SGM Journals
Proteobacteria

Stakelama pacifica gen. nov., sp. nov., a new member of the family Sphingomonadaceae isolated from the Pacific Ocean

Chuang Chen, Qiang Zheng, Ya-Nan Wang, Xiao-Jun Yan, Li-Kai Hao, Xun Du, Nianzhi Jiao

  • 1State Key Laboratory for Marine Environmental Science, Xiamen University, Xiamen 361005, PR China
  • 2Key Laboratory of Microbial Engineering at the Institute of Biology, Henan Academy of Sciences, Zhengzhou 450008, PR China
  • 3Key Laboratory of Applied Marine Biotechnology, Ministry of Education, Ningbo University, Ningbo 315211, PR China
  • Correspondence
    Nianzhi Jiao
    Jiao{at}xmu.edu.cn

    • International Journal of Systematic and Evolutionary Microbiology 2010; 60(12):2857–2861 · https://doi.org/10.1099/ijs.0.018945-0

    View at publisher PubMed

    Abstract

    A Gram reaction-negative, weakly motile, non-spore-forming, rod-shaped, aerobic bacterium designated strain JLT832T was isolated from surface water of the central Pacific Ocean and formed yellow colonies on rich organic (RO) medium. The strain was oxidase-negative and catalase-positive. Acid was produced from mannitol, glucose, sucrose, lactose, sorbitol, maltose, (+)-trehalose and d-fructose. No acid was produced from d-(+)-xylose. The major cellular fatty acids of strain JLT832T were C18 : 1ω7c, C14 : 0 2-OH and C16 : 0. The major polar lipids were sphingoglycolipid, diphosphatidylglycerol, phosphatidylglycerol and phosphatidylethanolamine. Ubiquinone-10 and spermidine were present as the major quinone and polyamine, respectively. The genomic DNA G+C content of strain JLT832T was 66.0±0.5 mol%. Phylogenetic analysis based on 16S rRNA gene sequences indicated that the new isolate formed a tight branch within the family Sphingomonadaceae but was clearly separate from established genera in this family. The sequence similarities between the new isolate and type strains of established genera ranged from 90.5 to 94.9 %. Based on these data, strain JLT832T constitutes a novel genus and species, for which the name Stakelama pacifica gen. nov., sp. nov. is proposed. The type strain of Stakelama pacifica is JLT832T (=CGMCC 1.7294T =LMG 24686T).

    • The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain JLT832T is EU581829.

    • A transmission electron micrograph of negatively stained cells and polar lipid profiles of strain JLT832T are available as supplementary material with the online version of this paper.

    The family Sphingomonadaceae was established by Kosako et al. (2000) and was further divided into families Sphingomonadaceae and Erythrobacteraceae by Lee et al. (2005). The genus Sphingomonas was first described by Yabuuchi et al. (1990) and was later divided into four genera, Sphingomonas, Sphingobium, Novosphingobium and Sphingopyxis (Takeuchi et al., 2001), based on both phylogenetic analysis of 16S rRNA gene sequences and chemotaxonomic and phenotypic differences. Many novel genera of this clade have been described, such as Sphingomonas, Sphingobium, Novosphingobium, Sphingopyxis, Zymomonas, Sandaracinobacter, Sphingosinicella, Sandarakinorhabdus and Blastomonas.

    Strain JLT832T was isolated from the surface of the Pacific Ocean at 17 ° 21.70′ E 2 ° 34.44′ N and formed a deep branch within the family Sphingomonadaceae. In this study, we characterized strain JLT832T by using a polyphasic approach.

    Strain JLT832T was initially isolated with seawater medium (SW) containing (per litre seawater filtered by 0.2 μm film) 1 g peptone, 1 g yeast extract, 20 μg vitamin B12 and 1 ml of a trace element solution (ES) as described by Drews (1983). The strain was identified by growing on modified rich organic (RO) medium (Yurkov et al., 1999) containing (per litre distilled water) 1 g peptone, 1 g yeast extract, 20 g NaCl, 1 g sodium acetate, 0.3 g KCl, 0.5 g MgSO4.7H2O, 0.05 g CaCl2.7H2O, 0.3 g NH4Cl, 0.3 g K2HPO4, 20 μg vitamin B12 and 1.0 ml ES, at pH 8.0 and 28 °C.

    Cell morphology was examined using light microscopy (BX61; Olympus) and TEM (H600; Hitachi). The Gram reaction was tested according to standard procedures (Gerhardt et al., 1994). Catalase activity was determined by adding drops of 3 % H2O2 to an overnight colony and assessed by the formation of bubbles. The pH and temperature ranges for growth were determined by adjusting the RO medium to a final pH of 5.5–9.5 (at intervals of 0.5 pH units) with HCl and NaOH, and by incubation at 0, 5, 10, 15, 25, 28, 30, 37 and 40 °C. Growth in NaCl 1–6 % (w/v) (increments of 0.5 %) was investigated in RO medium. Oxidase and urease activity, hydrolysis of casein, starch, gelatin and Tween 80 and reduction of nitrate and nitrite were determined as described by Dong & Cai (2001) on RO medium. Denitrification was tested by growing the strain anaerobically in the presence of nitrate (Zumft, 1992). H2S production was tested as described by Bruns et al. (2001). Indole production, methyl red (MR) test and Voges–Proskauer (VP) reaction were determined as described by Denner et al. (2001).

    The ability of strain JLT832T to utilize substrates as sole carbon and energy sources was examined on RO medium without organic compounds at 28 °C for 7–14 days (Williams et al., 1983). Acid production from carbohydrates was assessed using the procedures outlined by Cappuccino & Sherman (2002). Antibiotic susceptibility was tested by using the disc-diffusion plate (Kirby–Bauer) method according to Fraser & Jorgensen (1997) and Andrews (2008).

    Cellular fatty acid composition was determined as described previously (Hu et al., 2004) by growing cells of strain JLT832T on marine agar 2216e (MA; Difco) for 2 days at 28 °C. Polyamines were extracted and derivatized as described by Busse & Auling (1988) and analysed by HPLC using the equipment previously described by Stolz et al. (2007). Polar lipids were identified by two-dimensional TLC and detection with sulfuric acid/ethanol (1 : 2, v/v) followed by heating at 150 °C for 3 min, according to the procedures described by Minnikin et al. (1984). Isoprenoid quinones were analysed as described by Hiraishi et al. (1998) using ultra-performance liquid chromatography coupled with quadruple time-of-flight mass spectrometry (UPLC-Q-TOF-MS) in an electrospray ionization method (Romano et al., 2006).

    Genomic DNA was extracted following the method of Marmur (1961) from cells that were grown in RO medium for 2 days at 28 °C, washed and then resuspended in the buffer. Purity was assessed by measuring absorbance at A280/A260 and A230/A260 ratios (Johnson, 1994). The 16S rRNA gene was amplified and sequenced as described by Rainey et al. (1996) and sequences were aligned using the blast tool () (Altschul et al., 1990) to determine the approximate phylogenetic affiliation. Phylogenetic trees were constructed using neighbour-joining and maximum-parsimony algorithms in mega 4.0 software (Kumar et al., 2004). DNA G+C contents were determined by thermal denaturation (Marmur & Doty, 1962) using DNA of Escherichia coli DH5α as a standard for calibration of the Tm.

    Strain JLT832T cells were strictly aerobic, heterotrophic, Gram reaction-negative, short rods. Flagella were observed (Supplementary Fig. S1, available in IJSEM Online). Colonies were gold–yellow, glossy, opaque, circular and about 1 mm in diameter. JLT832T grew in the presence of 0–5 % (w/v) NaCl (optimum 2 %), at 5–37 °C (optimum 28 °C) and pH 6–9 (optimum pH 8). Strain JLT832T was positive for catalase, aerobic nitrate reduction, H2S production and VP reaction and negative for oxidase, urease, aerobic nitrite reduction, anaerobic nitrate reduction, indole production, MR test and hydrolysis of gelatin, starch, Tween 80 and casein. Acid was produced from mannitol, glucose, sucrose, lactose, sorbitol, maltose, (+)-trehalose and d-fructose. No acid was produced from (+)-d-xylose.

    JLT832T exhibited a polyamine pattern with the following compounds (per g dry weight): spermidine (17.6 μmol), putrescine (0.2 μmol), cadaverine (0.6 μmol) and traces (<0.1 μmol) of 1,3-diaminopropane and spermine. This polyamine pattern was in agreement with the traits reported for the genera Blastomonas, Sphingobium, Novosphingobium and Sphingopyxis, while the presence of homospermidine was restricted in species of Zymomonas, Sphingosinicella and Sphingomonas (Busse et al., 1999; Takeuchi et al., 2001; Hamana et al., 2003). The major polar lipids were sphingoglycolipid, diphosphatidylglycerol, phosphatidylglycerol and phosphatidylethanolamine (Supplementary Fig. S2). Q-10 was the sole respiratory quinone. Cellular fatty acids were C18 : 1ω7c (72.88 %), C14 : 0 2-OH (11.93 %), C16 : 0 (11.53 %), C18 : 1ω5c (2.02 %) and C16 : 1ω7c (1.64 %) (Supplementary Table S1).

    The DNA G+C content of JLT832T was 66±0.5 mol%. The complete 16S rRNA gene sequence (1412 bp) of strain JLT832T was determined. Phylogenetic trees based on 16S rRNA gene sequences (1374 bp compared) showed that strain JLT832T forms a distinct phylogentic lineage within the family Sphingomonadaceae (Fig. 1⇓) and exhibits the highest sequence similarity to Novosphingobium subarcticum SMCC B0478T (94.9 %), followed by Novosphingobium mathurense SM 117T (94.3 %) and Sphingosinicella microcystinivorans JCM 13185T (94.3 %).

    Figure image not available in archive
    Fig. 1.

    Neighbour-joining phylogenetic tree based on 16S rRNA gene sequences showing the relationship between strain JLT832T and representatives of the family Sphingomonadaceae. Bootstrap percentages (above 50 %) from both neighbour-joining (above nodes) and maximum-parsimony (below nodes) are shown. Bar, 0.005 substitutions per nucleotide position.

    No currently recognized species in the genera Novosphingobium, Sphingosinicella and Zymomonas utilize sorbitol except Novosphingobium panipatense and N. subarcticum. N. subarcticum only utilized mannitol, while strain JLT832T could utilize sorbitol, mannitol, myo-inositol and glycerol.

    A comparison of differential characteristics of strain JLT832T and members of related genera Novosphingobium, Sphingosinicella and Zymomonas are listed in Table 1⇓. The morphological, physiological and biochemical characteristics of strain JLT832T indicate that the new isolate represents a novel genus and species of the family Sphingomonadaceae, for which the name Stakelama pacifica gen. nov., sp. nov. is proposed.

    Table 1.

    Differentiation of physiological and biochemical characteristics between strain JLT832T and closely related species within the family Sphingomonadaceae

    Taxa: 1, JLT832T; 2, Sphingosinicella microcystinivorans JCM 13185T; 3, Sphingosinicella xenopeptidilytica DSM 17130T; 4, Sphingosinicella soli DSM 17328T; 5, Novosphingobium panipatense CCM 7472T; 6, Novosphingobium mathurense CCM 7473T; 7, Novosphingobium subarcticum SMCC B0478T; 8, Zymomonas species. Data in columns were taken from the following sources: 1–4, Maruyama et al. (2006), Geueke et al. (2007) and Yoon et al. (2008); 5–6, Gupta et al. (2009); 7, Nohynek et al. (1996) and Lim et al. (2007); 8, De Ley & Swings (1976) and Swings & De Ley (1977). All strains were negative for Gram stain, gelatin hydrolysis and indole production. Q-10 was the respiratory quinone for all the strains. w, Weak reaction; nd, no data.

    Description of Stakelama gen. nov.

    Stakelama (Sta.ke.la′ma. N.L. fem. n. Stakelama arbitrary name derived from State Key Laboratory of Marine Environment Science).

    Cells are Gram reaction-negative, weakly motile, non-spore-forming rods. Colonies are gold–yellow. Strictly aerobic. Positive for catalase and negative for oxidase. The major fatty acids are C18 : 1ω7c, C14 : 0 2-OH and C16 : 0. The sole respiratory quinone is Q-10. The DNA G+C content of the type strain of the type species is 66±0.5 mol%. The type species is Stakelama pacifica.

    Description of Stakelama pacifica sp. nov.

    Stakelama pacifica (pa.ci′fi.ca. L. fem. adj. pacifica peaceful, referring to the Pacific Ocean, the origin of the type strain).

    The following properties are shown in addition to those given in the genus description. Cells are 0.5–0.8×1.5–2.0 μm, and have a flagellum. Colonies on RO medium are circular, slightly raised, smooth and 1 mm in diameter after incubation for 24 h at 28 °C. Growth occurs at 5–37 °C (optimum 28 °C), pH 6–9 (optimum pH 8) and in the presence of 0–5  % (w/v) NaCl (optimum 2 %). Positive for catalase, aerobic nitrate reduction, H2S production and the VP reaction. Negative for oxidase, urease, aerobic nitrite reduction, anaerobic nitrate reduction, indole production, the MR test and hydrolysis of gelatin, starch, Tween 80 and casein. (+)-d-Xylose, maltose, sorbitol, lactose, (+)-trehalose, cellobiose, lactic acid, sodium d-gluconate, inositol, glycerol, mannitol, acetic acid, ethanol and malonic acid are utilized. Sucrose, glucose, (+)-d-galactose, (−)-l-malic acid, l-proline and l-threonine are used weakly. (−)-l-Sorbose, d-fructose, dextrin, trisodium citrate, oxalic acid, l-alanine, l-lysine, glycine and l-arginine are not utilized. The type strain was sensitive (μg per disc) to ampicillin (10), penicillin (10), tetracycline (30), chloramphenicol (30), erythromycin (15), cefoperazone (75), cefotaxime (30), ciprofloxacin (5), clarithromycin (15), gentamicin (120) and streptomycin (300); resistant to clindamycin (2), sulfamethoxazole (300), vancomycin (30) and cefalothin (30).

    The type strain, JLT832T (=CGMCC 1.7294T =LMG 24686T), was isolated from surface water at 17 ° 21.70′ E 2 ° 34.44′ N in the Pacific Ocean.

    Acknowledgments

    The authors would like to thank Dr Hans-Jürgen Busse, Institut für Bakteriologie, Mykologie und Hygiene, University of Veterinary Medicine Vienna, for the analysis of the polyamine pattern. This work was supported by the MOST 973 project (2007CB815904), the NSFC projects (40632013 and 40821063), the SOA project (200805068) and the COMRA project (DYXM-115-02-4-3).

    References

    1. Altschul, S. F., Gish, W., Miller, W., Myers, E. W. & Lipman, D. J. (1990). Basic local alignment search tool. J Mol Biol 215, 403–410.
    2. Andrews, J. M. (2008). BSAC standardized disc susceptibility testing method (Version 7). J Antimicrob Chemother 62, 256–278.
    3. Bruns, A., Rohde, M. & Berthe-Corti, L. (2001). Muricauda ruestringensis gen. nov., sp nov., a facultatively anaerobic, appendaged bacterium from German North Sea intertidal sediment. Int J Syst Evol Microbiol 51, 1997–2006.
    4. Busse, H.-J. & Auling, G. (1988). Polyamine pattern as a chemotaxonomic marker within the Proteobacteria. Syst Appl Microbiol 11, 1–8.
    5. Busse, H. J., Kämpfer, P. & Denner, E. B. (1999). Chemotaxonomic characterisation of Sphingomonas. J Ind Microbiol Biotechnol 23, 242–251.
    6. Cappuccino, J. G. & Sherman, N. (2002). Microbiology: a Laboratory Manual, 6th edn. San Francisco. : Pearson Education, Inc. and Benjamin Cummings.
    7. De Ley, J. & Swings, J. (1976). Phenotypic description, numerical analysis and a proposal of an improved taxonomy and nomenclature of the genus Zymomonas Kluyer and van Niel 1936. Int J Syst Bacteriol 26, 146–157.
    8. Denner, E. B., Paukner, S., Kämpfer, P., Moore, E. R., Abraham, W.-R., Busse, H.-J., Wanner, G. & Lubitz, W. (2001). Description of Sphingomonas pituitosa sp. nov., a novel polysaccharide-producing sphingomonad. Int J Syst Evol Microbiol 51, 827–841.
    9. Dong, X.-Z. & Cai, M.-Y. (2001). Determinative Manual for Routine Bacteriology. Beijing. : Scientific Press (in Chinese).
    10. Drews, G. (1983). Mikrobiologisches Praktikum. Berlin, Germany: Springer (in German).
    11. Fraser, S. L. & Jorgensen, J. H. (1997). Reappraisal of the antimicrobial susceptibilities of Chryseobacterium and Flavobacterium species and methods for reliable susceptibility testing. Antimicrob Agents Chemother 41, 2738–2741.
    12. Gerhardt, P., Murray, R. G. E., Wood, W. A. & Krieg, N. R. (editors) (1994). Methods for General and Molecular Bacteriology. Washington, DC. : American Society for Microbiology.
    13. Geueke, B., Busse, H.-J., Fleishmann, T., Kämpfer, P. & Kohler, H.-P. E. (2007). Description of Sphingosinicella xenopeptidilytica sp. nov., a β-peptide-degrading species, and emended descriptions of the genus Sphingosinicella and Sphingosinicella microcystinivorans. Int J Syst Evol Microbiol 57, 107–113.
    14. Gupta, S. K., Lal, D. & Lal, R. (2009). Novosphingobium panipatense sp. nov. and Novosphingobium matheurense sp. nov., from oil-contaminated soil. Int J Syst Evol Microbiol 59, 156–161.
    15. Hamana, K., Sakamoto, A., Tachiyanagi, S., Terauchi, E. & Takeuchi, M. (2003). Polyamine profiles of some members of the alpha subclass of the Proteobacteria: polyamine analysis of 20 recently described genera. Microbiol Cult Coll 19, 13–21.
    16. Hiraishi, A., Ueda, Y. & Ishihara, J. (1998). Quinone profiling of bacterial communities in natural and synthetic sewage activated sludge for enhanced phosphate removal. Appl Environ Microbiol 64, 992–998.
    17. Hu, Y.-T., Zhou, P.-J., Zhou, Y.-G., Liu, Z.-H. & Liu, S.-J. (2004). Xaccharothrix xingjiangensis sp. nov., a pyrene-degrading actinomycete isolated from Tianchi Lake, Xinjiang, China. Int J Syst Evol Microbiol 54, 2091–2094.
    18. Johnson, J. L. (1994). Similarity analysis of DNAs. In Methods for General and Molecular Bacteriology, pp. 655–681. Edited by Gerhardt, P. E., Murray, R. G., Wood, W. A. & Krieg, N. R.. Washington, DC. : American Society for Microbiology.
    19. Kosako, Y., Yabuuchi, E., Naka, T., Fujiwara, N. & Kobayashi, K. (2000). Proposal of Sphingomonadaceae fam. nov., consisting of Sphingomonas Yabuuchi et al. 1990, Erythrobacter Shiba and Shimidu 1982, Erythromicrobium Yurkove et al. 1994, Porphyrobacter Fuerst et al. 1993, Zymomonas Kluyver and van Niel 1936, and Sandaracinobacter Yurkove et al. 1997, with the type genus Sphingomonas Yabuuchi et al. 1990. Microbiol Immunol 44, 563–575.
    20. Kumar, S., Tamura, K. & Nei, M. (2004). mega3: Integrated software for molecular evolutionary genetics analysis and sequence alignment. Brief Bioinform 5, 150–163.
    21. Lee, K.-B., Liu, C.-T., Anzai, Y., Kim, H., Aono, T. & Oyaizu, H. (2005). The hierarchical system of the ‘Alphaproteobacteria’: description of Hyphomonadaceae fam. nov., Xanthobacteraceae fam. nov. and Erythrobacteraceae fam. nov. Int J Syst Evol Microbiol 55, 1907–1919.
    22. Lim, Y. W., Moon, E. Y. & Chun, J. (2007). Reclassification of Flavobacterium resinovorum Delaporte and Daste 1956 as Novosphingobium resinovorum comb. nov., with Novosphingobium subarcticum (Nohynek et al. 1996) Takeuchi et al. 2001 as a later heterotypic synonym. Int J Syst Evol Microbiol 57, 1906–1908.
    23. Marmur, J. (1961). A procedure for the isolation of deoxyribonucleic acid from microorganisms. J Mol Biol 3, 208–218.
    24. Marmur, J. & Doty, P. (1962). Determination of the base composition of deoxyribonucleic acid from thermal denaturation temperature. J Mol Biol 5, 109–118.
    25. Maruyama, T., Park, H.-D., Ozawa, K., Tanaka, Y., Sumino, T., Hamana, K., Hiraishi, A. & Kato, K. (2006). Sphingosinicella microcystinivorans gen. nov., sp. nov., a microcystin-degrading bacterium. Int J Syst Evol Microbiol 56, 85–89.
    26. Minnikin, D. E., O'Donnell, A. G., Goodfellow, M., Alderson, G., Athalye, M., Schaal, A. & Parlett, J. H. (1984). An integrated procedure for the extraction of bacterial isoprenoid quinones and polar lipids. J Microbiol Methods 2, 233–241.
    27. Nohynek, L. J., Nurmiaho-Lassila, E.-L., Suhonen, E. L., Busse, H.-J., Mohammadi, M., Hantula, J., Rainey, F. & Salkinoja-Salonen, M. S. (1996). Description of chlorophenol-degrading Pseudomonas sp. strains KF1T, KF3 and NKF1 as a new species of the genus Sphingomonas, Sphingomonas subarctica sp. nov. Int J Syst Bacteriol 46, 1042–1055.
    28. Rainey, F. A., Ward-Rainey, N., Kroppenstedt, R. M. & Stackebrandt, E. (1996). The genus Nocardiopsis represents a phylogenetically coherent taxon and a distinct actinomycete lineage: proposal of Nocardiopsaceae fam. nov. Int J Syst Bacteriol 46, 1088–1092.
    29. Romano, I., Lama, L., Nicolaus, B., Poli, A., Gambacorta, A. & Giordano, A. (2006). Halomonas alkaliphila sp. nov., a novel halotolerant alkaliphilic bacterium isolated from a salt pool in Campania (Italy). J Gen Appl Microbiol 52, 339–348.
    30. Stolz, A., Busse, H.-J. & Kämpfer, P. (2007). Pseudomonas knackmussii sp. nov. Int J Syst Evol Microbiol 57, 572–576.
    31. Swings, J. & De Ley, J. (1977). The biology of Zymomonas. Bacteriol Rev 41, 1–46.
    32. Takeuchi, M., Hamana, K. & Hiraishi, A. (2001). Proposal of the genus Sphingomonas sensu stricto and three new genera, Sphingobium, Novosphingobium and Sphingopyxis, on the basis of phylogenetic and chemotaxonomic analyses. Int J Syst Evol Microbiol 51, 1405–1417.
    33. Williams, S. T., Goodfellow, M., Alderson, G., Wellington, E. M. H., Sneath, P. H. A. & Sackin, M. J. (1983). Numerical classification of Streptomyces and related genera. J Gen Microbiol 129, 1743–1813.
    34. Yabuuchi, E., Yano, I., Oyaizu, H., Hashimoto, Y., Ezaki, T. & Yamamoto, H. (1990). Proposals of Sphingomonas paucimobilis gen. nov. and comb. nov., Sphingomonas parapaucimobilis sp. nov., Sphingomonas yanoikuyae sp. nov., Sphingomonas adhaesiva sp. nov., Sphingomonas capsulata comb. nov., and two genospecies of the genus Sphingomonas. Microbiol Immunol 34, 99–119.
    35. Yoon, J.-H., Kang, S.-J., Lee, J.-S., Nam, S.-W., Kim, W. & Oh, T.-K. (2008). Sphingosinicella soli sp. nov., isolated from an alkaline soil in Korea. Int J Syst Evol Microbiol 58, 173–177.
    36. Yurkov, V. V., Krieger, S., Stackebrandt, E. & Beatty, J. T. (1999). Cirtomicrobium bathyomarinum, a novel aerobic bacterium isolated from deep-sea hydrothermal vent plume waters that contains photosynthetic pigment-protein complexes. J Bacteriol 181, 4517–4525.
    37. Zumft, W. G. (1992). The denitrifying bacteria. In The Prokaryotes, 2nd edn. pp. 554–582. Edited by Balows, A., Trüper, H. G., Dworkin, M., Harder, W. & Schleifer, K. H.. New York. : Springer Verlag.