Abstract
Published online ahead of print on 20 May 2005 as DOI 10.1099/ijs.0.63690-0.
The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain GUM-KajiT is AB193101.
A scanning electron micrograph of cells of strain GUM-KajiT is available as a supplementary figure in IJSEM Online.
Footnotes
†Present address: Graduate School of Fisheries Science, Hokkaido University, 3-1-1 Minato-cho, Hakodate 041-8611, Japan.The genus Chryseobacterium was created by Vandamme et al. (1994) to accommodate several species formerly classified in the genus Flavobacterium, i.e. Chryseobacterium balustinum, C. gleum, C. indologenes, C. indoltheticum, C. meningosepticum and C. scophthalmum. Six species, Chryseobacterium defluvii (Kämpfer et al., 2003), C. joostei (Hugo et al., 2003), C. miricola (Li et al., 2003), C. daecheongense (Kim et al., 2005a), C. formosense (Young et al., 2005) and C. taichungense (Shen et al., 2005), have been added to the genus recently. However, C. meningosepticum and C. miricola have been transferred to the novel genus Elizabethkingia (Kim et al., 2005b) and the genus now consists of 10 species. Chryseobacterium proteolyticum (Yamaguchi & Yokoe, 2000) is a member of the genus, although the name has not been validly published. From a lactic acid beverage, we isolated a bacterial strain that is phylogenetically and phenotypically similar to Chryseobacterium species. In this paper, we report the characteristics of the novel bacterium and propose to classify it as a novel species of the genus Chryseobacterium.
The novel bacterium, designated strain GUM-KajiT (NCIMB Japan Culture Collection number BAMY 1001T), was isolated from a fresh lactic acid beverage (Yumai Co., Ltd, Kouga-gun, Shiga Prefecture, Japan) by the agar-plating method using nutrient agar (CM3; Oxoid). This bacterium has been considered as a part of the normal community in the lactic acid beverage. For testing, precultures were grown aerobically on the same medium. All test media were incubated at 30 °C unless otherwise specified. General cell morphology was studied using phase-contrast and light microscopy (BX50F4; Olympus). Morphological observations under a scanning electron microscope were performed at Hanaichi UltraStructure Research Institute (Okazaki, Japan). The Gram reaction was determined using a Favour G Gram-stain kit (Nissui) according to the manufacturer's instructions. A flexirubin-type pigment was detected by using the method of Reichenbach (1989). Catalase activity, oxidase activity, the oxidationfermentation reaction, growth on MacConkey agar and the alkaline reaction on Christensen's citrate were determined by using standard methods (Barrow & Feltham, 1993). Additional physiological and biochemical tests were performed using an API 20E kit (bioMérieux) according to the manufacturer's instructions. Fatty acid methyl esters were extracted and prepared by using the standard protocol of the Microbial Identification System (MIDI; Microbial ID). Extracts were analysed using a Hewlett Packard model HP6890A gas chromatograph equipped with a flame-ionization detector, according to the manufacturer's instructions. Respiratory quinones were detected by using the HPLC method as described previously (Nishijima et al., 1997). Genomic DNA was extracted and purified using the InstaGene Matrix kit (Bio-Rad). The G+C content (mol%) of the DNA was determined by using the HPLC method, as described previously (Katayama-Fujimura et al., 1984). 16S rRNA gene fragments were PCR-amplified and sequenced using the MicroSeq Full Gene 16S rDNA Bacterial Sequencing kit (Applied Biosystems) and an ABI Model 3100 Genetic Analyzer (Applied Biosystems). The 16S rRNA gene sequence determined was compared with those retrieved from the GenBank/EMBL/DDBJ databases. Sequence similarities were calculated and a neighbour-joining evolutionary distance tree (Saitou & Nei, 1987) was constructed using the programs CLUSTAL X (Thompson et al., 1997) and MEGA2 (Kumar et al., 2001). The topology of the tree was evaluated by using the bootstrap resampling method with 1000 replicates (Felsenstein, 1985).
Strain GUM-KajiT comprised straight and slightly curved rods measuring 0·60·7 µm in width and 1·01·5 µm in length (a scanning electron micrograph of cells is available as a supplementary figure in IJSEM Online). The cells were Gram-negative and non-motile. Colonies on nutrient agar were deep-yellow and shiny. A flexirubin-type pigment was detected. No growth occurred on MacConkey agar. The strain was an aerobic chemo-organotrophic bacterium with a strictly respiratory type of metabolism. Growth occurred at 530 °C but not at 37 °C (optimum, 2030 °C). The pH range for growth was 58. Catalase and oxidase activities were present. Casein, gelatin and starch were hydrolysed. Cells were negative for urease activity and nitrate reduction. Acid was produced from D-fructose and D-glucose. Other physiological and biochemical characteristics are shown in the species description.
Cellular fatty acid analysis showed that C15 : 0 iso was the most abundant component (39·7 %). Considerable proportions of summed feature 4 (2-OH-C15 : 0 iso and/or C16 : 1ω7c and/or C16 : 1ω7t) and C17 : 1ω9c iso were also found. 3-OH-C17 : 0 iso (19·6 %) was the major component of the 3-hydroxy acids. Analysis of the respiratory quinones revealed that menaquinone MK-6 was the sole quinone.
The 16S rRNA gene sequence of strain GUM-KajiT determined (1476 bp) was compared with those retrieved from the databases. The sequence of strain GUM-KajiT was most similar to that of C. joostei LMG 18212T (95·7 %). The neighbour-joining phylogenetic tree clearly showed that strain GUM-KajiT was positioned within the cluster of the genus Chryseobacterium, with C. joostei as its nearest phylogenetic neighbour (Fig. 1). The G+C content of the genomic DNA of strain GUM-KajiT was 36·6 mol%, the value being similar to that of C. joostei.
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On the basis of the phylogenetic data, it is clear that strain GUM-KajiT should be classified as a member of the genus Chryseobacterium. This phylogenetic assignment is supported unequivocally by the chemotaxonomic profiles, i.e. by the presence of C15 : 0 iso and 3-OH-C17 : 0 iso as the major fatty acids and MK-6 as the sole respiratory quinone. However, strain GUM-KajiT differs from all of the previously established species of Chryseobacterium in terms of a number of phenotypic characteristics (Table 1). For example, strain GUM-KajiT can be differentiated from its closest relative, C. joostei, by negative reactions for growth on MacConkey agar and for acid production from maltose and trehalose. Although genomic DNADNA reassociation studies were not performed in this study, the low similarities (below 96 %) of 16S rRNA gene sequences between our strain and any established Chryseobacterium species indicate a distinct position for strain GUM-KajiT within the genus, since genomic DNAs of bacterial strains having less than 97·0 % sequence similarity will usually not reassociate to more than 60 % hybridization (Stackebrandt & Goebel, 1994). In fact, Chryseobacterium species sharing 94·996·0 % 16S rRNA gene sequence similarities showed only 3·031·0 % DNADNA relatedness (Yamaguchi & Yokoe, 2000). DNA binding values as low as 16 and 23 % were even found between C. joostei and the two other Chryseobacterium species with which it shared 97·7 and 97·6 % 16S rRNA gene sequence similarity, respectively (Hugo et al., 2003). In view of these results, it is logical to conclude that strain GUM-KajiT should be classified as a novel species of the genus Chryseobacterium, for which we propose the name Chryseobacterium shigense sp. nov. Although only one strain of the novel species is available at this time, it is important to create a novel species because of the strain's unique origin and distinct phylogenetic position within the genus.
Table 1. Differential characteristics of strain GUM-KajiT and previously described Chryseobacterium species Taxa: 1, C. joostei; 2, C. balustinum; 3, C. daecheongense; 4, C. defluvii; 5, C. formosense; 6, C. gleum; 7, C. indoltheticum; 8, C. indologenes; 9, C. scophthalmum; 10, C. taichungense; 11, C. proteolyticum; 12, Elizabethkingia meningoseptica; 13, Elizabethkingia miricola. Symbols: +, positive; , negative; W, weakly positive; V, variable reaction; D, delayed reaction; NA, information not available. Data for reference species are based on information from Hugo et al. (2003), Li et al. (2003), Kim et al. (2005a, b), Young et al. (2005) and Shen et al. (2005).
Description of Chryseobacterium shigense sp. nov.
Chryseobacterium shigense (shi.gen'se. N.L. neut. adj. shigense pertaining to Shiga Prefecture in Japan, the geographical area of isolation of the type strain).
Cells are straight or slightly curved rods measuring 0·60·7x1·01·5 µm. Gram-negative and non-motile. Strictly aerobic and chemo-organotrophic. Colonies on nutrient agar are deep yellow (flexirubin-type pigment) and shiny. No growth occurs on MacConkey agar. Growth occurs at 530 °C (optimum 2030 °C) but not at 37 °C. The pH range for growth is 58. Catalase and oxidase are present. Indole is produced. Starch, casein and gelatin are hydrolysed. Negative reactions obtained for nitrate reduction, urease activity, hydrogen sulphide production and an alkaline reaction on Christensen's citrate. Acid is produced from D-fructose and D-glucose but not from L-arabinose, D-xylose, glycerol, lactose, maltose, trehalose, D-mannitol or glycerol. The major fatty acids are C15 : 0 iso and 3-OH-C17 : 0 iso. Considerable proportions of summed feature 4 (2-OH-C15 : 0 iso and/or C16 : 1ω7c and/or C16 : 1ω7t) and C17 : 1ω9c iso are also present. Menaquinone MK-6 is the sole respiratory quinone. The G+C content of the genomic DNA of the type strain is 36·6 mol%. The closest phylogenetic relative is C. joostei.
The type strain, strain GUM-KajiT (=BAMY 1001T=NCIMB 14047T=DSM 17126T), was isolated from a lactic acid beverage in Japan.
References
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