SGM Journals
Proteobacteria

Microbulbifer donghaiensis sp. nov., isolated from marine sediment of the East China Sea

Chun-Sheng Wang, Yu Wang, Xue-Wei Xu, Dong-Sheng Zhang, Yue-Hong Wu, Min Wu

  • 1Laboratory of Marine Ecosystem and Biogeochemistry, State Oceanic Administration, Hangzhou 310012, PR China
  • 2Second Institute of Oceanography, State Oceanic Administration, Hangzhou 310012, PR China
  • 3College of Life Sciences, Zhejiang University, Hangzhou 310058, PR China
  • Correspondence
    Chun-Sheng Wang
    wangsio{at}sio.org.cn

    • International Journal of Systematic and Evolutionary Microbiology 2009; 59(3):545–549 · https://doi.org/10.1099/ijs.0.004531-0

    View at publisher PubMed

    Abstract

    A Gram-negative, aerobic, neutrophilic, rod-shaped bacterium, designated strain CN85T, was isolated from a sediment sample collected from the East China Sea and was subjected to a polyphasic taxonomic characterization. This isolate grew in the presence of 0.5–6.0 % (w/v) NaCl and at 20–40 °C; optimum growth was observed with 3 % (w/v) NaCl and at 35 °C. Chemotaxonomic analysis showed that Q-8 was the predominant respiratory quinone and that iso-C15 : 0, iso-C11 : 0 3-OH, iso-C17 : 1ω9c, iso-C17 : 0, iso-C11 : 0 and C16 : 0 were the major fatty acids. The G+C content of the genomic DNA was 57.8 mol%. On the basis of 16S rRNA gene sequence analysis, the isolate was affiliated to the genus Microbulbifer. Strain CN85T exhibited most phylogenetic affinity with respect to the type strain of Microbulbifer maritimus (97.0 % sequence similarity) and showed less than 97 % sequence similarity with respect to other described Microbulbifer species with known 16S rRNA gene sequences. The DNA–DNA hybridization between strain CN85T and M. maritimus JCM 12187T was 44 %. On the basis of phenotypic and genotypic data, strain CN85T represents a novel species of the genus Microbulbifer, for which the name Microbulbifer donghaiensis sp. nov. is proposed. The type strain is CN85T (=CGMCC 1.7063T =JCM 15145T).

    • The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain CN85T is EU365694.

    • Fatty acid compositions and phenotypic characteristics of strain CN85T and related strains are provided in supplementary tables available with the online version of this paper.

    The genus Microbulbifer was first proposed in 1997 to accommodate a Gram-negative, rod-shaped, strictly aerobic gammaproteobacterium capable of utilizing a variety of hydrocarbons (González et al., 1997). The type species, Microbulbifer hydrolyticus, was isolated from enrichment cultures from a marine pulp-mill effluent associated with paper manufacture. Subsequently, four additional species, Microbulbifer salipaludis (Yoon et al., 2003a), Microbulbifer maritimus (Yoon et al., 2004), Microbulbifer agarilyticus and Microbulbifer thermotolerans (Miyazaki et al., 2008), have been described. Pseudomonas elongata was reclassified within the genus as Microbulbifer elongatus (Yoon et al., 2003b). Here, we present the results of a polyphasic study of a novel Microbulbifer strain isolated from sediment from the East China Sea.

    The sediment sample was collected using a multicorer from the East China Sea (2 ° 19′ 57″ N 12 ° 34′ 29″ E). A subsample (approx. 100 mg) was suspended in 3 ml sterile seawater and vortexed for 15 min. The dispersed sediment suspension was diluted and subsequently added to modified ZoBell medium (ZoBell, 1941). The modified ZoBell medium contained the following (l−1 distilled water): NaCl, 19.45 g; MgCl2, 8.8 g; Na2SO4, 3.24 g; CaCl2, 1.8 g; KCl, 0.55 g; NaHCO3, 0.16 g; ferric citrate, 0.1 g; KBr, 0.08 g; CsCl2, 34 mg; H3BO3, 22 mg; Na2SiO3, 4.0 mg; NaF, 2.4 mg; NH4NO3, 1.6 mg; Na3PO4, 8.0 mg; peptone (Difco), 0.5 g; yeast extract (Difco), 0.1 g; pH 7.4. After 3 days incubation aerobically at 37 °C, one colony, designated CN85T, was picked. The novel strain was purified by repeated restreaking and was maintained on marine agar 2216 (Difco).

    The optimal conditions for growth were determined in ZoBell medium and trypticase soy yeast extract medium (DSMZ medium 92) with different NaCl concentrations (0, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 10, 15 and 20 %, w/v). The pH range for growth was determined in marine broth 2216 and marine salts basal medium according to González et al. (1997). The marine salts basal medium (Mikhailov et al., 2006) contained the following (l−1 distilled water): NH4Cl, 1.0 g; K2HPO4, 0.044 g; FeSO4 . 7H2O, 0.028 g; artificial seawater, 500 ml; Tris/HCl (1 M, pH 7.5), 100 ml. The artificial seawater contained the following (l−1 distilled water): NaCl, 23.4 g; MgSO4 . 7H2O, 24.6 g; KCl, 1.5 g; CaCl2, 2.9 g. The temperature range for growth was determined by using incubation temperatures ranging from 4 to 48 °C. Cell morphology and motility were examined by means of optical microscopy (BX40; Olympus) and electron microscopy (S260; Cambridge; JEM-1230, JEOL).

    Oxidase activity was determined by assessing the oxidation of 1 % p-aminodimethylaniline oxalate. Catalase activity was determined by assessing bubble production in a 3 % (v/v) H2O2 solution. Tests to determine the assimilation of single carbon sources were performed using marine salts basal medium. The corresponding filter-sterilized sugar (0.2 %), alcohol (0.2 %), organic acid (0.1 %) or amino acid (0.1 %) was added into liquid medium. Acid production was tested using modified MOF medium supplemented with 0.5 % sugars or alcohols (Leifson, 1963; Xu et al., 2008). Biochemical and nutritional tests were performed in marine agar 2216, as described by Mata et al. (2002). API 20E, API 20NE and API ZYM tests (all from bioMérieux) were also used to determine physiological and biochemical characteristics.

    Fatty acid methyl esters obtained from cells grown in marine agar 2216 for 3 days at 37 °C were analysed using GC/MS (Kuykendall et al., 1988). Isoprenoid quinones were extracted from freeze-dried cells (200 mg) with chloroform/methanol (2 : 1) and were analysed using reversed-phase HPLC. Purified DNA was hydrolysed with P1 nuclease and the nucleotides were dephosphorylated with calf intestine alkaline phosphatase (Mesbah & Whitman, 1989). The G+C content of the resulting deoxyribonucleosides was determined using reversed-phase HPLC and was calculated from the ratio of deoxyguanosine and thymidine (Mesbah & Whitman, 1989).

    The 16S rRNA gene was amplified and analysed as described previously (Xu et al., 2007). The sequence was compared with closely related sequences of reference organisms from fasta and the EzTaxon service (Chun et al., 2007). Sequence data were aligned with clustal w, version 1.8 (Thompson et al., 1994). Phylogenetic trees were constructed using the neighbour-joining (Saitou & Nei, 1987) and maximum-parsimony methods (Fitch, 1971) (with the mega3 package; Kumar et al., 2004) and the maximum-likelihood method (Felsenstein, 1981) (with TreePuzzle, version 5.2). Evolutionary distances were calculated according to the algorithm of Kimura's two-parameter model (Kimura, 1980) for the neighbour-joining method.

    The cells of strain CN85T were long rods, 0.3–0.4 μm wide and 2.5–4.0 μm long (Fig. 1⇓). An electron micrograph of negatively stained cells did not reveal flagella. Strain CN85T was oxidase- and catalase-positive and could hydrolyse aesculin, casein, gelatin, starch and Tween 20. Detailed results from the phenotypic and biochemical tests are given in the species description or shown in Table 1⇓.

    Figure image not available in archive
    Fig. 1.

    Scanning electron micrograph (a) and transmission electron micrograph of an ultrathin section (b) of exponentially grown cells of strain CN85T. Bars, 5 μm (a) and 100 nm (b).

    Table 1.

    Differential phenotypic characteristics of strain CN85T and the type strains of related Microbulbifer species

    Strains: 1, strain CN85T; 2, M. maritimus JCM 12187T; 3, M. thermotolerans JAMB A94T (Miyazaki et al., 2008); 4, M. hydrolyticus IRE-31T (González et al., 1997; Yoon et al., 2003a, 2007); 5, M. salipaludis SM-1T (Yoon et al., 2003a, 2007); 6, M. elongatus DSM 6810T (Yoon et al., 2003b, 2007); 7, M. celer ISL-39T (Yoon et al., 2007); 8, M. agarilyticus JAMB A3T (Miyazaki et al., 2008). All strains are Gram-negative and catalase-positive. Results for M. maritimus JCM 12187T were determined in this study in parallel with tests for strain CN85T unless indicated. +, Positive; −, negative; w, weakly positive; nd, no data available.

    The almost-complete 16S rRNA gene sequence (1416 nt) of strain CN85T was obtained. Strain CN85T showed the highest sequence similarity with respect to M. maritimus TF-17T (97.0 %) and showed <97 % sequence similarity with respect to other described Microbulbifer species. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain CN85T formed a coherent cluster with types strain of M. maritimus and M. thermotolerans, with a high level of bootstrap support (100 % with the neighbour-joining method) (Fig. 2⇓).

    Figure image not available in archive
    Fig. 2.

    Neighbour-joining phylogenetic tree, based on 16S rRNA gene sequences, showing the relationships between strain CN85T and related taxa. Bootstrap percentages (based on 1000 replicates) are given at branch points; only values >50 % are shown. Filled circles indicate that nodes were recovered with bootstrap percentages >50 % in both maximum-likelihood and maximum-parsimony trees. Bar, 0.02 substitutions per nucleotide position.

    Comparisons of phenotypic properties (Table 1⇑) and cellular fatty acid compositions (Supplementary Table S1, available in IJSEM Online) also indicated differences between strain CN85T and known Microbulbifer species. Growth of strain CN85T was not observed at temperatures higher than 42 °C, which is lower than the optimal growth temperature for M. thermotolerans (43–49 °C) (Miyazaki et al., 2008). The 16 rRNA gene sequence divergence values between strain CN85T and the type strain of M. thermotolerans exceeded 3 %, an accepted value for the distinction of different genomospecies (Stackebrandt & Goebel, 1994).

    DNA–DNA hybridizations were performed using the thermal denaturation and renaturation method of De Ley et al. (1970), as modified by Huß et al. (1983), using a Beckman DU 800 spectrophotometer. The hybridization temperature used was 75 °C and the experiments were carried out in triplicate. The DNA relatedness between strain CN85T and M. maritimus JCM 12187T was 44.1 %. In addition, a comparison of phenotypic properties (Table 1⇑ and Supplementary Table S2) also indicated differences between strain CN85T and M. maritimus JCM 12187T, such as salt or temperature range for growth, nitrate reduction, glucose oxidation, hydrolysis of substrates, acid production from sugars and sensitivity to antibiotics.

    On the basis of phylogenetic, genotypic, chemotaxonomic and phenotypic data, therefore, strain CN85T represents a novel species of the genus Microbulbifer, for which the name Microbulbifer donghaiensis sp. nov. is proposed.

    Description of Microbulbifer donghaiensis sp. nov.

    Microbulbifer donghaiensis (dong.hai.en′sis. N.L. masc. adj. donghaiensis pertaining to Donghai, the Chinese name for the East China Sea).

    Cells are Gram-negative and rod-shaped (0.3–0.4×2.5–4.0 μm). Colonies on marine agar 2216 are 1–2 mm in diameter, circular, convex and light yellow in colour after 24 h at 35 °C. No growth occurs in the absence of salt. Growth occurs at NaCl concentrations of 0.5–6.0 % (w/v), with optimum growth at 3.0 %. The optimal pH for growth is between 7.0 and 8.0; growth occurs at pH 6.0, but not at pH 5.5. The temperature range for growth is 20–40 °C; optimum growth occurs at 35–37 °C. Oxidase- and catalase-positive. Nitrate is reduced. Aesculin, casein, gelatin, Tween 80 and starch are hydrolysed. DNA and tyrosine are not hydrolysed. Positive for N-acetyl-β-glucosaminidase. Negative for gluconate oxidation, indole production, lysine decarboxylase, ONPG (o-nitrophenyl β-d-galactopyranoside) hydrolysis, ornithine decarboxylase and urease. H2S is not produced from thiosulfate. The following substrates are utilized for growth: acetate, l-alanine, l-arginine, cellobiose, glucose, l-histidine, isoleucine, malate, maltose, propionate, succinate, trehalose and l-valine. The following compounds are not utilized as sole carbon sources: l-arabinose, l-aspartate, citrate, l-cysteine, ethanol, formate, d-fructose, fumarate, d-galactose, gluconate, l-glutamate, glycerol, glycine, myo-inositol, lactate, lactose, malonate, mannitol, d-mannose, melibiose, l-methionine, l-ornithine, raffinose, rhamnose, ribose, l-serine, sorbitol, sorbose, sucrose and d-xylose. Acid is produced from cellobiose, glucose and maltose. Acid is not produced from l-arabinose, d-fructose, d-galactose, lactose, mannitol, d-mannose, raffinose, rhamnose, sorbitol, sorbose, sucrose, trehalose or d-xylose. Susceptible to cefotaxime (30 μg), chloramphenicol (30 μg), kanamycin (30 μg), cefoxitin (30 μg), nitrofurantoin (300 μg) and novobiocin (30 μg), but not to ampicillin (10 μg), bacitracin (0.04 IU), cefalexin (30 μg), erythromycin (15 μg), penicillin (10 μg), streptomycin (10 μg) or tetracycline (30 μg). In the API ZYM system, acid and alkaline phosphatases, N-acetyl-β-glucosaminidase, α-chymotrypsin, cystine arylamidase, esterase (C4), esterase lipase (C8), leucine arylamidase, naphthol-AS-BI-phosphohydrolase and valine arylamidase activities are present, whereas α-fucosidase, α- and β-galactosidase, α- and β-glucosidase, β-glucuronidase, lipase (C14), α-mannosidase and trypsin activities are absent. Q-8 is the major respiratory quinone. The major fatty acids (>5 %) are iso-C15 : 0, iso-C11 : 0 3-OH, iso-C17 : 1ω9c, iso-C17 : 0, iso-C11 : 0 and C16 : 0. The DNA G+C content of the type strain is 57.8 mol% (HPLC).

    The type strain, CN85T (=CGMCC 1.7063T =JCM 15145T), was isolated from a sample of marine sediment from the East China Sea.

    Acknowledgments

    We thank Dr Aharon Oren for revising our English text. We also thank Jean Euzéby for his help with the specific etymology and nomenclature. This work was supported by grants from the Major State Basic Research Development Program of China (973 program) (2004CB719604-3), Chinese Offshore Investigation and Assessment (908-ZC-I-02) and the Scientific Research Fund of the Second Institute of Oceanography, State Oceanic Administration (PR China) (JT0709, JG0724).

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