HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) Supplementary Figure and Table Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via CrossRef Citing Articles via Google Scholar Google Scholar Articles by Kim, B.-Y. Articles by Go, S.-J. Search for Related Content PubMed PubMed Citation Articles by Kim, B.-Y. Articles by Go, S.-J. Agricola Articles by Kim, B.-Y. Articles by Go, S.-J.

Marinobacter koreensis sp. nov., isolated from sea sand in Korea

¹ Korean Agricultural Culture Collection (KACC), Microbial Genetics Division, National Institute of Agricultural Biotechnology, Rural Development Administration, Suwon 441-707, Korea
² Applied Microbiology Division, National Institute of Agricultural Science and Technology, Rural Development Administration, Suwon 441-707, Korea
³ Department of Environmental Sciences, University of California, Riverside, CA 92521, USA
⁴ DSMZ – Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Mascheroder Weg 1b, D-38124 Braunschweig, Germany

Correspondence
Soon-Wo Kwon
swkwon{at}rda.go.kr

	ABSTRACT

TOP ABSTRACT MAIN TEXT REFERENCES

 
A marine, Gram-negative, aerobic, motile, straight-rod-shaped, moderately halophilic bacterium, designated strain DD-M3^T, was isolated from sea sand in Pohang, Korea. A phylogenetic tree based on 16S rRNA gene sequences showed that the strain fell within the evolutionary radiation encompassed by the genus Marinobacter. The levels of 16S rRNA gene sequence similarity between the novel strain and the type strains of recognized Marinobacter species ranged from 94.2 to 97.6 %, the highest values being with Marinobacter flavimaris SW-145^T (97.6 %) and Marinobacter lipolyticus SM19^T (96.8 %). The values for DNA–DNA relatedness between isolate DD-M3^T and the type strains of the most closely related species, M. flavimaris and M. lipolyticus, were 41 and 36 %, respectively. Strain DD-M3^T was characterized as having Q-9 as the predominant respiratory quinone and 16 : 0, summed feature 3 and 18 : 19c as the main fatty acids. The DNA G+C content was 54.1 mol%. On the basis of its phenotypic and genotypic characteristics, DD-M3^T represents a novel species of the genus Marinobacter, for which the name Marinobacter koreensis sp. nov. is proposed, with DD-M3^T (=KACC 11513^T=DSM 17924^T) as the type strain.

A transmission electron micrograph of strain DD-M3^T and a table showing the cellular fatty acid composition of strain DD-M3^T and Marinobacter strains are available as supplementary material in IJSEM Online.

	MAIN TEXT

TOP ABSTRACT MAIN TEXT REFERENCES

 
The genus Marinobacter was first proposed, by Gauthier et al. (1992), as comprising halophiles from marine-related environments. The genus Marinobacter is a member of the Gammaproteobacteria, and currently contains 11 species with validly published names. The type species is Marinobacter hydrocarbonoclasticus.

Sea-shore sand was collected at Homi Cape (Pohang, Korea). The sample was serially diluted using 0.85 % (w/v) saline. The diluted solution was spread on marine agar 2216 (MA; Difco) and incubated for 7 days at 28 °C. The isolate was routinely grown on MA for 2 days at 28 °C and maintained as a glycerol suspension (20 %, w/v) at –80 °C.

The cell morphology was observed using cells grown for 48 h on MA. For observation using transmission electron microscopy (model 912AB; LEO), cells were negatively stained with 0.5 % (w/v) uranyl acetate (see Supplementary Fig. S1 available in IJSEM Online). Growth was tested on MacConkey agar, nutrient agar, R2A and trypticase soy agar (all from Difco). Tests for catalase, oxidase and hydrolysis of casein, cellulose, DNA, gelatin, Tween 80 and starch were conducted according to the methods of Smibert & Krieg (1994). Hydrolysis of alginic acid (0.5 %, w/v), chitin (1 %, w/v), pectin (0.5 %, w/v) and tyrosine (0.5 %, w/v) was also tested by observing the appearance of clear zones around colonies. The urease test was performed using the method described by MacFaddin (2000). Growth under anaerobic conditions was determined in BBL anaerobic agar (Becton Dickinson). Tolerance of various NaCl concentrations was determined using CASO broth (15.0 g peptone from casein, 5.0 g peptone from soymeal in 1000 ml deionized water) supplemented with appropriate NaCl concentrations (0, 0.5, 1, 3, 5, 8, 10, 12, 15, 18, 20 and 25 %, w/v). The pH range (pH 4.0–10.0, using increments of 1.0 pH unit) for growth was determined in marine broth buffered with citrate-phosphate buffer or Tris/HCl buffer (Breznak & Costilow, 1994). API 20NE, API 50 CH and API ZYM test strips (bioMérieux) were used according to the manufacturer's instructions except for the following: as inocula for the API 20NE and API 50 CH test strips, colonies suspended in a solution of sea salts (Sigma) and 0.85 % NaCl (w/v) were used. The utilization of organic substrates was tested using Biolog GN2 microplates; colonies suspended in half-strength sea-salts solution were used as the inocula.

The 16S rRNA gene was amplified by using a PCR with primers fD1 and rP2 (Weisburg et al., 1991) on colonies; the entire PCR fragment was directly sequenced (Hiraishi, 1992). For the phylogenetic analyses, the 16S rRNA gene sequences of all the type strains of the genus Marinobacter (and Oceanospirillum linum ATCC 11336^T as an outgroup) were used. The 16S rRNA gene sequences were aligned by using the MEGALIGN program (DNASTAR). A phylogenetic tree was reconstructed using the neighbour-joining method of Saitou & Nei (1987) on MEGA, version 2.1 (Kumar et al., 2001). The stability of relationships was assessed by performing bootstrap analyses of the neighbour-joining data, based on 1000 resamplings. The phylogenetic analyses were also assessed by using maximum-parsimony analysis.

DNA–DNA hybridization was determined using a membrane filter technique with the DIG High Prime DNA labelling and detection starter kit II (Roche Molecular Biochemicals) (Kwon et al., 2003). Isoprenoid quinones were analysed by HPLC as described previously (Groth et al., 1996). For quantitative analysis of the whole-cell fatty acids, strain DD-M3^T was cultivated on MA at 28 °C for 48 h. For preparation and analysis of the fatty acid methyl esters, the standard protocol of the MIDI/Hewlett Packard Microbial Identification System was used (Sasser, 1990). The DNA G+C content was determined according to Mesbah et al. (1989), using a reversed-phase column (Supelcosil LC-18-S; Supelco).

The cells of strain DD-M3^T were straight rods, 0.3–0.5 µm in diameter and 1.5–3.0 µm in length (Supplementary Fig. S1). Optimum growth was obtained at 28 °C, pH 6.0–8.0 and 3–8 % NaCl. Strain DD-M3^T showed poor growth on trypticase soy agar, and no growth was observed on R2A, nutrient agar or MacConkey agar. In the API 50 CH test strip, no colour changes were detected for any of the substrates. Details of the physiological and biochemical properties are given in Table 1 and in the species description.

In the phylogenetic tree (Fig. 1) constructed using almost-complete 16S rRNA gene sequences (1444 nt for strain DD-M3^T) on the basis of the neighbour-joining algorithm, strain DD-M3^T clustered with members of the genus Marinobacter. The sequence similarities between isolate DD-M3^T and the type strains of the genus Marinobacter ranged from 94.2 to 97.6 %. Strain DD-M3^T showed the highest levels of 16S rRNA gene sequence similarity with Marinobacter flavimaris SW-145^T (97.6 %) and Marinobacter lipolyticus SM19^T (96.8 %). In the light of the high sequence similarities, DNA–DNA hybridization was performed between strain DD-M3^T and reference strains (M. flavimaris SW-145^T and M. lipolyticus SM19^T). The values for DNA relatedness between the isolate and the type strains of the two most closely related species, M. flavimaris and M. lipolyticus, were 41 and 36 %, respectively, which means that this isolate is genotypically distinct from the type strains of these species (Wayne et al., 1987).

View larger version (31K): [in this window] [in a new window]   Fig. 1. Neighbour-joining tree, based on 16S rRNA gene sequences of Marinobacter species, showing the relationship between strain DD-M3T and members of the genus Marinobacter. Bootstrap values (expressed as percentages of 1000 replications) greater than 50 % are shown at branch points. O. linum ATCC 11336T was used as an outgroup. Bar, 0.01 substitutions per site.

Description of Marinobacter koreensis sp. nov.
Marinobacter koreensis (ko.re.en'sis. N.L. masc. adj. koreensis pertaining to Korea, where the type strain was isolated).

Cells are Gram-negative, aerobic, motile by means of a single polar flagellum, moderately halophilic, straight-rod-shaped (0.3–0.5x1.5–3.0 µm) and do not form spores. Colonies on MA are creamy in colour, circular (1–2 mm) and convex with entire edges after 48 h at 28 °C. Temperature, salt and pH ranges for growth are 10–45 °C, 0.5–20 % NaCl and pH 5–9. Tween 80 is hydrolysed, but alginic acid, casein, chitin, CM-cellulose, DNA, gelatin, pectin, tyrosine, starch and urea are not. Tweens 40 and 80, pyruvic acid methyl ester, acetic acid, -hydroxybutyric acid, -ketoglutaric acid, -ketovaleric acid, DL-lactic acid, succinic acid, bromosuccinic acid, L-glutamic acid and L-proline are utilized, and glycogen, succinic acid monomethyl ester and L-leucine are used weakly (Biolog GN2 microplate). Positive for nitrate reduction, but negative for indole production, glucose fermentation, arginine dihydrolase, urease, aesculin hydrolysis, gelatin hydrolysis and -galactosidase. Assimilates malic acid and phenylacetic acid, but does not assimilate D-glucose, L-arabinose, D-mannose, D-mannitol, N-acetylglucosamine, D-maltose, potassium gluconate, capric acid, adipic acid or trisodium citrate (API 20NE). Enzymic activity is observed for alkaline phosphatase, esterase (C4), esterase lipase (C8), leucine arylamidase, acid phosphatase, naphthol-AS-BI-phosphohydrolase and N-acetyl--glucosaminidase. No enzymic activity is observed for lipase (C14), valine arylamidase, cystine arylamidase, trypsin, -chymotrypsin, -galactosidase, -galactosidase, -glucuronidase, -glucosidase, -glucosidase, -mannosidase or -fucosidase (API ZYM). The predominant isoprenoid quinone is Q-9. The main fatty acids are 16 : 0, summed feature 3 and 18 : 19c. The DNA G+C content is 54.1 mol% (determined by HPLC).

The type strain, DD-M3^T (=KACC 11513^T=DSM 17924^T), was isolated from sea sand at Homo Cape, Pohang, Korea.

	ACKNOWLEDGEMENTS

 
This work was supported by a grant (203068-03-2-SB010) from the Agricultural R&D Promotion Center of the Republic of Korea.

	REFERENCES

TOP ABSTRACT MAIN TEXT REFERENCES

 
Breznak, J. A. & Costilow, R. N. (1994). Physicochemical factors in growth. In Methods for General and Molecular Bacteriology, pp. 137–154. Edited by P. Gerhardt, R. G. E. Murray, W. A. Wood & N. R. Krieg. Washington, DC: American Society for Microbiology.

Gauthier, M. J., Lafay, B., Christen, R., Fernandez, L., Acquaviva, M., Bonin, P. & Bertrand, J.-C. (1992). Marinobacter hydrocarbonoclasticus gen. nov., sp. nov., a new, extremely halotolerant, hydrocarbon-degrading marine bacterium. Int J Syst Bacteriol 42, 568–576.[Abstract/Free Full Text]

Gorshkova, N. M., Ivanova, E. P., Sergeev, A. F., Zhukova, N. V., Alexeeva, Y., Wright, J. P., Nicolau, D. V., Mikhailov, V. V. & Christen, R. (2003). Marinobacter excellens sp. nov., isolated from sediments of the Sea of Japan. Int J Syst Evol Microbiol 53, 2073–2078.[Abstract/Free Full Text]

Green, D. H., Bowman, J. P., Smith, E. A., Gutierrez, T. & Bolch, C. J. S. (2006). Marinobacter algicola sp. nov., isolated from laboratory cultures of paralytic shellfish toxin-producing dinoflagellates. Int J Syst Evol Microbiol 56, 523–527.[Abstract/Free Full Text]

Groth, I., Schumann, P., Weiss, N., Martin, K. & Rainey, F. A. (1996). Agrococcus jenensis gen. nov., sp. nov., a new genus of actinomycetes with diaminobutyric acid in the cell wall. Int J Syst Bacteriol 46, 234–239.[Abstract/Free Full Text]

Hiraishi, A. (1992). Direct automated sequencing of 16S rDNA amplified by polymerase chain reaction from bacterial cultures without DNA purification. Lett Appl Microbiol 15, 210–213.[Medline]

Kumar, S., Tamura, K., Jakobsen, I.-B. & Nei, M. (2001). MEGA2: molecular evolutionary genetics analysis software. Bioinformatics 17, 1244–1245.[Abstract/Free Full Text]

Kwon, S. W., Kim, J. S., Park, I. C., Yoon, S. H., Park, D. H., Lim, C. K. & Go, S. J. (2003). Pseudomonas koreensis sp. nov., Pseudomonas umsongensis sp. nov. and Pseudomonas jinjuensis sp. nov., novel species from farm soils in Korea. Int J Syst Evol Microbiol 53, 21–27.[Abstract/Free Full Text]

MacFaddin, J. F. (2000). Biochemical Tests for Identification of Medical Bacteria, 3rd edn, pp. 424–438. Baltimore: Lippincott Williams & Wilkins.

Martín, S., Márquez, M. C., Sánchez-Porro, C., Mellado, E., Arahal, D. R. & Ventosa, A. (2003). Marinobacter lipolyticus sp. nov., a novel moderate halophile with lipolytic activity. Int J Syst Evol Microbiol 53, 1383–1387.[Abstract/Free Full Text]

Mesbah, M., Premachandran, U. & Whitman, W. B. (1989). Precise measurement of the G+C content of deoxyribonucleic acid by high-performance liquid chromatography. Int J Syst Bacteriol 39, 159–167.

Romanenko, L. A., Schumann, P., Rohde, M., Zhukova, N. V., Mikhailov, V. V. & Stackebrandt, E. (2005). Marinobacter bryozoorum sp. nov. and Marinobacter sediminum sp. nov., novel bacteria from the marine environment. Int J Syst Evol Microbiol 55, 143–148.[Abstract/Free Full Text]

Saitou, N. & Nei, M. (1987). The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 4, 406–425.[Abstract]

Sasser, M. (1990). Identification of bacteria by gas chromatography of cellular fatty acids. Technical Note 101. Newark, DE: MIDI.

Shieh, W. Y., Jean, W. D., Lin, Y.-T. & Tseng, M. (2003). Marinobacter lutaoensis sp. nov., a thermotolerant marine bacterium isolated from a coastal hot spring in Lutao, Taiwan. Can J Microbiol 49, 244–252.[CrossRef][Medline]

Shivaji, S., Gupta, P., Chaturvedi, P., Suresh, K. & Delille, D. (2005). Marinobacter maritimus sp. nov., a psychrotolerant strain isolated from sea water off the subantarctic Kerguelen islands. Int J Syst Evol Microbiol 55, 1453–1456.[Abstract/Free Full Text]

Smibert, R. M. & Krieg, N. R. (1994). Phenotypic characterization. In Methods for General and Molecular Bacteriology, pp. 607–654. Edited by P. Gerhardt, R. G. E. Murray, W. A. Wood & N. R. Krieg. Washington, DC: American Society for Microbiology.

Wayne, L. G., Brenner, D. J., Colwell, R. R. & 9 other authors (1987). International Committee on Systematic Bacteriology. Report of the ad hoc committee on reconciliation of approaches to bacterial systematics. Int J Syst Bacteriol 37, 463–464.[Free Full Text]

Weisburg, W. G., Barns, S. M., Pelletier, D. A. & Lane, D. J. (1991). 16S ribosomal DNA amplification for phylogenetic study. J Bacteriol 173, 697–703.[Abstract/Free Full Text]

Yoon, J.-H., Shin, D.-Y., Kim, I.-G., Kang, K. H. & Park, Y.-H. (2003). Marinobacter litoralis sp. nov., a moderately halophilic bacterium isolated from sea water from the East Sea in Korea. Int J Syst Evol Microbiol 53, 563–568.[Abstract/Free Full Text]

Yoon, J.-H., Yeo, S.-H., Kim, I.-G. & Oh, T.-K. (2004). Marinobacter flavimaris sp. nov. and Marinobacter daepoensis sp. nov., slightly halophilic organisms isolated from sea water of the Yellow Sea in Korea. Int J Syst Evol Microbiol 54, 1799–1803.[Abstract/Free Full Text]

This article has been cited by other articles:

				C.-Y. Wang, C.-C. Ng, W.-S. Tzeng, and Y.-T. Shyu Marinobacter szutsaonensis sp. nov., isolated from a solar saltern Int J Syst Evol Microbiol, October 1, 2009; 59(10): 2605 - 2609. [Abstract] [Full Text] [PDF]

				M. Aguilera, M. L. Jimenez-Pranteda, K. Kharroub, A. Gonzalez-Paredes, J. J. Durban, N. J. Russell, A. Ramos-Cormenzana, and M. Monteoliva-Sanchez Marinobacter lacisalsi sp. nov., a moderately halophilic bacterium isolated from the saline-wetland wildfowl reserve Fuente de Piedra in southern Spain Int J Syst Evol Microbiol, July 1, 2009; 59(7): 1691 - 1695. [Abstract] [Full Text] [PDF]

				K. M. Handley, M. Hery, and J. R. Lloyd Marinobacter santoriniensis sp. nov., an arsenate-respiring and arsenite-oxidizing bacterium isolated from hydrothermal sediment Int J Syst Evol Microbiol, April 1, 2009; 59(4): 886 - 892. [Abstract] [Full Text] [PDF]

				S. W. Roh, Z.-X. Quan, Y.-D. Nam, H.-W. Chang, K.-H. Kim, S.-K. Rhee, H.-M. Oh, C. O. Jeon, J.-H. Yoon, and J.-W. Bae Marinobacter goseongensis sp. nov., from seawater Int J Syst Evol Microbiol, December 1, 2008; 58(12): 2866 - 2870. [Abstract] [Full Text] [PDF]

				Y.-Y. Huo, C.-S. Wang, J.-Y. Yang, M. Wu, and X.-W. Xu Marinobacter mobilis sp. nov. and Marinobacter zhejiangensis sp. nov., halophilic bacteria isolated from the East China Sea Int J Syst Evol Microbiol, December 1, 2008; 58(12): 2885 - 2889. [Abstract] [Full Text] [PDF]

				M. J. Montes, N. Bozal, and E. Mercade Marinobacter guineae sp. nov., a novel moderately halophilic bacterium from an Antarctic environment Int J Syst Evol Microbiol, June 1, 2008; 58(6): 1346 - 1349. [Abstract] [Full Text] [PDF]

				D.-C. Zhang, H.-R. Li, Y.-H. Xin, Z.-M. Chi, P.-J. Zhou, and Y. Yu Marinobacter psychrophilus sp. nov., a psychrophilic bacterium isolated from the Arctic Int J Syst Evol Microbiol, June 1, 2008; 58(6): 1463 - 1466. [Abstract] [Full Text] [PDF]

				X.-W. Xu, Y.-H. Wu, C.-S. Wang, J.-Y. Yang, A. Oren, and M. Wu Marinobacter pelagius sp. nov., a moderately halophilic bacterium Int J Syst Evol Microbiol, March 1, 2008; 58(3): 637 - 640. [Abstract] [Full Text] [PDF]

				B. Guo, J. Gu, Y.-G. Ye, Y.-Q. Tang, K. Kida, and X.-L. Wu Marinobacter segnicrescens sp. nov., a moderate halophile isolated from benthic sediment of the South China Sea Int J Syst Evol Microbiol, September 1, 2007; 57(9): 1970 - 1974. [Abstract] [Full Text] [PDF]

				J.-H. Yoon, M.-H. Lee, S.-J. Kang, and T.-K. Oh Marinobacter salicampi sp. nov., isolated from a marine solar saltern in Korea Int J Syst Evol Microbiol, September 1, 2007; 57(9): 2102 - 2105. [Abstract] [Full Text] [PDF]

				A. Antunes, L. Franca, F. A. Rainey, R. Huber, M. F. Nobre, K. J. Edwards, and M. S. da Costa Marinobacter salsuginis sp. nov., isolated from the brine-seawater interface of the Shaban Deep, Red Sea Int J Syst Evol Microbiol, May 1, 2007; 57(5): 1035 - 1040. [Abstract] [Full Text] [PDF]

				J. Gu, H. Cai, S.-L. Yu, R. Qu, B. Yin, Y.-F. Guo, J.-Y. Zhao, and X.-L. Wu Marinobacter gudaonensis sp. nov., isolated from an oil-polluted saline soil in a Chinese oilfield Int J Syst Evol Microbiol, February 1, 2007; 57(2): 250 - 254. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Supplementary Figure and Table Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via CrossRef Citing Articles via Google Scholar Google Scholar Articles by Kim, B.-Y. Articles by Go, S.-J. Search for Related Content PubMed PubMed Citation Articles by Kim, B.-Y. Articles by Go, S.-J. Agricola Articles by Kim, B.-Y. Articles by Go, S.-J.