International Journal of Systematic and Evolutionary Microbiology (2014), 64, 4073–4078
DOI 10.1099/ijs.0.067579-0
Pedobacter rivuli sp. nov., isolated from a freshwater stream Heeyoung Kang, Haneul Kim, Yochan Joung and Kiseong Joh Correspondence Kiseong Joh
Department of Bioscience and Biotechnology, Hankuk University of Foreign Studies, Gyeonggi 449-791, Republic of Korea
[email protected]
A Gram-staining-negative, rod-shaped, red strain, HME8457T, was isolated from a freshwater stream located in the Republic of Korea. A phylogenetic tree based on 16S rRNA gene sequences showed that strain HME8457T formed a lineage within the genus Pedobacter. Strain HME8457T was most closely related to species of the genus Pedobacter: Pedobacter daechungensis Dae 13T (96.4 % sequence similarity), Pedobacter lentus DS-40T (95.3 %), Pedobacter terricola DS-45T (94.9 %), Pedobacter glucosidilyticus 1-2T (94.2 %) and ‘Pedobacter soyangensis’ HME6451 (93.6 %). The major fatty acids were iso-C15 : 0 (28.8 %), summed feature 3 (comprising C16 : 1v6c and/or C16 : 1v7c; 21.7 %), iso-C17 : 0 3-OH (7.7 %) and anteiso-C15 : 0 (6.2 %). The only respiratory quinone was MK-7. Polar lipid analysis revealed the presence of phosphatidylethanolamine, one unidentified aminolipid and two unidentified polar lipids. Sphingolipid was present. The DNA G+C content was 33.3 mol%. On the basis of the evidence presented in this study, strain HME8457T represents a novel species of the genus Pedobacter, for which the name Pedobacter rivuli sp. nov. is proposed; the type strain is HME8457T (5KACC 17312T5CECT 8291T).
The genus Pedobacter, belonging to the family Sphingobacteriaceae, was proposed by Steyn et al. (1998) with the description of four species: Pedobacter heparinus, Pedobacter piscium, Pedobacter africanus and Pedobacter saltans. The description of the genus was subsequently emended by Vanparys et al. (2005), Hwang et al. (2006), Gallego et al. (2006), Zhou et al. (2012) and Farfa´n et al. (2014). Strains of species of the genus Pedobacter are Gram-staining negative, strictly aerobic, non-motile, rod-shaped and have MK-7 as the major menaquinone and DNA G+C contents of 33–45 mol%. The genus Pedobacter currently comprises 44 species with validly published names (http://www. bacterio.net/), including the recently identified species Pedobacter ginsengiterrae, Pedobacter ginsenosidimutans, Pedobacter luteus, Pedobacter ruber, Pedobacter tournemirensis, Pedobacter jejuensis, Pedobacter nutrimenti and Pedobacter antarcticus (Yang et al., 2013; Oh et al., 2013; Margesin & Zhang, 2013; Urios et al., 2013; Kook et al., 2014; Derichs et al., 2014; Hoang et al., 2013; Farfa´n et al., 2014). Strains of species of the genus Pedobacter have been isolated from different environments, such as soil, activated sludge, compost, chilled food, glaciers and water samples (Joung et al., 2014; Derichs et al., 2014). The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain HME8457T is JQ911707. One supplementary figure and one supplementary table are available with the online Supplementary Material.
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A water sample was collected from a stream (Dorimcheon) in Seoul, Republic of Korea (37u 309 190 N 126u 539 370 E). Strain HME8457T was isolated from the water sample by the dilution-plating method on R2A agar (Difco) at 30 uC. It was routinely cultivated on R2A at 30 uC and preserved at 280 uC in distilled water supplemented with 20 % (v/v) glycerol. The 16S rRNA gene sequence (1386 bp) of strain HME8457T was determined as described by Cho & Giovannoni (2003) with a pair of universal primers, 27F and 1492R. The 16S rRNA gene sequence was identified using the EzTaxon-e server (http://eztaxon-e.ezbiocloud.net/; Kim et al., 2012) and compared with sequences in GenBank using the BLAST program (http://www.ncbi.nlm.nih.gov/Blast.cgi). Phylogenetic analysis was carried out using MEGA version 6 (Tamura et al., 2013), after multiple alignments of the data with CLUSTAL W (Thompson et al., 1994). Phylogenetic trees were reconstructed using neighbour-joining (Saitou & Nei, 1987), maximum-parsimony (Fitch, 1971) and maximum-likelihood (Felsenstein, 1981) algorithms in MEGA 6 software. The topology of the neighbour-joining phylogenetic tree was evaluated via bootstrap analysis (Felsenstein, 1985), based on 1000 replications. Strain HME8457T showed the highest 16S rRNA gene sequence similarity with Pedobacter daechungensis Dae 13T (96.4 %), Pedobacter lentus DS-40T (95.3 %), Pedobacter terricola DS-45T (94.9 %), Pedobacter glucosidilyticus 1-2T (94.2 %) and ‘Pedobacter soyangensis’ HME6451 (93.6 %). The neighbour-joining tree (Fig. 1) 4073
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0.02
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100 Pedobacter caeni LMG 22862T (AJ786798) Pedobacter steynii WB 2.3-45T (AM491372) Pedobacter duraquae WB 2.1-25T (AM491368) Pedobacter metabolipauper WB 2.3-71T (AM491370) Pedobacter africanus DSM 12126T (AJ438171) Pedobacter nutrimenti J22T (HF536497) Pedobacter heparinus DSM 2366T (CP001681) Pedobacter nyackensis NWG-II14T (EU030686) Pedobacter panaciterrae Gsoil 042T (AB245368) 89 Pedobacter ginsengisoli Gsoil 104T (AB245371) Pedobacter piscium DSM 11725T (AJ438174) Pedobacter hartonius WB 3.3-3T (AM491371) 99 Pedobacter cryoconitis A37T (AJ438170) 86 Pedobacter westerhofensis WB 3.3-22T (AM491369) Pedobacter himalayensis HHS 22T (AJ583425) Pedobacter insulae DS-139T (EF100697) 85 91 Pedobacter koreensis WPCB189T (DQ092871) 83 Pedobacter aquatilis AR107T (AM114396) 99 Pedobacter jejuensis THG-DR3T (KC252614) Pedobacter ginsengiterrae DCY49T (HM776704) Pedobacter rhizosphaerae 01-96T (AM279214) 89 95 Pedobacter alluvionis NWER-II11T (EU030688) 82 Pedobacter roseus CL-GP80T (DQ112353) Pedobacter soli 15-51T (AM279215) Pedobacter sandarakinus DS-27T (DQ235228) Pedobacter agri PB92T (EF660751) Pedobacter terrae DS-57T (DQ889723) 99 Pedobacter suwonensis 15-52T (DQ097274) Pedobacter oryzae N7T (EU109726) Pedobacter luteus N7d-4T (FJ377314) 98 Pedobacter ruber W1T (HQ882803) 98 79 Pedobacter composti TR6-06T (AB267720) Pedobacter bauzanensis BZ42T (GQ161990) Pedobacter arcticus A12T (HM051286) ‘Pedobacter hainanensis’ 13-Q (JQ083404) 77 Pedobacter saltans DSM 12145T (CP002545) Pedobacter glucosidilyticus 1-2T (EU585748) ‘Pedobacter soyangensis’ HME6451 (HQ714329) Pedobacter rivuli HME8457T (JQ911707) Pedobacter terricola DS-45T (EF446147) Pedobacter daechungensis Dae 13T (AB267722) Pedobacter lentus DS-40T (EF446146) ‘Pedobacter zeaxanthinifaciens’ TDMA-5 (AB264126) ‘Pedobacter xinjiangensis’ 12157 (EU734803) Pedobacter tournemirensis TF5-37.2-LB10T (GU198945) Flavobacterium aquatile ATCC 11947T (M62797)
Fig. 1. Neighbour-joining phylogenetic tree based on 16S rRNA gene sequences, showing the phylogenetic position of strain HME8457T in the genus Pedobacter. Bootstrap percentages (.70 %) from neighbour-joining (above nodes) analyses are shown. GenBank accession numbers of 16S rRNA gene sequences are given in parentheses. Flavobacterium aquatile ATCC 11947T (M62797) was used as the outgroup. Bar, 0.02 substitutions per nucleotide position.
showed that strain HME8457T grouped with members of the genus Pedobacter. Strain HME8457T also clustered with the genus Pedobacter in the maximum-parsimony and maximum-likelihood trees (data not shown). This phylogenetic inference, together with the level 16S rRNA gene sequence similarity (,97 %) (Wayne et al., 1987)
between strain HME8457T and the five most closely related species of the genus Pedobacter, suggest that strain HME8457T represents a novel species of the genus Pedobacter.
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Cell morphology and size were determined by using light microscopy and transmission electron microscopy
Pedobacter rivuli sp. nov.
(JEM1010; JEOL). Gram staining was determined using the bioMe´rieux Gram Stain kit according to the manufacturer’s instructions. Gliding motility was examined using the hanging-drop technique on fresh R2A broth culture (Bernardet et al., 2002). Oxidase activity was evaluated via the oxidation of 1 % (w/v) N,N,N9,N9-tetramethyl-pphenylenediamine dihydrochloride. Catalase activity was determined by assessing bubble production in 3 % (v/v) H2O2. The presence of flexirubin-type pigments was investigated from colour shifts after exposure to 20 % (w/ v) KOH solution (Bernardet et al., 2002). Anaerobic growth was tested on R2A agar at 30 uC for 3 weeks using a GasPak EZ Anaerobic container System (BD). The temperature range and optimum for growth were determined on R2A agar at 4, 10, 15, 20, 25, 30, 37 and 42 uC. The pH range for growth was determined in R2A broth adjusted to various pH (initially pH 4.0–10.0 at intervals of 1.0 pH unit) and assessed after 2 days’ incubation. The pH of R2A broth was adjusted by adding 0.1 mM sodium acetate buffer (pH 4.0–6.0), 0.1 mM phosphate buffer (pH 7.0 and 8.0) or 0.1 mM sodium carbonate buffer (pH 9.0 and 10.0). Growth in the absence of NaCl and in the presence of 0.5 % (w/v) and 1.0–5.0 % (w/v) [at intervals of 1.0 % (w/v)] NaCl was assessed after 2 days’ incubation in R2A broth. Additional biochemical characteristics were determined using the API 20NE, API ZYM and API 50CH kits (bioMe´rieux) and carbon source utilization was evaluated by using a Biolog GN2 microplate according to the manufacturer’s instructions. Growth was assessed on nutrient (NA; Difco), marine 2216 (MA; Difco), tryptic soy (TSA; Difco), blood (Difco) and MacConkey (Difco) agars. DNase activity was assessed on DNase test agar (Difco). Hydrolysis of casein [3 % (w/v) skimmed milk (Difco)], CM-cellulose [1 % (w/v) CM-cellulose (Sigma)], cellulose [1 % (w/v) filter paper (Whatman no. 1)], dextrin [1 % (w/v) dextrin (Sigma)] and starch [1 % (w/v) soluble starch (Sigma)] was tested using 0.16 R2A agar as the basal medium. Pedobacter daechungensis KACC 14960T, Pedobacter lentus KCTC 12875T, Pedobacter terricola KACC 17433T, Pedobacter glucosidilyticus KCTC 22438T and ‘Pedobacter soyangensis’ HME6451 were used as reference strains in tests. The phenotypic characteristics of strain HME8457T are given in Table 1, Table S1 (available in the online Supplementary Material) and in the species description. Strain HME8457T exhibited a number of phenotypic similarities to species of the genus Pedobacter, including being Gram-staining-negative, aerobic, rod-shaped (Fig. S1), non-motile, and oxidase- and catalase-positive. These features of strain HME8457T are typical of members of the genus Pedobacter. However, several characteristics of HME8457T, such as its ability to utilize a-ketoglutaric acid and to produce acid from maltose and lactose distinguished this strain from other strains of species of the genus Pedobacter (Table 1). The genomic DNA of strain HME8457T was extracted, purified by using a Genomic DNA isolation kit (Solgent), and the DNA G+C content was determined by the thermal http://ijs.sgmjournals.org
Table 1. Characteristics differentiating strain HME8457T from related members of the genus Pedobacter Strains: 1, HME8457T; 2, Pedobacter daechungensis KACC 14960T; 3, Pedobacter lentus KCTC 12875T; 4, Pedobacter terricola KACC 17433T; 5, Pedobacter glucosidilyticus KCTC 22438T; 6, ‘Pedobacter soyangensis’ HME6451. Data for strains 1 to 6 were obtained from this study, except the DNA G+C content of the five reference strains (An et al., 2009; Yoon et al., 2007; Luo et al., 2010; Joung et al., 2014). All strains grew on R2A, TSA and blood agar but not on MacConkey agar. All strains were positive for oxidase and catalase activities, aesculin hydrolysis, b-galactosidase, alkaline phosphatase, esterase lipase (C8), leucine arylamidase, valine arylamidase and acid phosphatase activities and for the utilization of aesculin ferric citrate. All strains were negative for nitrate reduction, glucose fermentation, arginine dihydrolase, urease, gelatinase, lipase (C14), trypsin, a-galactosidase, b-glucuronidase and a-mannosidase activities. +, Positive; –, negative. Characteristic
1
2
3
4
5
Growth conditions 37 uC 2 2 2 2 2 pH 9 2 2 2 2 + 1.5 % (w/v) NaCl 2 2 + 2 2 Growth on NA + 2 + + + Growth on MA 2 2 + + + Degradation of DNA 2 + 2 2 + Enzyme activity (API ZYM) Esterase (C4) + + + 2 + Cystine arylamidase + + + 2 + a-Chymotrypsin 2 2 + 2 2 Naphthol-AS-BI+ 2 + + + phosphohydrolase b-Galactosidase + 2 + 2 2 a-Glucosidase 2 + + 2 + 2 + + + N-acetyl-b-glucosaminidase + DNA G+C content (mol%) 33.3 33.8 36.0 36.8 37.2
6 + + + + 2 2 + + 2 + 2 + + 36.0
denaturation fluorimetic method (Gonzalez & Saiz-Jimenez, 2002) using SYBR Green 1 (SG 1; Invitrogen) and a realtime PCR thermocycler (Bio-Rad). Genomic DNA from Polaribacter dokdonensis KCTC 12392T (30.6 mol%), Cyclobacterium marinum KCTC 2917 (38.1 mol%), Acinetobacter baumannii KACC 12454 (39.1 mol%), Leadbetterella byssophila KACC 11308 T (40.4 mol%), Bacillus subtilis subsp. spizizenii KACC 14741 (43.8 mol%), Runella slithyformis KACC 13042T (46.4 mol%), Corynebacterium glutamicum KACC 10784 (54.1 mol%), Pseudomonas syringae pv. actinidiae KACC 10772T (58.8 mol%), Aeromonas hydrophila ATCC 7966T (61.5 mol%) and Sphingopyxis alaskensis KACC 12337T (65.5 mol%), which have genomes that have been completely sequenced, were used as calibration references. The thermal cycler conditions consisted of a ramp from 25 to 99 uC at 1.2 uC min–1. Fluorescent DNA melting curves were generated in triplicate. The DNA G+C content of strain HME8457T was calculated using linear regression analysis of the melting temperature (Tm) plotted against calibration reference strains. 4075
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Fatty acid analysis was performed on strains HME8457T and the five reference strains. Cells were grown on R2A agar at 30 uC until they reached the mid-exponential growth phase. Fatty acids were saponified, methylated and extracted using the standard protocol of the Sherlock Microbial Identification System version 6.1 (MIDI). The fatty acids were analysed by GC (model 7890; Hewlett Packard) and identified using the RTSBA6 database of the Microbial Identification System (Sasser, 1990). For analysis of respiratory quinones and polar lipids, cells of strain HME8457T were harvested from R2A after incubation at 30 uC for 2 days and freeze-dried. The isoprenoid quinones of strain HME8457T were analysed by TLC according to the protocol of Minnikin et al. (1984). Polar lipids were extracted according to the method of Minnikin et al. (1984) and separated by two-dimensional TLC (plates were coated with silica gel, 10610 cm; Merck). Chromatography was performed by using chloroform/methanol/water (65 : 25 : 4, by vol.) in the first direction, followed by chloroform/ methanol/acetic acid/water (40 : 7.5 : 6 : 2, by vol.) in the second. Plates were sprayed with 10 % (v/v) ethanolic molybdophosphoric acid (Sigma) for detection of the total polar lipids, ninhydrin (Sigma) for aminolipids, a-naphthol for glycolipids and molybdenum blue (Sigma) for phospholipids. The sphingolipids of strain HME8457T and Pedobacter glucosidilyticus KCTC 22438T were extracted according to the method of Yabuuchi et al. (1990). The presence of sphingolipids was confirmed by two-dimensional TLC with 0.4 % (v/v) ninhydrin in butanol. The DNA G+C content of the genomic DNA of strain HME8457T was 33.3 mol%, which is in line with that reported for other species of the genus Pedobacter. The major fatty acids of strain HME8457T were iso-C15 : 0, summed feature 3 (comprising C16 : 1v6c and/or C16 : 1v7c), iso-C17 : 0 3-OH and anteiso-C15 : 0 (Table 2). The fatty acid composition of strain HME8457T was similar to those of the five reference strains, with only minor quantitative differences. The only isoprenoid quinone of strain HME8457T was menaquinone 7 (MK-7). The polar lipids of strain HME8457T were phosphatidylethanolamine, one unidentified aminolipid, and two unidentified polar lipids (Fig. 2). Strain HME8457T and the five reference strains had phosphatidylethanolamine, an unidentified aminolipid, and unidentified polar lipids in common. However, strain HME8457T differed from the five reference strains by the absence of the unidentified phospholipid and unidentified aminophospholipid (Zhou et al., 2012; Joung et al., 2014). Sphingolipid was present in strain HME8457T; sphingolipids are a distinct feature of members of the genus Pedobacter (Steyn et al., 1998).
Table 2. Cellular fatty acid composition of strain HME8457T and some other members of the genus Pedobacter Strains: 1, HME8457T; 2, Pedobacter daechungensis KACC 14960T; 3, Pedobacter lentus KCTC 12875T; 4, Pedobacter terricola KACC 17433T; 5, Pedobacter glucosidilyticus KCTC 22438T; 6, ‘Pedobacter soyangensis’ HME6451. Values are percentages of the total cellular fatty acids. Only fatty acids amounting to at least 1.0 % of the total cellular fatty acids of at least one of the strains are shown. tr, Trace(,1.0 %); ND, not detected. All data are from this study. Fatty acid
1
iso-C11 : 0 2.5 iso-C13 : 0 tr iso-C14 : 0 2.9 C14 : 0 1.4 28.8 iso-C15 : 0 anteiso-C15 : 0 6.2 3.5 C15 : 1v6c tr iso-C16 : 1 H iso-C16 : 0 tr 3.0 C16 : 1v5c 2.5 C16 : 0 3.1 iso-C15 : 0 3-OH C15 : 0 2-OH 1.2 2.2 C17 : 1v8c 2.0 iso-C16 : 0 3-OH 1.0 C16 : 0 3-OH iso-C17 : 0 3-OH 7.7 tr C17 : 0 2-OH Summed Feature 3* 21.7 Summed Feature 9* 3.0
2 1.4 tr ND
1.1 15.8 6.3 2.3 1.9 3.9 3.4 6.9 1.8 1.9 2.9 4.4 tr 10.5 2.0 27.6 4.4
3
4
5
6
tr
tr ND
1.7 tr
ND
ND
2.3 tr 27.8 3.4 1.4 tr 1.1 3.5 3.3 3.3 1.1 tr 1.2 tr 14.4 tr 26.5 3.0
ND
ND
ND
tr 14.2 4.0 2.0 tr 1.4 2.8 6.7 2.1 1.8 1.7 tr tr 10.4 1.1 40.3 2.8
1.0
tr tr 37.3 41.2 5.4 4.7 3.7 2.3 tr ND 1.1 tr 2.7 6.0 3.4 1.5 2.0 2.4 tr tr 1.1 tr tr tr tr 1.3 10.7 10.2 tr tr 17.9 23.0 5.0 1.3
*Summed features are groups of two or three fatty acids that could not be separated by GC with the MIDI system. Summed feature 3 comprised C16 : 1v6c and/or C16 : 1v7c; summed feature 9 comprised iso-C17 : 1v9c and/or 10-methyl C16 : 0.
suggest that strain HME8457T should be classified in the genus Pedobacter as a member of a novel species, for which the namehttp://dx.doi.org/10.1601/nm.8232Pedobacter rivuli sp. nov. is proposed. Description of Pedobacter rivuli sp. nov. Pedobacter rivuli (ri9vu.li. L. gen. n. rivuli of/from a small stream).
On the basis of the phylogenetic distances, morphological characteristics, fatty acid profiles and quinone system, strain HME8457T should be affiliated to the genus Pedobacter. However, several of the phenotypic characteristics (Table 1) and the polar lipid profile of strain HME8457T distinguish it from closely related type strains of species of the genus Pedobacter. Therefore, our data
Cells are Gram-staining-negative, non-motile, aerobic rods and approximately 1.2–1.4 mm60.3–0.4 mm after 2 days at 30 uC on R2A agar. Colonies on R2A agar are red, smooth, convex and circular with entire margins and 1.5 mm in diameter after 2 days at 30 uC. Growth occurs at 15–30 uC (optimum, 30 uC), at pH 7–8 (optimum, pH 7.0) and with 0–0.5 % (w/v) NaCl [optimum 0 % (w/v) NaCl]. Oxidase and catalase activities are positive. Flexirubin-type pigments are not produced. Growth occurs on NA, TSA, R2A and blood agars, and no growth occurs on MA or
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Pedobacter rivuli sp. nov.
Republic of Korea. The genomic DNA G+C content of the type strain is 33.3 mol%
Acknowledgements This work was supported by a grant from the National Institute of Biological Resources (NIBR), funded by the Ministry of Environment (MOE) of the Republic of Korea (NIBR no. 2013-02-001).
L1 L2 AL
PE
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nov. and comb. nov., Sphingomonas parapaucimobilis sp. nov., Sphingomonas yanoikuyae sp. nov., Sphingomonas adhaesiva sp. nov., Sphingomonas capsulata comb. nov., and two genospecies of the genus Sphingomonas. Microbiol Immunol 34, 99–119.
Steyn, P. L., Segers, P., Vancanneyt, M., Sandra, P., Kersters, K. & Joubert, J. J. (1998). Classification of heparinolytic bacteria into a new
Yang, J. E., Son, H. M., Lee, J. M., Shin, H. S., Park, S. Y., Lee, D. G., Kook, M. & Yi, T. H. (2013). Pedobacter ginsenosidimutans sp. nov.,
method for reconstructing phylogenetic trees. Mol Biol Evol 4, 406– 425. Sasser, M. (1990). Identification of bacteria by gas chromatography of
genus, Pedobacter, comprising four species: Pedobacter heparinus comb. nov., Pedobacter piscium comb. nov., Pedobacter africanus sp. nov. and Pedobacter saltans sp. nov. proposal of the family Sphingobacteriaceae fam. nov. Int J Syst Bacteriol 48, 165–177. Tamura, K., Stecher, G., Peterson, D., Filipski, A. & Kumar, S. (2013). MEGA6: molecular evolutionary genetics analysis version 6.0. Mol Biol Evol 30, 2725–2729.
with ginsenoside-converting activity. Int J Syst Evol Microbiol 63, 4396–4401. Yoon, J. H., Kang, S. J., Park, S. & Oh, T. K. (2007). Pedobacter lentus
sp. nov. and Pedobacter terricola sp. nov., isolated from soil. Int J Syst Evol Microbiol 57, 2089–2095. Zhou, Z., Jiang, F., Wang, S., Peng, F., Dai, J., Li, W. & Fang, C. (2012).
improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 22, 4673–4680.
Pedobacter arcticus sp. nov., a facultative psychrophile isolated from Arctic soil, and emended descriptions of the genus Pedobacter, Pedobacter heparinus, Pedobacter daechungensis, Pedobacter terricola, Pedobacter glucosidilyticus and Pedobacter lentus. Int J Syst Evol Microbiol 62, 1963–1969.
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Thompson, J. D., Higgins, D. G. & Gibson, T. J. (1994).
CLUSTAL W:
DOI 10.1099/ijs.0.067579-0
Pedobacter rivuli sp. nov., isolated from a freshwater stream Heeyoung Kang, Haneul Kim, Yochan Joung and Kiseong Joh Correspondence Kiseong Joh
Department of Bioscience and Biotechnology, Hankuk University of Foreign Studies, Gyeonggi 449-791, Republic of Korea
[email protected]
A Gram-staining-negative, rod-shaped, red strain, HME8457T, was isolated from a freshwater stream located in the Republic of Korea. A phylogenetic tree based on 16S rRNA gene sequences showed that strain HME8457T formed a lineage within the genus Pedobacter. Strain HME8457T was most closely related to species of the genus Pedobacter: Pedobacter daechungensis Dae 13T (96.4 % sequence similarity), Pedobacter lentus DS-40T (95.3 %), Pedobacter terricola DS-45T (94.9 %), Pedobacter glucosidilyticus 1-2T (94.2 %) and ‘Pedobacter soyangensis’ HME6451 (93.6 %). The major fatty acids were iso-C15 : 0 (28.8 %), summed feature 3 (comprising C16 : 1v6c and/or C16 : 1v7c; 21.7 %), iso-C17 : 0 3-OH (7.7 %) and anteiso-C15 : 0 (6.2 %). The only respiratory quinone was MK-7. Polar lipid analysis revealed the presence of phosphatidylethanolamine, one unidentified aminolipid and two unidentified polar lipids. Sphingolipid was present. The DNA G+C content was 33.3 mol%. On the basis of the evidence presented in this study, strain HME8457T represents a novel species of the genus Pedobacter, for which the name Pedobacter rivuli sp. nov. is proposed; the type strain is HME8457T (5KACC 17312T5CECT 8291T).
The genus Pedobacter, belonging to the family Sphingobacteriaceae, was proposed by Steyn et al. (1998) with the description of four species: Pedobacter heparinus, Pedobacter piscium, Pedobacter africanus and Pedobacter saltans. The description of the genus was subsequently emended by Vanparys et al. (2005), Hwang et al. (2006), Gallego et al. (2006), Zhou et al. (2012) and Farfa´n et al. (2014). Strains of species of the genus Pedobacter are Gram-staining negative, strictly aerobic, non-motile, rod-shaped and have MK-7 as the major menaquinone and DNA G+C contents of 33–45 mol%. The genus Pedobacter currently comprises 44 species with validly published names (http://www. bacterio.net/), including the recently identified species Pedobacter ginsengiterrae, Pedobacter ginsenosidimutans, Pedobacter luteus, Pedobacter ruber, Pedobacter tournemirensis, Pedobacter jejuensis, Pedobacter nutrimenti and Pedobacter antarcticus (Yang et al., 2013; Oh et al., 2013; Margesin & Zhang, 2013; Urios et al., 2013; Kook et al., 2014; Derichs et al., 2014; Hoang et al., 2013; Farfa´n et al., 2014). Strains of species of the genus Pedobacter have been isolated from different environments, such as soil, activated sludge, compost, chilled food, glaciers and water samples (Joung et al., 2014; Derichs et al., 2014). The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain HME8457T is JQ911707. One supplementary figure and one supplementary table are available with the online Supplementary Material.
067579 G 2014 IUMS
Printed in Great Britain
A water sample was collected from a stream (Dorimcheon) in Seoul, Republic of Korea (37u 309 190 N 126u 539 370 E). Strain HME8457T was isolated from the water sample by the dilution-plating method on R2A agar (Difco) at 30 uC. It was routinely cultivated on R2A at 30 uC and preserved at 280 uC in distilled water supplemented with 20 % (v/v) glycerol. The 16S rRNA gene sequence (1386 bp) of strain HME8457T was determined as described by Cho & Giovannoni (2003) with a pair of universal primers, 27F and 1492R. The 16S rRNA gene sequence was identified using the EzTaxon-e server (http://eztaxon-e.ezbiocloud.net/; Kim et al., 2012) and compared with sequences in GenBank using the BLAST program (http://www.ncbi.nlm.nih.gov/Blast.cgi). Phylogenetic analysis was carried out using MEGA version 6 (Tamura et al., 2013), after multiple alignments of the data with CLUSTAL W (Thompson et al., 1994). Phylogenetic trees were reconstructed using neighbour-joining (Saitou & Nei, 1987), maximum-parsimony (Fitch, 1971) and maximum-likelihood (Felsenstein, 1981) algorithms in MEGA 6 software. The topology of the neighbour-joining phylogenetic tree was evaluated via bootstrap analysis (Felsenstein, 1985), based on 1000 replications. Strain HME8457T showed the highest 16S rRNA gene sequence similarity with Pedobacter daechungensis Dae 13T (96.4 %), Pedobacter lentus DS-40T (95.3 %), Pedobacter terricola DS-45T (94.9 %), Pedobacter glucosidilyticus 1-2T (94.2 %) and ‘Pedobacter soyangensis’ HME6451 (93.6 %). The neighbour-joining tree (Fig. 1) 4073
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0.02
76
100 Pedobacter caeni LMG 22862T (AJ786798) Pedobacter steynii WB 2.3-45T (AM491372) Pedobacter duraquae WB 2.1-25T (AM491368) Pedobacter metabolipauper WB 2.3-71T (AM491370) Pedobacter africanus DSM 12126T (AJ438171) Pedobacter nutrimenti J22T (HF536497) Pedobacter heparinus DSM 2366T (CP001681) Pedobacter nyackensis NWG-II14T (EU030686) Pedobacter panaciterrae Gsoil 042T (AB245368) 89 Pedobacter ginsengisoli Gsoil 104T (AB245371) Pedobacter piscium DSM 11725T (AJ438174) Pedobacter hartonius WB 3.3-3T (AM491371) 99 Pedobacter cryoconitis A37T (AJ438170) 86 Pedobacter westerhofensis WB 3.3-22T (AM491369) Pedobacter himalayensis HHS 22T (AJ583425) Pedobacter insulae DS-139T (EF100697) 85 91 Pedobacter koreensis WPCB189T (DQ092871) 83 Pedobacter aquatilis AR107T (AM114396) 99 Pedobacter jejuensis THG-DR3T (KC252614) Pedobacter ginsengiterrae DCY49T (HM776704) Pedobacter rhizosphaerae 01-96T (AM279214) 89 95 Pedobacter alluvionis NWER-II11T (EU030688) 82 Pedobacter roseus CL-GP80T (DQ112353) Pedobacter soli 15-51T (AM279215) Pedobacter sandarakinus DS-27T (DQ235228) Pedobacter agri PB92T (EF660751) Pedobacter terrae DS-57T (DQ889723) 99 Pedobacter suwonensis 15-52T (DQ097274) Pedobacter oryzae N7T (EU109726) Pedobacter luteus N7d-4T (FJ377314) 98 Pedobacter ruber W1T (HQ882803) 98 79 Pedobacter composti TR6-06T (AB267720) Pedobacter bauzanensis BZ42T (GQ161990) Pedobacter arcticus A12T (HM051286) ‘Pedobacter hainanensis’ 13-Q (JQ083404) 77 Pedobacter saltans DSM 12145T (CP002545) Pedobacter glucosidilyticus 1-2T (EU585748) ‘Pedobacter soyangensis’ HME6451 (HQ714329) Pedobacter rivuli HME8457T (JQ911707) Pedobacter terricola DS-45T (EF446147) Pedobacter daechungensis Dae 13T (AB267722) Pedobacter lentus DS-40T (EF446146) ‘Pedobacter zeaxanthinifaciens’ TDMA-5 (AB264126) ‘Pedobacter xinjiangensis’ 12157 (EU734803) Pedobacter tournemirensis TF5-37.2-LB10T (GU198945) Flavobacterium aquatile ATCC 11947T (M62797)
Fig. 1. Neighbour-joining phylogenetic tree based on 16S rRNA gene sequences, showing the phylogenetic position of strain HME8457T in the genus Pedobacter. Bootstrap percentages (.70 %) from neighbour-joining (above nodes) analyses are shown. GenBank accession numbers of 16S rRNA gene sequences are given in parentheses. Flavobacterium aquatile ATCC 11947T (M62797) was used as the outgroup. Bar, 0.02 substitutions per nucleotide position.
showed that strain HME8457T grouped with members of the genus Pedobacter. Strain HME8457T also clustered with the genus Pedobacter in the maximum-parsimony and maximum-likelihood trees (data not shown). This phylogenetic inference, together with the level 16S rRNA gene sequence similarity (,97 %) (Wayne et al., 1987)
between strain HME8457T and the five most closely related species of the genus Pedobacter, suggest that strain HME8457T represents a novel species of the genus Pedobacter.
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Cell morphology and size were determined by using light microscopy and transmission electron microscopy
Pedobacter rivuli sp. nov.
(JEM1010; JEOL). Gram staining was determined using the bioMe´rieux Gram Stain kit according to the manufacturer’s instructions. Gliding motility was examined using the hanging-drop technique on fresh R2A broth culture (Bernardet et al., 2002). Oxidase activity was evaluated via the oxidation of 1 % (w/v) N,N,N9,N9-tetramethyl-pphenylenediamine dihydrochloride. Catalase activity was determined by assessing bubble production in 3 % (v/v) H2O2. The presence of flexirubin-type pigments was investigated from colour shifts after exposure to 20 % (w/ v) KOH solution (Bernardet et al., 2002). Anaerobic growth was tested on R2A agar at 30 uC for 3 weeks using a GasPak EZ Anaerobic container System (BD). The temperature range and optimum for growth were determined on R2A agar at 4, 10, 15, 20, 25, 30, 37 and 42 uC. The pH range for growth was determined in R2A broth adjusted to various pH (initially pH 4.0–10.0 at intervals of 1.0 pH unit) and assessed after 2 days’ incubation. The pH of R2A broth was adjusted by adding 0.1 mM sodium acetate buffer (pH 4.0–6.0), 0.1 mM phosphate buffer (pH 7.0 and 8.0) or 0.1 mM sodium carbonate buffer (pH 9.0 and 10.0). Growth in the absence of NaCl and in the presence of 0.5 % (w/v) and 1.0–5.0 % (w/v) [at intervals of 1.0 % (w/v)] NaCl was assessed after 2 days’ incubation in R2A broth. Additional biochemical characteristics were determined using the API 20NE, API ZYM and API 50CH kits (bioMe´rieux) and carbon source utilization was evaluated by using a Biolog GN2 microplate according to the manufacturer’s instructions. Growth was assessed on nutrient (NA; Difco), marine 2216 (MA; Difco), tryptic soy (TSA; Difco), blood (Difco) and MacConkey (Difco) agars. DNase activity was assessed on DNase test agar (Difco). Hydrolysis of casein [3 % (w/v) skimmed milk (Difco)], CM-cellulose [1 % (w/v) CM-cellulose (Sigma)], cellulose [1 % (w/v) filter paper (Whatman no. 1)], dextrin [1 % (w/v) dextrin (Sigma)] and starch [1 % (w/v) soluble starch (Sigma)] was tested using 0.16 R2A agar as the basal medium. Pedobacter daechungensis KACC 14960T, Pedobacter lentus KCTC 12875T, Pedobacter terricola KACC 17433T, Pedobacter glucosidilyticus KCTC 22438T and ‘Pedobacter soyangensis’ HME6451 were used as reference strains in tests. The phenotypic characteristics of strain HME8457T are given in Table 1, Table S1 (available in the online Supplementary Material) and in the species description. Strain HME8457T exhibited a number of phenotypic similarities to species of the genus Pedobacter, including being Gram-staining-negative, aerobic, rod-shaped (Fig. S1), non-motile, and oxidase- and catalase-positive. These features of strain HME8457T are typical of members of the genus Pedobacter. However, several characteristics of HME8457T, such as its ability to utilize a-ketoglutaric acid and to produce acid from maltose and lactose distinguished this strain from other strains of species of the genus Pedobacter (Table 1). The genomic DNA of strain HME8457T was extracted, purified by using a Genomic DNA isolation kit (Solgent), and the DNA G+C content was determined by the thermal http://ijs.sgmjournals.org
Table 1. Characteristics differentiating strain HME8457T from related members of the genus Pedobacter Strains: 1, HME8457T; 2, Pedobacter daechungensis KACC 14960T; 3, Pedobacter lentus KCTC 12875T; 4, Pedobacter terricola KACC 17433T; 5, Pedobacter glucosidilyticus KCTC 22438T; 6, ‘Pedobacter soyangensis’ HME6451. Data for strains 1 to 6 were obtained from this study, except the DNA G+C content of the five reference strains (An et al., 2009; Yoon et al., 2007; Luo et al., 2010; Joung et al., 2014). All strains grew on R2A, TSA and blood agar but not on MacConkey agar. All strains were positive for oxidase and catalase activities, aesculin hydrolysis, b-galactosidase, alkaline phosphatase, esterase lipase (C8), leucine arylamidase, valine arylamidase and acid phosphatase activities and for the utilization of aesculin ferric citrate. All strains were negative for nitrate reduction, glucose fermentation, arginine dihydrolase, urease, gelatinase, lipase (C14), trypsin, a-galactosidase, b-glucuronidase and a-mannosidase activities. +, Positive; –, negative. Characteristic
1
2
3
4
5
Growth conditions 37 uC 2 2 2 2 2 pH 9 2 2 2 2 + 1.5 % (w/v) NaCl 2 2 + 2 2 Growth on NA + 2 + + + Growth on MA 2 2 + + + Degradation of DNA 2 + 2 2 + Enzyme activity (API ZYM) Esterase (C4) + + + 2 + Cystine arylamidase + + + 2 + a-Chymotrypsin 2 2 + 2 2 Naphthol-AS-BI+ 2 + + + phosphohydrolase b-Galactosidase + 2 + 2 2 a-Glucosidase 2 + + 2 + 2 + + + N-acetyl-b-glucosaminidase + DNA G+C content (mol%) 33.3 33.8 36.0 36.8 37.2
6 + + + + 2 2 + + 2 + 2 + + 36.0
denaturation fluorimetic method (Gonzalez & Saiz-Jimenez, 2002) using SYBR Green 1 (SG 1; Invitrogen) and a realtime PCR thermocycler (Bio-Rad). Genomic DNA from Polaribacter dokdonensis KCTC 12392T (30.6 mol%), Cyclobacterium marinum KCTC 2917 (38.1 mol%), Acinetobacter baumannii KACC 12454 (39.1 mol%), Leadbetterella byssophila KACC 11308 T (40.4 mol%), Bacillus subtilis subsp. spizizenii KACC 14741 (43.8 mol%), Runella slithyformis KACC 13042T (46.4 mol%), Corynebacterium glutamicum KACC 10784 (54.1 mol%), Pseudomonas syringae pv. actinidiae KACC 10772T (58.8 mol%), Aeromonas hydrophila ATCC 7966T (61.5 mol%) and Sphingopyxis alaskensis KACC 12337T (65.5 mol%), which have genomes that have been completely sequenced, were used as calibration references. The thermal cycler conditions consisted of a ramp from 25 to 99 uC at 1.2 uC min–1. Fluorescent DNA melting curves were generated in triplicate. The DNA G+C content of strain HME8457T was calculated using linear regression analysis of the melting temperature (Tm) plotted against calibration reference strains. 4075
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Fatty acid analysis was performed on strains HME8457T and the five reference strains. Cells were grown on R2A agar at 30 uC until they reached the mid-exponential growth phase. Fatty acids were saponified, methylated and extracted using the standard protocol of the Sherlock Microbial Identification System version 6.1 (MIDI). The fatty acids were analysed by GC (model 7890; Hewlett Packard) and identified using the RTSBA6 database of the Microbial Identification System (Sasser, 1990). For analysis of respiratory quinones and polar lipids, cells of strain HME8457T were harvested from R2A after incubation at 30 uC for 2 days and freeze-dried. The isoprenoid quinones of strain HME8457T were analysed by TLC according to the protocol of Minnikin et al. (1984). Polar lipids were extracted according to the method of Minnikin et al. (1984) and separated by two-dimensional TLC (plates were coated with silica gel, 10610 cm; Merck). Chromatography was performed by using chloroform/methanol/water (65 : 25 : 4, by vol.) in the first direction, followed by chloroform/ methanol/acetic acid/water (40 : 7.5 : 6 : 2, by vol.) in the second. Plates were sprayed with 10 % (v/v) ethanolic molybdophosphoric acid (Sigma) for detection of the total polar lipids, ninhydrin (Sigma) for aminolipids, a-naphthol for glycolipids and molybdenum blue (Sigma) for phospholipids. The sphingolipids of strain HME8457T and Pedobacter glucosidilyticus KCTC 22438T were extracted according to the method of Yabuuchi et al. (1990). The presence of sphingolipids was confirmed by two-dimensional TLC with 0.4 % (v/v) ninhydrin in butanol. The DNA G+C content of the genomic DNA of strain HME8457T was 33.3 mol%, which is in line with that reported for other species of the genus Pedobacter. The major fatty acids of strain HME8457T were iso-C15 : 0, summed feature 3 (comprising C16 : 1v6c and/or C16 : 1v7c), iso-C17 : 0 3-OH and anteiso-C15 : 0 (Table 2). The fatty acid composition of strain HME8457T was similar to those of the five reference strains, with only minor quantitative differences. The only isoprenoid quinone of strain HME8457T was menaquinone 7 (MK-7). The polar lipids of strain HME8457T were phosphatidylethanolamine, one unidentified aminolipid, and two unidentified polar lipids (Fig. 2). Strain HME8457T and the five reference strains had phosphatidylethanolamine, an unidentified aminolipid, and unidentified polar lipids in common. However, strain HME8457T differed from the five reference strains by the absence of the unidentified phospholipid and unidentified aminophospholipid (Zhou et al., 2012; Joung et al., 2014). Sphingolipid was present in strain HME8457T; sphingolipids are a distinct feature of members of the genus Pedobacter (Steyn et al., 1998).
Table 2. Cellular fatty acid composition of strain HME8457T and some other members of the genus Pedobacter Strains: 1, HME8457T; 2, Pedobacter daechungensis KACC 14960T; 3, Pedobacter lentus KCTC 12875T; 4, Pedobacter terricola KACC 17433T; 5, Pedobacter glucosidilyticus KCTC 22438T; 6, ‘Pedobacter soyangensis’ HME6451. Values are percentages of the total cellular fatty acids. Only fatty acids amounting to at least 1.0 % of the total cellular fatty acids of at least one of the strains are shown. tr, Trace(,1.0 %); ND, not detected. All data are from this study. Fatty acid
1
iso-C11 : 0 2.5 iso-C13 : 0 tr iso-C14 : 0 2.9 C14 : 0 1.4 28.8 iso-C15 : 0 anteiso-C15 : 0 6.2 3.5 C15 : 1v6c tr iso-C16 : 1 H iso-C16 : 0 tr 3.0 C16 : 1v5c 2.5 C16 : 0 3.1 iso-C15 : 0 3-OH C15 : 0 2-OH 1.2 2.2 C17 : 1v8c 2.0 iso-C16 : 0 3-OH 1.0 C16 : 0 3-OH iso-C17 : 0 3-OH 7.7 tr C17 : 0 2-OH Summed Feature 3* 21.7 Summed Feature 9* 3.0
2 1.4 tr ND
1.1 15.8 6.3 2.3 1.9 3.9 3.4 6.9 1.8 1.9 2.9 4.4 tr 10.5 2.0 27.6 4.4
3
4
5
6
tr
tr ND
1.7 tr
ND
ND
2.3 tr 27.8 3.4 1.4 tr 1.1 3.5 3.3 3.3 1.1 tr 1.2 tr 14.4 tr 26.5 3.0
ND
ND
ND
tr 14.2 4.0 2.0 tr 1.4 2.8 6.7 2.1 1.8 1.7 tr tr 10.4 1.1 40.3 2.8
1.0
tr tr 37.3 41.2 5.4 4.7 3.7 2.3 tr ND 1.1 tr 2.7 6.0 3.4 1.5 2.0 2.4 tr tr 1.1 tr tr tr tr 1.3 10.7 10.2 tr tr 17.9 23.0 5.0 1.3
*Summed features are groups of two or three fatty acids that could not be separated by GC with the MIDI system. Summed feature 3 comprised C16 : 1v6c and/or C16 : 1v7c; summed feature 9 comprised iso-C17 : 1v9c and/or 10-methyl C16 : 0.
suggest that strain HME8457T should be classified in the genus Pedobacter as a member of a novel species, for which the namehttp://dx.doi.org/10.1601/nm.8232Pedobacter rivuli sp. nov. is proposed. Description of Pedobacter rivuli sp. nov. Pedobacter rivuli (ri9vu.li. L. gen. n. rivuli of/from a small stream).
On the basis of the phylogenetic distances, morphological characteristics, fatty acid profiles and quinone system, strain HME8457T should be affiliated to the genus Pedobacter. However, several of the phenotypic characteristics (Table 1) and the polar lipid profile of strain HME8457T distinguish it from closely related type strains of species of the genus Pedobacter. Therefore, our data
Cells are Gram-staining-negative, non-motile, aerobic rods and approximately 1.2–1.4 mm60.3–0.4 mm after 2 days at 30 uC on R2A agar. Colonies on R2A agar are red, smooth, convex and circular with entire margins and 1.5 mm in diameter after 2 days at 30 uC. Growth occurs at 15–30 uC (optimum, 30 uC), at pH 7–8 (optimum, pH 7.0) and with 0–0.5 % (w/v) NaCl [optimum 0 % (w/v) NaCl]. Oxidase and catalase activities are positive. Flexirubin-type pigments are not produced. Growth occurs on NA, TSA, R2A and blood agars, and no growth occurs on MA or
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Pedobacter rivuli sp. nov.
Republic of Korea. The genomic DNA G+C content of the type strain is 33.3 mol%
Acknowledgements This work was supported by a grant from the National Institute of Biological Resources (NIBR), funded by the Ministry of Environment (MOE) of the Republic of Korea (NIBR no. 2013-02-001).
L1 L2 AL
PE
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SL
2 1
daechungensis sp. nov., from freshwater lake sediment in South Korea. Int J Syst Evol Microbiol 59, 69–72. Bernardet, J. F., Nakagawa, Y., Holmes, B. & Subcommittee on the taxonomy of Flavobacterium and Cytophaga-like bacteria of the International Committee on Systematics of Prokaryotes (2002).
Proposed minimal standards for describing new taxa of the family Flavobacteriaceae and emended description of the family. Int J Syst Evol Microbiol 52, 1049–1070. Fig. 2. Two-dimensional TLC of the total polar lipids of strain HME8457T. PE, phosphatidylethanolamine; AL, unidentified aminolipids; L1–2, unidentified polar lipids; SL, sphingolipid.
Cho, J.-C. & Giovannoni, S. J. (2003). Parvularcula bermudensis gen. nov., sp. nov., a marine bacterium that forms a deep branch in the a-
Proteobacteria. Int J Syst Evol Microbiol 53, 1031–1036. Derichs, J., Ka¨mpfer, P. & Lipski, A. (2014). Pedobacter nutrimenti sp.
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MacConkey agars. Casein (skimmed milk), DNA, starch, dextrin, CM-cellulose and cellulose (filter paper) are not hydrolysed. With API 20NE strips, aesculin hydrolysis, bgalactosidase activity (PNPG test) and assimilation of Dmannose and N-acetylglucosamine are positive, but nitrate reduction, indole production, glucose fermentation, arginine dihydrolase, urease and gelatinase activities and assimilation of D-glucose, L-arabinose, D-mannitol, maltose, potassium gluconate, capric acid, adipic acid, malic acid, trisodium citrate and phenylacetic acid are negative. With API ZYM strips, alkaline phosphatase, esterase (C4), esterase lipase (C8), leucine arylamidase, valine arylamidase, cystine arylamidase, acid phosphatase, b-galactosidase, a-glucosidase and N-acetyl-b-glucosaminidase activities are present; lipase (C14), trypsin, a-chymotrypsin, naphthol-AS-BI-phosphohydrolase, a-galactosidase, b-glucuronidase, b-glucosidase, a-mannosidase and a-fucosidase activities are absent. With API 50CH strips, acid is produced from aesculin ferric citrate, maltose and lactose. The other substrates do not produce acid. With the GN2 Biolog MicroPlate, only a-ketobutyric acid and a-ketoglutaric acid are utilized. The other substrates are not utilized. The only isoprenoid quinone is menaquinone 7 (MK-7). The polar lipids present are phosphatidylethanolamine, one unidentified aminolipid and two unidentified lipid. Sphingolipid is present. The major cellular fatty acids (.5 %) are iso-C15 : 0, summed feature 3 (comprising C16 : 1v6c and/or C16 : 1v7c), iso-C17 : 0 3-OH and anteisoC15 : 0. The type strain, HME8457T (5KACC 17312T5CECT 8291T), was isolated from a freshwater stream in Seoul, http://ijs.sgmjournals.org
Farfa´n, M., Montes, M. J. & Marque´s, A. M. (2014). Reclassification
of Sphingobacterium antarcticum Shivaji et al. 1992 as Pedobacter antarcticus comb. nov. and Pedobacter piscium (Takeuchi and Yokota 1993) Steyn et al. 1998 as a later heterotypic synonym of Pedobacter antarcticus. Int J Syst Evol Microbiol 64, 863–868. Felsenstein, J. (1981). Evolutionary trees from DNA sequences: a
maximum likelihood approach. J Mol Evol 17, 368–376. Felsenstein, J. (1985). Confidence limits on phylogenies: an approach
using the bootstrap. Evolution 39, 783–791. Fitch, W. M. (1971). Toward defining the course of evolution: minimum change for a specific tree topology. Syst Zool 20, 406– 416. Gallego, V., Garcı´a, M. T. & Ventosa, A. (2006). Pedobacter aquatilis
sp. nov., isolated from drinking water, and emended description of the genus Pedobacter. Int J Syst Evol Microbiol 56, 1853–1858. Gonzalez, J. M. & Saiz-Jimenez, C. (2002). A fluorimetric method for
the estimation of G+C mol% content in microorganisms by thermal denaturation temperature. Environ Microbiol 4, 770–773. Hoang, V. A., Kim, Y. J., Nguyen, N. L., Min, J. W. & Yang, D. C. (2013).
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CLUSTAL W:
