Kaistella koreensis gen. nov., sp. nov., a novel member of the Chryseobacterium-Bergeyella-Riemerella branch

The CytophagaFlavobacteriumBacteroides (CFB) group, rRNA superfamily V, is recognized as a separate line of descent within the domain Bacteria (Segers et al., 1993; Woese et al., 1990). The family Flavobacteriaceae, emended by Bernardet et al. (1996), in the CFB group contains many species in the genera Chryseobacterium, Bergeyella, Riemerella, Ornithobacterium, Empedobacter, Weeksella and Flavobacterium (Holmes, 1992; Jooste & Hugo, 1999). The genera Bergeyella, Chryseobacterium and Riemerella form a separate branch, the so-called BergeyellaChryseobacteriumRiemerella branch, on the basis of rRNA cistron similarity (Bernardet et al., 1996; Vandamme et al., 1994) and phenotypic characteristics. The genus Chryseobacterium contains eight recognized species, the genus Bergeyella accommodates a single species (Holmes et al., 1986; Vandamme et al., 1994) and the genus Riemerella comprises two species (Segers et al., 1993; Vancanneyt et al., 1999). Members of these genera are yellow-pigmented rods and have very low G+C contents of 3038 mol%.

In the present study, three strains, Chj707^T, Ko2 and Ko10, were isolated from a freshwater stream in Korea and we studied their taxonomic parameters, including genotypic, chemotaxonomic and classical phenotypic parameters, in order to establish their phylogenetic affiliation and to describe comprehensively the taxonomic position of these strains.

Lack of flagellation, low G+C content, the presence of menaquinones as the predominant respiratory quinones, the presence of branched-chain fatty acids in high percentages, the absence of carbohydrate fermentation and similar hydrolytic enzyme patterns confirmed that these strains belong to the BergeyellaChryseobacteriumRiemerella branch. However, some characteristics such as phylogenetic distance from any type strains of species with validly published names and their phenotypic properties, higher G+C content and specific discriminatory fatty acids confirmed that the strains constitute a separate genus. Below, we show that these isolates constitute a novel genus and species, for which we propose the name Kaistella koreensis gen. nov., sp. nov.

Bacterial strains and growth conditions.
Unless specified otherwise, strains were grown on nutrient agar or broth and incubated at 30 °C. The strains and their sources are listed in Table 1.

Table 1. Strains studied KCTC, Korean Collection for Type Cultures, Daejeon, Korea; ATCC, American Type Culture Collection, Manassas, VA, USA; LMG, Culture collection, Laboratorium voor Microbiologie Gent, Universiteit Gent, Belgium.

Phenotypic characterization.
The API 20NE and API ID32 GN microtest systems were used according to the recommendations of the manufacturer (bioMérieux). Cell morphologies were observed under a light microscope (1000x magnification) with cells grown for 3 days. For determination of the Gram reaction, the non-staining method (3 % KOH; Buck, 1982) was used. Motility was examined by phase-contrast microscopy. Oxidase activity was tested using Bactident-Oxidase test strips (Merck), catalase activity with 3 % H₂O₂. Growth was investigated at different temperatures (15, 25, 30, 37 and 42 °C) and on MacConkey agar. Additional biochemical tests were performed: oxidative or fermentative acid production from 1 % glucose (Hugh & Leifson, 1953), H₂S production using peptone iron agar (Difco 289100) and starch hydrolysis using starch agar (Difco 272100).

DNA extraction and 16S rRNA gene sequencing.
Chromosomal DNA was extracted and purified using the DNeasy tissue kit (Qiagen). PCR amplification of extracted 16S rRNA genes was done as described previously by Yoon et al. (1997). Primers (Weisburg et al., 1991) annealing at the 5' and 3' ends of the 16S rRNA genes were 9F (5'-GAGTTTGATCCTGGCTCAG-3'; positions 927, Escherichia coli 16S rRNA numbering) and 1512R (5'-ACGGCTACCTTGTTACGACTT-3'; positions 15121492). PCR amplification was performed in a final reaction volume of 100 µl and the reaction mixture contained each primer at 1 µM, 100 ng extracted DNA, each dNTP at 0·1 µM, 10x reaction buffer and 2·5 U Taq DNA polymerase. PCR was run for 35 cycles and the following thermal profile was used: denaturation at 94 °C for 1 min, primer annealing at 60 °C for 1 min and extension at 72 °C for 2 min. The final cycle included extension for 10 min at 72 °C. Direct sequencing of amplified 16S rRNA genes was done using the following primers (Stackebrandt & Liesack, 1993; Yoon et al., 1998): 9F (5'-GAGTTTGATCCTGGCTCAG-3'; 927), 341F (5'-CCTACGGGAGGCAGCAG-3'; 341357), 519F (5'-CAGCAGCCGCGGTAATAC-3'; 519536), 907F (5'-AAACTCAAAKGAATTGACGG-3'; 907926), 536R (5'-GTATTACCGCGGCTGCTG-3'; 536519), 1100R (5'-GGGTTGCGCTCGTTG-3'; 11141100) and 1512R (5'-ACGGCTACCTTGTTACGACTT-3'; 15121492).

Phylogenetic analysis.
The full 16S rRNA gene sequences were compiled after sequencing using SeqMan software and sequences of the test strains were edited using the BioEdit program (Hall, 1999) and aligned using CLUSTAL X (Thompson et al., 1997). The distance matrix was calculated by the BioEdit program (Hall, 1999) after deleting regions containing ambiguous nucleotides. The phylogenetic tree was constructed by the neighbour-joining method (Saitou & Nei, 1987) using the Mega2 program (Kumar et al., 2001).

DNA base composition.
Genomic DNA was extracted and purified using the Qiagen Genomic-tip system 100/G and enzymically degraded into nucleosides as described previously (Mesbah et al., 1989; Tamaoka & Komagata, 1984). A 10 µl volume of a solution containing 10 µg DNA was heated in a boiling water bath for 5 min and rapidly cooled in an ice-water bath. The denatured DNA solution was mixed with 10 µl nuclease P1 solution (20 U ml¹) and incubated at 37 °C for 1 h. Then, 10 µl alkaline phosphatase (1 U ml¹) was added to the sample and incubated at 37 °C for 3 h. The nucleoside mixture obtained was then separated by HPLC using a Waters Nova-Pak C₁₈ column (3·9x300 mm) and eluted with a mixture of 0·2 M NH₄H₂PO₄ and acetonitrile (40 : 1, v/v) at a flow rate of 0·7 ml min¹ and detected by UV absorbance at 270 nm. DNA of E. coli (Sigma) and two other type strains, Chryseobacterium indologenes KCTC 2905^T and Chryseobacterium scophthalmum KCTC 2907^T, were used as the calibration reference.

Fatty acid methyl ester (FAME) analysis.
Strains were grown for 24 h and then two loops of well-grown cells were harvested. FAMEs were prepared, separated and identified using the Sherlock Microbial Identification System (MIS) by MIDI, Inc. (Sasser, 1990).

Respiratory isoprenoid quinone analysis.
Isoprenoid quinones were extracted with chloroform/methanol (2 : 1, v/v) and purified by TLC on Merck Kieselgel 60 F254 plates (20x20 cm, 0·5 cm thick) using petroleum benzene/diethyl ether (85 : 15, v/v) as the solvent and then analysed by HPLC as described previously (Collins & Jones, 1981; Shin et al., 1996).

Respiratory isoprenoid quinone analyses
Analyses of the respiratory isoprenoid quinones of isolates Chj707^T, Ko2 and Ko10 and other related taxa gave only one characteristic peak, which corresponded to menaquinone MK-6. As presented in Table 2, it is clear that all species of the genera Chryseobacterium, Bergeyella and Riemerella and the three novel isolates have MK-6 as their common respiratory isoprenoid quinone.

Table 2. Chemotaxonomic characteristics of the novel isolates with species in the ChryseobacteriumBergeyellaRiemerella branch Strains/species: 1, C. balustinum KCTC 2903T; 2, C. defluvii B2T; 3, C. gleum (n=5); 4, C. indologenes (n=45); 5, C. indoltheticum KCTC 2920T; 6, C. joostei (n=11); 7, C. meningosepticum KCTC 2906T; 8, C. scophthalmum (n=7); 9, B. zoohelcum KCTC 2910T; 10, R. anatipestifer (n=16); 11, R. columbina (n=13); 12, Chj707T; 13, Ko2; 14, Ko10. Values are percentages of total fatty acids; only means are given and standard deviations are not given. Fatty acids that account for less than 1 % of the total fatty acids are not shown; the percentages therefore do not add up to 100 %. tr, Trace (<1 %); , not detected; ECL, equivalent chain-length (i.e. the identity of the fatty acids is unknown). Summed feature 4 contains 15 : 0 iso 2-OH and/or 16 : 1ω7c/t. The respiratory quinones of C. gleum, C. indoltheticum, C. scophthalmum, B. zoohelcum were analysed in this study (ND, not determined). Data were taken from Bernardet et al. (1996), Dees et al. (1985), Holmes et al. (1984, 1986), Hugo et al. (2003), Jooste & Hugo (1999), Kämpfer et al. (2003), Mudarris et al. (1994), Segers et al. (1993), Vancanneyt et al. (1999) and Yabuuchi et al. (1983).

Phylogenetic relationships
Comparative 16S rRNA gene sequence analyses indicated the phylogenetic position of strains Chj707^T, Ko2 and Ko10 as members of the ChryseobacteriumBergeyellaRiemerella branch of the Flavobacteriaceae in rRNA superfamily V, the CFB phylum, which has been studied previously in detail by the DNArRNA hybridization technique. The 16S rRNA gene sequences of strains Chj707^T, Ko2 and Ko10, consisting of continuous stretches of 1466 bp (positions 281493, according to E. coli numbering), were used to search GenBank for related sequences. The sequences of strains Ko2 and Ko10 were identical and differed from the sequence of Chj707^T in only two nucleotide positions. Sequence searches showed that strains Chj707^T, Ko2 and Ko10 were phylogenetically most closely related to representatives of the family Flavobacteriaceae. The results of sequence similarity calculations indicated that the nearest relatives of strain Chj707^T are Chryseobacterium balustinum (94·3 % sequence similarity over 1430 bp sequence), C. scophthalmum (94·1 % over 1442 bp) and Chryseobacterium indoltheticum (93·7 % over 1418 bp). Sequence similarity of 92·694·3 % was observed to all species of the genus Chryseobacterium. Lower sequence similarities (87·692·9 %) were found to species from the genera Bergeyella and Riemerella. Phylogenetic analyses revealed that strains Chj707^T, Ko2 and Ko10 formed a distinct lineage that belonged to a well-supported cluster containing the genera Chryseobacterium, Bergeyella and Riemerella (Fig. 1). The strains form a distinctly separate cluster separate from all species of the closest genus, Chryseobacterium. This distinct branch in the phylogenetic tree provides strong support that these strains belong to a new genus. The isolates have a phylogenetically much more distant position from species of the genera Bergeyella and Riemerella. Members of Bergeyella and Riemerella are very different in that they are microaerophilic and can not grow on nutrient agar, unlike the novel isolates and all species of Chryseobacterium. More than 99·9 % 16S rRNA gene sequence similarity between Chj707^T, Ko2 and Ko10 showed the intraspecific relationship among the three strains (Rosselló-Mora & Amann, 2001).

(42K): Fig. 1. Phylogenetic tree of strains of Chj707T, Ko2 and Ko10 and related taxa of the ChryseobacteriumBergeyellaRiemerella branch of the Flavobacteriaceae, drawn with 1348 nt of the 16S rRNA gene by using the complete deletion mode in MEGA2. The tree was constructed using the neighbour-joining method. Bootstrap values indicated at branches were calculated from 1000 trees. Accession numbers are indicated in parentheses.

DNA base composition
G+C content analyses strongly support the proposal of a new genus. As shown in Table 3, species of the genus Chryseobacterium have G+C contents of 3038 mol% and strains Chj707^T, Ko2 and Ko10 have higher G+C contents of 41·242·3 mol%. It is now generally accepted that bacteria with DNA G+C contents differing by more than 5 mol% should not be assigned to the same species and those differing by more than 10 mol% should not be classified in the same genus (Busse et al., 1996; Rosselló-Mora & Amann, 2001; Schleifer & Stackebrandt, 1983; Stackebrandt & Liesack, 1993; Vandamme et al., 1996). The significantly higher G+C contents of strains Chj707^T, Ko2 and Ko10 compared with species of the genera Chryseobacterium, Bergeyella and Riemerella support the inclusion of these strains in a new genus.

Table 3. Differentiating phenotypic characteristics for the novel isolates and other related type species Strains/species: 1, C. balustinum; 2, C. defluvii; 3, C. gleum; 4, C. indologenes; 5, C. indoltheticum; 6, C. joostei; 7, C. meningosepticum; 8, C. scophthalmum; 9, B. zoohelcum; 10, R. anatipestifer; 11, R. columbina; 12, Chj707T; 13, Ko2; 14, Ko10. All strains were Gram-negative, non-motile, rod-shaped and oxidase- and catalase-positive. +, Present in all strains; , absent in all strains; (+), present in most strains; (), absent in most strains; V, variable; ND, not determined. See legend to Table 2 for sources of data for reference species.

FAME analysis
The long-chain fatty acid contents of strains Chj707^T, Ko2 and Ko10 and related taxa are presented in Table 2. FAME analyses revealed some differences between these strains and their nearest phylogenetic neighbours in the genera Chryseobacterium, Bergeyella and Riemerella. Dominant fatty acids for all strains investigated (mean levels above 1 %) are 15 : 0 iso and 17 : 0 iso 3-OH. All species of the genus Chryseobacterium, including CDC group IIb strains (Hugo et al., 1999), have additional characteristic dominant fatty acids summed feature 4 (15 : 0 iso 2-OH, 16 : 1ω7c and/or 16 : 1ω7t) and 17 : 1 iso ω9c. However, significant differences were observed between the genera Chryseobacterium, Bergeyella and Riemerella and the novel strains. In strains Chj707^T, Ko2 and Ko10, the fatty acid 15 : 0 iso was dominant, with significant levels of 15 : 0 anteiso, 17 : 0 iso 3-OH and 17 : 1 iso ω9c. Strains Chj707^T, Ko2 and Ko10 had larger relative amounts of 15 : 0 iso and smaller amounts of 17 : 0 iso 3-OH, summed feature 4 and 17 : 1 iso ω9c than members of the genus Chryseobacterium. The three strains all had distinct fatty acids 13 : 0 iso and 15 : 0 anteiso, similar to those of Bergeyella and Riemerella. Although representatives of the genera Bergeyella and Riemerella showed similar fatty acid profiles, differentiation on the basis of the amounts of several other fatty acids is possible (15 : 0 anteiso to Bergeyella and 17 : 1 iso ω9c to Riemerella).

Phenotypic characterization
Phenotypic features that distinguish the novel strains from their nearest relatives are summarized in Table 3. Strains Chj707^T, Ko2 and Ko10 are different from species of the genus Chryseobacterium in that they did not produce acid from glucose, did not have β-galactosidase activity, could not hydrolyse starch or assimilate glucose, arabinose or 3-hydroxybutyrate and could not grow on MacConkey agar. They differ from species of the genera Bergeyella and Riemerella in that they produced indole, had β-glucosidase activity and grew aerobically on nutrient agar. From the results of analyses of 16S rRNA gene sequence similarity, G+C content, fatty acid profiles and phenotype, it is evident that strains Chj707^T, Ko2 and Ko10 should be assigned to a species in a new genus, for which the name Kaistella koreensis gen. nov., sp. nov. is proposed.

Description of Kaistella gen. nov.
Kaistella (Ka.is.tel'la. L. dim. suff. -ella; N.L. fem. n. Kaistella arbitrary name after KAIST, Korea Advanced Institute of Science and Technology).

Cells are Gram-negative, non-sporulating rods. All strains grow aerobically. Colonies are circular, entire, low-convex, smooth, opaque and yellow. Catalase and oxidase positive. Nitrate is not reduced to N₂. Indole is produced and acid is not produced from glucose. Arginine dihydrolase and β-galactosidase are negative. Aesculin and gelatin are hydrolysed. At present, it is not possible to give a general biochemical profile for the genus as only a single species is known. The DNA base composition ranges from 41·2 to 41·6 mol% G+C. The major isoprenoid quinone is MK-6. The type species is Kaistella koreensis.

Description of Kaistella koreensis sp. nov.
Kaistella koreensis (ko.re.en'sis. N.L. fem. adj. koreensis of Korea, from where the novel organisms were isolated).

The description is the same as that of the genus. Mannitol, maltose, caprate, adipate, malate, citrate, phenylacetate, propionate, 4-hydroxybenzoate, L-proline, rhamnose, suberate, acetate, L-alanine and glycogen are assimilated. Glucose, arabinose, mannose, N-acetylglucosamine, salicin, melibiose, fucose, sorbitol, valerate, histidine, 2-ketogluconate, 3-hydroxybutyrate, D-ribose, inositol, D-sucrose, itaconate, malonate, DL-lactate, 5-ketogluconate, 3-hydroxybenzoate and L-serine are not assimilated. The G+C content of the DNA is 41·241·6 mol%.

Strains were isolated from natural mineral water from Daejeon City, Korea. The type strain is Chj707^T (=KCTC 12107^T=IAM 15050^T) and Ko2 (=KCTC 12108=IAM 15051) and Ko10 (=KCTC 12109=IAM 15052) are reference strains.

This work was supported by the 21C Frontier Microbial Genomics and Applications Center Program, Ministry of Science & Technology (grant MG02-0101-001-2-2-0), Republic of Korea.

Footnotes

†Present address: Dept of Biologie I, Fakultat fur Biologie, Ludwig-Maximilians-Universität München, Maria-Ward-Strasse 1a, D-80638 München, Germany.