bay, King George Island, west Antarctica.
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ACKNOWLEDGEMENTS |
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