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Int J Syst Evol Microbiol 57 (2007), 1906-1908; DOI  10.1099/ijs.0.64852-0
© 2007 International Union of Microbiological Societies

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

Young Woon Lim1, Eun Young Moon2 and Jongsik Chun1,2

1 Institute of Molecular Biology and Genetics, Seoul National University, 56-1 Shillim-dong, Kwanak-gu, Seoul 151-742, Republic of Korea
2 School of Biological Sciences and Institute of Microbiology, Seoul National University, 56-1 Shillim-dong, Kwanak-gu, Seoul 151-742, Republic of Korea

Correspondence
Jongsik Chun
jchun{at}snu.ac.kr


   ABSTRACT
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The taxonomic status of Flavobacterium resinovorum Delaporte and Daste 1956Go (Approved Lists 1980) was investigated using a polyphasic approach. The 16S rRNA gene sequence of F. resinovorum NCIMB 8767T was almost identical to that of the type strain of Novosphingobium subarcticum (Nohynek et al. 1996Go) Takeuchi et al. 2001Go, with 99.85 % sequence similarity. The DNA–DNA relatedness value between the type strains of these species was 100 %. Phenotypic comparison based on API 20E, API NE and API ZYM kits demonstrated that the type strains of F. resinovorum and N. subarcticum were also indistinguishable based on their biochemical properties. On the basis of genotypic and phenotypic evidence, it is therefore proposed to reclassify Flavobacterium resinovorum as Novosphingobium resinovorum comb. nov., with the type strain NCIMB 8767T =ATCC 33545T =DSM 7478T =LMG 8367T, and that Novosphingobium subarcticum is a later heterotypic synonym of Novosphingobium resinovorum.

The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of F. resinovorum NCIMB 8767T is EF029110.

Details of API test results and fatty acid compositions for F. resinovorum NCIMB 8767T and N. subarcticum KCTC 2890T are available as supplementary material with the online version of this paper.


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The genus Flavobacterium was created in 1923 to accommodate Gram-negative, non-spore-forming, yellow-pigmented rods that produce acid weakly from carbohydrates (Holmes et al., 1984Go). This broad definition has subsequently resulted in the inclusion of many phylogenetically heterogeneous species in the genus. Despite several attempts (Bernardet et al., 1996Go; Xie & Yokota, 2006Go), the genus still contains poorly characterized species. The aim of this study is to evaluate the taxonomic properties of Flavobacterium resinovorum. The species was initially proposed by Delaporte & Daste (1956)Go and included in the Approved Lists (Skerman et al., 1980Go), but it has never been included in comparative taxonomic study.

F. resinovorum NCIMB 8767T was obtained from the NCIMB and grown and maintained on nutrient agar (NA; Difco) at 25 °C. Its 16S rRNA gene sequence was determined and analysed as described previously (Chun & Goodfellow, 1995Go; Yi & Chun, 2006Go). The combination of a BLAST search and pairwise alignment analysis based on the jPHYDIT program (Jeon et al., 2005Go) indicated that the 16S rRNA gene sequence of F. resinovorum NCIMB 8767T was almost identical to that of the type strain of Novosphingobium subarcticum, KF1T (GenBank accession no. X94102), with 99.85 % sequence similarity (2 nucleotide differences out of 1373 bp). Phylogenetic analysis clearly showed that the phylogenetic position of F. resinovorum is within the clade encompassing the genus Novosphingobium (data not shown). The next closest phylogenetic neighbour to F. resinovorum was Novosphingobium pentaromativorans, with 95.98 % 16S rRNA gene sequence similarity to the type strain. Therefore, it is evident from 16S rRNA gene sequence analysis that F. resinovorum is a member of the genus Novosphingobium and shows a very close phylogenetic relationship to N. subarcticum.

Genomic DNA of test strains was extracted and purified according to Yi & Chun (2006)Go. DNA–DNA hybridization was carried out as described as described by De Ley et al. (1970)Go with modifications as described by Huß et al. (1983)Go, using a Cary 300 Bio model UV/Vis spectrophotometer equipped with a Peltier-thermostatted 6x6 multicell changer and a temperature controller (Varian). The DNA–DNA relatedness value between F. resinovorum NCIMB 8767T and N. subarcticum KCTC 2890T (obtained from the Korean Collection of Type Cultures) was 100 %. The combination of 16S rRNA gene sequence similarity and DNA–DNA relatedness clearly indicates that the two strains belong to the same species.

To elucidate further the taxonomic relationship between F. resinovorum and N. subarcticum, the phenotypic profile of N. subarcticum KCTC 2890T was compared with that of F. resinovorum NCIMB 8767T. Both type strains were grown and characterized in duplicate using the API 20E, API 20NE and API ZYM kits (bioMérieux) according to the manufacturer's instructions. The two strains showed identical results for 60 tests. Two tests (2-naphthyl caprylate utilization in API ZYM and beta-galactosidase activity in API 20NE) produced a weakly positive result in one strain with a positive result in the other, and assimilation of caprate (API 20NE) produced variable results for N. subarcticum KCTC 2890T. Cellular fatty acid compositions of the test strains were compared using the MIDI system as described by Yi & Chun (2006)Go. F. resinovorum NCIMB 8767T and N. subarcticum KCTC 2890T showed generally similar profiles, with major amounts of C18 : 1{omega}7c (45.28 and 59.95 %, respectively). The biochemical and fatty acid profiles of the two strains are given in Supplementary Tables S1 and S2 in IJSEM Online.

On the basis of molecular, chemotaxonomic and physiological comparison, it is clear that Novosphingobium subarcticum (Nohynek et al. 1996) Takeuchi et al. 2001Go is a later heterotypic synonym of Flavobacterium resinovorum Delaporte and Daste 1956 (Approved Lists 1980). In addition, Flavobacterium resinovorum should be transferred to the genus Novosphingobium as Novosphingobium resinovorum comb. nov.

Description of Novosphingobium resinovorum (Delaporte and Daste 1956) comb. nov.
Novosphingobium resinovorum [re.si.no.vo'rum. L. n. resina resin or gum of trees; L. v. vorare to devour; N.L. neut. adj. resinovorum (sic) resin-devouring].

Basonym: Flavobacterium resinovorum Delaporte and Daste 1956Go (Approved Lists 1980).

Heterotypic synonyms: Novosphingobium subarcticum (Nohynek et al. 1996Go) Takeuchi et al. 2001Go, Sphingomonas subarctica Nohynek et al. 1996Go.

In addition to the description given by Nohynek et al. (1996)Go for Sphingomonas subarctica, the organism shows positive reactions in the following biochemical tests: utilization of citrate, production of acetoin, cytochrome oxidase, catalase (API 20E), 2-naphthyl phosphate, 2-naphthyl butyrate, 2-naphthyl caprylate, L-leucyl 2-naphthylamide, L-valyl 2-naphthylamide, 2-naphthyl phosphate, naphthol-AS-BI-phosphate, 2-naphthyl {alpha}-D-glucopyranoside, 6-bromo-2-naphthyl beta-D-galactopyranoside (API ZYM), beta-glucosidase, beta-galactosidase and assimilation of glucose, arabinose, N-acetylglucosamine, maltose, malate and citrate (API 20NE). The organism does not react in the following biochemical tests: beta-galactosidase, arginine dihydrolase, lysine decarboxylase, ornithine decarboxylase, H2S production, urease, tryptophan deaminase, indole production, gelatinase, fermentation/oxidation of glucose, mannitol, inositol, sorbitol, rhamnose, sucrose, melibiose, amygdalin and arabinose (API 20E), 2-naphthyl myristate, L-cystyl 2-naphthylamide, N-benzoyl-DL-arginine 2-naphthylamide, N-glutaryl-phenylalanine 2-naphthylamide, 6-bromo-2-naphthyl {alpha}-D-galactopyranoside, 2-naphthyl beta-D-galactopyranoside, naphthol-AS-BI-beta-D-glucuronide, 1-naphthyl N-acetyl-beta-D-glucosaminide, 6-bromo-2-naphthyl {alpha}-D-mannopyranoside, 2-naphthyl {alpha}-L-fucopyranoside (API ZYM), reduction of nitrates to nitrites/nitrogen, indole production, acidification, arginine dihydrolase, urease, protease and assimilation of mannose, mannitol, gluconate, adipate and phenylacetate (API 20NE).

The type strain is NCIMB 8767T =ATCC 33545T =DSM 7478T =LMG 8367T.


   ACKNOWLEDGEMENTS
 
We are grateful to Dr J. P. Euzéby for help with nomenclature. This work was supported by the Korean Ministry of Science and Technology under the National Research Laboratory Program. Y. W. L. was supported by Korea Research Foundation grant 2005-005-J16001.


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Bernardet, J. F., Segers, P., Vancanneyt, M., Berthe, F., Kersters, K. & Vandamme, P. (1996). Cutting a Gordian knot: emended classification and description of the genus Flavobacterium, emended description of the family Flavobacteriaceae, and proposal of Flavobacterium hydatis nom. rev. (basonym, Cytophaga aquatilis Strohl and Tait 1978). Int J Syst Bacteriol 46, 128–148.[Abstract/Free Full Text]

Chun, J. & Goodfellow, M. (1995). A phylogenetic analysis of the genus Nocardia with 16S rRNA gene sequences. Int J Syst Bacteriol 45, 240–245.[Abstract/Free Full Text]

De Ley, J., Cattoir, H. & Reynaerts, A. (1970). The quantitative measurement of DNA hybridization from renaturation rates. Eur J Biochem 12, 133–142.[Medline]

Delaporte, B. & Daste, P. (1956). Une bactérie du sol capable de décomposer la fraction fixe de certaines oléorésines Flavobacterium resinovorum n. sp. C R Acad Sci 242, 831–834 (in French).

Holmes, B., Owen, R. J. & McMeekin, T. A. (1984). Genus Flavobacterium Bergey, Harrison, Breed, Hammer and Huntoon 1923, 97AL. In Bergey's Manual of Systematic Bacteriology, vol. 1, pp. 353–361. Edited by N. R. Krieg & J. G. Holt. Baltimore: Williams & Wilkins.

Huß, V. A. R., Festl, H. & Schleifer, K. H. (1983). Studies on the spectrophotometric determination of DNA hybridization from renaturation rates. Syst Appl Microbiol 4, 184–192.

Jeon, Y. S., Chung, H., Park, S., Hur, I., Lee, J. H. & Chun, J. (2005). jPHYDIT: a JAVA-based integrated environment for molecular phylogeny of ribosomal RNA sequences. Bioinformatics 21, 3171–3173.[Abstract/Free Full Text]

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Skerman, V. B. D., McGowan, V. & Sneath, P. H. A. (editors) (1980).. Approved lists of bacterial names. Int J Syst Bacteriol 30, 225–420.[Free Full Text]

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.[Abstract]

Xie, C. H. & Yokota, A. (2006). Reclassification of [Flavobacterium] ferrugineum as Terrimonas ferruginea gen. nov., comb. nov., and description of Terrimonas lutea sp. nov., isolated from soil. Int J Syst Evol Microbiol 56, 1117–1121.[Abstract/Free Full Text]

Yi, H. & Chun, J. (2006). Thalassobius aestuarii sp. nov., isolated from tidal flat sediment. J Microbiol 44, 171–176.[Medline]




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