SGM Journals
Bacteroidetes

Gramella marina sp. nov., isolated from the sea urchin Strongylocentrotus intermedius

Olga I. Nedashkovskaya, Seung Bum Kim, Kyung Sook Bae

  • 1Pacific Institute of Bioorganic Chemistry of the Far-Eastern Branch of the Russian Academy of Sciences, Prospekt 100 Let Vladivostoku 159, 690022, Vladivostok, Russia
  • 2Department of Microbiology, College of Bioscience and Biotechnology, Chungnam National University, 220 Gung-dong, Yuseong, Daejeon 305-764, Republic of Korea
  • 3Biological Resources Center, Korea Institute of Bioscience and Biotechnology, 52 Oun-dong, Yuseong, Daejeon 305-333, Republic of Korea
  • Correspondence
    Olga I. Nedashkovskaya
    olganedashkovska{at}piboc.dvo.ru
    or
    olganedashkovska{at}gmail.com

    • International Journal of Systematic and Evolutionary Microbiology 2010; 60(12):2799–2802 · https://doi.org/10.1099/ijs.0.013870-0

    View at publisher PubMed

    Abstract

    The taxonomic position of a novel Gram-stain-negative, aerobic, heterotrophic, gliding, yellow–orange-pigmented bacterium, isolated from the sea urchin Strongylocentrotus intermedius and designated strain KMM 6048T, was established. Phylogenetic analysis based on 16S rRNA gene sequencing revealed that the isolate was a member of the family Flavobacteriaceae affiliated with recognized species of the genus Gramella, forming a distinct lineage within the genus. 16S rRNA gene sequence similarities between strain KMM 6048T and the type strains of species of the genus Gramella were 97.4–98.4 %. In line with representative members of the genus Gramella, strain KMM 6048T was oxidase- and catalase-positive, hydrolysed gelatin and starch, utilized carbohydrates and possessed a DNA G+C content of 40.0 mol%. However, differentiating phenotypic traits and phylogenetic distinctiveness clearly indicated that the strain represented a novel species within the genus Gramella, for which the name Gramella marina sp. nov. is proposed; the type strain is KMM 6048T (=KCTC 12366T=LMG 25418T).

    • The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of Gramella marina KMM 6048T is AY753911.

    The genus Gramella, a member of the family Flavobacteriaceae (phylum ‘Bacteroidetes’), and the species Gramella echinicola were created to accommodate a gliding, strictly aerobic, yellow–orange-pigmented, Gram-stain-negative, and oxidase- and catalase-positive bacterial strain associated with the sea urchin Strongylocentrotus intermedius from the Gulf of Peter the Great (Nedashkovskaya et al., 2005). A further species in the genus, Gramella portivictoriae, isolated from a sediment sample from the South China Sea (Lau et al., 2005), was subsequently described.

    In this work, a novel strain, designated KMM 6048T, was isolated and identified using a polyphasic approach. As shown by phylogenetic analysis, strain KMM 6048T belonged to the family Flavobacteriaceae, phylum ‘Bacteroidetes’, and formed a distinct lineage within the genus Gramella. On the basis of phenotypic and molecular phylogenetic evidence, the isolate represents a novel species of the genus Gramella.

    Strain KMM 6048T was isolated from a sea urchin, Strongylocentrotus intermedius, collected at a depth of 3 m in Troitsa Bay, Gulf of Peter the Great, Sea of Japan (also known as the East Sea), Pacific Ocean, in September 2002. For strain isolation, 0.1 ml sea-urchin tissue homogenate was plated onto marine agar 2216 (Difco). After primary isolation and purification, the strain was cultivated at 28 °C on the same medium and stored at −80°C in marine broth (Difco) supplemented with 20 % (v/v) glycerol.

    DNA extraction, PCRs and sequencing of the 16S rRNA gene were carried out as described previously (Kim et al., 1998). The sequence obtained (1438 bp) was aligned with those of representative members of selected genera of the family Flavobacteriaceae by using phydit version 3.2 (). Phylogenetic trees were inferred by using suitable programs of the phylip package (Felsenstein, 1993). Phylogenetic distances were calculated from the Jukes–Cantor model (Jukes & Cantor, 1969) and trees were constructed on the basis of the neighbour-joining (Saitou & Nei, 1987) and maximum-likelihood (Felsenstein, 1993) algorithms. A bootstrap analysis was performed with 1000 resampled datasets by using the seqboot and consense programs of the phylip package.

    Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain KMM 6048T was a member of the family Flavobacteriaceae and formed a coherent cluster with species of the genus Gramella (Fig. 1⇓). The 16S rRNA gene sequence similarities between the isolate and the type strains of G. echinicola and G. portivictoriae were 97.5 and 98.4 %, respectively. According to Stackebrandt & Ebers (2006), DNA–DNA reassociation experiments between two bacterial strains are required to demonstrate whether or not they belong to the same species only when they share more than 98.7–99.0 % 16S rRNA gene sequence similarity. For this reason, DNA–DNA hybridization experiments were not performed.

    Figure image not available in archive
    Fig. 1.

    Neighbour-joining phylogenetic tree based on the 16S rRNA gene sequences of KMM 6048T and related members of the family Flavobacteriaceae. The same tree topology was recovered using the maximum-likelihood algorithm. Numbers at nodes are the levels of bootstrap support (%) from 1000 resampled datasets. Stenothermobacter spongiae UST030701-156T (DQ064789), Persicivirga dokdonensis DSW-6T (DQ017065) and Sandarakinotalea sediminis CKA-5T (AB206954) were used as the outgroups. Bar, 0.01 substitutions per nucleotide position.

    DNA was isolated following the method of Marmur (1961) and its G+C content was determined by the thermal denaturation method (Marmur & Doty, 1962). The DNA G+C content of strain KMM 6048T was 40.0 mol%, a value similar to those reported for the two other recognized species of the genus Gramella (Lau et al., 2005; Nedashkovskaya et al., 2005).

    Analysis of fatty acid methyl esters was performed according to the standard protocol of the Microbial Identification System (Microbial ID). Strain KMM 6048T was cultivated on marine agar 2216E at 25 °C for 48 h. The fatty acids of strain KMM 6048T accounting for greater than 5 % of the total fatty acids were: iso-C15 : 0, 17.9 %; iso-C17 : 0 3-OH, 12.4 %; summed feature 3 (C16 : 1ω7c and/or iso-C15 : 0 2-OH fatty acids), 11.6 %; anteiso-C15 : 0, 8.6 %; iso-C16 : 0, 6.3 %; iso-C17 : 1ω9c, 5.5 %; and C17 : 0 2-OH, 5.1 %. The fatty acid profile of the novel isolate was similar to those of recognized species of the genus Gramella (Lau et al., 2005; Nedashkovskaya et al., 2005), but it differed from them in the proportions of iso-C16 : 0, iso-C15 : 0 3-OH and C17 : 0 2-OH (Table 1⇓). Also, C15 : 0, iso-C15 : 1 G, iso-C16 : 1 G and anteiso-C17 : 1ω9c were detected only in cells of strains KMM 6048T and G. echinicola KMM 6050T, whereas C15 : 0 3-OH was found only in cells of G. portivictoriae UST040801-001T; however, these differences may be a result of different cultivation conditions. Isoprenoid quinones were extracted and analysed using a standard procedure (Minnikin et al., 1984). The major respiratory quinone of strain KMM 6048T was MK-6, which was in line with that of the two recognized species of the genus Gramella and all other members of the family Flavobacteriaceae (Bernardet et al., 2002; Lau et al., 2005; Nedashkovskaya et al., 2005).

    Table 1.

    Fatty acid profiles of species of the genus Gramella

    Strains: 1, KMM 6048T; 2, G. echinicola KMM 6050T; 3, G. portivictoriae UST040801-001T. Data from Lau et al. (2005), Nedashkovskaya et al. (2005) and this study. All strains were grown on marine agar 2216 for 48 h. Strain KMM 6048T and G. echinicola KMM 6050T were grown at 25 °C, whereas G. portivictoriae UST040801-001T was grown at 30 °C. Fatty acids amounting to <1 % of the total fatty acids in all strains are not shown. nd, Not detected; tr, trace (<1 %).

    Phenotypic analysis of strain KMM 6048T was performed by using previously described methods (Nedashkovskaya et al., 2003a, b). In addition, API 20E, API 20NE and API ZYM galleries (bioMérieux) and GN2 MicroPlates (Biolog) were used according to the manufacturers' instructions, except that the galleries were incubated at 28 °C. Susceptibility to antibiotics was tested using the diffusion method with discs containing ampicillin (10 μg), benzylpenicillin (10 U), carbenicillin (100 μg), cefalexin (30 μg), cefazolin (30 μg), chloramphenicol (30 μg), doxycycline (10 μg), erythromycin (15 μg), gentamicin (10 μg), kanamycin (30 μg), lincomycin (15 μg), nalidixic acid (30 μg), neomycin (30 μg), ofloxacin (5 μg), oleandomycin (15 μg), oxacillin (10 μg), polymixin (300 U), rifampicin (5 μg), streptomycin (30 μg), tetracycline (30 μg) and vancomycin (30 μg) (Research Center of Pharmacotherapy, Russia). Gliding motility was determined as described by Bowman (2000).

    In accordance with the two other recognized species of the genus Gramella, strain KMM 6048T was heterotrophic, strictly aerobic, yellow–orange-pigmented and motile by gliding. It was oxidase-, catalase- and alkaline phosphatase-positive, required NaCl or seawater for growth, hydrolysed aesculin, gelatin and starch, and utilized carbohydrates. However, strain KMM 6048T differed from the type strains of the two other species of the genus in a number of phenotypic properties (Table 2⇓).

    Table 2.

    Phenotypic characteristics that differentiate strain KMM 6048T from other species of the genus Gramella

    Strains: 1, KMM 6048T; 2, G. echinicola KMM 6050T; 3, G. portivictoriae UST040801-001T. All strains were positive for the following tests: respiratory metabolism; gliding motility; oxidase and catalase activities; requirement for Na+ ions for growth; growth with 1–6 % NaCl and at 4–36 °C; hydrolysis of aesculin, gelatin, starch, Tween 40 and Tween 80; utilization of arabinose, galactose, glucose and sucrose; acid phosphatase, alkaline phosphatase, α-chymotrypsin, cystine arylamidase, leucine arylamidase, valine arylamidase, esterase (C4), esterase lipase (C8), lipase (C14), α-galactosidase, α-glucosidase, β-glucosidase, trypsin and naphthol-AS-BI-phosphohydrolase activities; susceptibility to ampicillin, benzylpenicillin, chloramphenicol and tetracycline; and resistance to kanamycin. All strains were negative for the following tests: production of flexirubin-type pigments; nitrate reduction; α-fucosidase and urease activities; H2S and indole production; hydrolysis of agar, cellulose (CM-cellulose and filter paper) and chitin; acid production from fucose, lactose, rhamnose, sorbose, cellobiose, xylose, glycerol, adonitol, inositol, mannitol, sorbitol and citrate; and utilization of lactose, inositol and malonate. Data are from Lau et al. (2005), Nedashkovskaya et al. (2005) and this study. nd, Not determined.

    Consequently, phylogenetic distinctiveness and differential phenotypic characteristics warrant description of strain KMM 6048T as a representative of a novel species in the genus Gramella, for which the name Gramella marina sp. nov. is proposed.

    Description of Gramella marina sp. nov.

    Gramella marina (ma.ri′na. L. fem. adj. marina of the sea, marine, a bacterium isolated from a marine environment).

    The main characteristics are the same as those given for the genus. In addition, cells are 0.4–0.6 μm in width and 1.7–2.5 μm in length. On marine agar, colonies are 2–3 mm in diameter, circular, shiny with entire edges and yellow–orange-pigmented. Growth is observed at 4–37 °C (optimum, 28–30 °C) and with 1–15 % (w/v) NaCl (optimum, 2–5 % NaCl). Arginine dihydrolase, lysine decarboxylase, ornithine decarboxylase and tryptophan deaminase activities are absent. Acid is produced from amygdalin, but not from cellobiose. d-Mannose, adipate, gluconate, malate and phenylacetate are utilized, but caprate is not. The substrates utilized in the GN2 MicroPlate are listed in Table 1⇑. Susceptible to ampicillin, benzylpenicillin, carbenicillin, cefalexin, cefazolin, chloramphenicol, doxycycline, erythromycin, lincomycin, nalidixic acid, ofloxacin, oleandomycin, streptomycin, tetracycline, rifampicin and vancomycin, but resistant to gentamicin, kanamycin, neomycin, oxacillin and polymyxin. Other biochemical and physiological properties are shown in Table 2⇑. The predominant fatty acids (i.e. >5 % of the total fatty acids) are iso-C15 : 0, iso-C17 : 0 3-OH, anteiso-C15 : 0, iso-C16 : 0, iso-C17 : 1ω9c and summed feature 3 (comprising C16 : 1ω7c and/or iso-C15 : 0 2-OH and C17 : 0 2-OH).

    The type strain is KMM 6048T (=KCTC 12366T=LMG 25418T), isolated from the sea urchin Strongylocentrotus intermedius collected in Troitsa Bay, Gulf of Peter the Great, Sea of Japan (also known as the East Sea). The DNA G+C content of the type strain is 40.0 mol%.

    Acknowledgments

    This research was supported by grants from the Far-Eastern Branch of the Russian Academy of Sciences no. 09-III-A-06-228, Presidium of the Russian Academy of Sciences ‘Molecular and Cell Biology’ and State Contract 02.518.11.7169 from the Federal Agency for Science and Innovations of the Russian Federation. K. S. B. also acknowledges support from the KRIBB initiative program.

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