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Wangia profunda gen. nov., sp. nov., a novel marine bacterium of the family Flavobacteriaceae isolated from southern Okinawa Trough deep-sea sediment

Qi-Long Qin, Dian-Li Zhao, Jing Wang, Xiu-Lan Chen, Hong-Yue Dang, Tie-Gang Li, Yu-Zhong Zhang, Pei-Ji Gao
DOI: http://dx.doi.org/10.1111/j.1574-6968.2007.00694.x 53-58 First published online: 1 June 2007

Abstract

An orange-pigmented, Gram-negative, nonmotile, strictly aerobic and oxidase- and catalase-positive bacterium (SM-A87T) was isolated from the deep-sea sediment of the southern Okinawa Trough area. The main fatty acids were i15 : 0, i17 : 0 3OH, i15 : 1 G, i17 : 1ω9c, 15 : 0, i15 : 0 3OH and summed feature 3 (comprising i-15 : 0 2OH and/or 16 : 1ω7c). MK-6 was the predominant respiratory quinone. DNA G+C content was 35.8 mol%. Flexirubin-type pigments were absent. Phylogenetic analyses based on 16S rRNA gene sequences revealed that strain SM-A87T formed a distinct lineage within the family Flavobacteriaceae, with <93% sequence similarity to the nearest strain of genus Salegentibacter. Moreover, strain SM-A87T could be distinguished from the nearest phylogenetic neighbors by a number of chemotaxonomic and phenotypic properties. On the basis of polyphasic analyses, it is proposed that strain SM-A87T be classified in a novel genus and a new species in the family Flavobacteriaceae, designated Wangia profunda gen. nov., sp. nov. The type strain is SM-A87T (CCTCC AB 206139T=DSM 18752).

Keywords
  • a novel genus
  • Flavobacteriaceae
  • southern Okinawa Trough
  • deep-sea sediment

Introduction

The family Flavobacteriaceae, belonging to the phylum Bacteroidetes [formerly Cytophaga–Flavobacterium–Bacteroides, (CFB)], includes a number of marine bacteria (Bowman et al., 1998; Nedashkovskaya et al., 2003, 2005a, b). Some members of the family have been reported to be able to decompose complex polysaccharides and other biomacromolecules (Bernardet et al., 2002). In recent years, several new genera of the Flavobacteriaceae have been described, e.g. Mesonia, Gramella, Leeuwenhoekiella, Nonlabens, Stenothermobacter, Dokdonia and Sandarakinotalea (Nedashkovskaya et al., 2003, 2005a, b; Lau et al., 2005a, b, 2006; Yoon et al., 2005; Khan et al., 2006). In this study, the bacterial strain SM-A87T originating from marine sediment is proposed to represent a novel genus of the family Flavobacteriaceae.

Materials and methods

Isolation of the strain and culture condition

Bacteria were isolated on marine agar 2216 medium (Difco) from deep-sea sediment samples taken from near the southern Okinawa Trough at a water depth of 1245 m using core sampler. Strain SM-A87T was isolated from the subseafloor sediments at 2 mbsf (meters below seafloor). The in situ temperature, pH and chlorinity of the sediment or porewater were 4.7°C, 7.35, and 533 mmol kg−1, respectively. Besides strain SM-A87T, more than 300 other bacterial strains were also isolated from the surface and subseafloor (down to 8.6 mbsf) sediments. After primary isolation, the purified isolate was cultivated on an agar medium composed of 10 g L−1 peptone, 5 g L−1 yeast extract (both Oxoid), 15 g L−1 agar and artificial sea water (Bian et al., 2006) (referred to as marine agar hereafter) at 25°C. The isolate was stored at −70°C in marine broth (composing of 10 g L−1 peptone, 5 g L−1 yeast extract and artificial sea water) supplemented with 20% (v/v) glycerol.

DNA isolation, PCR amplification, sequencing of the 16S rRNA gene and phylogenetic analysis

Genomic DNA extraction, PCR and 16S rRNA gene sequencing followed the procedures of Kim (1998). The nearly complete 16S rRNA gene sequence of strain SM-A87T (1493 nucleotides) has been deposited in the GenBank database under accession number DQ855467. It was aligned with its nearest neighboring sequences retrieved from GenBank, and only the valid published strains were considered. Phylogenetic trees were constructed using the mega software package (version 3.1) with three different methods (neighbor-joining, maximum-parsimony and UPGMA). Phylogenetic distances were calculated from the model of Jukes & Cantor (1969) and bootstrap analysis was performed with 1000 replicates by mega package.

Phenotypic study

Cell morphology was examined using scanning electron microscopy (SEM) according to the method of Neu (2001) at different growth phases. Gliding motility was determined using the methods of Bowman (2000) and Bernardet (2002). The strain growth temperature (4–45°C) and pH (3–12) were tested in marine broth by measuring OD660 nm after 24 h incubation (Ivanova et al., 2004). The requirement for NaCl (0–15%) was determined on marine agar except that the artificial sea water was replaced by NaCl solutions at different concentrations. The presence of flexirubin-type pigments was examined using 20% KOH (w/v) as described by Bernardet (2002). Oxidative or fermentative utilization of glucose was determined according to the method of Lemos (1985). Sensitivity to antibiotics was tested using the disc-diffusion method as described by Ivanova (2004). Other physiological and biochemical properties were tested using standard procedures as described by Gerhardt (1994). The commercial systems API 20E, API ZYM (both from bioMerieux) and MicroPlate GN2 (Biolog) were used to test the substrate oxidation profile, nitrate reduction and production of H2S, indole and acetoin. The manufacturer's instructions were followed except that cells for inoculation of API 20E and MicroPlate GN2 systems were suspended in artificial sea water (Khan et al., 2006). The GC content of DNA was determined by HPLC (Mesbah et al., 1989). Chemotaxonomic analyses were carried out by Dr Brian Tindall (Identification Service of the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Braunschweig, Germany). The analysis of cellular fatty acids was carried out according to the standard protocol of the Sherlock microbial identification system. Analysis of quinones was performed by HPLC. Polar lipids were determined by thin layer chromatography.

Results and discussion

16S rRNA gene phylogeny

The neighbor-joining phylogenetic tree revealed that strain SM-A87T formed a distinct lineage within the family Flavobacteriaceae (Fig. 1). Trees based on maximum-parsimony and UPGMA methods showed essentially the same topology. Strain SM-A87T had 92.9% 16S rRNA gene sequence similarity (99 nucleotides differences) to its nearest neighbor Salegentibacter holothuriorum, and 91.8% and 91.5% to Mesonia algae and Gramella portivictoriae, respectively. Therefore, according to phylogenetic analyses, strain SM-A87T should be classified as a novel genus and species in the family Flavobacteriaceae.

1

Phylogenetic tree based on 16S rRNA gene sequences of strain SM-A87T and members of related genera in the family Flavobacteriaceae. The tree was generated by the neighbor-joining method (Saitou & Nei, 1987). Numbers at nodes indicate bootstrap values (%) (only values >50% are shown). Bar, 0.01 substitutions per nucleotide position.

Phenotypic characteristics

After 48 h cultivation at 28°C on marine agar, the colonies were yellow to orange and circular, about 1–3 mm in diameter, and were adherent to the agar. Cells were rod-shaped and ranged from 0.3 to 0.6 µm in width and from 1.5 to 3.3 µm in length and were nonmotile. Cells in old cultures might form coccoid bodies (Fig. 2).

2

Micrographs of strain SM-A87T cultured on marine agar at 28°C for 24 h taken by SEM (a) and coccoid bodies in aging culture taken by phase contrast microscopy (b).

The DNA G+C content of strain SM-A87T was 35.8 mol%, an intermediate value among members of the family Flavobacteriaceae (Bernardet et al., 2002). The main respiratory quinone was MK-6, in accordance with all members of the family Flavobacteriaceae. The strain synthesized mainly terminally branched iso- and anteiso-fatty acids together with diagnostic amounts of iso-branched 2-hydroxy and 3-hydroxy fatty acids. The dominant fatty acids were i-15 : 0 (22.73%), summed feature 3 (comprising i-15 : 0 2OH and/or 16 : 1ω7c) (14.59 %), i-17 : 0 3OH (14.52%), i-15 : 1 G (9.68 %), i-17 : 1ω9c (9.01%), 15 : 0 (7.45%) and i-15 : 0 3OH (3.92%) (total more than 77%). Strain SM-A87T contained the characteristic fatty acids of the family; however, the higher content of i-17 : 0 3OH, the absence of a-17 : 1ω9c and the presence of 15 : 0 3OH distinguished it from related genera (Table 1). The only identified phospholipid was phosphatidylethanolamine.

View this table:
1

Comparison of major cellular fatty acids of SM-A87T and related genera

Fatty acidsSM-A87TS. salegensM. algaeG. echinicolaP. torquis (n=4)L. aequorea (n=6)S. spongiaeN. tegetincola
15 : 07.454.54.87.14.2 ± 0.6
i-15 : 022.7323.721.214.41.1 ± 0.318.2 ± 1.138.1 ± 0.533.1 ± 6. 4
a-15 : 01.6711.64.27.635.2 ± 4.44.5 ± 0.46.2 ± 0.73.8 ± 2.3
i-15 : 1 G9.6823.77.91.27.6 ± 1.6
15 : 1ω6c2.440.61.81.9
15 : 0 3OH1.952.5 ± 0.7
i-15 : 0 3OH3.923.21.30.3 ± 0.22.1 ± 0.26.2 ± 0.35.4 ± 3.0
i-16 : 01.647.66.313.16.0 ± 1.23.8 ± 1.21.9 ± 0.46.8 ± 0.4
i-16 : 0 3OH1.610.36.05.915.4 ± 1.03.4 ± 0.93.4 ± 0.35.7 ± 3.1
i-17 : 0 3OH14.523.014.66.70.2 ± 0.112.7 ± 1.011.2 ± 0.313.7 ± 5.5
17 : 1ω6c1.520.82.43.62.1 ± 0.41.7 ± 0.32.2 ± 0.4
i-17 : 1ω9c9.015.13.518.8 ± 2.95.7 ± 1.43.5 ± 0.8
a-17 : 1ω9c1.92.01.6 ± 0.4
i-17 : 1ω7c13.2
Summed feature 314.599.45.111.49.4 ± 1.68.8 ± 1.27.5 ± 3.6
Unknown2.673.34.6tr4.0 ± 1.16.1 ± 1.8
  • Values are percentages of total fatty acids. Data for P. torquis, N. tegetincola, L. aequorea and S. spongiae are means ± SD. n, number of strains studied.

  • Summed feature 3, comprising i-15 : 0 2OH and/or 16 : 1ω7c.

  • −, not detected; tr, trace amount (<1%).

  • Data from Dobson et al. (1993), Nedashkovskaya et al. (2003, 2005a, b), Bowman et al. (1998), Lau et al. (2005a, b, 2006).

Strain SM-A87T could be differentiated from members of the nearest genus, Salegentibacter, by (1) the formation of coccoid bodies in aging cultures, (2) growth without Na+ and with 12% NaCl, (3) being able to hydrolyze casein and unable to hydrolyze starch, (4) producing acid from arabinose, (5) production of H2S and (6) being resistant to ampicillin. Other characteristics that differentiate strain SM-A87T from members of related genera are shown in Table 2. Results of polyphasic analysis supported the description of Strain SM-A87T as a new genus and species in the family Flavobacteriaceae for which the name Wangia profunda is proposed.

View this table:
2

Characteristics differentiating strain SM-A87T from related members of the family Flavobacteriaceae

Characteristic123456789
Coccoid bodies in aging cultures++
Gliding motilityv++++
Growth at
    4°C+v++++v
    37°C+vvvv++
Growth with
    0% NaCl++
    12% NaCl+v+vv+
Hydrolysis of
    Casein+vvvND++
    Gelatin+++++++
    Starch++++++
Acid from
    Glucose+vvvvv
    Arabinose+
    H2S production+vND
Susceptible to
    Ampicillin+++ND+ND+
    Tetracycline+++ND++++
    StreptomycinND+ND+
    DNA G+C content (mol%)35.836.8–40.432.7–36.139.6–39.932.6–3533.638.335–42.541.0
  • 1, SM-A87T; 2, Salegentibacter (4) (in parentheses is the number of species compared); 3, Mesonia (2); 4, Gramella (2); 5, Psychroflexus (2); 6, Nonlabens (1); 7, Dokdonia (1); 8, Leeuwenhoekiella (2) 9, Stenothermobacter (1).

  • −, negative; +, positive; ND, not determined; v, variable.

  • Data from Dobson et al. (1993), Nedashkovskaya et al. (2003, 2004, 2005a, b, c, 2006), Ivanova et al. (2006), Lau et al. (2005a, b, 2006), Bowman et al. (1998), Donachie et al. (2004), Yoon et al. (2005), Pinhassi et al. (2006).

Description of Wangia gen. nov.

Wangia [Wan'gi.a. N.L. fem. n. Wangia of Wang, named in honor of Zu-Nong Wang, who has made great contributions to the development of microbiology in China]

Cells are Gram-negative, rod-shaped, nonmotile, nonspore-forming, strictly aerobic, oxidase- and catalase-positive. Flexirubin-type pigments are absent. MK-6 is the predominant respiratory quinone. The main fatty acids are i15 : 0, Summed Feature 3 (comprising i-15 : 0 2OH and/or 16 : 1ω7c), i-17 : 0 3OH, i-15 : 1 G, i-17 : 1ω9c, 15 : 0 and i-15 : 0 3OH. On the basis of 16S rRNA gene sequence analysis, the genus Wangia is a member of the family Flavobacteriaceae in the phylum Bacteroidetes. The type species is Wangia profunda.

Description of Wangia profunda sp. nov.

Wangia profunda (pro.fun'da L. adj. description of the environment where the stain was isolated).

Description is as for the genus plus the following. On marine agar, colonies are circular, 1–3 mm in diameter, convex with smooth surfaces and yellow to orange in color. Rods are from 0.3 to 0.6 µm in width and from 1.5 to 3.3 µm in length, coccoid bodies appear in aging cultures. Nondiffusible yellow pigments are produced. Growth occurs at 4–38°C (25–30°C optimum), at pH 5.0–8.5 and in the presence of 0–12% NaCl (3%, optimum). Growth is not observed on MacConkey agar. Hydrolyzes gelatin, casein and Tweens 20, 40, 80, but not agar, starch, cellulose (CM-cellulose or filter paper) and chitin. Positive for the following enzyme activities: ONPG (2-nitrophenyl-β-d-galactopyranoside) and gelatinase (API 20E), alkaline and acid phosphatase, trypsin, leucine arylamidase, valine arylamidase, naphthol-AS-BI-phosphohydrolase, β-galactosidase, α-glucosidase, β-glucosidase, N-acetyl-β-glucosaminidase (API ZYM). Negative for the following enzyme activities: arginine dihydrolase, lysine decarboxylase, ornithine decarboxylase and tryptophan deaminase (API 20E), esterase (C4), esterase lipase (C8), lipase (C14), cystine arylamidase, α-chymotrypsin, α-galactosidase, β-glucuronidase, α-mannosidase and α-fucosidase (API ZYM). Acids are produced from glucose, sucrose and arabinose, but not from mannose, inositol, sorbitol, rhamnose, melibiose and amygdalin (API 20E). Oxidizes d-melibiose, acetic acid, α-cyclodextrin, d-fructose, dextrin, citric acid, d-galactose, d-raffinose, gentiobiose, α-d-glucose, d-sorbitol, d-galacturonic acid, sucrose, 2-aminoethanol, α-d-lactose, d-trehalose, 2,3-butanediol, lactulose, turanose, glycerol, l-arabinose, maltose, l-threonine, d,l-α-glycerol phosphate, pyruvic acid methyl ester, glycyl-l-aspartic acid, d,l-carnitine, α-d-glucose-1-phosphate, d-cellobiose, d-mannose and d-glucose-6-phosphate (MicroPlates). Does not oxidize i-erythritol, p-hydroxyphenyl-acetic acid, bromosuccinic acid, l-histidine, urocanic acid, β-methyl-d-glucoside, cis-aconitic acid, itaconic acid, succinamic acid, hydroxyl-l-proline, inosine, l-fucose, d-psicose, α-ketobutyric acid, glucuronamide, l-leucine, uridine, formic acid, α-ketoglutaric acid, l-alaninamide, l-ornithine, Tween 40, l-rhamnose, d-galactonic acid lactone, α-ketovaleric acid, d-alanine, l-phenylalanine, phenylethylamine, Tween 80, dl-lactic acid, l-alanine, l-proline, putrescine, N-acetyl-d-galactosamine, m-inositol, d-gluconic acid, malonic acid, l-alanyl-glycine, l-pyroglutamic acid, N-acetyl-d-glucosamine, d-glucosaminic acid, propionic acid, l-asparagine, d-serine, adonitol, d-glucuronic acid, quinic acid, l-aspartic acid, l-serine, xylitol, α-hydroxybutyric acid, d-saccharic acid, l-glutamic acid, d-arabitol, d-mannitol, β-hydroxybutyric acid, sebacic acid, succinic acid mono-methyl ester, γ-hydroxybutyric acid, succinic acid, glycyl-l-glutamic acid and γ-aminobutyric acid (MicroPlates). Sensitive to tetracycline, cephalosporin, erythromycin and resistant to streptomycin, ampicillin, gentamicin, neomycin and kanamycin. Nitrate is reduced to N2. H2S is not produced. Indole and acetoin (Voges–Proskauer reaction) production are negative. Phosphatidylethanolamine is the only phospholipid identified. DNA G+C content is 35.8 mol%.

The type strain is SM-A87T (CCTCC AB 206139T=DSM 18752), isolated from the deep-sea sediment of the southern Okinawa Trough area.

Author Contribution

Co first author, Zhao Dian-Li and Qin Qi-Long contributed equally to the work in this manuscript.

Acknowledgements

The deep-sea sediment sample used in this study was retrieved during the IMAGES XII, MD-147-Marco Polo Leg 2 cruise of the R/V Marion Dufresne of the French Polar Institute (IPEV). This work was financially supported by the Pilot Projects of Knowledge Innovation Project of Chinese Academy of Sciences grants (Nos. KZCX3-SW-233 and KZCX3-SW-223), and the National Natural Science Foundation of China grants (Nos. 40476058 and 40576069), Hi-Tech Research and Development Program of China (2006AA09Z414), the Science and Technology R&D Program of Shandong Province of China (2005JJ3205108), Natural Science Foundation of Shandong Province of China (Z2004D02), and Foundation for Young Excellent Scientists in Shandong Province (2006BS02002).

Footnotes

  • Editor: Aharon Oren

References

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