International Journal of Systematic and Evolutionary Microbiology (2014), 64, 851–857
DOI 10.1099/ijs.0.057398-0
Chryseobacterium camelliae sp. nov., isolated from green tea MooChang Kook,13 Heung-Min Son,23 Hien T. T. Ngo2 and Tae-Hoo Yi2 Correspondence
1
Tae-Hoo Yi
2
[email protected]
Department of Marine Biotechnology, Anyang University, Incheon 417-833, Republic of Korea Department of Oriental Medicinal Material & Processing College of Life Science, Kyung Hee University, 1 Seocheon, Kihung Yongin, Kyunggi 446-701, Republic of Korea
A Gram-staining-negative, strictly aerobic, non-motile, rod-shaped and flexirubin-type-pigmented strain, THG C4-1T, was isolated from green tea leaves in Jangheung-gun, Republic of Korea. Strain THG C4-1T grew well at 20–30 6C, at pH 7.0–7.5 and in the absence of NaCl on nutrient agar. Based on 16S rRNA gene sequence comparisons, strain THG C4-1T was most closely related to Chryseobacterium taiwanense Soil-3-27T (97.7 %), C. hagamense RHA2-9T (97.2 %), C. gregarium P 461/12T (97.2 %), C. ginsenosidimutans THG 15T (97.1 %), C. taeanense PHA3-4T (97.0 %) and C. daeguense K105T (97.0 %), but DNA–DNA relatedness between strain THG C4-1T and its closest phylogenetic neighbours was below 21 %. The DNA G+C content was 41.7 mol%. The only isoprenoid quinone detected in strain THG C4-1T was menaquinone 6 (MK-6). The major component of the polyamine pattern was sym-homospermidine. The major polar lipids were phosphatidylethanolamine and unidentified aminolipids. The major fatty acids were iso-C15 : 0, iso-C17 : 0 3-OH and iso-C17 : 1v9c. These data supported the affiliation of strain THG C4-1T to the genus Chryseobacterium. The results of physiological and biochemical tests enabled strain THG C4-1T to be differentiated genotypically and phenotypically from recognized species of the genus Chryseobacterium. Therefore, the novel isolate represents a novel species, for which the name Chryseobacterium camelliae sp. nov. is proposed, with THG C4-1T (5KACC 16985T5JCM 18745T) as the type strain.
The genus Chryseobacterium (family Flavobacteriaceae, phylum Bacteroidetes) was described by Vandamme et al. (1994). The description of the genus has since been emended by Tai et al. (2006), Weon et al. (2008) and Ka¨mpfer et al. (2009). The common characteristics of the genus Chryseobacterium include Gram-negative-staining reaction, flexirubin-type pigments, menaquinone 6 (MK-6) as the major predominant respiratory quinone, branched-chain fatty acids iso-C15 : 0, iso-C17 : 0 3-OH and iso-C17 : 1v9c as the major fatty acids, sym-homospermidine as the major polyamine and a DNA G+C content of 29–39 mol% (Vandamme et al., 1994; Ka¨mpfer et al., 2009; Bernardet et al., 2010). The membership of the genus has been growing rapidly, as demonstrated by the multitude of novel species that have been proposed in recent years. At the time of writing, the genus Chryseobacterium contains 72 species isolated from various environments such as soils (Weon et al., 2008), fresh water (Strahan et al., 2011), sewage and 3These authors equally contributed to this work. The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain THG C4-1T is JX843771. Three supplementary figures are available with the online version of this paper.
057398 G 2014 IUMS
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wastewater (Ka¨mpfer et al., 2003), compost (Ka¨mpfer et al., 2010), the plant rhizosphere (Park et al., 2006), diseased fish (Bernardet et al., 2005), meat (de Beer et al., 2005) and dairy environments (Hugo et al., 2003). In this study, we report the taxonomic characterization of a novel member of the genus Chryseobacterium, strain THG C4-1T. The taxonomic position of strain THG C4-1T was determined precisely by using a polyphasic approach. Strain THG C4-1T was isolated from leaves of green tea (Camellia sinensis) at Jangheung-gun (34.65–34.68u N 126.88–126.93u E) in the Republic of Korea. Green tea leaves were collected under aseptic conditions and 1 g leaves was suspended in 10 ml sterile 0.85 % (w/v) NaCl and stirred for 30 min. From this suspension, serially diluted samples were spread onto nutrient agar (NA; Oxoid). The plates were incubated at 28 uC for a week. Single colonies were purified by transferring them onto fresh medium plates and were incubated once again on NA. One isolate, THG C4-1T, was cultured routinely on NA at 28 uC and preserved as a suspension in nutrient broth (NB; Oxoid) with glycerol (25 %, w/v) at 270 uC. Extraction of genomic DNA was achieved using a commercial genomic DNA extraction kit (Solgent). The 16S rRNA gene was amplified from chromosomal DNA with 851
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0.02
Chryseobacterium daecheongense CPW406T (AJ457206) Chryseobacterium wajuense R2A10-2T (DQ256729) Chryseobacterium defluvii B2T (AJ309324) Chryseobacterium gambrini 5-1St1aT (AM232810) Chryseobacterium camelliae THG C4-1T (JX843771) Chryseobacterium taiwanense BCRC 17412T (DQ318789) Chryseobacterium taihuense THMBM1T (JQ283114) Chryseobacterium taeanense PHA3-4T (AY883416) Chryseobacterium taichungense CC-TWGS1T (AJ843132) 93 Chryseobacterium hagamense RHA2-9T (DQ673672) Chryseobacterium rigui CJ16T (JQ071497) Chryseobacterium daeguense K105T (EF076759) Chryseobacterium gregarium P461/12T (AM773820) 100 Chryseobacterium aquaticum 10-46T (AM748690) 76 Chryseobacterium greenlandense UMB34T (FJ932652) Chryseobacterium soli JS6-6T (EF591302) Chryseobacterium ginsenosidimutants THG 15T (GU138380) 70 Chryseobacterium ginsengisoli DCY 63T (JN852949) 98 Chryseobacterium indoltheticum LMG 4025T (AY468448) Chryseobacterium rhizosphearae RSB3-1T (DQ673670) Chryseobacterium vietnamense GIMN1.005T (HM212415) Chryseobacterium jejuense JS17-8T (EF591303) Chryseobacterium culicis R4-1AT (FN554975) Chryseobacterium indologense LMG 8337T (AM232813) 97 Chryseobacterium visecerum 687B-08T (FR871426) Chryseobacterium ureilyticum F-Fue-04IIIaaaatT (AM232806) 73 Chryseobacterium oncorhynchi 701B-08T (FN674441) 82 Chryseobacterium pallidum 26-3St2bT (AM232809) Chryseobacterium hominis NF802T (AM261868) 72 Chryseobacterium molle DW3T (AJ534853) 73 Chryseobacterium hungaricum CHB-20pT (EF685359) Flavobacterium haoranii LQY-7T (GQ988780)
Fig. 1. Neighbour-joining phylogenetic tree based on 16S rRNA gene sequences showing the relationships of strain THG C41T and related members of the genus Chryseobacterium. Filled circles at nodes indicate branches that were also recovered by using the maximum-parsimony algorithm. Bootstrap values (expressed as percentages of 1 000 replications) over 70 % are shown at branching points. Flavobacterium haoranii LQY-7T was used as an outgroup. Bar, 0.02 substitutions per nucleotide position.
the universal bacterial primer pair 27F and 1492R (Weisburg et al., 1991) and the purified PCR products were sequenced by Solgent (Daejeon, Korea). The 16S rRNA gene sequences of related taxa were obtained from the GenBank database and EzTaxon-e server (Kim et al., 2012). Multiple alignments were performed by using the CLUSTAL_X program (Thompson et al., 1997) and gaps were edited in the BioEdit program (Hall, 1999). Evolutionary distances were calculated using Kimura’s two-parameter model (Kimura, 1983). Phylogenetic trees were reconstructed using the neighbour-joining method (Saitou & Nei, 1987) and the maximum-likelihood method in the MEGA5 program (Kumar et al., 2008), with bootstrap values based on 1000 replications (Felsenstein, 1985).
taeanense PHA3-4T (97.0 %) and C. daeguense K105T (97.0 %). Lower sequence similarity (,97.0 %) was found to other recognized species of the family Flavobacteriaceae. This relationship between strain THG C4-1T and other members of the genus Chryseobacterium was also evident in the phylogenetic tree (Fig. 1 and Fig. S1, available in the online Supplementary Material).
The 16S rRNA gene sequence of strain THG C4-1T determined in this study was a continuous stretch of 1411 bp. Sequence similarity indicated that the closest relatives of strain THG C4-1T were Chryseobacterium taiwanense Soil-3-27T (97.7 % sequence similarity), C. hagamense RHA2-9T (97.2 %), C. gregarium P 461/12T (97.2 %), C. ginsenosidimutans THG 15T (97.1 %), C.
Gram-staining was determined using the bioMe´rieux Gram stain kit according to the manufacturer’s instructions. Anaerobic growth was tested in serum bottles containing NB supplemented with thioglycolate (1 g l–1), in which the air was replaced with nitrogen gas. Cell morphology and motility were observed at 61 000 magnification, with a light microscope (BX50; Olympus) using cells grown for 24 h at 28 uC on NA. Motility was assayed on sulfideindole motility (SIM) medium (Difco). Growth was tested using several bacterial culture media including NA, tryptone soya agar (TSA; Oxoid), R2A agar (Difco), Luria– Bertani (LB) agar (Oxoid) and MacConkey agar (Oxoid) at 28 uC. Growth at 4, 10, 15, 20, 25, 28, 30, 37, 40 and 45 uC and at pH 5.0–10.0 (at intervals of 0.5 pH units) was
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assessed in NB after 4 days of incubation at 28 uC. For the pH experiments, two different buffers were used (final concentration, 100 mM): acetate buffer was used for pH 5.0–6.5 and phosphate buffer was used for pH 7.0– 10.0. The pH was confirmed after autoclaving. Salt tolerance was tested in NB supplemented with 0–5.0 % (w/v) NaCl (at 0.5 % intervals) after 4 days of incubation at 28 uC. Growth was estimated by monitoring the OD600. Production of flexirubin-type pigments was determined by the reversible colour shift to red, purple or brown when yellow or orange colonies are covered with aqueous 20 % KOH solution (Fautz & Reichenbach, 1980). Catalase activity was determined by bubble production in 3 % (v/ v) H2O2 and oxidase activity was determined using 1 % (w/ v) N,N,N9,N9-tetramethyl 1,4-phenylenediamine reagent. Tests for degradation of DNA [DNase agar, Oxoid; DNase activity revealed by flooding the plates with 1 M HCl], casein [on NA supplemented with 2 % skimmed milk (Oxoid)], starch [on NA containing 1 % starch (Difco)], aesculin (bile aesculin agar; Difco) and Tween 80 [on NA containing 0.01 % CaCl2 . 2H2O and 1 % Tween 80 (Sigma)] were evaluated after 4 days of incubation at 28 uC. Carbon-source utilization and enzyme activities were tested by using API 20NE, ID 32 GN and API ZYM test kits according to the instructions of the manufacturer (bioMe´rieux). Cells of strain THG C4-1T were Gram-staining-negative, strictly aerobic, non-spore-forming, non-motile, rodshaped and oxidase- and catalase-positive. Colonies grown on NA plates for 2 days were smooth, circular, yellow and convex, 0.5–1.0 mm in diameter. The isolate also grew on NA, TSA, LB agar and R2A agar but not on MacConkey agar. Growth occurred at 4–37 uC (optimum 20–30 uC), at pH 5.5–8.0 (optimum pH 7.0–7.5) and at 0–3.0 % (w/v) NaCl. The isolate could hydrolyse starch, casein, aesculin, Tween 80 and DNA. Physiological characteristics of strain THG C4-1T are summarized in the species description and a comparison of selective characteristics of strain THG C41T and related type strains is given in Table 1. For determination of the DNA G+C content, genomic DNA was extracted, purified as described by Moore & Dowhan (1995) and degraded enzymically into nucleosides. The nucleosides were analysed using reversed-phase HPLC system (Alliance 2690 system; Waters) as described previously (Mesbah et al., 1989) with a reversed-phase SunFire C18 column (4.66250 mm65 mm), flow rate 1.0 ml min21, a solvent mixture of 200 mM (NH4)H2PO4/ acetonitrile (97 : 3, v/v) as mobile phase and detection at 270 nm. DNA–DNA hybridization was performed fluorometrically, according to the method developed by Ezaki et al. (1989), using photobiotin-labelled DNA probes and microdilution wells. DNA–DNA hybridization experiments were performed between strain THG C4-1T and closely related type strains of the genus Chryseobacterium. Optimum hybridization temperature was 41.4 uC. Hybridization was performed with five replications for each sample; the highest and lowest values obtained for each sample were http://ijs.sgmjournals.org
excluded and the means of the remaining three values were converted to percentage DNA–DNA relatedness values. The DNA G+C content of strain THG C4-1T was 41.7 mol%, which lies close to the expected range of G+C contents of members of the genus Chryseobacterium. In this study, DNA–DNA relatedness between strain THG C4-1T and the type strains of other Chryseobacterium species was in the range 13–21 % (Chryseobacterium taiwanense KACC 12985T 18.9±0.5 %, C. hagamense KCTC 22545T, 17.7 %, C. gregarium KACC 13435T 13.4±0.9 %, C. ginsenosidimutans KACC 14527T 17.8±0.5 %, C. taeanense KACC 15162T 15.1±0.2 % and C. daeguense KACC 17428T 20.5±0.4 %). These very low DNA relatedness values suggested that THG C4-1T represents a novel species of the genus Chryseobacterium (Wayne et al., 1987). The polar lipids of the strain were extracted from freezedried cells grown on NA for 48 h at 28 uC (Minnikin et al., 1977, 1984). Polar lipids were examined by two-dimensional TLC using Kieselgel 60 F254 plates (10610 cm; Merck). Chromatograms were developed in the first dimension with chloroform/methanol/water (65 : 25 : 4, by vol.) and in the second dimension with chloroform/ methanol/acetic acid/water (80 : 12 : 15 : 4, by vol.) as solvent systems. For detection of total lipids, TLC plates were sprayed with 5 % molybdophosphoric acid followed by charring at 120 uC for 15 min. Aminolipids were detected by spraying with 0.2 % ninhydrin and charring at 120 uC for 5 min. Phospholipids were detected by spraying with molybdenum blue reagent at room temperature. The cellular fatty acid profile was determined for strain THG C4-1T grown on NA for 2 days at 28 uC. The cellular fatty acids were saponified, methylated and extracted according to the protocol of the Sherlock Microbial Identification System (MIDI). Fatty acids were analysed by using a gas chromatograph (Hewlett Packard 6890) and the Sherlock aerobic bacterial database (TSBA60) (Sasser, 1990). The isoprenoid quinones of strain THG C4-1T were isolated from freeze-dried cells. Menaquinones were extracted with chloroform/methanol (2 : 1, v/v), evaporated under vacuum conditions and re-extracted in n-hexane. The crude hexane–quinone solution was purified using Sep-Pak Vac silica cartridges (Waters) and subsequently analysed by reversed-phase HPLC (Alliance 2690 system; Waters) [solvent; methanol/2-propanol (7 : 5, v/v), flow rate; 1.0 ml min21] as described previously (Hiraishi et al., 1996; Collins & Jones, 1981; Tamaoka et al., 1983). The polyamines of strain THG C4-1T were extracted as described by Busse & Auling (1988) and Taibi et al. (2000). Polyamines were extracted from approximately 100 mg freeze-dried biomass with 2 ml 0.2 M HClO4 at 100 uC for 30 min with occasional shaking. Each mixture contained the internal standard l,8-diamino-octane (10 mmol per 100 mg cells). Samples were analysed using a Waters Alliance 2690 HPLC fitted with a reversed-phase column (Watchers 120 ODS-AP 4.66250 mm65 mm) and using an analyte flow rate of 1.0 ml min21, detection at 234 nm and 60 % methanol as mobile phase. 853
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Table 1. Physiological characteristics of strain THG C4-1T and related type strains of species of the genus Chryseobacterium Strains: 1, THG C4-1T; 2, C. taiwanense Soil-3-27T; 3, C. hagamense RHA2-9T; 4, C. gregarium P 461/12T; 5, C. ginsenosidimutans THG 15T; 6, C. taeanense PHA3-4T; 7, C. daeguense K105T. Data for reference strains were taken from Tai et al. (2006), Cho et al. (2010), Behrendt et al. (2008), Im et al. (2011), Yoon et al. (2007) and Park et al. (2006). All species are positive for a-glucosidase, assimilation of maltose (not reported for C. taiwanense) and hydrolysis of starch, casein (2 % skimmed milk), Tween 80, aesculin and gelatin. All strains except C. taeanense PHA3-4T are positive for alkaline phosphatase, esterase lipase (C8), leucine arylamidase, valine arylamidase, acid phosphatase (not reported for C. taiwanense, C. hagamense or C. daeguense) and naphthol-AS-BI-phosphohydrolase. All strains are negative for lipase (C14), b-glucuronidase, a-fucosidase, amannosidase (not reported for C. taeanense), reduction of nitrates to nitrogen (not reported for C. ginsenosidimutans) and assimilation of N-acetylglucosamine (not reported for C. taiwanense). All strains produce yellow pigments. No growth on MacConkey agar (except C. taiwanense). +, Positive; W, weakly positive; 2, negative; ND, no data available. Characteristic Catalase Oxidase Growth temperature (uC) pH for growth Growth with NaCl (%, w/v) Indole production Nitrate reduction Hydrolysis of: Urea DNA Arginine dihydrolase Assimilation of: Histidine D-Glucose L-Arabinose D-Mannose D-Mannitol Gluconate Citrate Histidine L-Rhamnose Sucrose Enzyme activity Esterase (C4) Cystine arylamidase Trypsin a-Chymotrypsin a-Galactosidase b-Galactosidase b-Glucosidase N-Acetyl-b-glucosaminidase DNA G+C content (mol%)
1 + + 4–37 5.5–8.0 0–3.0 + 2 2 + 2 + + + 2 2 2 2 + 2 + + + + + 2 W
+ + 41.7
2 + + 5–42 5–10.0 0–4.0 + +
3 ND ND
10–37 5.0–10.0 0–2.0 2 2
4 + + 4–30 ND
0–2.0 2 2
5
6
ND
ND
10–37 5.5–10.0 0–1.0 2 2
+ 5–35 5.0–9.0 0–6.0 2 2
+ 2 +
2 2 2 2 + + + + + 2 + + + 2 2 2 2 2 + 2 32.1
ND
2 +
2 +
ND
ND
ND
ND
ND
2 + + + 2 + + 2 + +
ND
2 + + + 2 2 2 2 2 2
2 2 2 2 2 2 2 2 36.8
+ + 2 2 + + + + 36.9
2 2 2 2 2 2 + + 38.4
+ 2 2 2 2 2 + + 35.7
ND ND ND ND ND ND ND ND
ND
2 2
2 2 2 2 + 2 ND ND
W
7 + + 10–37 5.0–9.5 0–2.0 2 2 2 W
2 ND
+ + + 2 ND ND ND
+ 2 + + 2 2 + W
2 + 36.8
The polar lipid profile of strain THG C4-1T is shown in Fig. S2. The polar lipids were phosphatidylethanolamine and two unidentified aminolipids (AL1 and AL3); significant amounts of unidentified aminolipids and unidentified lipids were also present. The fatty acid profiles of this strain and type strains of related species of the genus Chryseobacterium are shown in Table 2. The major cellular fatty acids were iso-C15 : 0 (48.7 %), iso-C17 : 0 3-OH (17.4 %) and iso-C17 : 1v9c (7.8 %). The presence of large amounts of iso-C15 : 0, iso-C17 : 0 3-OH and iso-C17 : 1v9c in particular is typical for the genus Chryseobacterium (Park et al., 2006; Behrendt et al., 2008). The only respiratory
quinone detected in strain THG C4-1T was MK-6, in line with all other members of the family Flavobacteriaceae. The major polyamine was sym-homospermidine (Fig. S3). Unlike eukaryotes, where the predominant polyamine is spermidine, bacteria may instead contain an alternative polyamine, sym-homospermidine. Although the role of polyamines in the bacterial cell is not entirely clear, they seem to be important in bacterial metabolism (Vandamme et al., 1996; Prakash et al., 2007). In summary, the characteristics of strain THG C4-1T were consistent with descriptions of the genus Chryseobacterium with regard to morphological, biochemical and
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Table 2. Cellular fatty acid profiles of strain THG C4-1T and type strains of phylogenetically related species of the genus Chryseobacterium Strains: 1, THG C4-1T; 2, C. taiwanense Soil-3-27T; 3, C. hagamense RHA2-9T; 4, C. gregarium P 461/12T; 5, C. ginsenosidimutans THG 15T; 6, C. taeanense PHA3-4T; 7, C. daeguense K105T. Data for reference strains were taken from Tai et al. (2006), Cho et al. (2010), Behrendt et al. (2008), Im et al. (2011), Yoon et al. (2007) and Park et al. (2006). Cells of strain THG C4-1T were cultured on NA for 2 days at 28 uC. Data are percentages of total fatty acids. Fatty acids accounting for less than 0.5 % of the total fatty acids in all strains are not listed. ND, Not detected/not reported; TR, traces (,1.0 %). Fatty acid Saturated C16 : 0 Unsaturated C18 : 1v5c iso-C17 : 1v9c Branched-chain iso-C13 : 0 iso-C15 : 0 iso-C16 : 0 iso-C17 : 0 anteiso-C15 : 0 iso-C15 : 0 3-OH iso-C16 : 0 3-OH iso-C17 : 0 3-OH Hydroxy C16 : 0 3-OH C17 : 0 2-OH Summed features* Summed feature 3
1
2
3
1.7
1.4
2.8
TR
ND
7.8 17.5
4
5
6
7
ND
ND
1.8
1.5
ND
7.2
1.2
ND
TR
ND
ND
9.3 15.8 19.8
1.4 1.0 1.2 1.3 ND TR 1.2 48.7 43.2 24.1 35.1 50.3 36.1 32.8 TR ND 1.0 TR ND ND ND TR 1.3 ND TR ND 1.1 ND 5.0 1.0 2.5 9.1 3.8 1.1 2.0 4.0 3.8 3.9 2.8 5.2 3.1 3.2 TR ND 3.9 TR ND TR ND 17.4 16.6 13.8 10.0 21.9 18.8 16.7 1.0 1.0
TR
1.8
1.2
ND
TR
1.2
TR
ND
TR
ND
ND
ND
6.6
7.9 12.2
ND
ND
11.2 10.9
*Summed features represent groups of two or three fatty acids that could not be separated by GLC with the MIDI system. Summed feature 3 contained C16 : 1v7c and/or iso-C15 : 0 2-OH.
chemotaxonomic properties. On the basis of phylogenetic distances between strain THG C4-1T and recognized species of the genus Chryseobacterium indicated by 16S rRNA gene sequence similarity, the combination of unique phenotypic characteristics (Table 1) and low levels of DNA–DNA relatedness, it is demonstrable that strain THG C4-1T should be assigned to the genus Chryseobacterium as the type strain of a novel species, for which the name Chryseobacterium camelliae sp. nov. is proposed. Description of Chryseobacterium camelliae sp. nov. Chryseobacterium camelliae (ca.mel9li.ae. N.L. gen. n. camelliae of the tea plant, Camellia sinensis). Cells are a Gram-staining-negative, strictly aerobic, nonmotile, non-spore-forming and rod-shaped. After culture on NA for 2 days, colonies are smooth, circular, yellow and http://ijs.sgmjournals.org
convex, 0.5–1.0 mm in diameter. Grows on NA at 4–37 uC and at pH 5.5–8.0, but not at 40 uC. Optimum growth occurs at 20–30 uC and at pH 7.0–7.5. Growth occurs in the absence of NaCl and in the presence of 0.5–3.0 % (w/v) NaCl, but not 3.5 % (w/v) NaCl. Catalase and oxidase activities are positive. In API ZYM tests, positive for alkaline phosphatase, esterase (C4), esterase lipase (C8), leucine arylamidase, valine arylamidase, cystine arylamidase, trypsin, a-chymotrypsin, acid phosphatase, naphtholAS-BI-phosphohydrolase, a-glucosidase, b-glucosidase and N-acetyl-b-glucosaminidase; weakly positive for b-galactosidase and negative for lipase (C14), a-galactosidase, bglucuronidase, a-mannosidase and a-fucosidase. According to API 20NE tests, positive results are obtained for indole production and gelatin hydrolysis; weakly positive for bgalactosidase (PNPG) activity and negative for arginine dihydrolase, urease, nitrate reduction, glucose acidification and assimilation of D-glucose, L-arabinose, D-mannose, Dmannitol, N-acetylglucosamine, maltose, gluconate, caprate, adipate, malate, citrate and phenylacetate. According to the API ID32 GN test, positive results are obtained for utilization of L-histidine, sucrose and glycogen; weakly positive results are obtained with salicin and L-proline and negative results are obtained with melibiose, L-fucose, D-sorbitol, propionate, valerate, 2-ketogluconate, 3-hydroxybutyrate, 4-hydroxybenzoate, L-rhamnose, N-acetylglucosamine, D-ribose, inositol, itaconate, suberate, malonate, acetate, lactate, L-alanine, 5-ketogluconate, 3-hydroxybenzoate and L-serine. The predominant menaquinone is MK-6, and iso-C15 : 0, iso-C17 : 0 3-OH and iso-C17 : 1v9c are the major cellular fatty acids (.7 %). The major polar lipid is phosphatidylethanolamine. The polyamine is symhomospermidine. The type strain, THG C4-1T (5KACC 14527T5JCM 18745T), was isolated from green tea leaves at Jangheunggun in the Republic of Korea. The G+C content of genomic DNA of the type strain is 41.7 mol%.
Acknowledgements This work was supported by a grant from Kyung Hee University, Republic of Korea, in 2012 (20120596).
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DOI 10.1099/ijs.0.057398-0
Chryseobacterium camelliae sp. nov., isolated from green tea MooChang Kook,13 Heung-Min Son,23 Hien T. T. Ngo2 and Tae-Hoo Yi2 Correspondence
1
Tae-Hoo Yi
2
[email protected]
Department of Marine Biotechnology, Anyang University, Incheon 417-833, Republic of Korea Department of Oriental Medicinal Material & Processing College of Life Science, Kyung Hee University, 1 Seocheon, Kihung Yongin, Kyunggi 446-701, Republic of Korea
A Gram-staining-negative, strictly aerobic, non-motile, rod-shaped and flexirubin-type-pigmented strain, THG C4-1T, was isolated from green tea leaves in Jangheung-gun, Republic of Korea. Strain THG C4-1T grew well at 20–30 6C, at pH 7.0–7.5 and in the absence of NaCl on nutrient agar. Based on 16S rRNA gene sequence comparisons, strain THG C4-1T was most closely related to Chryseobacterium taiwanense Soil-3-27T (97.7 %), C. hagamense RHA2-9T (97.2 %), C. gregarium P 461/12T (97.2 %), C. ginsenosidimutans THG 15T (97.1 %), C. taeanense PHA3-4T (97.0 %) and C. daeguense K105T (97.0 %), but DNA–DNA relatedness between strain THG C4-1T and its closest phylogenetic neighbours was below 21 %. The DNA G+C content was 41.7 mol%. The only isoprenoid quinone detected in strain THG C4-1T was menaquinone 6 (MK-6). The major component of the polyamine pattern was sym-homospermidine. The major polar lipids were phosphatidylethanolamine and unidentified aminolipids. The major fatty acids were iso-C15 : 0, iso-C17 : 0 3-OH and iso-C17 : 1v9c. These data supported the affiliation of strain THG C4-1T to the genus Chryseobacterium. The results of physiological and biochemical tests enabled strain THG C4-1T to be differentiated genotypically and phenotypically from recognized species of the genus Chryseobacterium. Therefore, the novel isolate represents a novel species, for which the name Chryseobacterium camelliae sp. nov. is proposed, with THG C4-1T (5KACC 16985T5JCM 18745T) as the type strain.
The genus Chryseobacterium (family Flavobacteriaceae, phylum Bacteroidetes) was described by Vandamme et al. (1994). The description of the genus has since been emended by Tai et al. (2006), Weon et al. (2008) and Ka¨mpfer et al. (2009). The common characteristics of the genus Chryseobacterium include Gram-negative-staining reaction, flexirubin-type pigments, menaquinone 6 (MK-6) as the major predominant respiratory quinone, branched-chain fatty acids iso-C15 : 0, iso-C17 : 0 3-OH and iso-C17 : 1v9c as the major fatty acids, sym-homospermidine as the major polyamine and a DNA G+C content of 29–39 mol% (Vandamme et al., 1994; Ka¨mpfer et al., 2009; Bernardet et al., 2010). The membership of the genus has been growing rapidly, as demonstrated by the multitude of novel species that have been proposed in recent years. At the time of writing, the genus Chryseobacterium contains 72 species isolated from various environments such as soils (Weon et al., 2008), fresh water (Strahan et al., 2011), sewage and 3These authors equally contributed to this work. The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain THG C4-1T is JX843771. Three supplementary figures are available with the online version of this paper.
057398 G 2014 IUMS
Printed in Great Britain
wastewater (Ka¨mpfer et al., 2003), compost (Ka¨mpfer et al., 2010), the plant rhizosphere (Park et al., 2006), diseased fish (Bernardet et al., 2005), meat (de Beer et al., 2005) and dairy environments (Hugo et al., 2003). In this study, we report the taxonomic characterization of a novel member of the genus Chryseobacterium, strain THG C4-1T. The taxonomic position of strain THG C4-1T was determined precisely by using a polyphasic approach. Strain THG C4-1T was isolated from leaves of green tea (Camellia sinensis) at Jangheung-gun (34.65–34.68u N 126.88–126.93u E) in the Republic of Korea. Green tea leaves were collected under aseptic conditions and 1 g leaves was suspended in 10 ml sterile 0.85 % (w/v) NaCl and stirred for 30 min. From this suspension, serially diluted samples were spread onto nutrient agar (NA; Oxoid). The plates were incubated at 28 uC for a week. Single colonies were purified by transferring them onto fresh medium plates and were incubated once again on NA. One isolate, THG C4-1T, was cultured routinely on NA at 28 uC and preserved as a suspension in nutrient broth (NB; Oxoid) with glycerol (25 %, w/v) at 270 uC. Extraction of genomic DNA was achieved using a commercial genomic DNA extraction kit (Solgent). The 16S rRNA gene was amplified from chromosomal DNA with 851
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0.02
Chryseobacterium daecheongense CPW406T (AJ457206) Chryseobacterium wajuense R2A10-2T (DQ256729) Chryseobacterium defluvii B2T (AJ309324) Chryseobacterium gambrini 5-1St1aT (AM232810) Chryseobacterium camelliae THG C4-1T (JX843771) Chryseobacterium taiwanense BCRC 17412T (DQ318789) Chryseobacterium taihuense THMBM1T (JQ283114) Chryseobacterium taeanense PHA3-4T (AY883416) Chryseobacterium taichungense CC-TWGS1T (AJ843132) 93 Chryseobacterium hagamense RHA2-9T (DQ673672) Chryseobacterium rigui CJ16T (JQ071497) Chryseobacterium daeguense K105T (EF076759) Chryseobacterium gregarium P461/12T (AM773820) 100 Chryseobacterium aquaticum 10-46T (AM748690) 76 Chryseobacterium greenlandense UMB34T (FJ932652) Chryseobacterium soli JS6-6T (EF591302) Chryseobacterium ginsenosidimutants THG 15T (GU138380) 70 Chryseobacterium ginsengisoli DCY 63T (JN852949) 98 Chryseobacterium indoltheticum LMG 4025T (AY468448) Chryseobacterium rhizosphearae RSB3-1T (DQ673670) Chryseobacterium vietnamense GIMN1.005T (HM212415) Chryseobacterium jejuense JS17-8T (EF591303) Chryseobacterium culicis R4-1AT (FN554975) Chryseobacterium indologense LMG 8337T (AM232813) 97 Chryseobacterium visecerum 687B-08T (FR871426) Chryseobacterium ureilyticum F-Fue-04IIIaaaatT (AM232806) 73 Chryseobacterium oncorhynchi 701B-08T (FN674441) 82 Chryseobacterium pallidum 26-3St2bT (AM232809) Chryseobacterium hominis NF802T (AM261868) 72 Chryseobacterium molle DW3T (AJ534853) 73 Chryseobacterium hungaricum CHB-20pT (EF685359) Flavobacterium haoranii LQY-7T (GQ988780)
Fig. 1. Neighbour-joining phylogenetic tree based on 16S rRNA gene sequences showing the relationships of strain THG C41T and related members of the genus Chryseobacterium. Filled circles at nodes indicate branches that were also recovered by using the maximum-parsimony algorithm. Bootstrap values (expressed as percentages of 1 000 replications) over 70 % are shown at branching points. Flavobacterium haoranii LQY-7T was used as an outgroup. Bar, 0.02 substitutions per nucleotide position.
the universal bacterial primer pair 27F and 1492R (Weisburg et al., 1991) and the purified PCR products were sequenced by Solgent (Daejeon, Korea). The 16S rRNA gene sequences of related taxa were obtained from the GenBank database and EzTaxon-e server (Kim et al., 2012). Multiple alignments were performed by using the CLUSTAL_X program (Thompson et al., 1997) and gaps were edited in the BioEdit program (Hall, 1999). Evolutionary distances were calculated using Kimura’s two-parameter model (Kimura, 1983). Phylogenetic trees were reconstructed using the neighbour-joining method (Saitou & Nei, 1987) and the maximum-likelihood method in the MEGA5 program (Kumar et al., 2008), with bootstrap values based on 1000 replications (Felsenstein, 1985).
taeanense PHA3-4T (97.0 %) and C. daeguense K105T (97.0 %). Lower sequence similarity (,97.0 %) was found to other recognized species of the family Flavobacteriaceae. This relationship between strain THG C4-1T and other members of the genus Chryseobacterium was also evident in the phylogenetic tree (Fig. 1 and Fig. S1, available in the online Supplementary Material).
The 16S rRNA gene sequence of strain THG C4-1T determined in this study was a continuous stretch of 1411 bp. Sequence similarity indicated that the closest relatives of strain THG C4-1T were Chryseobacterium taiwanense Soil-3-27T (97.7 % sequence similarity), C. hagamense RHA2-9T (97.2 %), C. gregarium P 461/12T (97.2 %), C. ginsenosidimutans THG 15T (97.1 %), C.
Gram-staining was determined using the bioMe´rieux Gram stain kit according to the manufacturer’s instructions. Anaerobic growth was tested in serum bottles containing NB supplemented with thioglycolate (1 g l–1), in which the air was replaced with nitrogen gas. Cell morphology and motility were observed at 61 000 magnification, with a light microscope (BX50; Olympus) using cells grown for 24 h at 28 uC on NA. Motility was assayed on sulfideindole motility (SIM) medium (Difco). Growth was tested using several bacterial culture media including NA, tryptone soya agar (TSA; Oxoid), R2A agar (Difco), Luria– Bertani (LB) agar (Oxoid) and MacConkey agar (Oxoid) at 28 uC. Growth at 4, 10, 15, 20, 25, 28, 30, 37, 40 and 45 uC and at pH 5.0–10.0 (at intervals of 0.5 pH units) was
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assessed in NB after 4 days of incubation at 28 uC. For the pH experiments, two different buffers were used (final concentration, 100 mM): acetate buffer was used for pH 5.0–6.5 and phosphate buffer was used for pH 7.0– 10.0. The pH was confirmed after autoclaving. Salt tolerance was tested in NB supplemented with 0–5.0 % (w/v) NaCl (at 0.5 % intervals) after 4 days of incubation at 28 uC. Growth was estimated by monitoring the OD600. Production of flexirubin-type pigments was determined by the reversible colour shift to red, purple or brown when yellow or orange colonies are covered with aqueous 20 % KOH solution (Fautz & Reichenbach, 1980). Catalase activity was determined by bubble production in 3 % (v/ v) H2O2 and oxidase activity was determined using 1 % (w/ v) N,N,N9,N9-tetramethyl 1,4-phenylenediamine reagent. Tests for degradation of DNA [DNase agar, Oxoid; DNase activity revealed by flooding the plates with 1 M HCl], casein [on NA supplemented with 2 % skimmed milk (Oxoid)], starch [on NA containing 1 % starch (Difco)], aesculin (bile aesculin agar; Difco) and Tween 80 [on NA containing 0.01 % CaCl2 . 2H2O and 1 % Tween 80 (Sigma)] were evaluated after 4 days of incubation at 28 uC. Carbon-source utilization and enzyme activities were tested by using API 20NE, ID 32 GN and API ZYM test kits according to the instructions of the manufacturer (bioMe´rieux). Cells of strain THG C4-1T were Gram-staining-negative, strictly aerobic, non-spore-forming, non-motile, rodshaped and oxidase- and catalase-positive. Colonies grown on NA plates for 2 days were smooth, circular, yellow and convex, 0.5–1.0 mm in diameter. The isolate also grew on NA, TSA, LB agar and R2A agar but not on MacConkey agar. Growth occurred at 4–37 uC (optimum 20–30 uC), at pH 5.5–8.0 (optimum pH 7.0–7.5) and at 0–3.0 % (w/v) NaCl. The isolate could hydrolyse starch, casein, aesculin, Tween 80 and DNA. Physiological characteristics of strain THG C4-1T are summarized in the species description and a comparison of selective characteristics of strain THG C41T and related type strains is given in Table 1. For determination of the DNA G+C content, genomic DNA was extracted, purified as described by Moore & Dowhan (1995) and degraded enzymically into nucleosides. The nucleosides were analysed using reversed-phase HPLC system (Alliance 2690 system; Waters) as described previously (Mesbah et al., 1989) with a reversed-phase SunFire C18 column (4.66250 mm65 mm), flow rate 1.0 ml min21, a solvent mixture of 200 mM (NH4)H2PO4/ acetonitrile (97 : 3, v/v) as mobile phase and detection at 270 nm. DNA–DNA hybridization was performed fluorometrically, according to the method developed by Ezaki et al. (1989), using photobiotin-labelled DNA probes and microdilution wells. DNA–DNA hybridization experiments were performed between strain THG C4-1T and closely related type strains of the genus Chryseobacterium. Optimum hybridization temperature was 41.4 uC. Hybridization was performed with five replications for each sample; the highest and lowest values obtained for each sample were http://ijs.sgmjournals.org
excluded and the means of the remaining three values were converted to percentage DNA–DNA relatedness values. The DNA G+C content of strain THG C4-1T was 41.7 mol%, which lies close to the expected range of G+C contents of members of the genus Chryseobacterium. In this study, DNA–DNA relatedness between strain THG C4-1T and the type strains of other Chryseobacterium species was in the range 13–21 % (Chryseobacterium taiwanense KACC 12985T 18.9±0.5 %, C. hagamense KCTC 22545T, 17.7 %, C. gregarium KACC 13435T 13.4±0.9 %, C. ginsenosidimutans KACC 14527T 17.8±0.5 %, C. taeanense KACC 15162T 15.1±0.2 % and C. daeguense KACC 17428T 20.5±0.4 %). These very low DNA relatedness values suggested that THG C4-1T represents a novel species of the genus Chryseobacterium (Wayne et al., 1987). The polar lipids of the strain were extracted from freezedried cells grown on NA for 48 h at 28 uC (Minnikin et al., 1977, 1984). Polar lipids were examined by two-dimensional TLC using Kieselgel 60 F254 plates (10610 cm; Merck). Chromatograms were developed in the first dimension with chloroform/methanol/water (65 : 25 : 4, by vol.) and in the second dimension with chloroform/ methanol/acetic acid/water (80 : 12 : 15 : 4, by vol.) as solvent systems. For detection of total lipids, TLC plates were sprayed with 5 % molybdophosphoric acid followed by charring at 120 uC for 15 min. Aminolipids were detected by spraying with 0.2 % ninhydrin and charring at 120 uC for 5 min. Phospholipids were detected by spraying with molybdenum blue reagent at room temperature. The cellular fatty acid profile was determined for strain THG C4-1T grown on NA for 2 days at 28 uC. The cellular fatty acids were saponified, methylated and extracted according to the protocol of the Sherlock Microbial Identification System (MIDI). Fatty acids were analysed by using a gas chromatograph (Hewlett Packard 6890) and the Sherlock aerobic bacterial database (TSBA60) (Sasser, 1990). The isoprenoid quinones of strain THG C4-1T were isolated from freeze-dried cells. Menaquinones were extracted with chloroform/methanol (2 : 1, v/v), evaporated under vacuum conditions and re-extracted in n-hexane. The crude hexane–quinone solution was purified using Sep-Pak Vac silica cartridges (Waters) and subsequently analysed by reversed-phase HPLC (Alliance 2690 system; Waters) [solvent; methanol/2-propanol (7 : 5, v/v), flow rate; 1.0 ml min21] as described previously (Hiraishi et al., 1996; Collins & Jones, 1981; Tamaoka et al., 1983). The polyamines of strain THG C4-1T were extracted as described by Busse & Auling (1988) and Taibi et al. (2000). Polyamines were extracted from approximately 100 mg freeze-dried biomass with 2 ml 0.2 M HClO4 at 100 uC for 30 min with occasional shaking. Each mixture contained the internal standard l,8-diamino-octane (10 mmol per 100 mg cells). Samples were analysed using a Waters Alliance 2690 HPLC fitted with a reversed-phase column (Watchers 120 ODS-AP 4.66250 mm65 mm) and using an analyte flow rate of 1.0 ml min21, detection at 234 nm and 60 % methanol as mobile phase. 853
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Table 1. Physiological characteristics of strain THG C4-1T and related type strains of species of the genus Chryseobacterium Strains: 1, THG C4-1T; 2, C. taiwanense Soil-3-27T; 3, C. hagamense RHA2-9T; 4, C. gregarium P 461/12T; 5, C. ginsenosidimutans THG 15T; 6, C. taeanense PHA3-4T; 7, C. daeguense K105T. Data for reference strains were taken from Tai et al. (2006), Cho et al. (2010), Behrendt et al. (2008), Im et al. (2011), Yoon et al. (2007) and Park et al. (2006). All species are positive for a-glucosidase, assimilation of maltose (not reported for C. taiwanense) and hydrolysis of starch, casein (2 % skimmed milk), Tween 80, aesculin and gelatin. All strains except C. taeanense PHA3-4T are positive for alkaline phosphatase, esterase lipase (C8), leucine arylamidase, valine arylamidase, acid phosphatase (not reported for C. taiwanense, C. hagamense or C. daeguense) and naphthol-AS-BI-phosphohydrolase. All strains are negative for lipase (C14), b-glucuronidase, a-fucosidase, amannosidase (not reported for C. taeanense), reduction of nitrates to nitrogen (not reported for C. ginsenosidimutans) and assimilation of N-acetylglucosamine (not reported for C. taiwanense). All strains produce yellow pigments. No growth on MacConkey agar (except C. taiwanense). +, Positive; W, weakly positive; 2, negative; ND, no data available. Characteristic Catalase Oxidase Growth temperature (uC) pH for growth Growth with NaCl (%, w/v) Indole production Nitrate reduction Hydrolysis of: Urea DNA Arginine dihydrolase Assimilation of: Histidine D-Glucose L-Arabinose D-Mannose D-Mannitol Gluconate Citrate Histidine L-Rhamnose Sucrose Enzyme activity Esterase (C4) Cystine arylamidase Trypsin a-Chymotrypsin a-Galactosidase b-Galactosidase b-Glucosidase N-Acetyl-b-glucosaminidase DNA G+C content (mol%)
1 + + 4–37 5.5–8.0 0–3.0 + 2 2 + 2 + + + 2 2 2 2 + 2 + + + + + 2 W
+ + 41.7
2 + + 5–42 5–10.0 0–4.0 + +
3 ND ND
10–37 5.0–10.0 0–2.0 2 2
4 + + 4–30 ND
0–2.0 2 2
5
6
ND
ND
10–37 5.5–10.0 0–1.0 2 2
+ 5–35 5.0–9.0 0–6.0 2 2
+ 2 +
2 2 2 2 + + + + + 2 + + + 2 2 2 2 2 + 2 32.1
ND
2 +
2 +
ND
ND
ND
ND
ND
2 + + + 2 + + 2 + +
ND
2 + + + 2 2 2 2 2 2
2 2 2 2 2 2 2 2 36.8
+ + 2 2 + + + + 36.9
2 2 2 2 2 2 + + 38.4
+ 2 2 2 2 2 + + 35.7
ND ND ND ND ND ND ND ND
ND
2 2
2 2 2 2 + 2 ND ND
W
7 + + 10–37 5.0–9.5 0–2.0 2 2 2 W
2 ND
+ + + 2 ND ND ND
+ 2 + + 2 2 + W
2 + 36.8
The polar lipid profile of strain THG C4-1T is shown in Fig. S2. The polar lipids were phosphatidylethanolamine and two unidentified aminolipids (AL1 and AL3); significant amounts of unidentified aminolipids and unidentified lipids were also present. The fatty acid profiles of this strain and type strains of related species of the genus Chryseobacterium are shown in Table 2. The major cellular fatty acids were iso-C15 : 0 (48.7 %), iso-C17 : 0 3-OH (17.4 %) and iso-C17 : 1v9c (7.8 %). The presence of large amounts of iso-C15 : 0, iso-C17 : 0 3-OH and iso-C17 : 1v9c in particular is typical for the genus Chryseobacterium (Park et al., 2006; Behrendt et al., 2008). The only respiratory
quinone detected in strain THG C4-1T was MK-6, in line with all other members of the family Flavobacteriaceae. The major polyamine was sym-homospermidine (Fig. S3). Unlike eukaryotes, where the predominant polyamine is spermidine, bacteria may instead contain an alternative polyamine, sym-homospermidine. Although the role of polyamines in the bacterial cell is not entirely clear, they seem to be important in bacterial metabolism (Vandamme et al., 1996; Prakash et al., 2007). In summary, the characteristics of strain THG C4-1T were consistent with descriptions of the genus Chryseobacterium with regard to morphological, biochemical and
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Chryseobacterium camelliae sp. nov.
Table 2. Cellular fatty acid profiles of strain THG C4-1T and type strains of phylogenetically related species of the genus Chryseobacterium Strains: 1, THG C4-1T; 2, C. taiwanense Soil-3-27T; 3, C. hagamense RHA2-9T; 4, C. gregarium P 461/12T; 5, C. ginsenosidimutans THG 15T; 6, C. taeanense PHA3-4T; 7, C. daeguense K105T. Data for reference strains were taken from Tai et al. (2006), Cho et al. (2010), Behrendt et al. (2008), Im et al. (2011), Yoon et al. (2007) and Park et al. (2006). Cells of strain THG C4-1T were cultured on NA for 2 days at 28 uC. Data are percentages of total fatty acids. Fatty acids accounting for less than 0.5 % of the total fatty acids in all strains are not listed. ND, Not detected/not reported; TR, traces (,1.0 %). Fatty acid Saturated C16 : 0 Unsaturated C18 : 1v5c iso-C17 : 1v9c Branched-chain iso-C13 : 0 iso-C15 : 0 iso-C16 : 0 iso-C17 : 0 anteiso-C15 : 0 iso-C15 : 0 3-OH iso-C16 : 0 3-OH iso-C17 : 0 3-OH Hydroxy C16 : 0 3-OH C17 : 0 2-OH Summed features* Summed feature 3
1
2
3
1.7
1.4
2.8
TR
ND
7.8 17.5
4
5
6
7
ND
ND
1.8
1.5
ND
7.2
1.2
ND
TR
ND
ND
9.3 15.8 19.8
1.4 1.0 1.2 1.3 ND TR 1.2 48.7 43.2 24.1 35.1 50.3 36.1 32.8 TR ND 1.0 TR ND ND ND TR 1.3 ND TR ND 1.1 ND 5.0 1.0 2.5 9.1 3.8 1.1 2.0 4.0 3.8 3.9 2.8 5.2 3.1 3.2 TR ND 3.9 TR ND TR ND 17.4 16.6 13.8 10.0 21.9 18.8 16.7 1.0 1.0
TR
1.8
1.2
ND
TR
1.2
TR
ND
TR
ND
ND
ND
6.6
7.9 12.2
ND
ND
11.2 10.9
*Summed features represent groups of two or three fatty acids that could not be separated by GLC with the MIDI system. Summed feature 3 contained C16 : 1v7c and/or iso-C15 : 0 2-OH.
chemotaxonomic properties. On the basis of phylogenetic distances between strain THG C4-1T and recognized species of the genus Chryseobacterium indicated by 16S rRNA gene sequence similarity, the combination of unique phenotypic characteristics (Table 1) and low levels of DNA–DNA relatedness, it is demonstrable that strain THG C4-1T should be assigned to the genus Chryseobacterium as the type strain of a novel species, for which the name Chryseobacterium camelliae sp. nov. is proposed. Description of Chryseobacterium camelliae sp. nov. Chryseobacterium camelliae (ca.mel9li.ae. N.L. gen. n. camelliae of the tea plant, Camellia sinensis). Cells are a Gram-staining-negative, strictly aerobic, nonmotile, non-spore-forming and rod-shaped. After culture on NA for 2 days, colonies are smooth, circular, yellow and http://ijs.sgmjournals.org
convex, 0.5–1.0 mm in diameter. Grows on NA at 4–37 uC and at pH 5.5–8.0, but not at 40 uC. Optimum growth occurs at 20–30 uC and at pH 7.0–7.5. Growth occurs in the absence of NaCl and in the presence of 0.5–3.0 % (w/v) NaCl, but not 3.5 % (w/v) NaCl. Catalase and oxidase activities are positive. In API ZYM tests, positive for alkaline phosphatase, esterase (C4), esterase lipase (C8), leucine arylamidase, valine arylamidase, cystine arylamidase, trypsin, a-chymotrypsin, acid phosphatase, naphtholAS-BI-phosphohydrolase, a-glucosidase, b-glucosidase and N-acetyl-b-glucosaminidase; weakly positive for b-galactosidase and negative for lipase (C14), a-galactosidase, bglucuronidase, a-mannosidase and a-fucosidase. According to API 20NE tests, positive results are obtained for indole production and gelatin hydrolysis; weakly positive for bgalactosidase (PNPG) activity and negative for arginine dihydrolase, urease, nitrate reduction, glucose acidification and assimilation of D-glucose, L-arabinose, D-mannose, Dmannitol, N-acetylglucosamine, maltose, gluconate, caprate, adipate, malate, citrate and phenylacetate. According to the API ID32 GN test, positive results are obtained for utilization of L-histidine, sucrose and glycogen; weakly positive results are obtained with salicin and L-proline and negative results are obtained with melibiose, L-fucose, D-sorbitol, propionate, valerate, 2-ketogluconate, 3-hydroxybutyrate, 4-hydroxybenzoate, L-rhamnose, N-acetylglucosamine, D-ribose, inositol, itaconate, suberate, malonate, acetate, lactate, L-alanine, 5-ketogluconate, 3-hydroxybenzoate and L-serine. The predominant menaquinone is MK-6, and iso-C15 : 0, iso-C17 : 0 3-OH and iso-C17 : 1v9c are the major cellular fatty acids (.7 %). The major polar lipid is phosphatidylethanolamine. The polyamine is symhomospermidine. The type strain, THG C4-1T (5KACC 14527T5JCM 18745T), was isolated from green tea leaves at Jangheunggun in the Republic of Korea. The G+C content of genomic DNA of the type strain is 41.7 mol%.
Acknowledgements This work was supported by a grant from Kyung Hee University, Republic of Korea, in 2012 (20120596).
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