International Journal of Systematic and Evolutionary Microbiology (2014), 64, 607–612
DOI 10.1099/ijs.0.055749-0
Cnuella takakiae gen. nov., sp. nov., a member of the phylum Bacteroidetes isolated from Takakia lepidozioides Ran Zhao, Xin Yao Chen, Xue Dong Li, Yang Tian, Bi He Kong, Zhi Ling Chen and Yan Hong Li Correspondence
College of Life Science, Capital Normal University, Beijing 100048, PR China
Yan Hong Li [email protected]
A Gram-staining-negative, rod-shaped and non-spore-forming bacterium, designated strain RG11T, was isolated from Takakia lepidozioides collected from Gawalong glacier in Tibet, China, and characterized by using a polyphasic taxonomic approach. The predominant fatty acids of strain RG1-1T were iso-C15 : 0 (19.8 %), summed feature 3 (C16 : 1v7c and/or C16 : 1v6c, 17.0 %), C16 : 0 (9.9 %) and iso-C17 : 0 3-OH (9.4 %); its major polar lipids were phosphatidylethanolamine, four unidentified aminolipids, one unidentified phospholipid, one unidentified aminoglycolipid, one unidentified glycolipid, and three unidentified lipids. Strain RG1-1T contained MK-7 as the dominant menaquinone, and the G+C content of its genomic DNA was 49.1 mol%. Strain RG11T exhibited the highest 16S rRNA gene sequence similarity (91.8 %) with Flavisolibacter ginsengiterrae Gsoil 492T and Flavisolibacter ginsengisoli Gsoil 643T. Phylogenetic analysis showed that strain RG1-1T was a member of the family Chitinophagaceae, phylum Bacteroidetes. On the basis of 16S rRNA gene sequence analysis, and phenotypic and chemotaxonomic data, strain RG1-1T is considered to represent a novel species of a novel genus, for which the name Cnuella takakiae gen. nov., sp. nov. is proposed. The type strain is RG1-1T (5CGMCC 1.12492T5DSM 26897T).
During the course of a study on the bacterial diversity in mosses from Gawalong glacier in Tibet, China, a novel orange-pigmented bacterium was isolated, named RG1-1T. On the basis of 16S rRNA gene sequence analysis, strain RG1-1T was considered to be a member of the family Chitinophagaceae, within the phylum Bacteroidetes. According to previous reports, the family Chitinophagaceae includes 14 genera, Chitinophaga (Ka¨mpfer et al., 2006), Ferruginibacter (Lim et al., 2009), Filimonas (Shiratori et al., 2009), Flavihumibacter (Zhang et al., 2010a), Flavisolibacter (Yoon & Im, 2007), Flavitalea (Wang et al., 2011), Hydrotalea (Ka¨mpfer et al., 2011), Lacibacter (Qu et al., 2009), Niabella (Kim et al., 2007), Niastella (Weon et al., 2006), Parasegetibacter (Zhang et al., 2009), Sediminibacterium (Qu & Yuan, 2008), Segetibacter (An et al., 2007) and Terrimonas (Xie & Yokota, 2006). In this paper, the results of a polyphasic study show that strain RG1-1T represents a novel species of a new genus in the phylum Bacteroidetes. Strain RG1-1T was originally isolated from plates of R2A agar (Niuniu) by inoculating homogenized Takakia The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain RG1-1T is KC560021. Two supplementary figures are available with the online version of this paper.
055749 G 2014 IUMS
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lepidozioides, which was sampled from Gawalong glacier at latitude 3900 m (29u 389 58.660 N 91u 069 56.340 E) in Tibet, on 13 October 2012. The R2A agar contained the following (l21): yeast extract 0.5 g, proteose peptone 0.5 g, casein hydrolysate 0.5 g, glucose 0.5 g, starch 0.5 g, sodium pyruvate 0.3 g, dipotassium hydrogen phosphate 0.3 g, magnesium sulphate anhydrous 0.024 g and agar 15.0 g. The pH was adjusted to pH 7.2±0.2. About 1 g of Takakia lepidozioides was collected and transported to the laboratory for surface disinfection as previously described (Li et al., 2011), then the disinfected plant individuals were homogenized in 0.85 % NaCl and spread on plates of R2A agar after serial dilution. The plates were incubated at 28 uC for 2–3 days. Single colonies from the plates were purified by transferring them onto new plates and were incubated again on R2A agar. The strain, designated RG11T, was isolated and purified after incubation at 28 uC for 3 days, and was then maintained on R2A agar at 4 uC and preserved as glycerol suspensions (30 %, v/v) at 280 uC. Biomass for chemotaxonomic and molecular systematic studies was prepared by growing the strain in R2A broth at 28 uC for 3 days. Cells were harvested by centrifugation and used in the physiological and biochemical studies. Preparation of genomic DNA was carried out according to the method of Marmur (1961). The 16S rRNA gene sequence of strain RG1-1T was determined by PCR 607
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amplification using the primers 27f and 1492r (Lane, 1991). The amplification product was purified and cloned into a pMD18-T vector (Takara). Sequencing was performed by using an ABI Big Dye Primer cycle sequencing ready reaction kit and an Applied Biosystems 3730 DNA sequencer. The 16S rRNA gene sequence of strain RG1-1T was first analysed using the EzTaxon-e server (Kim et al., 2012) to determine the most closely related type strains and calculate the sequence similarities. The sequence was then aligned with those of related species and phylogenetic trees were reconstructed using the neighbour-joining (Saitou & Nei, 1987) and maximum-likelihood methods in the MEGA 5.0 software (Tamura et al., 2011). Bootstrap analysis (1000 replications) was used to evaluate the topology of the trees. An almost complete 16S rRNA gene sequence (1493 bp) of strain RG1-1T was obtained. Comparative 16S rRNA gene sequence analysis showed that it exhibited the highest 16S rRNA gene sequence similarity with Flavisolibacter ginsengiterrae Gsoil 492T and Flavisolibacter ginsengisoli Gsoil 643T, but the value was only 91.8 %; it also shared lower sequence similarity with other species, such as Terrimonas rubra M-8T (91.4 %), Niastella populi THYL44T (91.2 %), Flavitalea populi HY-50RT (91.1 %) and Lacibacter cauensis NJ-8T (90.5 %). A phylogenetic tree based on neighbour-joining method is shown in Fig. 1. It showed that strain RG1-1T was not clustered together with any type species of other genera, but affiliated with species of the family Chitinophagaceae with a bootstrap value of 80 %. The phylogenetic tree made by the maximumlikelihood method also showed essentially the same topology (Fig. S1, available in IJSEM Online). Considering the 16S rRNA gene sequence similarity and phylogenetic analyses together, strains Flavisolibacter ginsengiterrae Gsoil 492T, Terrimonas rubra M-8T, Niastella populi THYL-44T, Flavitalea populi HY-50RT and Lacibacter cauensis NJ-8T were considered as representatives of related genera, and they were obtained from culture collections and used as the references for further taxonomic studies. Cellular morphology of strain RG1-1T was studied by Zeiss light microscope at 61000 and scanning electron microscopy (Quanta scanning fibre-optic microscope; FEI) at 65000– 20 000 after 3 days of growth in R2A broth at 28 uC. Colonial properties were examined after 3 days of growth at 28 uC on R2A agar. Cell motility was assessed by observing the growth spread of cells in test tubes containing semi-solid R2A agar after incubation for 72 h at 28 uC. Growth at various temperatures (4, 15, 20, 28, 30, 37, 40 and 45 uC) was tested on R2A agar for 5 days. The pH range (pH 4–11 at intervals of 0.5 pH unit) for growth and the requirement for and tolerance to NaCl [0, 1, 2, 3, 5 and 7 % (w/v)] were determined in R2A broth. Anaerobic growth was tested on R2A agar for up to 5 days in a 2.5 l jar containing an AnaeroPack-Anaero (Mitsubishi Gas Chemical), which works as an oxygen absorber and CO2 generator. Phenotypic characteristics such as Gram staining, catalase activity, oxidase activity and hydrolysis of casein, Tween 20, Tween 80, egg yolk and starch were performed using the methods of Smibert & Krieg (1994) 608
after 72 h of growth on R2A agar or in R2A broth at 28 uC. Methyl red reaction was assessed as described by Dong & Cai (2001). Antibiotic susceptibility of strain RG1-1T was examined by placing different antibiotic discs on R2A agar as previously described (Zhang et al., 2012). The antibiotic discs contained: kanamycin (5 mg), streptomycin (10 mg), chloramphenicol (30 mg), tetracycline (30 mg) or rifampicin (30 mg). Utilization of a variety of substrates as sole carbon sources of strain RG1-1T was tested using the GN2 MicroPlate Gram Negative Identification test panel (Biolog) by the service of China Center of Industrial Culture Collection (CICC) and the test was read after incubation at 30 uC for 24 h. Additional physiological and biochemical properties were determined using API 20NE, 20E and ZYM strips (bioMe´rieux) according to the manufacturer’s instructions and read after 24–48 h at 28 uC, 18–24 h at 37 uC and 4 h at 37 uC, respectively. All tests were run in duplicate. Cells of strain RG1-1T were Gram-staining-negative, strictly aerobic, non-spore-forming, motile and rodshaped. The other phenotypic characteristics are given in the species description, and Table 1 shows the features that differentiate the novel strain from other representatives of the family Chitinophagaceae. To determine the whole-cell fatty acid composition, strain RG1-1T and the five reference strains were grown on R2A agar at 28 uC for 3 days. Standardization of physiological age of all strains was made by choice of the second and third sector from a quadrant streak when colonies became visible in the fourth sector, as described by Sasser (1990). Extraction and analysis of the cellular fatty acids were performed according to the standard protocol of the Sherlock Microbial Identification System (Sasser, 1990) version 6.0 and peaks were identified using the peak naming table TSBA6 6.00 at China General Microbiological Culture Collection Center (CGMCC). The major fatty acid profile of strain RG1-1T comprised (.8 %): iso-C15 : 0 (19.8 %), summed feature 3 (comprising C16 : 1v7c and/or C16 : 1v6c, 17.0 %), C16 : 0 (9.9 %) and iso-C17 : 0 3-OH (9.4 %). In addition, iso-C13 : 0, iso-C13 : 0 3-OH, anteisoC15 : 0, C15 : 1v6c, iso-C16 : 0, iso-C16 : 1 H, iso-C17 : 0, C17 : 1v6c, summed feature 1 (comprising iso-C15 : 1 H and/or C13 : 0 3-OH), summed feature 4 (comprising isoC17 : 1 I and/or anteiso-C17 : 1 B) and summed feature 9 (comprising iso-C17 : 1 v9c and/or 10-methyl C16 : 0) also exceeded 1 % of the total fatty acid content. Comparisons of all fatty acid profiles (shown in Table 2) showed that strain RG1-1T contained a relatively high proportion of C16 : 0, iso-C16 : 0 and summed feature 9, but exhibited relatively low amounts of iso-C15 : 1 G, which distinguished strain RG1-1T from other reference strains of other genera. Polar lipids extracted from 200 mg of cells were separated by two-dimensional TLC. This was carried out by the Identification Service of DSMZ (Braunschweig, Germany). Cellular isoprenoid quinones were extracted and purified using the methods described by Minnikin et al. (1984), and purified quinones were determined by reverse-phase HPLC International Journal of Systematic and Evolutionary Microbiology 64
Cnuella takakiae gen. nov., sp. nov.
99 100
0.01
99
Terrimonas ferruginea DSM 30193T (AM230484) Terrimonas aquatica RIB1-6T (FJ347757) 98 Terrimonas rubra M-8T (JF803808) Terrimonas pekingensis QHT (JF834159) Terrimonas lutea DYT (AB192292) 100 100 Flavisolibacter ginsengiterrae Gsoil 492T (AB267476) Flavisolibacter ginsengisoli Gsoil 643T (AB267477) Flavitalea populi HY-50RT (HM130561) Flavitalea gansuensis JCN-23T (GU295962) 100 Niastella populi THYL-44T (EU877262) Niastella koreensis GR20-10T (CP003178) 100 Niastella yeongjuensis GR20-13T (DQ244076) 100 Parasegetibacter luojiensis RHYL-37T (EU877263) Segetibacter aerophilus 6424S-61T (GQ421847) Segetibacter koreensis Gsoil 664T (AB267478) 100 Flavihumibacter petaseus T41T (EU854577) Sediminibacterium ginsengisoli DCY13T (EF067860) Chitinophaga skermanii CC-SG1BT (DQ062743) 80 95 Chitinophaga eiseniae YC6729T (FJ750951) 91 Chitinophaga terrae KP01T (AB278570) 100 Chitinophaga niastensis JS16-4T (EU714260) 100 Chitinophaga rupis CS5-B1T (FM865977) Chitinophaga ginsengisoli Gsoil 052T (AB245374) 84 84 100 Chitinophaga filiformis IFO 15056T (AB078049) Cnuella takakiae RG1-1T (KC560021) 100 Lacibacter cauensis NJ-8T (EU521690) Uncultured bacterium clone MRA2015 (FN428761) Uncultured bacterium clone BIjii2 (AJ318153) ‘Portibacter lacus’ YM8-076 (AB675658) Aureispira marina 24T (AB245933) 99 ‘Rubidimonas crustatorum’ AK17-053 (AB602438) 75 Mucilaginibacter jinjuensis YC7004T (JQ765855) Pedobacter ruber W1T (HQ882803) 100 Barnesiella viscericola C46T (AB267809)
Fig. 1. Neighbour-joining phylogenetic tree based on 16S rRNA gene sequences showing the relationships of strain RG1-1T and related species. GenBank accession numbers are given in parentheses. Numbers at branching points are bootstrap values .70 %. Bar, 0.01 substitutions per nucleotide position. Barnesiella viscericola C46T (AB267809) was used as an outgroup.
(Kroppenstedt, 1982). The DNA G+C content was determined using the thermal denaturation method (Marmur & Doty, 1962) with Escherichia coli K-12 as a control. The polar lipid profile of strain RG1-1T contained phosphatidylethanolamine as the major compound. In addition, traces of one unidentified phospholipid, one unidentified aminoglycolipid, one unidentified glycolipid, four unidentified aminolipids and three unidentified lipids were also detected (Fig. S2). Strain RG1-1T contained MK7 as the major respiratory quinone, which is consistent with other species of the family Chitinophagaceae. The DNA G+C content was found to be 49.1 mol%, which is a little higher than that of other type strains from different genera of family Chitinophagaceae (shown in Table 1). In view of the combined morphological, physiological and chemotaxonomic data, and of the phylogenetic inference presented here, it is evident that strain RG1-1T represents a novel species of a new genus in the family Chitinophagaceae, phylum Bacteroidetes, for which the name Cnuella takakiae gen. nov., sp. nov. is proposed. http://ijs.sgmjournals.org
Description of Cnuella gen. nov. Cnuella (Cnu.el9la. L. dim. suff. -ella; N.L. fem. n. Cnuella arbitrary name after CNU, Capital Normal University, where taxonomic studies of this taxon were conducted). Cells are Gram-staining-negative, strictly aerobic, nonspore-forming, motile and rods. Major cellular fatty acids include large amounts of iso-C15 : 0 and summed feature 3 (comprising C16 : 1v7c and/or C16 : 1v6c). The most dominant polar lipids are phosphatidylethanolamine and some unknown lipids. The dominant menaquinone is MK-7. Phylogenetically, the genus Cnuella is a member of the family Chitinophagaceae, phylum Bacteroidetes. The type strain is Cnuella takakiae. Description of Cnuella takakiae sp. nov. Cnuella takakiae (ta.ka9ki.ae. N.L. gen. n. takakiae of Takakia, referring to Takakia lepidozioides, the plant from which the type strain was isolated). 609
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Table 1. Differential characteristics of strain RG1-1T and related type strains of members of the family Chitinophagaceae Strains: 1, RG1-1T; 2, Flavisolibacter ginsengiterrae KCTC 12656T; 3, Terrimonas rubra CCTCC AB 2010401T; 4, Niastella populi KCTC 22560T; 5, Flavitalea populi CCTCC AB 208255T; 6, Lacibacter cauensis NBRC 104930T. +, Positive; 2, negative; W, weak or delayed reaction. All data are from this study, except the DNA G+C content of the reference strains. In API 20E and API 20NE test strips, all strains are positive for aesculin hydrolysis, and urease and b-galactosidase activity (PNPG test), and negative for reduction of nitrates to nitrites/nitrogen, arginine dihydrolase, lysine decarboxylase and ornithine decarboxylase activities, citrate utilization, glucose fermentation, and indole and H2S production. All of strains can assimilate D-glucose and maltose, but cannot assimilate D-mannitol, potassium gluconate, capric acid, adipic acid, phenylacetic acid or Dsorbitol. In API ZYM test strips, all of the strains are positive for alkaline phosphatase, leucine arylamidase, valine arylamidase, cystine arylamidase, acid phosphatase, naphthol-AS-BI-phosphohydrolase and N-acetyl-b-glucosaminidase activities, but negative for lipase and b-glucuronidase activities. Characteristic Pigmentation pH range for growth Temperature range for growth (uC) Tolerance to NaCl (%) Catalase Oxidase Voges–Proskauer test ONPG test Tryptophan deaminase Enzyme activities: Esterase (C4) Esterase lipase (C8) Trypsin a-Chymotrypsin a-Galactosidase b-Galactosidase a-Glucosidase b-Glucosidase a-Mannosidase a-Fucosidase Hydrolysis of: Starch Gelatin Assimilation of: L-Arabinose D-Mannose N-Acetylglucosamine Malic acid Trisodium citrate Rhamnose Melibiose DNA G+C content (mol%)
1
2
3
4
5
6
Orange 6.0–10.0 20–30 0–2 + 2 + + +
Yellow 6.0–8.5 15–30 0–3 2 + 2 2 2
Salmon Red 5.0–10.0 20–30 0–2 + 2 + + 2
Yellow 5.0–8.0 17–37 0–1 2 2 2 + +
Yellow 5.0–8.0 15–30 0–2 + 2 + + 2
Orange 5.5–8.5 20–37 0–1 + + – + 2
+ + + 2 + + + + 2 +
+ 2 2 + 2 2 2 2 2 W
+ 2 + + 2 2 2 2 2 2
+ + + + +
+ 2 2 2 2 2 2 2 2 2
2 + 2 2 2 2 2 2 2
2 +
2 2
2 +
2 +
2 2
+
+ 2 2 2 + 2 + 49.1
+ + 2 2 2 2 + 42.5–43.3a*
+ + + + 2 2 + 47b
+ + + 2 2 + + 45.2c
2 + + 2 2 2 + 46.8d
2 + + 2 2 2 2 46.6e
W W
2 2 W
W
W
*Data from: a, Yoon & Im (2007); b, Zhang et al. (2012); c, Zhang et al. (2010b); d, Zhang et al. (2013); e, Qu et al. (2009).
Cells are Gram-staining-negative, aerobic, non-sporeforming, motile rods about 0.5–0.9 mm in diameter and 0.8–1.6 mm in length. Colonies are circular with regular edges, convex, opaque, moist, orange-pigmented and 2.0– 4.0 mm in diameter after 3 days of cultivation at 28 uC on R2A agar. Growth also occurs on NA agar, but not on TSA or MacConkey agar. Growth occurs at 20–30 uC (optimal growth at 28–30 uC) and at pH 6.0–10.0 (optimal growth at pH 7.0–7.5) in R2A broth. Growth occurs with 0–2 % NaCl (optimal growth without NaCl). Degrades Tween 80
and casein, but does not degrade Tween 20, starch or egg yolk. Positive for catalase activity, but negative for oxidase activity and methyl red test. According to tests of API 20E and 20NE, positive for aesculin and gelatin hydrolysis, urease, b-galactosidase (PNPG and ONPG tests) and tryptophan deaminase activity, and Voges–Proskauer reaction, but negative for arginine dihydrolase, lysine decarboxylase and ornithine decarboxylase activity, citrate utilization, glucose fermentation, and indole and H2S production. Nitrate cannot be reduced to nitrite or
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International Journal of Systematic and Evolutionary Microbiology 64
Cnuella takakiae gen. nov., sp. nov.
Table 2. Fatty acid composition (%) of strain strain RG1-1T and related type strains of members of family Chitinophagaceae Strains: 1, RG1-1T; 2, Flavisolibacter ginsengiterrae KCTC 12656T; 3, Terrimonas rubra CCTCC AB 2010401T; 4, Niastella populi KCTC 22560T; 5, Flavitalea populi CCTCC AB 208255T; 6, Lacibacter cauensis NBRC 104930T. TR, Traces (less than 1 % of the total); –, not present. All data are from this study. Fatty acid
1
2
3
4
5
6
iso-C13 : 0 iso-C13 : 0 3-OH C13 : 1 iso-C14 : 0 C14 : 0 iso-C15 : 0 iso-C15 : 0 3-OH anteiso-C15 : 0 C15 : 0 2-OH C15 : 0 3-OH iso-C15 : 1 G anteiso-C15 : 1 A C15 : 1v6c iso-C16 : 0 iso-C16 : 0 3-OH C16 : 0 C16 : 0 3-OH iso-C16 : 1 H C16 : 1v5c C16 : 1v11c iso-C17 : 0 iso-C17 : 0 3-OH anteiso-C17 : 0 anteiso-C17 : 1 C17 : 1v6c Summed features* 1 3 4 9
5.1 2.7
1.1
TR
TR
TR
TR
– 1.0 –
7.0 3.5 – – – 10.2 – 5.0
1.2 –
TR
TR
1.8
TR
1.5
TR
TR
TR
19.8
29.9 TR
1.0 19.0 2.3
10.0
TR
TR
TR
–
– 7.0 5.0
1.1 1.1 26.8
TR
2.5
TR
37.8 TR
6.6 TR
TR
– 15.6 7.1 – 1.5
2.3 7.4
TR
–
– 25.0 1.9 –
2.1
TR
TR
TR
TR
TR
9.9
4.6
3.5 3.8
TR
TR
1.8
1.4
TR
TR
– 1.3 9.4
– 1.2 7.0
TR
TR
– 2.6
– 3.0
4.5 17.0 1.2 3.0
3.7 14.1 1.9
TR TR
TR
TR
2.0 4.3 – – – – 12.2 – – – – 23.3 – –
TR
– – 1.0 TR
9.7 TR
– – – 2.9 – –
TR
1.8 – – 6.6 – 6.7 12.7 2.7 1.4 TR
3.5 4.4 6.4 –
TR TR TR
21.4 2.8 1.2 2.7 TR
15.6 TR
– 2.4
The type strain, RG1-1T (5CGMCC 1.12492T5DSM 26897T), was isolated from Takakia lepidozioides collected from Gawalong glacier in Tibet, China. The DNA G+C content of the type strain is 49.1 mol%.
TR
3.1 1.5 – 3.8 –
Acknowledgements
TR
References
10.0
This work was funded by the Scientific Research Program of National Natural Science Foundation of China (no. 31100004).
TR
An, D.-S., Lee, H.-G., Im, W.-T., Liu, Q. M. & Lee, S.-T. (2007).
– 1.0
Segetibacter koreensis gen. nov., sp. nov., a novel member of the phylum Bacteroidetes, isolated from the soil of a ginseng field in South Korea. Int J Syst Evol Microbiol 57, 1828–1833.
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Dong, X.-Z. & Cai, M.-Y. (2001). Determination of biochemical
*Summed features represent groups of two or three fatty acids that could not be separated by GLC using the MIDI system. Summed feature 1 contained iso-C15 : 1 H and/or C13 : 0 3-OH; summed feature 3 contained C16 : 1v7c and/or C16 : 1v6c; summed feature 4 contained iso-C17 : 1 I and/or anteiso-C17 : 1 B; summed feature 9 contained isoC17 : 1v9c and/or 10-methyl C16 : 0.
nitrogen. Assimilates D-glucose, L-arabinose, trisodium citrate and melibiose; does not assimilate D-mannose, Dmannitol, N-acetylglucosamine, malic acid, potassium gluconate, capric acid, adipic acid, phenylacetic acid, Dsorbitol and rhamnose. In API ZYM tests, positive for alkaline phosphatase, esterase (C4), esterase lipase (C8), leucine arylamidase, valine arylamidase, cystine arylamidase, trypsin, acid phosphatase, naphthol-AS-BI-phosphohydrolase, b-galactosidase, a-glucosidase, b-glucosidase, Nacetyl-b-glucosaminidase, a-galactosidase and a-fucosidase http://ijs.sgmjournals.org
activities, but negative for lipase, a-chymotrypsin, amannosidase and b-glucuronidase activities. The following carbon sources are utilized in the GN2 MicroPlate: dextrin, Tween 40, Tween 80, cellobiose, D-fructose, gentiobiose, aD-glucose, a-lactose, D-psicose, acetic acid, citric acid, Dgluconic acid, D-glucuronic acid, b-hydroxybutyric acid, a-ketobutyric acid, D-saccharic acid, bromosuccinic acid, L-alaninamide, D-alanine, L-aspartic acid, L-leucine, Lphenylalanine, L-proline and D-serine; other substrates are not utilized. Susceptible to all the tested antibiotics: kanamycin (5 mg), streptomycin (10 mg), chloramphenicol (30 mg), tetracycline (30 mg) and rifampicin (30 mg). The fatty acid profile is composed mainly of iso-C15 : 0, summed feature 3 (C16 : 1v7c and/or C16 : 1v6c), C16 : 0 and iso-C17 : 0 3-OH; the major polar lipids are phosphatidylethanolamine, four unidentified aminolipids, one unidentified phospholipid, one unidentified aminoglycolipid, one unidentified glycolipid and three unidentified lipids. The dominant menaquinone is MK-7.
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of deoxyribonucleic acid from its thermal denaturation temperature. J Mol Biol 5, 109–118. Minnikin, D. E., Odonnell, A. G., Goodfellow, M., Alderson, G., Athalye, M., Schaal, A. & Parlett, J. H. (1984). An integrated
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nov., sp. nov., a member of the phylum Bacteroidetes isolated from sediment of a eutrophic reservoir. Int J Syst Evol Microbiol 58, 2191– 2194. Qu, J.-H., Yuan, H.-L., Yang, J.-S., Li, H.-F. & Chen, N. (2009).
Lacibacter cauensis gen. nov., sp. nov., a novel member of the phylum Bacteroidetes isolated from sediment of a eutrophic lake. Int J Syst Evol Microbiol 59, 1153–1157.
Euphrates poplar (Populus euphratica) forest. Int. J. Syst. Evol. Microbiol. 61, 1554–1560. Weon, H.-Y., Kim, B.-Y., Yoo, S.-H., Lee, S.-Y., Kwon, S.-W., Go, S.-J. & Stackebrandt, E. (2006). Niastella koreensis gen. nov., sp. nov. and
Niastella yeongjuensis sp. nov., novel members of the phylum Bacteroidetes, isolated from soil cultivated with Korean ginseng. Int J Syst Evol Microbiol 56, 1777–1782. Xie, C. H. & Yokota, A. (2006). Reclassification of [Flavobacterium]
ferrugineum as Terrimonas ferruginea gen. nov., comb. nov., and description of Terrimonas lutea sp. nov., isolated from soil. Int J Syst Evol Microbiol 56, 1117–1121. Yoon, M.-H. & Im, W.-T. (2007). Flavisolibacter ginsengiterrae gen. nov., sp. nov. and Flavisolibacter ginsengisoli sp. nov., isolated from ginseng cultivating soil. Int J Syst Evol Microbiol 57, 1834–1839. Zhang, K., Tang, Y., Zhang, L., Dai, J., Wang, Y., Luo, X., Liu, M., Luo, G. & Fang, C. (2009). Parasegetibacter luojiensis gen. nov., sp. nov., a
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Smibert, R. M. & Krieg, N. R. (1994). Phenotypic characterization. In
DOI 10.1099/ijs.0.055749-0
Cnuella takakiae gen. nov., sp. nov., a member of the phylum Bacteroidetes isolated from Takakia lepidozioides Ran Zhao, Xin Yao Chen, Xue Dong Li, Yang Tian, Bi He Kong, Zhi Ling Chen and Yan Hong Li Correspondence
College of Life Science, Capital Normal University, Beijing 100048, PR China
Yan Hong Li [email protected]
A Gram-staining-negative, rod-shaped and non-spore-forming bacterium, designated strain RG11T, was isolated from Takakia lepidozioides collected from Gawalong glacier in Tibet, China, and characterized by using a polyphasic taxonomic approach. The predominant fatty acids of strain RG1-1T were iso-C15 : 0 (19.8 %), summed feature 3 (C16 : 1v7c and/or C16 : 1v6c, 17.0 %), C16 : 0 (9.9 %) and iso-C17 : 0 3-OH (9.4 %); its major polar lipids were phosphatidylethanolamine, four unidentified aminolipids, one unidentified phospholipid, one unidentified aminoglycolipid, one unidentified glycolipid, and three unidentified lipids. Strain RG1-1T contained MK-7 as the dominant menaquinone, and the G+C content of its genomic DNA was 49.1 mol%. Strain RG11T exhibited the highest 16S rRNA gene sequence similarity (91.8 %) with Flavisolibacter ginsengiterrae Gsoil 492T and Flavisolibacter ginsengisoli Gsoil 643T. Phylogenetic analysis showed that strain RG1-1T was a member of the family Chitinophagaceae, phylum Bacteroidetes. On the basis of 16S rRNA gene sequence analysis, and phenotypic and chemotaxonomic data, strain RG1-1T is considered to represent a novel species of a novel genus, for which the name Cnuella takakiae gen. nov., sp. nov. is proposed. The type strain is RG1-1T (5CGMCC 1.12492T5DSM 26897T).
During the course of a study on the bacterial diversity in mosses from Gawalong glacier in Tibet, China, a novel orange-pigmented bacterium was isolated, named RG1-1T. On the basis of 16S rRNA gene sequence analysis, strain RG1-1T was considered to be a member of the family Chitinophagaceae, within the phylum Bacteroidetes. According to previous reports, the family Chitinophagaceae includes 14 genera, Chitinophaga (Ka¨mpfer et al., 2006), Ferruginibacter (Lim et al., 2009), Filimonas (Shiratori et al., 2009), Flavihumibacter (Zhang et al., 2010a), Flavisolibacter (Yoon & Im, 2007), Flavitalea (Wang et al., 2011), Hydrotalea (Ka¨mpfer et al., 2011), Lacibacter (Qu et al., 2009), Niabella (Kim et al., 2007), Niastella (Weon et al., 2006), Parasegetibacter (Zhang et al., 2009), Sediminibacterium (Qu & Yuan, 2008), Segetibacter (An et al., 2007) and Terrimonas (Xie & Yokota, 2006). In this paper, the results of a polyphasic study show that strain RG1-1T represents a novel species of a new genus in the phylum Bacteroidetes. Strain RG1-1T was originally isolated from plates of R2A agar (Niuniu) by inoculating homogenized Takakia The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain RG1-1T is KC560021. Two supplementary figures are available with the online version of this paper.
055749 G 2014 IUMS
Printed in Great Britain
lepidozioides, which was sampled from Gawalong glacier at latitude 3900 m (29u 389 58.660 N 91u 069 56.340 E) in Tibet, on 13 October 2012. The R2A agar contained the following (l21): yeast extract 0.5 g, proteose peptone 0.5 g, casein hydrolysate 0.5 g, glucose 0.5 g, starch 0.5 g, sodium pyruvate 0.3 g, dipotassium hydrogen phosphate 0.3 g, magnesium sulphate anhydrous 0.024 g and agar 15.0 g. The pH was adjusted to pH 7.2±0.2. About 1 g of Takakia lepidozioides was collected and transported to the laboratory for surface disinfection as previously described (Li et al., 2011), then the disinfected plant individuals were homogenized in 0.85 % NaCl and spread on plates of R2A agar after serial dilution. The plates were incubated at 28 uC for 2–3 days. Single colonies from the plates were purified by transferring them onto new plates and were incubated again on R2A agar. The strain, designated RG11T, was isolated and purified after incubation at 28 uC for 3 days, and was then maintained on R2A agar at 4 uC and preserved as glycerol suspensions (30 %, v/v) at 280 uC. Biomass for chemotaxonomic and molecular systematic studies was prepared by growing the strain in R2A broth at 28 uC for 3 days. Cells were harvested by centrifugation and used in the physiological and biochemical studies. Preparation of genomic DNA was carried out according to the method of Marmur (1961). The 16S rRNA gene sequence of strain RG1-1T was determined by PCR 607
R. Zhao and others
amplification using the primers 27f and 1492r (Lane, 1991). The amplification product was purified and cloned into a pMD18-T vector (Takara). Sequencing was performed by using an ABI Big Dye Primer cycle sequencing ready reaction kit and an Applied Biosystems 3730 DNA sequencer. The 16S rRNA gene sequence of strain RG1-1T was first analysed using the EzTaxon-e server (Kim et al., 2012) to determine the most closely related type strains and calculate the sequence similarities. The sequence was then aligned with those of related species and phylogenetic trees were reconstructed using the neighbour-joining (Saitou & Nei, 1987) and maximum-likelihood methods in the MEGA 5.0 software (Tamura et al., 2011). Bootstrap analysis (1000 replications) was used to evaluate the topology of the trees. An almost complete 16S rRNA gene sequence (1493 bp) of strain RG1-1T was obtained. Comparative 16S rRNA gene sequence analysis showed that it exhibited the highest 16S rRNA gene sequence similarity with Flavisolibacter ginsengiterrae Gsoil 492T and Flavisolibacter ginsengisoli Gsoil 643T, but the value was only 91.8 %; it also shared lower sequence similarity with other species, such as Terrimonas rubra M-8T (91.4 %), Niastella populi THYL44T (91.2 %), Flavitalea populi HY-50RT (91.1 %) and Lacibacter cauensis NJ-8T (90.5 %). A phylogenetic tree based on neighbour-joining method is shown in Fig. 1. It showed that strain RG1-1T was not clustered together with any type species of other genera, but affiliated with species of the family Chitinophagaceae with a bootstrap value of 80 %. The phylogenetic tree made by the maximumlikelihood method also showed essentially the same topology (Fig. S1, available in IJSEM Online). Considering the 16S rRNA gene sequence similarity and phylogenetic analyses together, strains Flavisolibacter ginsengiterrae Gsoil 492T, Terrimonas rubra M-8T, Niastella populi THYL-44T, Flavitalea populi HY-50RT and Lacibacter cauensis NJ-8T were considered as representatives of related genera, and they were obtained from culture collections and used as the references for further taxonomic studies. Cellular morphology of strain RG1-1T was studied by Zeiss light microscope at 61000 and scanning electron microscopy (Quanta scanning fibre-optic microscope; FEI) at 65000– 20 000 after 3 days of growth in R2A broth at 28 uC. Colonial properties were examined after 3 days of growth at 28 uC on R2A agar. Cell motility was assessed by observing the growth spread of cells in test tubes containing semi-solid R2A agar after incubation for 72 h at 28 uC. Growth at various temperatures (4, 15, 20, 28, 30, 37, 40 and 45 uC) was tested on R2A agar for 5 days. The pH range (pH 4–11 at intervals of 0.5 pH unit) for growth and the requirement for and tolerance to NaCl [0, 1, 2, 3, 5 and 7 % (w/v)] were determined in R2A broth. Anaerobic growth was tested on R2A agar for up to 5 days in a 2.5 l jar containing an AnaeroPack-Anaero (Mitsubishi Gas Chemical), which works as an oxygen absorber and CO2 generator. Phenotypic characteristics such as Gram staining, catalase activity, oxidase activity and hydrolysis of casein, Tween 20, Tween 80, egg yolk and starch were performed using the methods of Smibert & Krieg (1994) 608
after 72 h of growth on R2A agar or in R2A broth at 28 uC. Methyl red reaction was assessed as described by Dong & Cai (2001). Antibiotic susceptibility of strain RG1-1T was examined by placing different antibiotic discs on R2A agar as previously described (Zhang et al., 2012). The antibiotic discs contained: kanamycin (5 mg), streptomycin (10 mg), chloramphenicol (30 mg), tetracycline (30 mg) or rifampicin (30 mg). Utilization of a variety of substrates as sole carbon sources of strain RG1-1T was tested using the GN2 MicroPlate Gram Negative Identification test panel (Biolog) by the service of China Center of Industrial Culture Collection (CICC) and the test was read after incubation at 30 uC for 24 h. Additional physiological and biochemical properties were determined using API 20NE, 20E and ZYM strips (bioMe´rieux) according to the manufacturer’s instructions and read after 24–48 h at 28 uC, 18–24 h at 37 uC and 4 h at 37 uC, respectively. All tests were run in duplicate. Cells of strain RG1-1T were Gram-staining-negative, strictly aerobic, non-spore-forming, motile and rodshaped. The other phenotypic characteristics are given in the species description, and Table 1 shows the features that differentiate the novel strain from other representatives of the family Chitinophagaceae. To determine the whole-cell fatty acid composition, strain RG1-1T and the five reference strains were grown on R2A agar at 28 uC for 3 days. Standardization of physiological age of all strains was made by choice of the second and third sector from a quadrant streak when colonies became visible in the fourth sector, as described by Sasser (1990). Extraction and analysis of the cellular fatty acids were performed according to the standard protocol of the Sherlock Microbial Identification System (Sasser, 1990) version 6.0 and peaks were identified using the peak naming table TSBA6 6.00 at China General Microbiological Culture Collection Center (CGMCC). The major fatty acid profile of strain RG1-1T comprised (.8 %): iso-C15 : 0 (19.8 %), summed feature 3 (comprising C16 : 1v7c and/or C16 : 1v6c, 17.0 %), C16 : 0 (9.9 %) and iso-C17 : 0 3-OH (9.4 %). In addition, iso-C13 : 0, iso-C13 : 0 3-OH, anteisoC15 : 0, C15 : 1v6c, iso-C16 : 0, iso-C16 : 1 H, iso-C17 : 0, C17 : 1v6c, summed feature 1 (comprising iso-C15 : 1 H and/or C13 : 0 3-OH), summed feature 4 (comprising isoC17 : 1 I and/or anteiso-C17 : 1 B) and summed feature 9 (comprising iso-C17 : 1 v9c and/or 10-methyl C16 : 0) also exceeded 1 % of the total fatty acid content. Comparisons of all fatty acid profiles (shown in Table 2) showed that strain RG1-1T contained a relatively high proportion of C16 : 0, iso-C16 : 0 and summed feature 9, but exhibited relatively low amounts of iso-C15 : 1 G, which distinguished strain RG1-1T from other reference strains of other genera. Polar lipids extracted from 200 mg of cells were separated by two-dimensional TLC. This was carried out by the Identification Service of DSMZ (Braunschweig, Germany). Cellular isoprenoid quinones were extracted and purified using the methods described by Minnikin et al. (1984), and purified quinones were determined by reverse-phase HPLC International Journal of Systematic and Evolutionary Microbiology 64
Cnuella takakiae gen. nov., sp. nov.
99 100
0.01
99
Terrimonas ferruginea DSM 30193T (AM230484) Terrimonas aquatica RIB1-6T (FJ347757) 98 Terrimonas rubra M-8T (JF803808) Terrimonas pekingensis QHT (JF834159) Terrimonas lutea DYT (AB192292) 100 100 Flavisolibacter ginsengiterrae Gsoil 492T (AB267476) Flavisolibacter ginsengisoli Gsoil 643T (AB267477) Flavitalea populi HY-50RT (HM130561) Flavitalea gansuensis JCN-23T (GU295962) 100 Niastella populi THYL-44T (EU877262) Niastella koreensis GR20-10T (CP003178) 100 Niastella yeongjuensis GR20-13T (DQ244076) 100 Parasegetibacter luojiensis RHYL-37T (EU877263) Segetibacter aerophilus 6424S-61T (GQ421847) Segetibacter koreensis Gsoil 664T (AB267478) 100 Flavihumibacter petaseus T41T (EU854577) Sediminibacterium ginsengisoli DCY13T (EF067860) Chitinophaga skermanii CC-SG1BT (DQ062743) 80 95 Chitinophaga eiseniae YC6729T (FJ750951) 91 Chitinophaga terrae KP01T (AB278570) 100 Chitinophaga niastensis JS16-4T (EU714260) 100 Chitinophaga rupis CS5-B1T (FM865977) Chitinophaga ginsengisoli Gsoil 052T (AB245374) 84 84 100 Chitinophaga filiformis IFO 15056T (AB078049) Cnuella takakiae RG1-1T (KC560021) 100 Lacibacter cauensis NJ-8T (EU521690) Uncultured bacterium clone MRA2015 (FN428761) Uncultured bacterium clone BIjii2 (AJ318153) ‘Portibacter lacus’ YM8-076 (AB675658) Aureispira marina 24T (AB245933) 99 ‘Rubidimonas crustatorum’ AK17-053 (AB602438) 75 Mucilaginibacter jinjuensis YC7004T (JQ765855) Pedobacter ruber W1T (HQ882803) 100 Barnesiella viscericola C46T (AB267809)
Fig. 1. Neighbour-joining phylogenetic tree based on 16S rRNA gene sequences showing the relationships of strain RG1-1T and related species. GenBank accession numbers are given in parentheses. Numbers at branching points are bootstrap values .70 %. Bar, 0.01 substitutions per nucleotide position. Barnesiella viscericola C46T (AB267809) was used as an outgroup.
(Kroppenstedt, 1982). The DNA G+C content was determined using the thermal denaturation method (Marmur & Doty, 1962) with Escherichia coli K-12 as a control. The polar lipid profile of strain RG1-1T contained phosphatidylethanolamine as the major compound. In addition, traces of one unidentified phospholipid, one unidentified aminoglycolipid, one unidentified glycolipid, four unidentified aminolipids and three unidentified lipids were also detected (Fig. S2). Strain RG1-1T contained MK7 as the major respiratory quinone, which is consistent with other species of the family Chitinophagaceae. The DNA G+C content was found to be 49.1 mol%, which is a little higher than that of other type strains from different genera of family Chitinophagaceae (shown in Table 1). In view of the combined morphological, physiological and chemotaxonomic data, and of the phylogenetic inference presented here, it is evident that strain RG1-1T represents a novel species of a new genus in the family Chitinophagaceae, phylum Bacteroidetes, for which the name Cnuella takakiae gen. nov., sp. nov. is proposed. http://ijs.sgmjournals.org
Description of Cnuella gen. nov. Cnuella (Cnu.el9la. L. dim. suff. -ella; N.L. fem. n. Cnuella arbitrary name after CNU, Capital Normal University, where taxonomic studies of this taxon were conducted). Cells are Gram-staining-negative, strictly aerobic, nonspore-forming, motile and rods. Major cellular fatty acids include large amounts of iso-C15 : 0 and summed feature 3 (comprising C16 : 1v7c and/or C16 : 1v6c). The most dominant polar lipids are phosphatidylethanolamine and some unknown lipids. The dominant menaquinone is MK-7. Phylogenetically, the genus Cnuella is a member of the family Chitinophagaceae, phylum Bacteroidetes. The type strain is Cnuella takakiae. Description of Cnuella takakiae sp. nov. Cnuella takakiae (ta.ka9ki.ae. N.L. gen. n. takakiae of Takakia, referring to Takakia lepidozioides, the plant from which the type strain was isolated). 609
R. Zhao and others
Table 1. Differential characteristics of strain RG1-1T and related type strains of members of the family Chitinophagaceae Strains: 1, RG1-1T; 2, Flavisolibacter ginsengiterrae KCTC 12656T; 3, Terrimonas rubra CCTCC AB 2010401T; 4, Niastella populi KCTC 22560T; 5, Flavitalea populi CCTCC AB 208255T; 6, Lacibacter cauensis NBRC 104930T. +, Positive; 2, negative; W, weak or delayed reaction. All data are from this study, except the DNA G+C content of the reference strains. In API 20E and API 20NE test strips, all strains are positive for aesculin hydrolysis, and urease and b-galactosidase activity (PNPG test), and negative for reduction of nitrates to nitrites/nitrogen, arginine dihydrolase, lysine decarboxylase and ornithine decarboxylase activities, citrate utilization, glucose fermentation, and indole and H2S production. All of strains can assimilate D-glucose and maltose, but cannot assimilate D-mannitol, potassium gluconate, capric acid, adipic acid, phenylacetic acid or Dsorbitol. In API ZYM test strips, all of the strains are positive for alkaline phosphatase, leucine arylamidase, valine arylamidase, cystine arylamidase, acid phosphatase, naphthol-AS-BI-phosphohydrolase and N-acetyl-b-glucosaminidase activities, but negative for lipase and b-glucuronidase activities. Characteristic Pigmentation pH range for growth Temperature range for growth (uC) Tolerance to NaCl (%) Catalase Oxidase Voges–Proskauer test ONPG test Tryptophan deaminase Enzyme activities: Esterase (C4) Esterase lipase (C8) Trypsin a-Chymotrypsin a-Galactosidase b-Galactosidase a-Glucosidase b-Glucosidase a-Mannosidase a-Fucosidase Hydrolysis of: Starch Gelatin Assimilation of: L-Arabinose D-Mannose N-Acetylglucosamine Malic acid Trisodium citrate Rhamnose Melibiose DNA G+C content (mol%)
1
2
3
4
5
6
Orange 6.0–10.0 20–30 0–2 + 2 + + +
Yellow 6.0–8.5 15–30 0–3 2 + 2 2 2
Salmon Red 5.0–10.0 20–30 0–2 + 2 + + 2
Yellow 5.0–8.0 17–37 0–1 2 2 2 + +
Yellow 5.0–8.0 15–30 0–2 + 2 + + 2
Orange 5.5–8.5 20–37 0–1 + + – + 2
+ + + 2 + + + + 2 +
+ 2 2 + 2 2 2 2 2 W
+ 2 + + 2 2 2 2 2 2
+ + + + +
+ 2 2 2 2 2 2 2 2 2
2 + 2 2 2 2 2 2 2
2 +
2 2
2 +
2 +
2 2
+
+ 2 2 2 + 2 + 49.1
+ + 2 2 2 2 + 42.5–43.3a*
+ + + + 2 2 + 47b
+ + + 2 2 + + 45.2c
2 + + 2 2 2 + 46.8d
2 + + 2 2 2 2 46.6e
W W
2 2 W
W
W
*Data from: a, Yoon & Im (2007); b, Zhang et al. (2012); c, Zhang et al. (2010b); d, Zhang et al. (2013); e, Qu et al. (2009).
Cells are Gram-staining-negative, aerobic, non-sporeforming, motile rods about 0.5–0.9 mm in diameter and 0.8–1.6 mm in length. Colonies are circular with regular edges, convex, opaque, moist, orange-pigmented and 2.0– 4.0 mm in diameter after 3 days of cultivation at 28 uC on R2A agar. Growth also occurs on NA agar, but not on TSA or MacConkey agar. Growth occurs at 20–30 uC (optimal growth at 28–30 uC) and at pH 6.0–10.0 (optimal growth at pH 7.0–7.5) in R2A broth. Growth occurs with 0–2 % NaCl (optimal growth without NaCl). Degrades Tween 80
and casein, but does not degrade Tween 20, starch or egg yolk. Positive for catalase activity, but negative for oxidase activity and methyl red test. According to tests of API 20E and 20NE, positive for aesculin and gelatin hydrolysis, urease, b-galactosidase (PNPG and ONPG tests) and tryptophan deaminase activity, and Voges–Proskauer reaction, but negative for arginine dihydrolase, lysine decarboxylase and ornithine decarboxylase activity, citrate utilization, glucose fermentation, and indole and H2S production. Nitrate cannot be reduced to nitrite or
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International Journal of Systematic and Evolutionary Microbiology 64
Cnuella takakiae gen. nov., sp. nov.
Table 2. Fatty acid composition (%) of strain strain RG1-1T and related type strains of members of family Chitinophagaceae Strains: 1, RG1-1T; 2, Flavisolibacter ginsengiterrae KCTC 12656T; 3, Terrimonas rubra CCTCC AB 2010401T; 4, Niastella populi KCTC 22560T; 5, Flavitalea populi CCTCC AB 208255T; 6, Lacibacter cauensis NBRC 104930T. TR, Traces (less than 1 % of the total); –, not present. All data are from this study. Fatty acid
1
2
3
4
5
6
iso-C13 : 0 iso-C13 : 0 3-OH C13 : 1 iso-C14 : 0 C14 : 0 iso-C15 : 0 iso-C15 : 0 3-OH anteiso-C15 : 0 C15 : 0 2-OH C15 : 0 3-OH iso-C15 : 1 G anteiso-C15 : 1 A C15 : 1v6c iso-C16 : 0 iso-C16 : 0 3-OH C16 : 0 C16 : 0 3-OH iso-C16 : 1 H C16 : 1v5c C16 : 1v11c iso-C17 : 0 iso-C17 : 0 3-OH anteiso-C17 : 0 anteiso-C17 : 1 C17 : 1v6c Summed features* 1 3 4 9
5.1 2.7
1.1
TR
TR
TR
TR
– 1.0 –
7.0 3.5 – – – 10.2 – 5.0
1.2 –
TR
TR
1.8
TR
1.5
TR
TR
TR
19.8
29.9 TR
1.0 19.0 2.3
10.0
TR
TR
TR
–
– 7.0 5.0
1.1 1.1 26.8
TR
2.5
TR
37.8 TR
6.6 TR
TR
– 15.6 7.1 – 1.5
2.3 7.4
TR
–
– 25.0 1.9 –
2.1
TR
TR
TR
TR
TR
9.9
4.6
3.5 3.8
TR
TR
1.8
1.4
TR
TR
– 1.3 9.4
– 1.2 7.0
TR
TR
– 2.6
– 3.0
4.5 17.0 1.2 3.0
3.7 14.1 1.9
TR TR
TR
TR
2.0 4.3 – – – – 12.2 – – – – 23.3 – –
TR
– – 1.0 TR
9.7 TR
– – – 2.9 – –
TR
1.8 – – 6.6 – 6.7 12.7 2.7 1.4 TR
3.5 4.4 6.4 –
TR TR TR
21.4 2.8 1.2 2.7 TR
15.6 TR
– 2.4
The type strain, RG1-1T (5CGMCC 1.12492T5DSM 26897T), was isolated from Takakia lepidozioides collected from Gawalong glacier in Tibet, China. The DNA G+C content of the type strain is 49.1 mol%.
TR
3.1 1.5 – 3.8 –
Acknowledgements
TR
References
10.0
This work was funded by the Scientific Research Program of National Natural Science Foundation of China (no. 31100004).
TR
An, D.-S., Lee, H.-G., Im, W.-T., Liu, Q. M. & Lee, S.-T. (2007).
– 1.0
Segetibacter koreensis gen. nov., sp. nov., a novel member of the phylum Bacteroidetes, isolated from the soil of a ginseng field in South Korea. Int J Syst Evol Microbiol 57, 1828–1833.
– 25.1 – –
Dong, X.-Z. & Cai, M.-Y. (2001). Determination of biochemical
*Summed features represent groups of two or three fatty acids that could not be separated by GLC using the MIDI system. Summed feature 1 contained iso-C15 : 1 H and/or C13 : 0 3-OH; summed feature 3 contained C16 : 1v7c and/or C16 : 1v6c; summed feature 4 contained iso-C17 : 1 I and/or anteiso-C17 : 1 B; summed feature 9 contained isoC17 : 1v9c and/or 10-methyl C16 : 0.
nitrogen. Assimilates D-glucose, L-arabinose, trisodium citrate and melibiose; does not assimilate D-mannose, Dmannitol, N-acetylglucosamine, malic acid, potassium gluconate, capric acid, adipic acid, phenylacetic acid, Dsorbitol and rhamnose. In API ZYM tests, positive for alkaline phosphatase, esterase (C4), esterase lipase (C8), leucine arylamidase, valine arylamidase, cystine arylamidase, trypsin, acid phosphatase, naphthol-AS-BI-phosphohydrolase, b-galactosidase, a-glucosidase, b-glucosidase, Nacetyl-b-glucosaminidase, a-galactosidase and a-fucosidase http://ijs.sgmjournals.org
activities, but negative for lipase, a-chymotrypsin, amannosidase and b-glucuronidase activities. The following carbon sources are utilized in the GN2 MicroPlate: dextrin, Tween 40, Tween 80, cellobiose, D-fructose, gentiobiose, aD-glucose, a-lactose, D-psicose, acetic acid, citric acid, Dgluconic acid, D-glucuronic acid, b-hydroxybutyric acid, a-ketobutyric acid, D-saccharic acid, bromosuccinic acid, L-alaninamide, D-alanine, L-aspartic acid, L-leucine, Lphenylalanine, L-proline and D-serine; other substrates are not utilized. Susceptible to all the tested antibiotics: kanamycin (5 mg), streptomycin (10 mg), chloramphenicol (30 mg), tetracycline (30 mg) and rifampicin (30 mg). The fatty acid profile is composed mainly of iso-C15 : 0, summed feature 3 (C16 : 1v7c and/or C16 : 1v6c), C16 : 0 and iso-C17 : 0 3-OH; the major polar lipids are phosphatidylethanolamine, four unidentified aminolipids, one unidentified phospholipid, one unidentified aminoglycolipid, one unidentified glycolipid and three unidentified lipids. The dominant menaquinone is MK-7.
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