Antonie van Leeuwenhoek (2014) 105:915–924 DOI 10.1007/s10482-014-0146-z

ORIGINAL PAPER

Variibacter gotjawalensis gen. nov., sp. nov., isolated from soil of a lava forest Kwang Kyu Kim • Keun Chul Lee • Mi Kyung Eom • Jong-Shik Kim Dae-Shin Kim • Suk-Hyung Ko • Byung-Hyuk Kim • Jung-Sook Lee

•

Received: 6 December 2013 / Accepted: 25 February 2014 / Published online: 6 March 2014 Ó Springer International Publishing Switzerland 2014

Abstract A novel bacterial strain designated GJW-30T was isolated from soil of the lava forest, Gotjawal, located in Aewol, Jeju, Korea. Strain GJW-30T was found to be strictly aerobic, Gram-negative and to form pleomorphic, non-motile rods and white colonies on R2A agar. The major fatty acids were identified as C18:1x7c, C16:0 and C17:0, the predominant isoprenoid quinone as Q-10, the polar lipids as diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, phosphatidylcholine, an unidentified aminolipid and an unidentified lipid. The cell-wall sugar pattern of strain GJW-30T was found to be composed of glucose, ribose and rhamnose and mesoDAP as the diagnostic diamino acid in the cell-wall peptidoglycan. The DNA G?C content of strain GJW30T is 62.2 mol%. Phylogenetic analysis, based on 16S

rRNA gene sequence similarities, showed that strain GJW-30T forms a deep branch within the order Rhizobiales, sharing the highest level of sequence homology with Bradyrhizobium oligotrophicum LMG 10732T (93.6 %). On the basis of the phenotypic, chemotaxonomic and phylogenetic characteristics, strain GJW-30T is considered to represent a novel genus and species, for which the name Variibacter gotjawalensis gen. nov., sp. nov. (the type strain is GJW-30T = KCTC 32391T = CECT 8514T = LMG 28093T) is proposed. Keywords Variibacter gotjawalensis gen. nov., sp. nov.  Proteobacteria  Taxonomy

Introduction Electronic supplementary material The online version of this article (doi:10.1007/s10482-014-0146-z) contains supplementary material, which is available to authorized users. The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of Variibacter gotjawalensis GJW-30T is HF970589. K. K. Kim  K. C. Lee  M. K. Eom  J.-S. Lee (&) Korean Collection for Type Cultures, Biological Resource Center, Korea Research Institute of Bioscience and Biotechnology, 125 Gwahak-ro, Yuseong-gu, Daejeon 305-806, Republic of Korea e-mail: [email protected] J.-S. Kim Gyeongbuk Institute for Marine Bioindustry, Uljin 767-813, Republic of Korea

The order Rhizobiales represented by a phenotypically heterogeneous group of Proteobacteria (Kuykendall 2005). At the time of writing, the order Rhizobiales in the class Alphaproteobacteria is composed of 13 families according to 16S rRNA gene sequence D.-S. Kim  S.-H. Ko Research Institute for Hallasan, Jeju-si, Jeju Special SelfGoverning Province 690-816, Republic of Korea B.-H. Kim Environmental Biotechnology Research Center, Korea Research Institute of Bioscience and Biotechnology, 125 Gwahak-ro, Yuseong-gu, Daejeon 305-806, Republic of Korea

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analysis: ‘Aurantimonadaceae’, Bartonellaceae, Beijerinckiaceae, Bradyrhizobiaceae, Brucellaceae, Cohaesibacteraceae, Hyphomicrobiaceae, Methylobacteriaceae, Methylocystaceae, Phyllobacteriaceae, Rhizobiaceae, Rhodobiaceae and Xanthobacteraceae. The family Bradyrhizobiaceae of the order Rhizobiales was proposed by Garrity et al. (2005) on the basis of phylogenetic analysis of 16S rRNA gene sequence similarities and included nine phenotypically, metabolically and ecologically diverse genera, Afipia, Agromonas, Blastobacter, Bosea, Bradyrhizobium, Nitrobacter, Oligotropha, Rhodoblastus and Rhodopseudomonas. Later on three genera, Balneimonas, Salinarimonas and Tardiphaga, were also included to the family Bradyrhizobiaceae. One species of the genus Agromonas, Agromonas oligotrophica was reclassified as Bradyrhizobium oligotrophicum (Ramı´rez-Bahena et al. 2013), and species Balneimonas flocculans of the genus Balneimonas was reclassified as Microvirga flocculans within the family Methylobacteriaceae (Weon et al. 2010). Three species of the genus Blastobacter, Blastobacter aggregatus, Blastobacter denitrificans and Blastobacter natatorius were transferred to three different genera as Rhizobium aggregatum (Kaur et al. 2011), Bradyrhizobium denitrificans (Van Berkum et al. 2006), and Blastomonas natatoria (Hiraishi et al. 2000), respectively. The remaining species, Blastobacter capsulatus is closely related to the genus Rhizobium. Liu et al. (2010) reported that the genera Bosea and Salinarimonas are closely related to the genus Methylobacterium in the family Methylobacteriaceae and Rhodoblastus acidophilus is closely related to the genus Methylocystis in the family Methylocystaceae. The genus Tardiphaga that is closely related to the genus Rhodopseudomonas was proposed by De Meyer et al. (2012). Gotjawal (from Jeju’s dialect ‘got’ means a forest and ‘jawal’ means rocks and pebbles), is a unique biologically diverse ecological niche with the trees, vines and rocks jumbled and tangled all together as a result of the volcanic activity (Yang et al. 2006). During the investigation of the bacterial diversity of soil of the Gotjawal forest, strain GJW-30T was recovered. On the basis of the phenotypic and chemotaxonomic characterization and 16S rRNA gene sequence analysis we show that the new bacterium isolated from lava forest soil belongs to the order Rhizobiales, the family Bradyrhizobiaceae and represents a novel genus and species.

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Materials and methods Isolation and maintenance of organism A soil sample was collected from the Gotjawal forest located in Aewol, Jeju, Republic of Korea in 2013. A portion of 1 g was diluted serially in sterile distilled water, and 100 ll of each serial dilution was spread on R2A (BD). Plates were incubated aerobically at 25 °C for 4 weeks. A strain, designated GJW-30T, was isolated from a single colony and purified by subculturing on the same medium. The bacterial culture was stored at -70 °C in water suspension supplemented with 20 % (v/v) of glycerol. For most experiments, strain GJW-30T was grown on R2A at 25 °C for 5 days, unless stated otherwise. The reference strains Bradyrhizobium japonicum KCTC 2422T, Bradyrhizobium oligotrophicum LMG 10732T, Bradyrhizobium betae NBRC 103048T, Bradyrhizobium cytisi LMG 25866T, Nitrobacter winogradskyi DSM 10237T, Afipia felis CIP 103515T, Bosea thiooxidans KACC 10745T, Oligotropha carboxidovorans DSM 1227T, Rhodopseudomonas palustris NBRC 100419T, Pseudolabrys taiwanensis CCUG 51779T, Rhodoplanes roseus DSM 5909T, Rhodoplanes elegans NBRC 16660T and Tardiphaga robiniae LMG 26467T were grown and maintained under identical conditions. Salinarimonas rosea KCTC 22346T was grown on Marine Agar 2216 (BD) at 25 °C for 5 days and maintained under glycerol/diluted marine broth 2216 (BD) suspensions (10 %, v/v) at -70 °C. The reference strains were received from CIP (Collection de L’Institut Pasteur), CCUG (Culture Collection University of Goteborg), DSM (Leibniz-Institut DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH), KACC (Korean Agricultural Culture Collection), KCTC (Korean Collection for Type Cultures), LMG (Belgian Coordinated Collections of Microorganisms/LMG Bacteria Collection) and NBRC (NBRC Culture Collection), respectively. Phenotypic characterization Gram staining was performed by using Hucker’s modification (La´nyı´ 1987). Cell morphology and motility were observed under a phase-contrast microscope (Eclipse 80i; Nikon). Motility was tested using the hanging-drop technique (Skerman 1967). For the observation of flagella or pili, cells were grown on

Antonie van Leeuwenhoek (2014) 105:915–924

R2A at 25 °C for 5 days, negatively stained with 1 % (w/v) uranyl acetate and examined using a model CM20 transmission electron microscope (Philips). Oxidase activity was tested by using the oxidase reagent (bioMe´rieux) and catalase activity was determined by production of bubbles after the addition of a drop of 3 % H2O2. Growth was investigated at 5, 10, 15, 20, 25, 30, 37, 42 and 45 °C, in the presence of 0.5, 1, 2, 3, and 5 % NaCl, and at pH 5–10 (in increments of 0.5 pH units). The following biological buffers were used to adjust the pH: Na2HPO4/NaH2PO4 for pH below 9 and Na2CO3/NaHCO3 for pH 9–10 (Gomori 1955). Acid production tests, enzyme activity tests and additional phenotypic tests were performed using API 50CH (with CHB/E medium), API ZYM, API 20E, API 20NE, and ID 32 GN galleries according to the instructions of the manufacturer (bioMe´rieux). Detection of nifH gene To confirm the nitrogen-fixing activity, a 370 bp DNA fragment of the nifH gene corresponding to positions 111F and 483R of the Bradyrhizobium japonicum nifH sequence (GenBank accession no. E01169) was amplified using following two primers: forward 50 -GGAATT CTGTGATCCTAAAGCTGA-30 and reverse 50 -AGC ATACATTGCCATCATTTCACC-30 (modified from Zehr et al. 1995). The presence of nifH gene was checked using the agarose gel electrophoresis. For comparative purposes, Bradyrhizobium japonicum KCTC 2422T, Bradyrhizobium oligotrophicum LMG 10732T and Escherichia coli KCTC 2441T were used in the same experiment. Acetylene-reduction test Acetylene-reduction test was performed according to Omar et al. (1989). The strains were grown for 3 days on solid NFb medium [(per liter) 5.0 g malic acid, 0.5 g K2HPO4, 0.2 g MgSO47H2O, 0.1 g NaCl, 0.02 g CaCl2, bromthymol blue 0.5 % in 0.2 N KOH, 1 ml vitamin solution, 2 ml microelement solution, 1.64 % Fe EDTA, 4.5 g KOH]. The vitamin solution contained in 100 ml: 10 mg biotin, 20 mg pyridoxol-HCl and the microelement solution contained in 1L: 0.4 g CuSO4, 0.12 g ZnSO47H2O, 1.4 g H2BO3, 1.0 g Na2MoO4 2H2O and 1.5 g MnSO4H2O. The pH was adjusted to 6.8 and 1.9 g L-1 of agar was added. For comparative purposes, Bradyrhizobium japonicum KCTC 2422T,

917

Bradyrhizobium oligotrophicum LMG 10732T and Escherichia coli KCTC 2441T were used. They were incubated under 1.8 % (v/v) acetylene/air for 4 days, and then analyzed for ethylene production by GC (Omar et al. 1989). The ethylene and acetylene were analyzed by injecting a 100 ll headspace sample into a GC–FID (GC 2010 plus; Shimadzu, Kyoto, Japan) fitted with a 50 m 9 0.52 mm I.D. HP–AL column (Agilent Technologies, CA, USA). Helium was used as carrier gas at 1 ml min-1. The injection and detection ports were maintained at 200 and 250 °C, respectively, and the column temperature programmed to hold at 40 °C for 2.5 min. Chemotaxonomy Isoprenoid quinones were extracted with chloroform/ methanol (2:1, v/v) and purified by using TLC on Kieselgel 60 F254 plates (20920 cm, 0.25 mm thickness; Merck) with petroleum ether/diethyl ether (9:1, v/v) as the solvent. Quinones were identified by using reversed-phase HPLC as described according to Shin et al. (1996). For analysis of fatty acids, cells of strain GJW-30T and reference strains except for Salinarimonas rosea KCTC 22346T were grown on R2A medium at 25 °C for 5 days. Salinarimonas rosea KCTC 22346T was grown on Marine Agar 2216 (BD) at 25 °C for 5 days. Fatty acid methyl esters were prepared according to the standard protocol of the Microbial Identification System (MIS; Microbial ID Inc.) and identified by using MIDI (Sasser 1990) with the TSBA database version 4.02. Purified cell-wall preparations were obtained as described by Schleifer and Kandler (1972). Cell-wall sugars were analysed according to the procedures of Staneck and Roberts (1974). Polar lipids were extracted, examined by twodimensional TLC and identified by using procedures as described elsewhere (Minnikin et al. 1977). Molecular and phylogenetic analysis Extraction of genomic DNA, PCR-mediated amplification of the 16S rRNA gene and sequencing of the purified PCR product were carried out according to Lee et al. (2011). The 16S rRNA gene sequence was compiled using the SeqMan software (DNASTAR) and aligned with published sequences retrieved from GenBank/EMBL/DDBJ using CLUSTAL X (Thompson et al. 1997); the resulting multiple alignment was

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edited manually using the program BioEdit (Hall 1999) according to the 16S rRNA secondary structure obtained from the Ribosomal Database Project II (RDP; release 10). The neighbour-joining (Saitou and Nei 1987), maximum-parsimony (Fitch 1971) and maximum-likelihood (Felsenstein 1981) algorithms were used in phylogenetic tree-building. Phylogenetic dendrograms were constructed by using the DNADIST, NEIGHBOR, DNAPARS and DNAML programs in the PHYLIP package (Felsenstein 2009). The distance matrix was produced on the basis of JukesCantor model (1969). Bootstrap analysis (Felsenstein 1985) was done with 1,000 replicates. For the measurement of DNA G?C content, genomic DNA was extracted and purified using a Blood & Cell Culture DNA Midi kit (Qiagen), and degraded enzymically into nucleosides. DNA G?C content was determined by reversed-phase HPLC, as described by Tamaoka and Komagata (1984).

Results and discussion Strain GJW-30T was observed to form visible white colonies (about 1 mm in diameter) within 5 days on R2A incubated at 25 °C. Good growth is observed at temperatures ranging from 20 to 30 °C. Colonies were observed to be creamy white, raised, semi-translucent, smooth and circular with undulate margins. Cells were found to be Gram-negative, oxidase- and catalasepositive, pleomorphic (oval to short rods or rods with wrinkled surfaces), non-motile rods (see Supplementary Fig. S1). The flagella or pili were not observed from transmission electron micrographs of cells of strain GJW-30T. The scanning (a) and transmission (b) electron micrographs of cells of strain GJW-30T presented in Supplementary Fig. S1. Strain GJW-30T was found to be negative for nitrogenase and acetylene-reduction (see Supplementary Fig. S2). The major fatty acids compositions of the strain GJW-30T, together with Bradyrhizobium japonicum KCTC 2422T, Bradyrhizobium oligotrophicum LMG 10732T, Bradyrhizobium betae NBRC 103048T, Bradyrhizobium cytisi LMG 25866T, Nitrobacter winogradskyi DSM 10237T, Afipia felis CIP 103515T, Bosea thiooxidans KACC 10745T, Oligotropha carboxidovorans DSM 1227T, Rhodopseudomonas palustris NBRC 100419T, Pseudolabrys taiwanensis CCUG 51779T, Rhodoplanes roseus DSM 5909T, Rhodoplanes elegans

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Antonie van Leeuwenhoek (2014) 105:915–924

NBRC 16660T and Tardiphaga robiniae LMG 26467T are shown in Tables 1 and 2. The major fatty acids were identified as C18:1x7c, C16:0, and C17:0 which were also the predominant fatty acids of the members of the order Rhizobiales. Strain GJW-30T was found to contain Q-10 as the predominant ubiquinone. The polar lipid profile of strain GJW-30T consists of diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, phosphatidylcholine, an unidentified aminolipid and an unidentified lipid (see Supplementary Fig. S3). The cell-wall sugar pattern of strain GJW30T was found to compose glucose, ribose and rhamnose and meso-DAP as the diagnostic diamino acid in the cell-wall peptidoglycan. Detailed physiological, biochemical and chemotaxonomic characteristics of strain GJW-30T are shown in Table 1 and the species description. The almost-complete 16S rRNA gene sequence of 1,407 nucleotides was determined for strain GJW-30T. Comparison of this sequence with those of all known representative species of the order Rhizobiales indicated that strain GJW-30T belongs to the order Rhizobiales. As shown in Fig. 1, the phylogenetic analysis revealed that strain GJW-30T forms a robust cluster with the family Bradyrhizobiaceae, this being supported by three tree-making algorithms (neighbour-joining, maximum-parsimony and maximumlikelihood) and a bootstrap value of 100 % (Fig. 1). Strain GJW-30T shows the highest 16S rRNA gene sequence similarity to that of Bradyrhizobium oligotrophica LMG 10732T (93.6 %) and to other representative members of the family Bradyrhizobiaceae and the order Rhizobiales (93.5 % or less). The physiological and biochemical characteristics that serve to differentiate strain GJW-30T from closely related taxa are summarized in Table 1. Strain GJW30T could be clearly distinguished from the other related genera by differences in motility, fatty acid profiles, catalase activity, growth at 10, 37 and 42 °C, nitrate reduction, citrate utilization, lysine decarboxylase, arginine dihydrolase, urease activity, assimilation of glucose, arabinose, mannose, mannitol, gluconate, adipate, malate and citrate, and acid from mannose, inositol and sorbitol. The phylogenetic analysis based on the 16S rRNA gene sequence similarity, phenotypic and chemotaxonomic properties revealed that strain GJW-30T can be clearly differentiated from other members of the genera of the family Bradyrhizobiaceae representing a novel

?

Catalase

?

?

?

–

–

w

?

w

–

20 °C

25 °C

30 °C

37 °C

42 °C

Arginine dihydrolase

Urease

Citrate utilization

Lysine decarboxylase

–

–

Inositol

Sorbitol

–

–

Malate

–

Mannitol

–

–

Mannose

Gluconate

–

Arabinose

Adipate

–

Glucose

Assimilation of:

–

Mannose

Acid from:

?

?

10 °C

15 °C

Growth at/with:

–

None

–

0.6–0.8 by 1.0–2.0

Pleomorphic rods

1

Nitrate reduction

Flagellar arrangementa

Motilitya

Cell size (lm)a

Cell shape

a

Characteristic

–

?

?

?

–

?

–

–

–

–

?

?

?

?

–

–

?

?

?

?

–

–

?

Polar or subpolar

?

0.5–0.9 by 1.2–3.0

Pleomorphic rods

2

?

–

?

?

?

?

?

?

?

?

?

?

?

?

–

?

?

?

?

?

w

?

?

Polar

?

0.6–1.0 by 2.0–7.0

Bent, branched rods

3

–

–

–

–

–

–

–

–

–

–

–

–

–

–

–

–

–

?

–

–

–

–

?

Single subpolar or lateral

?

0.6–0.8 by1.0–2.0

Pleomorphic rods

4

?

–

–

–

–

–

–

–

–

–

?

?

?

?

–

–

?

?

?

?

w

–

?

Polar, subpolar or lateral

?

0.2–0.5 by 0.2–2.5

Straight rods

5

–

–

–

–

–

–

–

–

–

–

–

–

–

–

–

?

?

?

?

w

–

?

–

Subpolar

?

0.4–0.7 by 1.0–3.0

Slightly curved rods

6

–

?

–

–

–

–

–

–

–

w

w

–

?

?

–

w

?

?

?

?

w

–

–

Subpolar

?

0.4–1.0 by 1.5–3.0

Rods, ovoids or vibrioids

7

a

?a ?a –a

a

?a –a –a

?

–

–

–

–

–

–

–

–

–

?

?

?

?

–

–

–

–

?

–

?

–

–

?

–

–

?

?

?

?a

–a ?

–a ?a

–a

?

?

Single polar

?

0.4–1.0 by 1.2–1.45

Rods

9

–a

?

?

Single polar

?

0.85 by 1.4–1.6

Straight rods

8

Table 1 Physiological, biochemical and chemotaxonomic characteristics that differentiate strain GJW-30T from closely related taxa

–

–

?

–

–

–

–

?

–

–

?

?

?

?

–a

–a

?a

?a

?a

?a

–a

?

?

None

–

0.4–1.0 by 1.2–1.45

Rods

10

–

–

–

–

–

–

–

–

–

w

w

w

?

?

?

?

?

?

?

w

–

–

?

None

–

0.4–0.6 by 1.2–2.2

Short rods

11

–

–

–

–

–

–

–

–

–

–

?

?

?

?

?

?

?

?

?

?

w

–

?

Polar

?

1.0 by 1.82.5

Rods or elongateovoid

12

Antonie van Leeuwenhoek (2014) 105:915–924 919

123

a Data from Das et al. (1996), De Meyer et al. (2012), Jordan (1982), Ohta and Hattori (1983), Auling et al. (1988), Brenner et al. (1991), Liu et al. (2010), Meyer et al. (1993), Hiraishi and Ueda (1994), Spieck and Bock (2005),.Kampfer et al. (2006), Ramana et al. (2012), and Ramı´rez-Bahena et al. (2013)

?, Positive; (?), weakly positive; -, negative; NR, not reported

Strains: 1, Variibacter gotjawalensis sp. nov. GJW-30T; 2, Bradyrhizobium japonicum KCTC 2422T; 3, Bradyrhizobium oligotrophicum LMG 10732T; 4, Nitrobacter winogradskyi DSM 10237T; 5, Afipia felis CIP 103515T; 6, Oligotropha carboxidovorans DSM 1227T; 7, Rhodopseudomonas palustris NBRC 100419T; 8, Bosea thiooxidans KACC 10745T; 9, Salinarimonas rosea KCTC 22346T; 10, Tardiphaga robiniae LMG 26467T; 11, Pseudolabrys taiwanensis CCUG 51779T; 12, Rhodoplanes roseus DSM 5909T. All strains tested were positive for oxidase. All strains tested were negative for Gram staining, indole production, trytophane deaminase, fermentation of glucose and hydrolysis of gelatin. Data are from this study, except where marked

67.1 67.3 60.8* 71.8* 65.8 62.2 DNA G?C content (mol%)

62.6

65.6

61.7

62.0

62.5

68.2*

C18:1, C16:0 C18:1, C19:0 cyclo, C18:1 2-OH C18:1, C16:0, C17:0 cyclo C18:1, C19:0 cyclo C18:1, C16:0, 11-methyl C18:1 C18:1, C18:0, C19:0 cyclo C18:1, C16:0, C17:0 Major fatty acids

C18:1, C16:0

C18:1, C16:0

C18:1, C16:0

C18:1, C16:1, C17:0 cyclo, C19:0 cyclo

C18:1, C16:0, C19:0 cyclo

–

Q-10, RQ10a

– ?

Q-10a Q-10a

? ?

Q-10a

–

Q-10a

–

Q-10a

–

Q-10a

– ?

Q-10a

–

Q-10a Q-10

Citrate

123

Isoprenoid quinone(s)

–

NR

11 10 9 8 7 6 5 4 3 2 1 Characteristic

Table 1 continued

NR

Antonie van Leeuwenhoek (2014) 105:915–924

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species in the new genus, for which the name Variibacter gotjawalensis gen nov., sp. nov. is proposed.

Description of Variibacter gen. nov Variibacter (Va.ri.i.bac’ter. L. adj. varius, varying; N.L. masc. n. bacter a rod; N.L. masc. n. Variibacter, a rod of varying morphology). Cells are non-motile pleomorphic (oval to short rods or rods with wrinkled surfaces) rods (approximately 0.6–0.8 9 1.0–2.0 lm), strictly aerobic, Gram-negative. Colonies on R2A agar are creamy white, raised, semi-translucent, smooth and circular with undulate margins. Oxidase and catalase test is positive. No nitrogenase activity. Contains Q-10 as the predominant ubiquinone. The major fatty acids are C18:1 x7c, C16:0, and C17:0, and diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, phosphatidylcholine, an unidentified aminolipid and an unidentified lipid are the polar lipids. Whole-cell sugars are glucose, ribose and rhamnose. The DNA G?C content of the type strain of the type species is 62.2 mol%. The type species is Variibacter gotjawalensis.

Description of Variibacter gotjawalensis sp. nov Variibacter gotjawalensis (got.ja.wal.en’sis: N.L. masc. adj. got.ja.wal.en’sis, of or belonging to Gotjawal, a forest located in Jeju, Korea). Cells are non-motile pleomorphic (oval to short rods or rods with wrinkled surfaces) rods (approximately 0.6–0.8 9 1.0–2.0 lm), strictly aerobic, Gram-negative. Colonies on R2A agar are creamy white, raised, semitranslucent, smooth and circular with undulate margins. Oxidase and catalase test is positive. No nitrogenase activity. Growth occurs at 10–30 °C (optimum 25 °C) and at pH 6.0–9.0 (optimum pH 7.0). Growth occurs in the presence of up to 1 % NaCl, but very week. In API 20E and 20NE, production of indole and H2S, lysine decarboxylase, reduction of nitrate, gelatinase, tryptophan deaminase, onitrophenyl-b-D-galactopyranoside, q-nitrophenly-b-Dgalactopyranosidase are negative, but production of urease and Voges–Proskauer test are positive. Utilization of citrate, ornithine decarboxylase and arginine dihaydrolase are weakly positive. In the API 50CH gallery, acid is not produced from any carbohydrates tested. According to API ZYM test results, alkaline phosphatase, leucine

Antonie van Leeuwenhoek (2014) 105:915–924

921

Table 2 Cellular fatty acid profiles of strain GJW-30T and closely related taxa Fatty acids (% of total)a

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

–

–

C13:1 at 12–13

–

–

–

–

–

–

–

–

–

0.4

–

–

C12:0 3-OH

0.6

–

–

–

–

–

–

0.8

–

–

–

–

–

–

–

C14:1 x5c C14:0

– –

– 0.6

0.5 –

– –

1.0 0.8

1.2 –

– –

– –

– –

– 0.7

– –

0.6 0.6

– 0.2

– 1.2

– 0.9

C15:0

1.2

–

–

–

–

–

–

–

–

–

–

3.9

–

–

–

Summed feature 2

–

–

–

–

–

–

–

–

1.1

–

0.8

–

–

–

–

Summed feature 3

5.6

1.5

7.9

2.1

6.5

–

16.3

7.0

5.0

3.5

1.9

4.8

0.7

3.4

1.6

C16:1 x5c

–

0.7

–

3.0

10.4

–

–

–

–

–

–

–

–

1.2

4.3 9.7

C16:0

11.3

14.0

10.1

14.2

13.1

11.2

3.4

3.1

15.9

19.9

17.6

5.4

10.9

Iso-C15:0 3-OH

–

–

–

–

–

–

–

–

–

–

–

–

2.0

–

Summed feature 9

–

–

–

–

–

–

–

–

–

–

–

–

0.4

–

–

Summed feature 4

0.5

0.6

–

0.7

3.7

–

–

–

–

–

–

–

–

1.3

0.8

Iso-C17:1 x5c

–

–

–

–

–

–

–

–

–

–

–

1.2

–

–

–

C17:1 x8c

3.8

–

–

–

–

–

–

–

–

–

0.3

4.7

–

–

–

C17:1 x6c

1.0

–

–

–

–

–

–

C17:0 cyclo

–

–

–

–

–

–

11.3

Iso-C17:0

6.0

–

–

–

–

4.4

–

4.1-

3.3

–

–

–

–

–

–

–

–

–

–

C17:0

10.1

–

–

–

–

–

–

–

–

C16:0 3-OH Summed feature 8

– 53.7

– 71.3

– 74.7

– 75.9

– 60.9

– 84.7

– 47.5

– 62.3

– 61.9

C18:1 x5c

–

–

–

–

–

–

–

C18:0

2.7

1.7

–

0.7

–

–

7.1

Iso-C17:0 3-OH

–

–

–

–

–

–

–

11-methyl C18:1 x7c

9.2

9.6

6.8

3.5

2.6

–

C19:0 cyclo x8c

–

–

–

–

1.0

2.9

C18:1 2-OH

–

–

–

–

–

–

–

4.1 51.4

–

–

–

–

–

10.9

–

–

– –

–

–

0.7

–

1.0

5.6

0.4

–

–

– 53.1

– 32.9

– 61.3

0.9 79.0

1.0 78.9

–

–

–

–

–

–

0.6

5.2

0.9

4.6

1.9

1.2

0.5

1.6

–

–

–

–

–

0.6

–

–

3.8

–

11.0

0.3-

3.4

8.3

–

1.1

0.8

10.8

10.4

–

14.0

24.4

7.0

15.5

–

–

–

–

–

–

–

–

10.3

–

– –

11.3

10-methyl C19:0

–

–

–

–

–

–

–

–

–

0.7

–

–

0.5

–

C18:0 3-OH

0.5

–

–

–

–

–

–

0.7

–

–

0.8

–

0.8

–

–

C20:1 x7c

–

–

–

–

–

–

–

–

–

–

0.4

–

–

–

–

1, Variibacter gotjawalensis sp. nov. GJW-30T; 2, Bradyrhizobium japonicum KCTC 2422T; 3, Bradyrhizobium oligotrophicum LMG 10732T; 4, Bradyrhizobium betae NBRC 103048T; 5, Bradyrhizobium cytisi LMG 25866T; 6, Nitrobacter winogradskyi DSM 10237T; 7, Afipia felis CIP 103515T; 8, Oligotropha carboxidovorans DSM 1227T; 9, Rhodopseudomonas palustris NBRC 100419T; 10, Bosea thiooxidans KACC 10745T; 11, Salinarimonas rosea KCTC 22346T; 12, Tardiphaga robiniae LMG 26467T; 13, Pseudolabrys taiwanensis CCUG 51779T; 14, Rhodoplanes roseus DSM 5909T; 15, Rhodoplanes elegans NBRC 16660T a Data from this study. Fatty acids are listed using standard abbreviations (number of carbon atoms: number of double bonds). Summed features represent groups of two or three fatty acids that could not be separated by GLC with the MIDI system: summed feature 2 comprises iso-C16:1 I and/or C14:0 3-OH; summed feature 3 comprises C16:1 x7c and/or C16:1 x6c; summed feature 4 comprises anteiso-C17:1 B and/or iso-C17:1 I; summed feature 8 comprises C18:1 x7c and/or C18:1 x6c; summed feature 9 comprises iso-C17:1 x9c and/or 10-methyl C16:0

arylamidase, trypsin, acid phosphatase, and naphthol-ASBI-phosphohydrolase are present, but esterase (C4), esterase lipase (C8), lipase (C14), valine arylamidase, cystine arylamidase, a-chymotrypsin, a-galactosidase, b-galactosidase, b-glucuronidase, a-glucosidase, b-glucosidase, Nacetyl-b-glucosaminidase, a-mannosidase and a-

fucosidase are absent. In API 20NE and ID 32 GN, the assimilation of L-arabinose, D-mannose, potassium gluconate, L-rhamnose, D-ribose, L-fucose, itaconate, suberate, Dlactate, potassium 2-ketogluconate, potassium 5-ketogluconate and 3-hydroxybutyrate are positive, trisodium citrate is weakly positive, but D-glucose, D-mannitol, inositol, D-

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922

Antonie van Leeuwenhoek (2014) 105:915–924

Fig. 1 Phylogenetic tree based on 16S rRNA gene sequences, reconstructed with the maximumlikelihood method, showing the position of strain GJW30T among related taxa within the family Bradyrhizobiaceae and the order Rhizobiales. Numbers at nodes are levels of bootstrap support ([50 %) based on 1,000 resamplings. Filled circles indicate nodes that are also found with the maximum-parsimony and neighbour-joining algorithms. Open circle indicates a node that is also found with the maximumparsimony algorithm. Escherichia coli (J01695) was used as an outgroup. Bar 0.05 substitutions per nucleotide position

sorbitol, D-sucrose, D-melibiose, N-acetyl-glucosamine, Dmaltose, caprate, adipate, malate, phenylacetate, salicin, propionate, valerate, L-histidine, 3-hydroxybenzoate, 4-hydroxybenzoate, L-proline, L-serine, glycogen, L-alanine, sodium acetate, and sodium malonate are negative. Contains Q-10 as the predominant ubiquinone. The major fatty acids are C18:1 x7c, C16:0, and C17:0, and diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, phosphatidylcholine, an unidentified aminolipid and an unidentified lipid are the polar lipids. Whole-cell sugars are glucose, ribose and rhamnose. The DNA G?C content of the type strain is 62.2 mol%. The type strain of the species is GJW-30T (=KCTC 32391T = CECT 8514T = LMG 28093T), which was isolated from lava forest soil collected in Gotjawal, Jeju, Korea. The GenBank (EMBL) accession number for the 16S rRNA gene sequence of strain GJW-30T is HF970589. Acknowledgments This work was supported by Mid-career Researcher Program through NRF grant funded by the Ministry of Science, ICT and Future Planning (MSIFP) of the Republic of Korea and a grant from the KRIBB Research Initiative Program.

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I thank Professors J. P. Euzeby and Bernhard Schink for his advice in naming the novel strain.

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