International Journal of Systematic and Evolutionary Microbiology (2009), 59, 1720–1726 DOI 10.1099/ijs.0.000158-0

Pedobacter nyackensis sp. nov., alluvionis sp. nov. and Pedobacter borealis sp. nov., isolated from Montana flood-plain sediment and forest soil

Nathan S. Gordon, Alejandra Valenzuela, Sandra M. Adams, Philip W. Ramsey, Jarrod L. Pollock, William E. Holben and James E. Gannon

Correspondence Microbial Ecology Program, Division of Biological Sciences, The University of Montana, Missoula, Nathan S. Gordon MT 59812-1006, USA [email protected]

Three Gram-negative, rod-shaped, non-spore-forming eubacterial strains were isolated in western Montana, USA, and subjected to taxonomic studies. Strains NWG-II14T and NWER- II11T were isolated from hyporheic sediments of a large alluvial flood plain, whereas strain G-1T was isolated from a conifer forest soil. On the basis of 16S rRNA gene sequence similarity, strains NWG-II14T, NWER-II11T and G-1T were shown to belong to the family and are most closely related to various species of the genus Pedobacter. The results of molecular, physiological and biochemical tests allowed genotypic and phenotypic differentiation of these three strains from 23 Pedobacter species with validly published names. The three isolates therefore represent novel species, for which the names Pedobacter nyackensis sp. nov. (type strain NWG-II14T 5DSM 19625T 5LMG 24260T), Pedobacter alluvionis sp. nov. (type strain NWER-II11T 5DSM 19624T 5LMG 24258T) and Pedobacter borealis sp. nov. (type strain G-1T 5DSM 19626T 5LMG 24259T) are proposed.

The genus Pedobacter was initially described by Steyn et al. cryoconite, glacial water, drinking water, fresh water, a (1998) and, at the time of writing, included 23 species: hypertrophic pond and a nitrifying enrichment of surface Pedobacter heparinus, P. piscium, P. africanus and P. saltans water. This range of habitats suggests that Pedobacter (Steyn et al., 1998), P. cryoconitis (Margesin et al., 2003), P. species are generalists, possessing a wide array of enzymes himalayensis (Shivaji et al., 2005), P. caeni (Vanparys et al., capable of degrading a diverse set of carbon structures for 2005), P. sandarakinus (Yoon et al., 2006), P. roseus energy. Such metabolic diversity suggests that Pedobacter (Hwang et al., 2006), P. aquatilis (Gallego et al., 2006), P. species have the potential to dominate aerobic hetero- ginsengisoli (Ten et al., 2006), P. panaciterrae (Yoon et al., trophic microbial communities in various terrestrial and 2007a), P. suwonensis (Kwon et al., 2007), P. insulae (Yoon aquatic environments. In this report, the taxonomic et al., 2007b), P. lentus and P. terricola (Yoon et al., 2007c), characterization of three novel strains that group at the P. koreensis (Baik et al., 2007), P. duraquae, P. wester- species level with other members of the genus Pedobacter is hofensis, P. metabolipauper, P. hartonius and P. steynii reported. One of these strains, NWG-II14T, showed (Muurholm et al., 2007) and P. terrae (Yoon et al., 2007d). seasonal domination of the readily cultured heterotrophic All of these species are aerophilic or microaerophilic, sediment community of an alluvial flood plain in western Gram-negative rods with MK-7 as the predominant Montana. isoprenoid quinone and a DNA G+C content of 36– Sediment and interstitial water samples were collected in 45 mol% (T ). Four species exhibit gliding motility, five m sterile tubes from the hyporheic zone of the Nyack flood exhibit non-gliding motility and four species have been plain along the Middle Fork of the Flathead River, reported to produce heparinase. Pedobacter species have Montana, USA. Soil samples were collected from the been isolated from soil, fish, activated sludge, glacier rhizosphere of a western Montana conifer forest. During the isolation of organisms extracted from hyporheic The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene T T T T T sediments and soil, strains NWG-II14 , NWER-II11 and sequences of strains NWG-II14 , G-1 and NWER-II11 are EU030686, T EU030687 and EU030688, respectively. G-1 were recovered on environmental water agar (EWA), T T T a Gelrite (Sigma) layer over dilute one-tenth-strength R2A Fatty acid profiles of strains NWG-II14 , G-1 and NWER-II11 and agar (Difco) and undiluted R2A agar, respectively. EWA strains of related Pedobacter species are available as supplementary material with the online version of this paper. was composed of 1 % agarose (Sigma) and filter-sterilized

1720 000158 G 2009 IUMS Printed in Great Britain Pedobacter nyackensis, P. alluvionis and P. borealis spp. nov. formation water from wells on the flood plain. EWA was PAUP was also used to generate bootstrap support for all used to provide the bacterial community with nutrient tree topologies using 1000 random replicates from the levels that most closely represented in situ conditions. The original sequence data (Felsenstein, 1985). Strains NWG- Gelrite layer was used to encapsulate bacterial cells in a 3D II14T, NWER-II11T and G-1T clustered with members of matrix and solidified using divalent cations contained in the genus Pedobacter with a bootstrap resampling value of the underlying dilute R2A agar. All plates were incubated 100 % (Fig. 1). On the basis of the phylogenetic data, it is aerobically for 2–4 weeks at 10 uC in the dark (in situ clear that the three novel strains should be classified in the temperature ranges seasonally from 4 to 12 uC). Initial genus Pedobacter. colonies of strain NWG-II14T on EWA were 0.2–0.5 mm in For chemotaxonomic analyses, cell biomass was obtained diameter, round and transparent, with creamy white from cells cultured in one-fifth-strength R2A broth at centres developing after 6 weeks of incubation. Initial 20 uC. Isoprenoid quinones were extracted and purified colonies of strains NWER-II11T and G-1T on Gelrite and according to Collins et al. (1977) with the TLC modifica- R2A agar, respectively, were 0.3–1.0 mm in diameter, tion as noted by Tamaoka et al. (1983). Purified isoprenoid round and produced a reddish pigment. All isolates were quinones were analysed by reversed-phase HPLC using a subsequently cultivated on one-fifth-strength R2A agar at Phenomenex Synergi 4u Fusion-RP 80A (25064.6 mm) 10 uC for 7 days. On this medium, all strains were able to column. Phospholipid fatty acids were extracted from cell grow at 4–32 uC, but not at 0 or 37 uC. Growth of all pellets and analysed by the method of White & Ringelberg strains was also observed at 10 and 30 uC on tryptic soy (1998) as described previously by Rillig et al. (2006). The agar (TSA; Difco) and nutrient agar (NA; Difco). No G+C content of the genomic DNA of strains NWG-II14T, growth was observed on MacConkey agar (BBL). NWER-II11T and G-1T was determined by HPLC analysis Nearly full-length 16S rRNA gene sequences of strains of deoxyribonucleosides according to the method of NWG-II14T, NWER-II11T and G-1T were obtained by direct Mesbah et al. (1989), using a reversed-phase column PCR amplification of 1 ml culture (grown as described (Synergi 4u Fusion-RP 80A; Phenomenex). above) using the primers 27f, 907r and 1492r (Lane, 1991) DNA–DNA hybridization experiments were conducted and 536f (Holben et al., 2004). PCR products were purified between strain NWER-II11T and P. roseus JCM 13399T using the QIAquick PCR purification kit (Qiagen) according (98.9 % 16S rRNA gene sequence similarity) and between to the manufacturer’s instructions and then subjected to strain G-1T and P. suwonensis DSM 18130T (98.2 % 16S direct bidirectional DNA sequence analysis (Murdock rRNA gene sequence similarity) by the Deutsche Molecular Biology Facility, University of Montana, Sammlung von Mikroorganismen und Zellkulturen Missoula). The Sequence Match version 3 component of (Braunschweig, Germany). DNA–DNA relatedness values the Ribosomal Database Project was used to determine S ab were determined in duplicate and mean values were scores (similarity a versus b) that indicate the closest calculated. DNA was isolated using a French pressure cell matching relatives of the three strains. S scores are ab (ThermoSpectronic) and was purified by chromatography generated by calculating the number of unique 7 bp on hydroxyapatite as described by Cashion et al. (1977). oligomers shared between two partial 16S rRNA sequences DNA–DNA hybridization experiments were carried out as divided by the smallest number of unique 7-mers in either described by De Ley et al. (1970) with the modifications sequence (Cole et al., 2007). BLAST 2 SEQUENCES was used to described by Huß et al. (1983) using a Cary 100 Bio UV/ generate 16S rRNA gene sequence similarities (Tatusova & VIS-spectrophotometer equipped with a Peltier-thermo- Madden, 1999). Similarity scores indicated that strain statted 666 multicell changer and a temperature controller NWG-II14T was most closely related to the type strains of with in situ temperature probe (Varian). P. caeni (Sab50.895; 97.2 % sequence similarity), P. steynii (Sab50.890; 97.7 %) and P. heparinus (Sab50.884; 97.4 %). Cell morphology and motility were determined using a Strain NWER-II11T was most closely related to the type Zeiss Axioskop light microscope at 61000 with cells grown strains of P. roseus (Sab50.927; 98.9 %), P. sandarakinus for 3 days at 10 uC on TSA. Gram-staining reaction and (Sab50.882; 97.3 %) and P. suwonensis (Sab50.870; 97.5 %). catalase and oxidase activities were tested according to the Strain G-1T was most closely related to the type strains of P. methods of Gerhardt et al. (1994). Heparinase activity was sandarakinus (Sab50.895; 98.1 %), P. suwonensis detected by the method of Joubert et al. (1984). Carbon (Sab50.894; 98.2 %) and P. roseus (Sab50.883; 98.0 %). assimilation and enzyme production tests were conducted using API 50CH, API 20NE and API ZYM strips Multiple alignments were performed with the sequences of (bioMe´rieux) under aerobic conditions. these novel strains and the type strains of species with validly published names obtained from the NCBI GenBank Strains NWG-II14T, NWER-II11T and G-1T possessed database (Wheeler et al., 2000; Benson et al., 2000) for the biochemical characteristics that associated them with genera and Pedobacter using CLUSTAL_X members of the genus Pedobacter and differentiated them (Thompson et al., 1997). PAUP version 4.0 beta 10 from members of the genus Sphingobacterium (Steyn et al., (Swofford, 2003) was used to generate phylogenetic trees 1998). They lacked urease activity and did not assimilate using the neighbour-joining (Saitou & Nei, 1987) and melezitose. The major isoprenoid quinone of the three maximum-parsimony (Kluge & Farris, 1969) algorithms. strains was MK-7. The DNA G+C contents of strains http://ijs.sgmjournals.org 1721 N. S. Gordon and others

Fig. 1. Neighbour-joining tree based on 16S rRNA sequences showing the phylogenetic positions of strains NWG-II14T, NWER-II11T and G-1T in relation to the type strains of 23 Pedobacter and two Sphingobacterium species. Cytophaga hutchinsonii ATCC 33406T was used as a monophyletic outgroup. Bootstrap support values greater than 50 % (percentages of 1000 replicates) are shown at branch points. Filled circles indicate nodes that were also recovered using maximum-parsimony algorithms. Bar, 0.01 substitutions per nucleotide site.

NWG-II14T, NWER-II11T and G-1T were 41.1±0.4, alluvionis sp. nov. and Pedobacter borealis sp. nov., 39.3±0.6 and 39.7±0.5 mol%, respectively. These values respectively, are proposed. are consistent with the 36–45 mol% range previously suggested for the genus Pedobacter (Steyn et al., 1998). All Description of Pedobacter nyackensis sp. nov. three strains contained iso-C15 : 0, iso-C17 : 0 3-OH, Pedobacter nyackensis (ny.ack.en9sis. N.L. masc. adj. nyack- C16 : 1v5c and C16 : 1v7c as major cellular fatty acids, which ensis from Nyack, a region of north-western Montana, is also in accordance with the description of the genus USA). Pedobacter (Steyn et al., 1998). The fatty acid compositions of strains NWG-II14T, NWER-II11T, G-1T and strains of Colonies grown on TSA for 48 h at 20 uC are 0.5–5.0 mm related Pedobacter species are detailed in Supplementary in diameter, round, convex, opaque and beige, darkening Table S1, available in IJSEM Online. Strains NWER-II11T with age. Cells are Gram-negative, oxidase- and catalase- and G-1T exhibited low levels of DNA–DNA relatedness to positive, non-motile, non-spore-forming rods (0.2– P. roseus JCM 13399T (6.3 %) and P. suwonensis DSM 0.761.0–4.0 mm), with an oxidative metabolism. Good 18130T (4.4 %), respectively, indicating that both strains growth is observed after 96 h at 10 and 30 uC on TSA, NA are unique at the species level according to the recom- and R2A. Growth does not occur at 37 uCoron mended threshold of 70 % DNA–DNA similarity (Wayne MacConkey agar. Growth occurs at pH 5–10 (optimal et al., 1987). In contrast to other members of the genus pH 7–8). It can be differentiated from other Pedobacter Pedobacter (except P. suwonensis), strains NWG-II14T, species with validly published names by its ability to NWER-II11T and G-1T demonstrated a-fucosidase activity. assimilate L-sorbose; it can be differentiated from its closest phylogenetic relatives, P. caeni, P. heparinus and P. On the basis of the phylogenetic, phenotypic and africanus, by its ability to assimilate glycogen and 2- T chemotaxonomic data presented, strains NWG-II14 , ketogluconate. The fatty acid profile is composed of C14 : 0, T T NWER-II11 and G-1 are proposed as the type strains iso-C15 : 0, iso-C15 : 0 2-OH, C16 : 1v7c and C16 : 1v5c. The of three novel species in the genus Pedobacter, for which major isoprenoid quinone is MK-7. Additional phenotypic the names Pedobacter nyackensis sp. nov., Pedobacter properties are shown in Table 1.

1722 International Journal of Systematic and Evolutionary Microbiology 59 T T T http://ijs.sgmjournals.org Table 1. Differential characteristics of strains NWGII14 , NWERII11 and G-1 and phylogenetically related species of the genus Pedobacter

Taxa: 1, strain NWGII14T; 2, strain NWERII11T; 3, strain G-1T;4,P. heparinus;5,P. africanus;6,P. piscium;7,P. cryoconitis;8,P. himalayensis;9,P. caeni; 10, P. sandarakinus; 11, P. roseus; 12, P. aquatilis; 13, P. ginsengisoli; 14, P. suwonensis; 15, P. steynii. Unless otherwise indicated, data were compiled from Steyn et al. (1998), Margesin et al. (2003), Shivaji et al. (2005), Vanparys et al. (2005), Yoon et al. (2006), Hwang et al. (2006), Gallego et al. (2006), Ten et al. (2006), Muurholm et al. (2007), Kwon et al. (2007) and the present study. +, Positive; (+), weak reaction; 2, negative; V, variable; NA, data not available. All taxa are positive for the following features: aerobic growth, presence of catalase, oxidase and alkaline phosphatase (NA for P. ginsengisoli) and assimilation of D-glucose, D-mannose, D-lactose, cellobiose, N-acetylglucosamine, amygdalin (NA for P. himalayensis and P. roseus), salicin (V for P. africanus), melibiose (V for P. saltans; NA for P. roseus), sucrose (V for P. saltans) and trehalose (V for P. saltans). All taxa are negative for the following features: Gram-staining, sporulation, indole production, nitrate reduction, urease activity and assimilation of D-lyxose (NA for P. himalayensis and P. roseus), D-fucose (NA for P. himalayensis and P. roseus), erythritol (NA for P. roseus and P. ginsengisoli), inositol and dulcitol.

Characteristic 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

Source Flood-plain Flood-plain Soil Soil Soil, activated Fish Glacier Glacier Nitrifying Soil Hypertrophic Drinking Soil Cabbage rhi- Hard-water sediment sediment sludge cryoconite water inoculum freshwater water zosphere creek Growth temperature range (uC) ,2–32 4–30 4–30 5–30 NA 5–30 1–25 4–25 NA 4–33 5–33 4–30 4–30 1–37 10–30 DNA G+C content (mol%) 41.1 39.3 39.7 42–43 43–45 40–43 43.4 41.0 42.7 39.7 41.3 38 43.6 44.2 NA Gelatin hydrolysis 2 ++2 V 2 ++22 + 22 + 2 Arginine dihydrolase 2 ++22NA 2 + 22 2 22 2 2 Heparinase + 22++22 + NA NA 2 NA NA 22 Assimilation of (API 50CH): Glycerol 2 222222+ 22 + 2 + 2 + D-Arabinose 2 + 22 V 22 + 22 NA 22 2 2 L-Arabinose +++2 V 2 +++2 ++2 + 2 D-Ribose ++2 + V + 2 + 22 2 22 2 2 D-Xylose + 2 ++ V 2 ++22 NA 2 ++ + L-Xylose 2 222222++2 + 2 + 2 NA D-Adonitol 2 + 2 + 222+ 22 NA 22 2 2 eoatrnyackensis Pedobacter Methyl b-D-xyloside 2 + 22222NA 22 NA 2 NA NA 2 D-Galactose +++++V ++2 ++ +++ 2 D-Fructose ++++V ++ + 22 +++(+) 2 L-Sorbose ++22222222NA 22 2 NA L-Rhamnose +++++V 2 + 22 ++2 + 2 D-Mannitol 222+ 222+ 22 2 22 2 2 D-Sorbitol 222+ 222+ 22 2 22 2 2

Methyl a-D-mannoside +++++22 ++(+) NA + NA + 2 , .alluvionis P. Methyl a-D-glucoside + +++++(+) ++(+) NA + NA ++ Arbutin +++VV++ + +2 NA + NA ++ Aesculin + ++++++++++ +2 ++ Maltose ++++V ++ + ++ + +++ + and Inulin ++2222(+) ++2 + 2 + 22 Melezitose 2 222222222NA + NA + 2 borealis P. Raffinose +++22++ + +2 +++++ Starch +++2 V ++ + ++ NA + 2 ++ Glycogen + 22222+ 22++ 22 ++ p.nov. spp. Xylitol 2 2222222+ 2 NA 22 2 2 1723 b-Gentiobiose ++++V ++ NA ++ NA + NA ++ 1724 others and Gordon S. N.

Table 1. cont.

Characteristic 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

Turanose + ++++++NA 22 NA + NA ++ D-Tagatose 2 222222NA 22 NA 2 NA NA 2 L-Fucose 222+ V 22 + 22 NA 2 NA 22 D-Arabitol 2 222222NA 22 NA 2 NA NA 2 L-Arabitol 2 222222NA 22 NA 2 NA NA 2 Gluconate 2 222222+ 22 2 22 NA 2 2-Ketogluconate + 22222+ NA 22 NA 2 NA NA 2 5-Ketogluconate + 2 + 2222NA 2 + NA 2 NA NA 2 Enzyme activity (API ZYM) Esterase +++V 2 VNANA 2 ++ +NA ++ Esterase lipase + +++++2 NA ++ + +NA ++ Lipase 22222V 22222 222 2 Leucine arylamidase + +++++NA NA ++ + +NA ++

nentoa ora fSseai n vltoayMicrobiology Evolutionary and Systematic of Journal International Valine arylamidase +++2 V +* NA NA 22 ++NA ++ Cystine arylamidase +++V 2 +* NA NA 2 (+) 2 + NA 22 Trypsin 2 ++22+* NA NA 22 ++NA + NA Chymotrypsin 222V 2 +* NA NA 22 + 2 NA 2 NA Acid phosphatase + +++++NA ++++ +NA + NA Naphthol-AS-BI-phosphohy- +++++NA NA NA 2 ++ +NA ++ drolase a-Galactosidase +++V 2 +* NA NA 22 + 2 NA 2 NA b-Galactosidase + +++++++++V + 2 ++ b-Glucuronidase 22+ 22V* NA NA 22 2 2NA 2 NA a-Glucosidase + +++++NA NA 2 ++ +NA ++ b-Glucosidase ++VV V +* ++2 + 2 + NA ++ N-Acetyl-b-glucosaminidase + +++++NA NA 2 ++ +NA ++ a-Mannosidase + 22VV+* NA NA 22 2 + NA 22 a-Fucosidase +++222* NA NA 22 2 2NA + 2

*Data from Takeuchi & Yokota (1992). 59 Pedobacter nyackensis, P. alluvionis and P. borealis spp. nov.

The type strain is NWG-II14T (5DSM 19625T 5LMG Montana, USA. The DNA G+C content of the type strain 24260T), isolated from hyporheic sediments of the Nyack is 35.5±0.5 mol%. flood plain in north-western Montana, USA. The DNA G+C content of the type strain is 36.1 mol%. Acknowledgements

Description of Pedobacter alluvionis sp. nov. Special thanks go to the Dalimata family, Brian Reid and Jack Stanford’s team at the Flathead Lake Biological Station for their help Pedobacter alluvionis (al.lu.vi.o9nis. L. gen. n. alluvionis of in facilitating this research. This work was supported by National alluvial land). Science Foundation Microbial Observatory grant no. MCB-0348773. Colonies grown on TSA for 48 h at 20 uC are 0.2–2.0 mm in diameter, round, convex, opaque and have a reddish- References pink pigment. Cells are Gram-negative, oxidase- and catalase-positive, non-motile, non-spore-forming rods Baik, K. S., Park, Y.-D., Kim, M. S., Park, S. C., Moon, E. Y., Rhee, M. S., Choi, J. H. & Seong, C. N. (2007). Pedobacter koreensis 6 m (0.3–0.6 1.0–3.0 m), with an oxidative metabolism. sp. nov., isolated from fresh water. Int J Syst Evol Microbiol 57, 2079– Good growth is observed after 96 h at 10 and 30 uCon 2083. u TSA, NA and R2A. Growth does not occur at 37 Coron Benson, D. A., Karsch-Mizrachi, I., Lipman, D. J., Ostell, J., Rapp, MacConkey agar. Growth occurs at pH 5–10 (optimal B. A. & Wheeler, D. L. (2000). GenBank. Nucleic Acids Res 28, 15–18. pH 7–8). It can be differentiated from other Pedobacter Cashion, P., Holder-Franklin, M. A., McCully, J. & Franklin, M. (1977). species with validly published names by its ability to A rapid method for base ratio determination of bacterial DNA. Anal assimilate methyl b-D-xyloside and L-sorbose; it can be Biochem 81, 461–466. differentiated from its closest phylogenetic relatives, P. Cole, J. R., Chai, B., Farris, R. J., Wang, Q., Kulam-Syed-Mohideen, roseus, P. sandarakinus and P. aquatilis, by the presence of A. S., McGarrell, D. M., Bandela, A. M., Cardenas, E., Garrity, G. M. & arginine dihydrolase activity and its ability to assimilate D- Tiedje, J. M. (2007). The ribosomal database project (RDP-II): arabinose, D-ribose and D-adonitol. The fatty acid profile is introducing myRDP space and quality controlled public data. Nucleic Acids Res 35, D169–D172. composed of C14 : 0, iso-C15 : 0, iso-C15 : 0 2-OH, C16 : 1v7c and C16 : 1v5c. The major isoprenoid quinone is MK-7. Collins, M. D., Pirouz, T., Goodfellow, M. & Minnikin, D. E. (1977). Additional phenotypic properties are shown in Table 1. Distribution of menaquinones in actinomycetes and corynebacteria. J Gen Microbiol 100, 221–230. T T The type strain is NWER-II11 (5DSM 19624 5LMG De Ley, J., Cattoir, H. & Reynaerts, A. (1970). T The quantitative 24258 ), isolated from hyporheic sediments of the Nyack measurement of DNA hybridization from renaturation rates. Eur J flood plain in north-western Montana, USA. The DNA Biochem 12, 133–142. + ± G C content of the type strain is 34.4 0.2 mol%. Felsenstein, J. (1985). Confidence limits on phylogenies: an approach using the bootstrap. Evolution 39, 783–791. Description of Pedobacter borealis sp. nov. Gallego, V., Garcı´a, M. T. & Ventosa, A. (2006). Pedobacter aquatilis sp. nov., isolated from drinking water, and emended description of Pedobacter borealis (bo.re.a9lis. M.L. masc. adj. borealis the genus Pedobacter. Int J Syst Evol Microbiol 56, 1853–1858. related to the north, boreal). Gerhardt, P., Murray, R. G. E., Wood, W. A. & Krieg, N. R. (editors) (1994). Methods for General and Molecular Bacteriology. Washington, Colonies grown on TSA for 48 h at 20 uC are 0.2–2.0 mm DC: American Society for Microbiology. in diameter, round, convex, opaque and have a reddish- pink pigment. Cells are Gram-negative, oxidase- and Holben, W. E., Feris, K. P., Kettunen, A. & Apajalahti, J. H. A. (2004). GC fractionation enhances microbial community diversity assessment catalase-positive, non-motile, non-spore-forming rods and detection of minority populations of by denaturing (0.3–0.560.7–3.0 mm), with an oxidative metabolism. gradient gel electrophoresis. Appl Environ Microbiol 70, 2263–2270. u Good growth is observed after 96 h at 10 and 30 Con Huß, V. A. R., Festl, H. & Schleifer, K. H. (1983). Studies on the TSA, NA and R2A. Growth does not occur at 37 uCoron spectrophotometric determination of DNA hybridization from MacConkey agar. Growth occurs at pH 5–7 (optimal renaturation rates. Syst Appl Microbiol 4, 184–192. pH 7). It can be differentiated from other Pedobacter Hwang, C. Y., Choi, D. H. & Cho, B. C. (2006). Pedobacter roseus sp. species with validly published names by the presence of the nov., isolated from a hypertrophic pond, and emended description of enzyme b-glucuronidase; it can be differentiated from its the genus Pedobacter. Int J Syst Evol Microbiol 56, 1831–1836. closest phylogenetic relatives, P. sandarakinus, P. roseus and Joubert, J. J., van Rensburg, E. J. & Pitout, M. J. (1984). A plate P. aquatilis, by the presence of arginine dihydrolase activity method for demonstrating the breakdown of heparin and chondroitin and its ability to assimilate D-xylose. The fatty acid profile sulfate by bacteria. J Microbiol Methods 2, 197–202. is composed of C14 : 0, iso-C15 : 0, iso-C15 : 0 2-OH, C16 : 1v7c Kluge, A. G. & Farris, J. S. (1969). Quantitative phyletics and the and C16 : 1v5c. The major isoprenoid quinone is MK-7. evolution of anurans. Syst Zool 18, 1–32. Additional phenotypic properties are shown in Table 1. Kwon, S.-W., Kim, B.-Y., Lee, K.-H., Jang, K.-Y., Seok, S.-J., Kwon, J.-S., Kim, W.-G. & Weon, H.-Y. (2007). Pedobacter suwonensis sp. nov., T T T The type strain is G-1 (5DSM 19626 5LMG 24259 ), isolated from the rhizosphere of Chinese cabbage (Brassica campes- isolated from the rhizosphere of a conifer forest in western tris). Int J Syst Evol Microbiol 57, 480–484. http://ijs.sgmjournals.org 1725 N. S. Gordon and others

Lane, D. J. (1991). 16S/23S rRNA sequencing. In Nucleic Acid Tatusova, T. A. & Madden, T. L. (1999). BLAST 2 Sequences, a new tool Techniques in Bacterial Systematics, pp. 115–175. Edited by E. for comparing protein and nucleotide sequences. FEMS Microbiol Lett Stackebrandt & M. Goodfellow. Chichester: Wiley. 174, 247–250. Margesin, R., Spro¨ er, C., Schumann, P. & Schinner, F. (2003). Ten, L. N., Liu, Q.-M., Im, W.-T., Lee, M., Yang, D.-C. & Lee, S.-T. sp. nov., a facultative psychrophile from alpine (2006). Pedobacter ginsengisoli sp. nov., a DNase-producing bacterium glacier cryoconite. Int J Syst Evol Microbiol 53, 1291–1296. isolated from soil of a ginseng field in South Korea. Int J Syst Evol Mesbah, M., Premachandran, U. & Whitman, W. B. (1989). Precise Microbiol 56, 2565–2570. measurement of the G+C content of deoxyribonucleic acid by high- Thompson, J. D., Gibson, T. J., Plewniak, F., Jeanmougin, F. & performance liquid chromatography. Int J Syst Bacteriol 39, 159–167. Higgins, D. G. (1997). The CLUSTAL_X windows interface: flexible Muurholm, S., Cousin, S., Pa¨uker, O., Brambilla, E. & Stackebrandt, E. strategies for multiple sequence alignment aided by quality analysis (2007). Pedobacter duraquae sp. nov., Pedobacter westerhofensis sp. nov., tools. Nucleic Acids Res 25, 4876–4882. Pedobacter metabolipauper sp. nov., Pedobacter hartonius sp. nov. and Vanparys, B., Heylen, K., Lebbe, L. & De Vos, P. (2005). Pedobacter Pedobacter steynii sp. nov., isolated from a hard-water rivulet. Int J Syst caeni sp. nov., a novel species isolated from a nitrifying inoculum. Int Evol Microbiol 57, 2221–2227. J Syst Evol Microbiol 55, 1315–1318. Rillig, M. C., Mummey, D. L., Ramsey, P. W., Klironomos, J. N. & Wayne, L. G., Brenner, D. J., Colwell, R. R., Grimont, P. A. D., Kandler, O., Gannon, J. E. (2006). Phylogeny of arbuscular mycorrhizal fungi Krichevsky, M. I., Moore, L. H., Moore, W. E. C., Murray, R. G. E. & other predicts community composition of symbiosis-associated bacteria. authors (1987). International Committee on Systematic Bacteriology. FEMS Microbiol Ecol 57, 389–395. Report of the ad hoc committee on reconciliation of approaches to Saitou, N. & Nei, M. (1987). The neighbor-joining method: a new bacterial systematics. Int J Syst Bacteriol 37, 463–464. method for reconstructing phylogenetic trees. Mol Biol Evol 4, 406–425. Wheeler, D. L., Chappey, C., Lash, A. E., Leipe, D. D., Madden, T. L., Shivaji, S., Chaturvedi, P., Reddy, G. S. N. & Suresh, K. (2005). Schuler, G. D., Tatusova, T. A. & Rapp, B. A. (2000). Database Pedobacter himalayensis sp. nov., from the Hamta glacier located in resources of the National Center for Biotechnology Information. the Himalayan mountain ranges of India. Int J Syst Evol Microbiol 55, Nucleic Acids Res 28, 10–14. 1083–1088. White, D. C. & Ringelberg, D. B. (1998). Signature lipid biomarker Steyn, P. L., Segers, P., Vancanneyt, M., Sandra, P., Kersters, K. & analysis. In Techniques in Microbial Ecology, pp. 255–272. Edited by Joubert, J. J. (1998). Classification of heparinolytic bacteria into a new R. S. Burlage, R. Atlas, D. Stahl, G. Geesey & G. Sayler. Oxford: genus, Pedobacter, comprising four species: Pedobacter heparinus Oxford University Press. comb. nov., Pedobacter piscium comb. nov., Pedobacter africanus sp. Yoon, J.-H., Lee, M.-H., Kang, S.-J., Park, S.-Y. & Oh, T.-K. (2006). nov. and Pedobacter saltans sp. nov. Proposal of the family Pedobacter sandarakinus sp. nov., isolated from soil. Int J Syst Evol Sphingobacteriaceae fam. nov. Int J Syst Bacteriol 48, 165–177. Microbiol 56, 1273–1277. Swofford, D. L. (2003). PAUP*: Phylogenetic analysis using parsimony Yoon, M.-H., Ten, L. N., Im, W.-T. & Lee, S.-T. (2007a). Pedobacter (*and other methods), version 4.0B10. Sunderland, MA: Sinauer panaciterrae sp. nov., isolated from soil in South Korea. Int J Syst Evol Associates. Microbiol 57, 381–386. Takeuchi, M. & Yokota, A. (1992). Proposals of Sphingobacterium Yoon, J.-H., Kang, S.-J., Oh, H. W. & Oh, T.-K. (2007b). faecium sp. nov., Sphingobacterium piscium sp. nov., Sphingobacterium Pedobacter heparinum comb. nov., Sphingobacterium thalpophilum comb. nov., insulae sp. nov., isolated from soil. Int J Syst Evol Microbiol 57, 1999– and two genospecies of the genus Sphingobacterium and synonymy of 2003. Flavobacterium yabuuchiae and Sphingobacterium spiritivorum. J Gen Yoon, J.-H., Kang, S.-J., Park, S. & Oh, T.-K. (2007c). Pedobacter lentus Appl Microbiol 38, 465–482. sp. nov. and Pedobacter terricola sp. nov., isolated from soil. Int J Syst Tamaoka, J., Katayama-Fujimura, Y. & Kuraishi, H. (1983). Analysis Evol Microbiol 57, 2089–2095. of bacterial menaquinone mixtures by high performance liquid Yoon, J.-H., Kang, S.-J. & Oh, T.-K. (2007d). Pedobacter terrae sp. nov., chromatography. J Appl Bacteriol 54, 31–36. isolated from soil. Int J Syst Evol Microbiol 57, 2462–2466.

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