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1 Department of Microbiology and Biotechnology Research Institute, Chungbuk National University, 12 Gaeshin-dong, Cheongju 361-763, Republic of Korea
2 Deep Ocean Water Application Research Center, Korea Ocean Research and Development Institute (KORDI), 245-7 Oho-ri, Jugwang-myeon, Goseong-gun 219-822, Republic of Korea
Correspondence
Sung-Keun Rhee
rhees{at}chungbuk.ac.kr
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ABSTRACT |
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7c and/or iso-C15 : 0 2-OH) and summed feature 7 (C18 : 19c/12t/7c) as the major cellular fatty acids. DNA–DNA relatedness data indicated that strain NO2T represents a distinct species that is separate from G. mesophila and G. polaris. On the basis of polyphasic evidence, it is proposed that strain NO2T (=KCTC 12755T=LMG 23762T) represents the type strain of a novel species, Glaciecola agarilytica sp. nov.
A transmission electron micrograph and a comparison of the fatty acid profile of strain NO2T and related strains are available as supplementary material in IJSEM Online.
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), Glaciecola mesophila (Romanenko et al., 2003) and Glaciecola polaris (Van Trappen et al., 2004). During screening, a novel agar-digesting bacterial strain, NO2T, detected by the occurrence of pitting around colonies, was isolated from marine sediment of the East Sea, Korea, and selected for further characterization by a polyphasic approach. Strain NO2T was isolated using a natural seawater agar medium, prepared using seawater of the East Sea. For solidification, 1.5 % (w/v) agarose was added to seawater before autoclaving. A sediment sample was placed in a sterile conical tube and serially diluted with filter-sterilized (0.22 µm pore size, Millipore) natural seawater. An aliquot of each dilution was spread onto the agar medium and incubated at 17 °C for 4 weeks. Agar-digesting colonies were transferred onto new plates and subjected to an additional incubation for 2 days at 25 °C. Cultured strains were preserved at –70 °C as suspensions in marine broth (Difco) containing 20 % (w/v) glycerol.
Bacterial genomic DNA was extracted using a commercial genomic DNA extraction kit (Bioneer). The 16S rRNA gene was amplified from chromosomal DNA using the universal bacterial primer set 9F and 1512R and the purified PCR products were sequenced by Solgent Co. Ltd (Daejeon, Korea) (Yoon et al., 1998). The full 16S rRNA gene sequences were compiled using SeqMan software (DNASTAR). 16S rRNA gene sequences of related taxa were obtained from the GenBank database. Sequence alignments were performed using the CLUSTAL_X program (Thompson et al., 1997) and gaps were edited in the BioEdit program (Hall, 1999). Evolutionary distances were calculated using the two-parameter model of Kimura (1983). Phylogenetic trees were constructed by using the neighbour-joining method (Saitou & Nei, 1987) using the MEGA3 program (Kumar et al., 2004) with bootstrap values based on 500 replications (Felsenstein, 1985). In the analysis of phylogeny, strain NO2T was determined to belong to the Gammaproteobacteria, and the highest degrees of sequence similarity were found with G. mesophila KMM 241T (98.6 % sequence similarity) and G. polaris LMG 21857T (98.0 % sequence similarity) (Fig. 1).
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. Cell morphology was examined by light microscopy (Nikon) and transmission electron microscopy (Carl Zeiss) after negative staining with 1 % (w/v) phosphotungstic acid. Catalase activity was determined by bubble production in 3 % (v/v) H2O2 and oxidase activity was determined using 1 % (w/v) tetramethyl-p-phenylenediamine. Strain NO2T is Gram-negative, aerobic and forms curved cells. Cells possess a polar flagellum (Supplementary Fig. S1 available in IJSEM Online) and form aggregates when cultivated in liquid media. Colonies are white, circular and convex, with regular edges and a diameter of 2–3 mm when grown on marine agar (Difco) plates for 2 days at 25 °C.
Cellular fatty acids were analysed from the isolate and reference organisms (G. mesophila KMM 241T and G. polaris LMG 21857T) grown on trypticase soy agar (Difco) for 2 days. The cellular fatty acids were saponified, methylated and extracted according to the protocol of the Sherlock Microbial Identification System (MIDI, 1999). The fatty acids were analysed by GC (Hewlett Packard 6890) and identified using the Microbial Identification software package. Chromosomal DNA extracted for 16S rRNA gene amplification was also used for determination of G+C content. RNA in the DNA solution was removed by incubation with a mixture of RNase A and RNase T1 (each at 20 U ml–1) at 30 °C for 1 h. The G+C content of the chromosomal DNA was analysed as described by Mesbah et al. (1989) using reversed-phase HPLC. Phospholipid analysis was performed as described previously (Komagata & Suzuki, 1987; Vaskovsky et al., 1975).
The major cellular fatty acids of strain NO2T were C16 : 0, summed feature 4 (C16 : 17c and/or iso-C15 : 0 2-OH) and summed feature 7 (C18 : 19c/12t/7c). This fatty acid profile is similar to those of G. mesophila KMM 241T and G. polaris LMG 21857T (Supplementary Table S1). The G+C content of genomic DNA of strain NO2T was 45 mol%. This value is consistent with the G+C content of the genus Glaciecola, which ranges between 40 and 47 mol% (Bowman et al., 1998; Romanenko et al., 2003; Van Trappen et al., 2004). The phospholipid composition of strain NO2T included phosphatidylethanolamine (71.8 %) and phosphatidylglycerol (23.2 %) as the main phospholipids. The polar lipid composition of NO2T was similar to that reported for G. mesophila KMM 241T (Romanenko et al., 2003).
Utilization of various substrates as sole carbon sources and some physiological characteristics were determined with API 20NE, API 32GN and API ZYM galleries according to the instructions of the manufacturer (bioMérieux). Bacterial suspensions were made in sterile, chilled artificial seawater medium (Levring, 1946). After inoculation, the galleries were incubated for 2 days at 25 °C and reactions were read. Growth at different temperatures was assessed after 5 days incubation on marine agar (Difco). Strain NO2T was able to grow at 7–30 °C, but not at 4 or above 37 °C. Salt tolerance was tested on R2A broth supplemented with 0–12 % (w/v) NaCl after 3 days incubation at 25 °C. Strain NO2T required sodium ions for growth and grew in 2–8 % NaCl. A comparison of selected physiological characteristics with G. mesophila, G. polaris, G. punicea and G. pallidula is shown in Table 1.
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. Hybridization levels of strain NO2T with G. mesophila KMM 241T and G. polaris LMG 21857T were respectively 27.7 and 28.4 %. On the basis of morphological, physiological and chemotaxonomic characteristics, together with the 16S rRNA gene sequence comparison described above, strain NO2T should be placed into a novel species, for which we propose the name Glaciecola agarilytica sp. nov.
Description of Glaciecola agarilytica sp. nov.
Glaciecola agarilytica (a.ga.ri.ly'ti.ca. N.L. n. agarum agar-agar, algal polysaccharide; N.L. adj. lyticus -a -um from Gr. adj. lutikos loosening, dissolving; N.L. fem. adj. agarilytica agar-dissolving).
Cells are Gram-negative, oxidase-positive, catalase-positive, agar-digesting and ovoid or curved rod-shaped (1.0–1.5 µm) and possess a single polar flagellum. Slightly halophilic and grows in 2–8 % NaCl at 7–30 °C. Favourable growth occurs aerobically, with circular colonies with regular edges formed within 2 days, with diameters of approximately 2.0–3.0 mm. Requires Na+ for growth. Negative for arginine dihydrolase and utilization of citrate and positive for degradation of starch, aesculin and DNA and utilization of dextrin, cellobiose, D-fructose, D-galactose, D-mannitol and D-mannose. No acid is produced from glucose, inositol, sorbitol, rhamnose, sucrose, melibiose or arabinose. Other phenotypic and biochemical tests are listed in Table 1
. The major cellular fatty acids are C16 : 0, summed feature 4 (C16 : 17c and/or iso-C15 : 0 2-OH) and summed feature 7 (C18 : 19c/12t/7c). Polar lipids include phosphatidylethanolamine (71.8 %) and phosphatidylglycerol (23.2 %). DNA G+C content is 45 mol% (as determined by HPLC).
The type strain is NO2T (=KCTC 12755T=LMG 23762T), isolated from marine sediment of the East Sea, Korea.
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ACKNOWLEDGEMENTS |
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