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Department of Microbiology and Parasitology, Faculty of Pharmacy, University of Sevilla, 41012 Sevilla, Spain
Correspondence
M. C. Márquez
cmarquez{at}us.es
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ABSTRACT |
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, to place a hydrocarbon-degrading marine bacterium belonging to the 
-subclass of the Proteobacteria. Marinobacter aquaeolei Nguyen et al. 1999, the second described species of the genus, was proposed for a strain isolated from an oil-producing well on an offshore platform in southern Vietnam, on the basis of differences in the whole-cell protein pattern and lipopolysaccharide composition, although its phenotypic and genotypic characteristics were very similar to those of the type species, M. hydrocarbonoclasticus. In the present paper, literature data concerning the two species were reviewed. Fatty acid composition, G+C content and DNA–DNA hybridization studies were performed in order to clarify the taxonomic positions of these two species. Based on the results obtained in this study and phenotypic and phylogenetic traits available from the literature, it is proposed that the two species should be united under the same name; according to the rules of priority, M. hydrocarbonoclasticus, the first described species, is the earlier synonym and M. aquaeolei is the later synonym.
A maximum-parsimony tree based on 16S rRNA gene sequences showing the relationship of M. hydrocarbonoclasticus and M. aquaeolei strains is available as supplementary material in IJSEM Online.
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MAIN TEXT |
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to accommodate Gram-negative, aerobic, motile, rod-shaped bacteria able to utilize a variety of hydrocarbons as the sole source of carbon and energy. Currently this genus includes eight species: Marinobacter hydrocarbonoclasticus Gauthier et al. 1992 (type species), M. aquaeolei Nguyen et al. 1999, M. litoralis Yoon et al. 2003, M. lipolyticus Martín et al. 2003, M. excellens Gorshkova et al. 2003, M. lutaoensis Shieh et al. 2003, M. daepoensis Yoon et al. 2004 and M. flavimaris Yoon et al. 2004. The description of M. hydrocarbonoclasticus was originally based on a single strain, DSM 8798T (Gauthier et al., 1992); however, a later study showed that DSM 50418, which was the type strain of the species Pseudomonas nautica Baumann et al. 1972, was also a member of this species and, thus, it was transferred to the genus Marinobacter (Spröer et al., 1998). The second described species of this genus, M. aquaeolei, was proposed by Nguyen et al. (1999) for a moderately halophilic organism, strain VT8T, isolated from an oil-producing well on an offshore oil/gas platform near the coastal town of Vung Tau in southern Vietnam. 16S rRNA gene sequence analysis indicated a high level of similarity to the sequences of M. hydrocarbonoclasticus DSM 8798T (99·4 %) and M. hydrocarbonoclasticus DSM 50418 (99·8 %). Its phenotypic characteristics, antibiotic resistance, isoprenoid quinones and fatty acids were also similar to those of the species M. hydrocarbonoclasticus. DNA–DNA hybridization studies showed the following levels of hybridization: 65 % between strain VT8T and M. hydrocarbonoclasticus DSM 8798T and 75·4 % between strain VT8T and M. hydrocarbonoclasticus DSM 50418 (Nguyen et al., 1999). Despite these results, the creation of the species M. aquaeolei for strain VT8T was proposed on the basis of its whole-cell protein pattern and lipopolysaccharide composition, different from those of M. hydrocarbonoclasticus. In current bacterial systematics, a bacterial species is defined as a group of strains that exhibit approximately 70 % or greater DNA–DNA hybridization at an optimal incubation temperature, with 5 °C or less 
Tm, together with phylogenetic inference based on 16S rRNA gene sequence comparison. Furthermore, their phenotypic characteristics should agree with this definition (Wayne et al., 1987; Stackebrandt & Goebel, 1994). In the present note, we report on a polyphasic taxonomic study that shows that M. aquaeolei and M. hydrocarbonoclasticus belong to the same species.
M. hydrocarbonoclasticus DSM 8798T, M. hydrocarbonoclasticus DSM 50418 and M. aquaeolei DSM 11845T were obtained from the DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen, Braunschweig, Germany). These strains were cultured on a saline medium (SW-5) with a final concentration of 5 % (w/v) total salts, supplemented with 0·5 % (w/v) yeast extract (Ventosa et al., 1982). All cultures were cultivated at 37 °C in an orbital shaker (New Brunswick Scientific) at 200 r.p.m. When necessary, solid media were prepared by adding 20 g Bacto-agar l–1 (Difco).
Phenotypically, the three strains have very similar features: they are Gram-negative, non-spore-forming rods, non-motile, aerobic and oxidase- and catalase-positive. The optimum temperature, pH and NaCl concentration for growth are respectively about 30 °C (32, 25 and 30 °C for strains DSM 8798T, DSM 50418 and DSM 11845T), near neutral (pH 7 to 7·5, 7·6 and 7·3) and about 0·7 M NaCl (0·6–0·85 M). They reduce nitrate to nitrite and do not hydrolyse aesculin. Indole and arginine dihydrolase tests are negative (Gauthier et al., 1992; Spröer et al., 1998; Nguyen et al., 1999). The sole differentiating biochemical characteristic between the two species is urease activity, this test being negative for the two strains of M. hydrocarbonoclasticus and positive in the case of M. aquaeolei.
The cellular fatty acids of the type strains of M. aquaeolei and M. hydrocarbonoclasticus were analysed with the MIDI system. The two strains showed similar fatty acid compositions. The predominant fatty acids of these species were 16 : 0, 18 : 1
9c, 12 : 0 3-OH and 16 : 19c (Table 1). These results are in accordance to those reported previously by Spröer et al. (1998) and Nguyen et al. (1999).
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) and 57·3 mol% (Spröer et al., 1998), these values respectively being determined by the thermal denaturation and HPLC methods. On the other hand, the values reported by Spröer et al. (1998) and Nguyen et al. (1999) for M. hydrocarbonoclasticus DSM 50418 and M. aquaeolei (both determined by HPLC) were respectively 57·7 and 55·7 mol%. In this study, we have determined the G+C contents of DNA from these three strains by the same method (Tm) as described previously (Márquez et al., 1990). The values obtained for M. hydrocarbonoclasticus DSM 8798T, M. hydrocarbonoclasticus DSM 50418 and M. aquaeolei DSM 11845T were respectively 57·3, 57·7 and 55·5 mol%. These values are similar to those described previously by Spröer et al. (1998) and Nguyen et al. (1999), but different from that described by Gauthier et al. (1992) for M. hydrocarbonoclasticus DSM 8798T (52·7 mol%). Thus, on the basis of the similar result obtained by us and by Spröer et al. (1998) for M. hydrocarbonoclasticus DSM 8798T, we consider that the result obtained by Gauthier et al. (1992) is erroneous. A maximum-parsimony phylogenetic tree was generated in order to show the relationship between the three strains. Similar tree topologies were also found in trees generated with maximum-likelihood and neighbour-joining algorithms. The type strain of M. aquaeolei exhibited the highest level of 16 rRNA gene sequence similarity to M. hydrocarbonoclasticus DSM 50418 (99·8 %), and the similarity to the type strain of M. hydrocarbonoclasticus was 99·7 %. The sequence similarity between M. hydrocarbonoclasticus DSM 8798T and M. hydrocarbonoclasticus DSM 50418 was 99·5 %. The levels of 16S rRNA gene sequence similarity between these three strains and other strains used in the phylogenetic analysis were lower than 97·7 % (results available as a supplementary figure in IJSEM Online).
To verify the species status of the two Marinobacter species, DNA–DNA hybridization studies were performed between the two strains included in the species M. hydrocarbonoclasticus and the type strain of M. aquaeolei. DNA was extracted and purified by the method of Marmur (1961). DNA–DNA hybridization was studied by the competition procedure of Johnson (1994), described in detail elsewhere (Arahal et al., 2001). The hybridization experiments were carried out under optimal conditions, at a temperature of 54·6 °C, which is within the limits of validity for the filter method (De Ley & Tijtgat, 1970). The percentage of hybridization was calculated as described by Johnson (1994). Our results reveal a high level of DNA–DNA hybridization among the three strains studied, ranging from 78 to 100 % (Table 2). Since the three strains are phylogenetically very closely related, they have very similar phenotypic features and show a high degree of DNA–DNA hybridization, all these data clearly indicate that these strains belong to the same species (Wayne et al., 1987; Stackebrandt & Goebel, 1994). As mentioned before, the proposal of Nguyen et al. (1999) of the species M. aquaeolei was based on differences found in the whole-cell protein pattern and lipopolysaccharide composition. They reported that the protein patterns of M. aquaeolei and M. hydrocarbonoclasticus DSM 8798T were markedly different. However, they also described very different protein patterns as well as differences in the lipopolysaccharide content between M. hydrocarbonoclasticus DSM 8798T and M. hydrocarbonoclasticus DSM 50418 (referred to as P. nautica in their article), two strains that are currently widely accepted as members of the same species (Spröer et al., 1998).
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), the name M. hydrocarbonoclasticus is the earlier synonym and the name M. aquaeolei is the later synonym.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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TOPABSTRACTMAIN TEXTREFERENCES |
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Baumann, L., Baumann, P., Mandel, M. & Allen, R. D. (1972). Taxonomy of aerobic marine eubacteria. J Bacteriol 110, 402–429.
De Ley, J. & Tijtgat, R. (1970). Evaluation of membrane filter methods for DNA-DNA hybridization. Antonie van Leeuwenhoek 36, 461–474.[CrossRef][Medline]
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.
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.
Johnson, J. L. (1994). Similarity analysis of DNAs. In Methods for General and Molecular Bacteriology, pp. 655–681. Edited by P. Gerhardt, R. G. E. Murray, W. A. Wood & N. R. Krieg. Washington, DC: American Society for Microbiology.
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Marmur, J. (1961). A procedure for the isolation of deoxyribonucleic acid from micro-organisms. J Mol Biol 3, 208–218.
Márquez, M. C., Ventosa, A. & Ruíz-Berraquero, F. (1990). Marinococcus hispanicus, a new species of moderately halophilic Gram-positive cocci. Int J Syst Bacteriol 40, 165–169.
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.
Nguyen, B. H., Denner, E. B. M., Dang, T. C. H., Wanner, G. & Stan-Lotter, H. (1999). Marinobacter aquaeolei sp. nov., a halophilic bacterium isolated from a Vietnamese oil-producing well. Int J Syst Bacteriol 49, 367–375.
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]
Spröer, C., Lang, E., Hobeck, P., Burghardt, J., Stackebrandt, E. & Tindall, B. J. (1998). Transfer of Pseudomonas nautica to Marinobacter hydrocarbonoclasticus. Int J Syst Bacteriol 48, 1445–1448.
Stackebrandt, E. & Goebel, B. M. (1994). Taxonomic note: a place for DNA-DNA reassociation and 16S rRNA sequence analysis in the present species definition in bacteriology. Int J Syst Bacteriol 44, 846–849.
Ventosa, A., Quesada, E., Rodríguez-Valera, F., Ruiz-Berraquero, F. & Ramos-Cormenzana, A. (1982). Numerical taxonomy of moderately halophilic Gram-negative rods. J Gen Microbiol 128, 1959–1968.
Wayne, L. G., Brenner, D. J., Colwell, R. R. & 9 other authors (1987). Report of the ad hoc committee on reconciliation of approaches to bacterial systematics. Int J Syst Bacteriol 37, 463–464.
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.
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.
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