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Int J Syst Evol Microbiol 57 (2007), 1765-1769; DOI  10.1099/ijs.0.65046-0
© 2007 International Union of Microbiological Societies

Chryseobacterium flavum sp. nov., isolated from polluted soil

Yu Zhou1,{dagger}, Jing Dong1,{dagger}, Xu Wang1, Xing Huang1, Ke-Yun Zhang1, Yu-Qin Zhang2,3, Yu-Feng Guo4, Ren Lai1,5 and Wen-Jun Li2

1 Key Laboratory of Microbiological Engineering of Agricultural Environment, Ministry of Agriculture, Life Sciences College of Nanjing Agricultural University, Nanjing, Jiangsu 210095, China
2 Laboratory for Conservation and Utilization of Bio-Resources, Yunnan Institute of Microbiology and Yunnan University, Kunming, Yunnan 650091, China
3 Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, China
4 Department of Environmental Science and Engineering, Tsinghua University, Beijing 100084, China
5 Biotoxin Department of Key Laboratory of Animal Models and Human Disease Mechanisms, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming, Yunnan 650223, China

Correspondence
Ren Lai
rlai72{at}njau.edu.cn
Wen-Jun Li
wjli{at}ynu.edu.cn


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A Gram-negative, non-motile, rod-shaped bacterial strain, designated CW-E 2T, was isolated from a polluted soil sample collected from Jiangsu Province, China. A taxonomic study of the isolate, including phylogenetic analysis based on 16S rRNA gene sequences and phenotypic characteristics, was carried out. The predominant menaquinone was MK-6 and the major fatty acids were i-C15 : 0, i-C17 : 0 3-OH, i-C17 : 1{omega}9c and summed feature 4. The G+C content of the DNA was 37.2 mol%. Based on phenotypic and genotypic characteristics, strain CW-E 2T represents a novel species of the genus Chryseobacterium for which the name Chryseobacterium flavum sp. nov. is proposed. The type strain is CW-E 2T (=KCTC 12877T=CCTCC AB 206147T).

Abbreviations: TEM, transmission electron microscopy

{dagger}These authors contributed equally to this work. Back

The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain CW-E 2T is EF154516.


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The family Flavobacteriaceae represents the main bacterial lineage in the phylum Bacteroidetes (formerly the Cytophaga–Flexibacter–Bacteroides group) (Bernardet et al., 1996Go, 2002Go). Among the genera currently included in the family, Chryseobacterium, Bergeyella, Ornithobacterium, Empedobacter, Weeksella, Wautersiella, Elizabethkingia, Sejongia and Kaistella form a separate branch in phylogenetic trees (Vandamme et al., 1994Go; Kim et al., 2004Go, 2005bGo; Bernardet et al., 2002Go, 2006Go). At the time of writing, the genus Chryseobacterium contains 18 species with validly published names (Shimomura et al., 2005Go; Kim et al., 2005aGo; Young et al., 2005Go; de Beer et al., 2005Go, 2006Go; Bernardet et al., 2006Go; Quan et al., 2007Go). Members of the genus Chryseobacterium are strictly aerobic, non-motile, Gram-negative rods that produce flexirubin-type pigments and have a DNA G+C content of about 29–39 mol%. Members of the genus Chryseobacterium occur in a variety of environments, including soil, fresh- and seawater, raw milk and chicken, diseased fish, bioreactor sludge and clinical samples (Yamaguchi & Yokoe, 2000Go; Hugo et al., 2003Go; Kim et al., 2005aGo; de Beer et al., 2005Go, 2006Go; Bernardet et al., 2006Go; Quan et al., 2007Go). In the course of an investigation of the bacterial community in a soil sample contaminated by atrazine, ethametsulfuron, glyphosate and other herbicides from a chemical factory in the suburb of Nanjing City, Jiangsu Province, China, one bacterial strain, CW-E 2T, which had the ability to decompose atrazine (data not shown), was isolated and subjected to a polyphasic taxonomy study. It is proposed that this strain represents a novel species in the genus Chryseobacterium, Chryseobacterium flavum sp. nov.

Strain CW-E 2T was isolated by plating dilutions of a soil sample on LB agar (Sambrook & Russell, 2002Go) incubated at 30 °C for 3 days. The strain was preserved in a 20 % (v/v) glycerol solution in distilled water at –80 °C. Biomass for molecular systematic and chemotaxonomic studies was obtained from LB broth (pH adjusted to 7.0) cultures incubated at 30 °C for 2 days in shake flasks (about 180 r.p.m.).

Cellular morphology of the isolate was observed by transmission electron microscopy (TEM) (model H-7650; Hitachi) examination of bacterial cells grown for 18 h on LB agar and prepared as described by Nedashkovskaya et al. (2005)Go (Fig. 1Go). Acid production from carbohydrates was investigated using the media and methods described by Yamaguchi & Yokoe (2000)Go. Utilization of various carbon sources was investigated by using modified ammonia salt-sugar medium [0.2 % (NH4)2SO4, 0.05 % NaH2PO4 . H2O, 0.02 % MgSO4 . 7H2O, 0.01 % CaCl2 . 2H2O and 0.05 % K2HPO4] containing 1 % of each tested carbon source. Growth was tested at 0, 5, 10, 25, 30, 35, 37, 40, 42, 45 and 50 °C on LB agar. NaCl and pH tolerance were tested using LB broth adjusted to various pH values (3.0, 6.0, 7.0, 7.8, 8.0, 10.0, 11.0 and 12.0) and NaCl concentrations (1, 4, 7 and 10 %) (Zhou et al., 2007Go). Growth was tested on cetrimide agar, beta-hydroxybutyrate agar, MacConkey agar and Simmons' citrate agar. Susceptibility to antibiotics was examined as described by Nedashkovskaya et al. (2003Go, 2005)Go. Flexirubin-type pigments were identified using the KOH test as described by Bernardet et al. (2002)Go. Gram-staining and other phenotypic characteristics were determined according to the methods of Gerhardt et al. (1994)Go. Detailed morphological, physiological and biochemical characteristics of strain CW-E 2T are given in Table 1Go and the species description.


Figure 1
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  		Fig. 1. TEMs of cells of strain CW-E 2T grown on LB agar for 18 h at 30 °C. Arrows indicate fimbriae. Bars, 200 nm.

 

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  		Table 1. Phenotypic characteristics that differentiate Chryseobacterium flavum CW-E 2T from its closest relatives

Species: 1, C. flavum (n=1); 2, C. joostei (n=11); 3, C. gleum (n=5); 4, C. indologenes (n=45); 5, C. shigense (n=1); 6, C. vrystaatense (n=36). +, Positive; –, negative; V, variable; D, delayed; NA, no data available; PS, polluted soil; RM, raw milk; RC, raw chicken; CS, clinical samples; LAB, lactic acid beverage. For all species, indole production was positive. Data from Hugo et al. (2003)Go, Shimomura et al. (2005)Go and de Beer et al. (2005)Go.

 
Menaquinones were extracted from lyophilized cells and determined according to Hu et al. (2001)Go. The predominant menaquinone was MK-6. The fatty acids were extracted, methylated and analysed using the standard MIDI (Microbial Identification) system as described by Sasser (1990)Go. The major fatty acids of strain CW-E 2T were i-C15 : 0, i-C17 : 0 3-OH, i-C17 : 1{omega}9c and summed feature 4 (comprising i-C15 : 0 2-OH and/or C16 : 1{omega}7c and/or C16 : 1{omega}7t). Detailed fatty acid profiles of strain CW-E 2T and its closest relatives are given in Table 2Go.


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  		Table 2. Cellular fatty acid content (%) of Chryseobacterium flavum CW-E 2T and its closest relatives

Species: 1, C. flavum (n=1); 2, C. joostei (n=11); 3, C. gleum (n=5); 4, C. indologenes (n=45); 5, C. shigense (n=1); 6, C. vrystaatense (n=36). Some of the Chryseobacterium species included in this table were cultivated under different conditions. Means±SD are given where more than one strain was tested. ND, Not detected; TR, trace (fatty acids amounting to less than 1 %). Data from Hugo et al. (2003)Go, Shimomura et al. (2005)Go and de Beer et al. (2005)Go.

 
Genomic DNA of strain CW-E 2T was prepared following the method of Marmur (1961)Go and the DNA G+C content was determined using the thermal denaturation method (Mandel & Marmur, 1968Go). The DNA G+C content of strain CW-E 2T was 37.2 mol%.

PCR amplification of the 16S rRNA gene was performed as described by Xu et al. (2003)Go. Phylogenetic analysis was performed using the software packages PHYLIP (Felsenstein, 1993Go) and MEGA version 3.1 (Kumar et al., 2001Go) after multiple alignment of data by CLUSTAL_X (Thompson et al., 1997Go). Distances (distance options according to the Kimura two-parameter model; Kimura, 1980Go, 1983Go) and clustering were based on the neighbour-joining (Saitou & Nei, 1987Go) and maximum-likelihood (Felsenstein, 1981Go) methods. Bootstrap analysis (1000 resamplings) was used to evaluate the topology of the neighbour-joining tree (Felsenstein, 1985Go).

The almost complete 16S rRNA gene sequence (1440 bp in length) of strain CW-E 2T and reference sequences selected from GenBank following BLAST searches were used to draw the phylogenetic tree. Phylogenetic analyses showed that strain CW-E 2T belongs to the genus Chryseobacterium but shares only 94.9, 93.9 and 93.9 % 16S rRNA gene sequence similarity with its closest relatives Chryseobacterium joostei LMG 18212T, Chryseobacterium gleum ATCC 35910T and Chryseobacterium indologenes ATCC 29897T, respectively (Fig. 2Go).


Figure 2
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  		Fig. 2. Neighbour-joining phylogenetic tree based on 16S rRNA gene sequences of strain CW-E 2T, other members of the genus Chryseobacterium, and representatives of the family Flavobacteriaceae. Numbers at nodes indicate bootstrap values (%) greater than 50 %. Bar, 0.02 substitutions per nucleotide position. The maximum-likelihood tree showed essentially the same topology (data not shown).

 
Differential phenotypic characteristics (Table 1Go) could be used to distinguish the isolate from other Chryseobacterium species. Hence, strain CW-E 2T represents a novel species for which the name Chryseobacterium flavum sp. nov. is proposed.

Description of Chryseobacterium flavum sp. nov.
Chryseobacterium flavum (fla'vum. L. neut. adj. flavum yellow, the colour of colonies or pigment).

Cells are Gram-negative, non-motile, non-spore-forming, aerobic and chemo-organotrophic rods (0.5–0.7x1.1–1.3 µm) that are covered by fimbriae. Colonies on LB agar plates are circular, convex, shiny, bright-yellow-coloured and smooth, 1.0–1.5 mm in diameter, with a ropy consistency. Growth occurs on trypticase soy agar, cetrimide agar, MacConkey agar and beta-hydroxybutyrate (beta-hydroxybutyrate inclusions are not produced). No growth occurs on Simmons' citrate agar. Growth occurs at 5–42 °C, pH 5.0–9.0 and with 0–6 % NaCl (optimum growth at 30–35 °C, pH 6.0–7.0 and 1 % NaCl). Non-diffusible flexirubin-type yellow pigments are produced. Casein, gelatin, starch and Tween 20 are hydrolysed. DNA, cellulose and chitin are not hydrolysed. Acid is produced from D-fructose, maltose, trehalose and D-xylose; but not from adonitol, D-arabinose, cellobiose, dulcitol, L-fucose, D-galactose, D-glucose, inositol, D-lactose, D-mannitol, D-melezitose, D-melibiose, L-raffinose, L-rhamnose, D-sorbitol, L-sorbose or sucrose. Adonitol, aesculin, D-arabinose, cellobiose, dextrin, D-fructose, D-galactose, glucosamine, D-glucose, inositol, inulin, D-lactose, maltose, D-mannose, D-melezitose, D-melibiose, D-ribose, D-sorbitol, sucrose, trehalose, turanose, xylitol and D-xylose are utilized. Citrate, acetate, adonitol, dulcitol, D-erythrose, malonate, D-mannitol, melezitose, D-raffinose, tartrate, salicin and D-sorbitose are not utilized. Oxidase, catalase, beta-galactosidase, phenylalanine decarboxylase, arginine dihydrolase and lipase activities are present. Methyl {alpha}-D-glucosidase, ornithine decarboxylase, lysine decarboxylase, arginine decarboxylase, urease and DNase activities are absent. Nitrate and nitrite are not reduced. H2S (triple-sugar iron test) is not produced. Methyl red and Voges–Proskauer tests are negative. Indole is produced. KCN (0.0075 %) is not tolerated. Susceptible to carbenicillin, tetracycline, doxycycline, gentamicin, penicillin G, kanamycin, vancomycin, erythromycin, rifampicin, neomycin, streptomycin and chloramphenicol; resistant to ampicillin, lincomycin and polymixin B. The predominant menaquinone is MK-6. The major fatty acids are i-C15 : 0 (35.1 %), i-C17 : 0 3-OH (18.5 %), i-C 17 : 1{omega}9c (16.8 %) and summed feature 4 (13.3 %, comprising i-C15 : 0 2-OH and/or C16 : 1{omega}7c and/or C16 : 1{omega}7t). The DNA G+C content is 37.2 mol%.

The type strain is CW-E 2T (=KCTC 12877T=CCTCC AB 206147T), isolated from the surface layer of polluted soil from Nanjing, Jiangsu Province, China.


   ACKNOWLEDGEMENTS
 
The authors are grateful to the editor and two anonymous reviewers for helpful comments. This work was supported by grants from the National Natural Science Foundation of China (Project no. 30600001), the Jiangsu Natural Science Foundation (BK2005422) and the Yunnan Science and Technology Commission (2005C0054M). W.-J. L. was also supported by the Program for New Century Excellent Talent in University (NCET).


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