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Int J Syst Evol Microbiol 56 (2006), 1501-1504; DOI  10.1099/ijs.0.64179-0
© 2006 International Union of Microbiological Societies

Chryseobacterium wanjuense sp. nov., isolated from greenhouse soil in Korea

Hang-Yeon Weon1, Byung-Yong Kim2, Seung-Hee Yoo2, Soon-Wo Kwon2, Yang-Hee Cho2, Seung-Joo Go2 and Erko Stackebrandt3

1 Korean Agricultural Culture Collection (KACC), Microbial Genetics Division, National Institute of Agricultural Biotechnology, Rural Development Administration (RDA), Suwon 441-707, Republic of Korea
2 Applied Microbiology Division, National Institute of Agricultural Science and Technology, RDA, Suwon 441-707, Republic of Korea
3 Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Mascheroder Weg 1b, D-38124 Braunschweig, Germany

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


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A taxonomic study was performed on strain R2A10-2T, isolated from greenhouse soil cultivated with lettuce (Lactuca sativa L.), collected in Wanju Province, Korea. The bacterial cells were Gram-negative, aerobic, short rods. The growth temperature and pH were 5–35 °C and 5.0–9.0, respectively. Phylogenetic analysis based on 16S rRNA gene sequences revealed that this isolate had 93.3–97.7 % similarity to Chryseobacterium species: the highest sequence similarities were to the type strains of Chryseobacterium daecheongense (97.7 %), Chryseobacterium formosense (97.1 %) and Chryseobacterium defluvii (96.9 %). Low levels of DNA–DNA relatedness were found between strain R2A10-2T and the type strains of these three species (<28 %). Differences in phenotypic properties were found with respect to Chryseobacterium species with validly published names. The predominant cellular fatty acids were iso-15 : 0 (40.0 %), iso-17 : 0 3-OH (21.9 %), iso-17 : 1{omega}9c (11.7 %) and summed feature 4 (iso-15 : 0 2-OH and/or 16 : 1{omega}7c/t, 11.0 %). Menaquinone MK-6 was detected as the sole respiratory quinone. The G+C content of the genomic DNA was 37.8 mol%. On the basis of the genomic and phenotypic evidence, this isolate represents a novel species of the genus Chryseobacterium, for which the name Chryseobacterium wanjuense sp. nov. is proposed. The type strain is R2A10-2T (=KACC 11468T=DSM 17724T).

The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain R2A10-2T is DQ256729.


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As a result of a thorough study of the genus Flavobacterium, the genus Chryseobacterium was proposed by Vandamme et al. (1994)Go. Recently, Chryseobacterium meningosepticum and Chryseobacterium miricola were transferred to the novel genus Elizabethkingia on the basis of a polyphasic taxonomic approach (Kim et al., 2005bGo). Hence, 12 species with validly published names are currently classified in the genus Chryseobacterium. The type species is Chryseobacterium gleum. All members of the genus are characterized by the presence of MK-6 as the major respiratory quinone and by the presence of large amounts of iso-15 : 0, iso-17 : 1{omega}9c and iso-17 : 0 3-OH fatty acids.

Strain R2A10-2T was isolated from greenhouse soil cultivated with lettuce (Lactuca sativa L.) in the Wanju region of Korea. A diluted soil sample was spread on R2A medium (Difco) at 28 °C. Colonial properties were observed after 48 h incubation on R2A medium. Phenotypic tests such as Gram staining, catalase and oxidase activities, indole production and hydrolysis of agar, casein, DNA, gelatin and starch were performed according to the methods of Gerhardt et al. (1994)Go. The presence of flexirubin-type pigments was determined by flooding the cell mass (taken from agar plates) with 20 % (w/v) KOH (Bernardet et al., 2002Go). Hydrolysis of carboxymethylcellulose (Sigma) (0.1 %), alginic acid (0.5 %, w/v), chitin from crab shells (1 %, w/v), pectin (0.5 %, w/v) and tyrosine (0.5 %, w/v) was tested according to the procedures of Gerhardt et al. (1994)Go. Temperature and salinity tolerances were assessed at 5, 10, 15, 20, 25, 30, 33, 35, 37 and 40 °C and NaCl concentrations of 0, 1, 2, 3, 5 and 7 % (w/v) in R2A agar and broth, respectively. R2A broth adjusted to initial pH values of 4, 5, 6, 7, 8, 9 and 10 with citrate/phosphate buffer or Tris/HCl buffer (Breznak & Costilow, 1994Go) was used to assess the ability of strains to grow at different pH values. Enzyme activities were determined by using the API ZYM system (bioMérieux). Additional physiological and biochemical tests were performed with the API 20NE, API 50 CH and API ID 32 GN systems (bioMérieux).

The 16S rRNA gene was amplified by using a PCR with two universal primers, as described by Kwon et al. (2003)Go. Sequencing of the amplified 16S rRNA gene and phylogenetic analysis were performed as described by Kwon et al. (2003)Go. Neighbour-joining and maximum-parsimony methods were carried out using MEGA, version 2.1 (Kumar et al., 2001Go). The resulting trees and topology were evaluated by bootstrap analyses (Felsenstein, 1985Go) based on 1000 resamplings.

Isoprenoid quinones were extracted and analysed as described by Groth et al. (1996)Go. DNA–DNA hybridization was carried out as described by Seldin & Dubnau (1985)Go. Probe labelling was conducted by using the non-radioactive DIG High Prime system (Roche); hybridized DNA was visualized using the DIG luminescent detection kit (Roche). DNA–DNA relatedness was quantified by using a densitometer (Bio-Rad). For fatty acid methyl ester analysis, cell mass was harvested from trypticase soy agar (Difco) after cultivation for 24 h at 28 °C. The fatty acid methyl esters were extracted and prepared according to the standard protocol of the MIDI/Hewlett Packard Microbial Identification System (Sasser, 1990Go). The G+C content of the DNA was determined according to Mesbah et al. (1989)Go, using a reverse-phase column.

Comparison of the almost complete 16S rRNA gene sequence (1462 nt) of strain R2A10-2T with sequences deposited in the public databases showed the organism to be a member of the genus Chryseobacterium. The isolate shared sequence similarities of 97.7 and 96.9 % with Chryseobacterium daecheongense DSM 15235T and Chryseobacterium defluvii DSM 14219T, respectively. The organism also showed a high level of 16S rRNA gene sequence similarity (97.1 %) with Chryseobacterium formosense KCTC 12435T. The type strains of all other Chryseobacterium species showed sequence similarities of less than 96 % with respect to strain R2A10-2T. On the basis of neighbour-joining and maximum-parsimony analyses, strain R2A10-2T forms a clade with C. daecheongense DSM 15235T and C. defluvii DSM 14219T. The intraclade branching, as well as the branching point with a sister clade, was supported by high bootstrap values (see Fig. 1Go for the neighbour-joining analysis). Because of the relatively high 16S rRNA gene sequence similarity values, DNA–DNA hybridization was carried out between the novel isolate and C. daecheongense DSM 15235T, C. defluvii DSM 14219T and C. formosense KCTC 12435T. Low levels of DNA–DNA relatedness were found between strain R2A10-2T and DSM 15235T (28 %), DSM 14219T (38 %) and KCTC 12435T (16 %), supporting the presence of a novel genospecies.


Figure 1
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  		Fig. 1. Unrooted neighbour-joining tree, based on almost-complete 16S rRNA gene sequences, showing the phylogenetic relationships of strain R2A10-2T within the genus Chryseobacterium. Bootstrap values (>50 %) based on 1000 replications are shown at the nodes of the tree. Bar, 0.005 substitutions per nucleotide position.

 
The phenotypic characteristics of strain R2A10-2T are indicated in the species description and in Table 1Go. The predominant cellular fatty acids observed in strain R2A10-2T were iso-15 : 0 (40.0 %), iso-17 : 0 3-OH (21.9 %), iso-17 : 1{omega}9c (11.7 %) and summed feature 4 (iso-15 : 0 2-OH and/or 16 : 1{omega}7c/t, 11.0 %). Table 2Go shows the detailed fatty acid composition of strain R2A10-2T in comparison with those of all other Chryseobacterium species. Menaquinone MK-6 was detected as the sole respiratory quinone. The G+C content of the genomic DNA was 37.8 mol%. Although the phenotypic record is poor for a range of Chryseobacterium type strains, the data available indicate that strain R2A10-2T differs in terms of several properties, which, in combination, allow its differentiation from other members of the genus Chryseobacterium.


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  		Table 1. Phenotypic comparisons among type strains of Chryseobacterium species

Strains: 1, strain R2A10-2T; 2, C. daecheongense DSM 15235T; 3, C. defluvii DSM 14219T; 4, C. formosense KCTC 12435T; 5, C. balustinum LMG 8329T; 6, C. gleum LMG 8334T; 7, C. indologenes LMG 8337T; 8, C. indoltheticum LMG 4025T; 9, C. joostei LMG 18212T; 10, C. scophthalmum LMG 13028T; 11, C. shigense DSM 17126T; 12, C. taichungense CCUG 50001T; 13, C. vrystaatense LMG 22846T. Data are from Yabuuchi et al. (1983)Go, Holmes et al. (1984aGo, bGo), Mudarris et al. (1994)Go, Vandamme et al. (1994)Go, Hugo et al. (2003)Go, Kämpfer et al. (2003)Go, de Beer et al. (2005)Go, Kim et al. (2005a)Go, Shen et al. (2005)Go, Young et al. (2005)Go and this study. All strains tested were positive for the presence of flexirubin-type pigments and for the hydrolysis of aesculin, casein and gelatin. +, Positive; W, weak; –, negative; NA, no data available.

 

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  		Table 2. Fatty acid composition of strain R2A10-2T in comparison with other members of the genus Chryseobacterium

Taxa: 1, strain R2A10-2T; 2, C. daecheongense (n=1); 3, C. defluvii (n=1); 4, C. formosense (n=1); 5, C. balustinum (n=1); 6, C. gleum (n=5); 7, C. indologenes (n=45); 8, C. indoltheticum (n=1); 9, C. joostei (n=11); 10, C. scophthalmum (n=2); 11, C. taichungense (n=1); 12, C. vrystaatense (n=7). Data are from Hugo et al. (2003)Go, Kämpfer et al. (2003)Go, de Beer et al. (2005)Go, Kim et al. (2005a)Go, Shen et al. (2005)Go, Young et al. (2005)Go and this study. Values are mean percentages of total fatty acids; fatty acids amounting to less than 2 % of the total fatty acids in all strains were not included. Major fatty acids are in bold. tr, Trace amount (less than 2 %); –, not detected.

 
On the basis of the results of DNA–DNA hybridization, phenotypic characterization and 16S rRNA gene sequencing, we propose that strain R2A10-2T represents a novel species within the genus Chryseobacterium, for which we propose the name Chryseobacterium wanjuense sp. nov.

Description of Chryseobacterium wanjuense sp. nov.
Chryseobacterium wanjuense (wan.ju.en'se. N.L. neut. adj. wanjuense pertaining to Wanju, a province in Korea).

Cells are Gram-negative rods (0.7–0.8x2.0–3.5 µm). Yellow colonies with entire edges are formed on R2A agar plates. Grows at 5–37 °C (optimum 28 °C), with 0–2 % NaCl (optimum 0–1 % NaCl) and at pH 5–9 (optimum pH 7). Growth occurs on R2A, trypticase soy agar and nutrient agar (Difco), but weak growth is observed on MacConkey agar. Degrades casein, gelatin, starch and tyrosine, and weakly degrades DNA. Urea, chitin and carboxymethylcellulose are not degraded. Utilizes malonate. The type strain shows positive reactions for aesculin and gelatin hydrolysis and for beta-galactosidase activity, but shows negative reactions for nitrate reduction, indole production, glucose fermentation and for arginine dihydrolase and urease activities (API 20NE). Assimilates D-glucose, D-mannose, D-maltose, sodium acetate, glycogen and L-proline (API 20NE and API ID 32 GN). Does not assimilate L-arabinose, D-mannitol, N-acetylglucosamine, potassium gluconate, capric acid, adipic acid, malic acid, trisodium citrate, phenylacetic acid, L-rhamnose, D-ribose, inositol, D-sucrose, itaconic acid, suberic acid, sodium malonate, lactic acid, L-alanine, potassium 5-ketogluconate, 3-hydroxybenzoic acid, L-serine, salicin, D-melibiose, L-fucose, D-sorbitol, propionic acid, valeric acid, L-histidine, potassium 2-ketogluconate, 3-hydroxybutyric acid or 4-hydroxybenzoic acid (API 20NE and API ID 32 GN). Activity is observed for alkaline phosphatase, esterase lipase (C8), leucine arylamidase, valine arylamidase, acid phosphatase, naphthol-AS-BI-phosphohydrolase and N-acetyl-beta-glucosaminidase, weak activity is observed for esterase (C4), cystine arylamidase, {alpha}-chymotrypsin and {alpha}-glucosidase and no activity is observed for lipase (C14), trypsin, {alpha}-galactosidase, beta-galactosidase, beta-glucuronidase, beta-glucosidase, {alpha}-mannosidase and {alpha}-fucosidase (API ZYM). Acids are produced only from D-glucose, D-mannose, D-trehalose and gentiobiose (API 50 CH). The predominant cellular fatty acids are iso-15 : 0, iso-17 : 0 3-OH, iso-17 : 1{omega}9c and summed feature 4. Menaquinone MK-6 is the sole respiratory quinone. The G+C content of the genomic DNA of the type strain is 37.8 mol%. The closest phylogenetic relative is C. daecheongense.

The type strain, R2A10-2T (=KACC 11468T=DSM 17724T), was isolated from greenhouse soil cultivated with lettuce (L. sativa L.), collected from Wanju Province, Korea.


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