International Journal of Systematic and Evolutionary Microbiology (2014), 64, 1679–1684
DOI 10.1099/ijs.0.052894-0
Sphingomonas oligoaromativorans sp. nov., an oligotrophic bacterium isolated from a forest soil Song-Ih Han,1 Jae-Chan Lee,2 Hiroyuki Ohta3 and Kyung-Sook Whang1,2 Correspondence Kyung-Sook Whang [email protected]
1
Department of Biotechnology, Mokwon University, Doan-dong 800, Seo-gu, Daejeon 302-729, Republic of Korea
2
Institute of Microbial Ecology and Resources, Mokwon University, Doan-dong 800, Seo-gu, Daejeon 302-729, Republic of Korea
3
Department of Bioresource Science, College of Agriculture, Ibaraki University; Ami-machi, Ibaraki 300-0393, Japan
A halo- and organo-sensitive oligotrophic bacterium, designated strain SY-6T, was isolated from humus forest soil at Gyeryong mountain in Korea. Cells of the strain were Gram-negative, strictly aerobic, non-motile rods and the strain formed yellow-pigmented colonies on 100-fold-diluted nutrient broth. Strain SY-6T grew at pH 6.0–7.0 (optimal growth at pH 7.0), at 10–37 6C (optimal growth at 28 6C) and at salinities of 0–0.5 % (w/v) NaCl, growing optimally at 0.01 % (w/v) NaCl. On the basis of 16S rRNA gene sequence analysis, strain SY-6T was shown to belong to the genus Sphingomonas and showed the closest phylogenetic similarity to Sphingomonas polyaromaticivorans B2-7T (96.7 %). The major polar lipids were phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol, phosphatidylcholine and sphingoglycolipid. The predominant ubiquinone and polyamine were Q-10 and sym-homospermidine, respectively. The major fatty acids were C18 : 1v7c and C16 : 0. The DNA G+C content of the novel isolate was 65.3 mol%. On the basis of the evidence from this polyphasic study, strain SY-6T represents a novel species of the genus Sphingomonas, for which the name Sphingomonas oligoaromativorans sp. nov. is proposed. The type strain is SY-6T (5KACC 12948T5NBRC 105508T).
The genus Sphingomonas, belonging to the Alphaproteobacteria (Anzai et al., 2000; Lee et al., 2005), was described by Yabuuchi et al. (1990), with the type species Sphingomonas paucimobilis, and the description of the genus was subsequently emended by Takeuchi et al. (2001). At the time of writing, the genus Sphingomonas comprised 79 recognized species (Euze´by, 2013), including some recently described species (An et al., 2013; Chen et al., 2013; Niharika et al., 2012). Members of the genus Sphingomonas are rod-shaped, yellow, non-sporulating, aerobic, chemoheterotrophic, Gram-staining-negative and non-motile or motile by means of a single polar flagellum (Busse et al., 2003). They are characterized chemotaxonomically by the presence of ubiquinone 10 (Q-10) and 2hydroxy fatty acids and by the absence of 3-hydroxy fatty acids (Zhang et al., 2005), containing sym-homospermidine as the major polyamine and sphingoglycolipids as major components of their outer membrane (Kawahara et al., 1999). The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain SY-6T is FJ434127. Four supplementary figures and a supplementary table are available with the online version of this paper.
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Strain SY-6T was isolated from humus forest soil of Gyeryong Mountain National Park (36u 219 50.920 N 127u 119 34.230 E) in Korea during studies focused on the isolation of oligotrophic bacteria. It was isolated by diluting a soil sample in sterile water, plating on 100fold-diluted nutrient broth (1022 NB) containing 1.2 % (w/v) agar followed by aerobic incubation at 28 uC for 10 days. NB comprised 1 % (w/v) beef extract (Junsei), 1 % (w/v) polypeptone (Junsei) and 0.5 % (w/v) NaCl. The medium was adjusted to pH 7.0 with 1 M NaOH. The strain was subsequently purified three times by plating on 1022 NB agar at 28 uC for 5 days and maintained on the same medium. The strain was stored at 280 uC on this medium without agar and supplemented with 20 % (v/v) glycerol. In order to characterize strain SY-6T phenotypically, the isolate was routinely grown aerobically on 1022 NB agar for 5 days at 28 uC and pH 7.0, except where indicated otherwise. The morphology of the isolate was observed by Gram staining and scanning electron microscopy and motility was observed by phase-contrast microscopy (Nikon Eclipse 80i) using cells from exponentially growing cultures. Gram staining was performed by the Burke method (Murray et al., 1994). Catalase, oxidase, nitrate reduction, hydrolysis of 1679
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aesculin and production of indole were tested as recommended by Smibert & Krieg (1994) with 0.01 % (w/v) substrate. The substrate utilization profile was tested in minimal medium supplemented with carbon sources corresponding to the API 50CH gallery ingredients, including adipic acid, caproic acid, citric acid, malic acid and phenylacetic acid. All compounds were sterilized by filtration and then added to the medium. Cells were suspended in sterile distilled water. To test the profile of aromatic compound degradation, strain SY-6T was grown at 28 uC in 1022 NB supplemented with the following compounds (1 mM): benzoic acid, p-coumaric acid, ferulic acid, p-hydroxybenzoic acid, p-anistic acid, phenol and vanillic acid. Cultures were left for 10 days and degradation of the added compound was evaluated by the change in the UV absorption spectrum. Enzyme activities were tested using the API ZYM kit according to the instructions of the manufacturer (bioMe´rieux). To determine the optimal temperature and pH for growth of the strain, broth cultures were incubated in 1022 NB at 5–50 uC at intervals of 5 uC and at 28 and 37 uC and at pH 5–11 at intervals of 0.5 pH units. pH ,6, 6–9 and .9 was obtained by using sodium acetate/acetic acid, Tris/HCl and glycine/NaOH, respectively. Growth at 0, 0.01, 0.03. 0.05, 0.1, 0.5, 1.0, 3.0 and 5.0 % (w/v) NaCl was tested in 1022 NB medium at pH 7.0. Growth was monitored by turbidity at OD600 by using a spectroscopic method (model UV-1650PC; Shimadzu). Strain SY-6T showed a range of phenotypic properties typical of members of the genus Sphingomonas (Yabuuchi et al., 1990, 2002; Takeuchi et al., 2001; Busse et al., 2003). It was Gram-negative, non-motile and strictly aerobic and colonies were yellow, round and convex with entire margins when grown for 5 days at 28 uC on 1022 NB agar. Cells were rods, 0.5–0.6 mm wide and 1.0–1.3 mm long (Fig. S1, available in the online Supplementary Material). Strain SY-6T was able to grow at 10–37 uC, at pH 6.0–7.0 and in 0–0.5 % (w/v) NaCl. Optimal growth was observed at 28 uC, at pH 7.0 and in 0.01 % (w/v) NaCl. Cells were catalase- and oxidase-positive. It grew on R2A (Difco) and 1021 R2A agar. Growth was suppressed by fullstrength NB and was sensitive to 1021 NB. Strain SY-6T was a halo- and organo-sensitive bacterium, but a drastic change in the outer membrane structure was not observed with respect to salt sensitivity. Strain SY-6T was able to degrade benzoic acid, ferulic acid and p-coumaric acid. Other phenotypic features are included in the species description and characteristics that differentiate strain SY6T from its closest relative Sphingomonas polyaromaticivorans B2-7T are summarized in Table 1. Genomic DNA from strain SY-6T was prepared by using the method described by Tamaoka & Komagata (1984). The 16S rRNA gene was amplified by PCR with the forward primer Eubac 27F and reverse primer 1492R (DeLong, 1992). Direct sequence determination of the PCR-amplified DNA was carried out using an automated DNA sequencer (model ABI 3730XL; Applied Biosystems). 1680
The 16S rRNA gene sequence was aligned with the published sequences of closely related bacteria with the CLUSTAL W 2.0 software (Larkin et al., 2007). Gaps at the 59 and 39 ends of the alignment were omitted from further analyses. Phylogenetic trees were reconstructed by using three different methods, the neighbour-joining (Saitou & Nei, 1987), maximum-likelihood (Felsenstein, 1981) and maximum-parsimony (Fitch, 1971) algorithms, within the MEGA5 program (Tamura et al., 2011). Evolutionary distance matrices for the neighbour-joining method were calculated using the algorithm of Kimura’s two-parameter model (Kimura, 1980). To evaluate the stability of the phylogenetic tree, a bootstrap analysis (1000 replications) was performed (Felsenstein, 1985). The 16S rRNA gene sequences used for phylogenetic comparisons were obtained from GenBank and their strain designations and accession numbers are shown in Fig. 1. The almost-complete 16S rRNA gene sequence (1402 bp) of strain SY-6T was obtained and used for initial BLAST searches in GenBank and phylogenetic analysis. Identification of phylogenetic neighbours and calculation of pairwise 16S rRNA gene sequence similarity were achieved by using the EzTaxon-e server (http://www.eztaxon-e. ezcloud.net/; Kim et al., 2012). The neighbour-joining (Fig. 1), maximum-likelihood and maximum-parsimony methods (Figs S2 and S3) resulted in highly similar tree topologies, with strain SY-6T forming a distinct phyletic line with Sphingomonas polyaromaticivorans B2-7T with a bootstrap value of 100 %. DNA–DNA hybridization between strain SY-6T and its nearest phylogenetic neighbour, Sphingomonas polyaromaticivorans KCCM 42951T, was not attempted, as strains differing by .3 % in their 16S rRNA gene sequences are unlikely to exhibit .70 % relatedness at the whole-genome level (Stackebrandt & Goebel, 1994; Rossello´-Mora & Amann, 2001; Stackebrandt et al., 2002; Stackebrandt & Ebers, 2006). For analysis of fatty acids, strain SY-6T was cultured on 100-fold-diluted tryptic soy agar (1022 TSA; BD Difco) at 28 uC for 5 days and the closest type strain, Sphingomonas polyaromaticivorans KCCM 42951T, was cultured under the same conditions for 1 day. A different cultivation time had to be used because there was no single growth condition that allowed growth of both strains. Cells of the same physiological age were used in the comparison. Cellular fatty acids were extracted and analysed by GC (Agilent Technologies 6890N) according to the standard protocol of the Sherlock Microbial Identification System (version 4.5; MIDI database TSBA40 4.10). For analysis of quinones and polar lipids, cells were harvested in the late-exponential phase and freeze-dried. Isoprenoid quinones were extracted and analysed by HPLC (Shimadzu SPD-10AV), as described by Collins & Jones (1981). For polar lipid analysis, the cellular lipids were extracted twice, washed and hydrolysed with 0.5 M NaOH as described by Yabuuchi et al. (1990, 1999). Total lipids were examined by two-dimensional TLC and identified International Journal of Systematic and Evolutionary Microbiology 64
Sphingomonas oligoaromativorans sp. nov.
Table 1. Phenotypic characteristics for that differentiate strain SY-6T from the closest related type strain, Sphingomonas polyaromaticivorans KCCM 42951T Both strains are Gram-negative, non-motile rods and form yellow colonies. The two strains share the following characteristics: negative for nitrate reduction, catalase-positive, hydrolyse aesculin but not gelatin or arginine, assimilate sucrose and cellobiose but not D-adonitol, rhamnose, inositol, D-mannitol, D-sorbitol, N-acetylglucosamine, melibiose, xylitol, L-fucose, D-arabitol, D-gluconic acid, L-malate or phenylacetate. Both strains show optimum growth at pH 7.0. Both strains are positive for esterase (C4), esterase lipase (C8), leucine arylamidase, acid phosphatase, naphthol-AS-BIphosphohydrolase and a-glucosidase but negative for a-galactosidase, b-glucuronidase, N-acetyl-b-glucosaminidase, a-mannosidase and afucosidase. Data for Sphingomonas polyaromaticivorans B2-7T were taken from Luo et al. (2012) unless indicated. Characteristic NaCl concentration for growth (%, w/v) Range Optimum Growth temperature (uC) Range Optimum pH range for growth Oxidase activity Assimilation of: L-Arabinose D-Glucose Trehalose Raffinose Gentiobiose Malate API ZYM tests Alkaline phosphatase b-Glucosidase b-Galactosidase DNA G+C content (mol%)
SY-6T
Sphingomonas polyaromaticivorans KCCM 42951T
0–0.5 0.01
0.5–2.5 0.5
10–37 28 6.0–7.0 +
20–35 26 4.0–7.5 –
– – – + – –
+* +* +* –* +* +*
– – + 65.3
+* +* –* 61.8
*Data from this study.
using the method of Minnikin et al. (1984). To detect spots and their colour reactions, ethanolic molybdatophosphoric acid, Dittmer–Lester reagent, ninhydrin solution and 1-naphthol/sulfuric acid were used for total lipids, phospholipids, aminolipids and glycolipids, respectively. For polyamine analysis, strain SY-6T was grown aerobically in 6-fold-diluted PYE broth (DSM medium 655) at 30 uC and harvested in the late-exponential phase. Polyamines were extracted as described by Busse & Auling (1988) and analysed as described by Busse et al. (1997) using the HPLC equipment reported by Stolz et al. (2007). Isolation of DNA (Saito & Miura, 1963) and determination of the DNA G+C content were performed by HPLC (Shimadzu SPD-10AV), as described by Mesbah et al. (1989). Major fatty acids in strain SY-6T were C18 : 1v7c (56.9 %), C16 : 0 (10.1 %) and summed feature 4 (21.0 %; iso-C15 : 0 2OH and/or C16 : 1v7c). 2-Hydroxy fatty acids were present (C14 : 0 2-OH; 1.6 %), but 3-hydroxy fatty acid were not present. These are common characteristic features of members of the genus Sphingomonas. The presence of a large amount of summed feature 4 (iso-C15 : 0 2-OH and/or C16 : 1v7c) and the absence of C12 : 0 2-OH distinguished http://ijs.sgmjournals.org
strain SY-6T from the closely related Sphingomonas polyaromaticivorans B2-7T (Table S1). The major isoprenoid quinone of isolate SY-6T was ubiquinone 10 (Q-10), as in all known members of the Sphingomonadaceae. Polar lipids of SY-6T were phosphatidylethanolamine, phosphatidylglycerol, sphingoglycolipid, diphosphatidylglycerol, phosphatidylcholine, an unknown glycolipid, unknown aminolipids and unidentified lipids (Fig. S4). The presence of sphingoglycolipid in strain SY-6T indicates that the isolate is a member of the family Sphingomonadaceae. The polyamine pattern of strain SY-6T consisted predominantly of symhomospermidine [55.0 mmol (g dry weight)21], with spermidine [7.6 mmol (g dry weight)21] and spermine [2.1 mmol (g dry weight)21] present in smaller amounts, and putrescine [0.4 mmol (g dry weight)21], cadaverine [0.2 mmol (g dry weight)21] and 1,3 diaminopropane [0.1 mmol (g dry weight)21] as minor components. This polyamine pattern is in agreement with the assignment of strain SY-6T to the genus Sphingomonas sensu stricto (Busse et al., 1999, 2003; Geueke et al., 2007; Takeuchi et al., 2001). The G+C content of the DNA of strain SY-6T was 65.3 mol%, which is in the range (60.7–69.9 mol%) reported previously for members of the genus Sphingomonas. 1681
S.-I. Han and others
0.005
96
Sphingomonas sanguinis ISO 13937T (D84529) Sphingomonas paucimobilis ATCC 29837T (U37337) Sphingomonas pseudosanguinis G1-2T (AM412238)
99
Sphingomonas parapaucimobilis NBRC 15100T (D13724) 95
71
Sphingomonas roseiflava MK341T (D84520)
97 Sphingomonas yabuuchiae GTC 868T (AB071955) Sphingomonas ginsenosidimutnans Gsoil 1429T (HM204925) Sphingomonas yunnanensis YIM 003T (AY894691)
99 77
99
Sphingomonas phyllosphaerae FA2T (AY453855) 99 Sphingomonas endophytica YIM 65583T (HM629444) Sphingomonas japonica KC7T (AB428568) Sphingomonas xinjiangensis 10-1-84T (FJ754464)
71
Sphingomonas pituitosa EDIVT (AJ243751) 100 Sphingomonas trueperi LMG 2142T (X97776) 86 Sphingomonas azotifigens NBRC 15497T (AB033947)
68
Sphingomonas oligoaromativorans SY-6T (FJ434127) 100
Sphingomonas polyaromaticivorans B2-7T (EF467848) Sphingomonas ginsengisoli Gsoil 634T (AB245347)
94
Sphingomonas indica Dd16T (HQ449390) Sphingomonas starnbergensis 382T (JN591314)
81
Sphingomonas laterariae LNB2T (HM159118)
Sphingobium yanoikuyae NBRC 15102T (D13728)
Fig. 1. Rooted neighbour-joining tree based on 16S rRNA gene sequences of strain SY-6T and related bacteria in the class Alphaproteobacteria. Bootstrap values, expressed as percentages of 1000 replications, are given at branching points when .50 %. Filled circles at nodes indicate generic branches that were also recovered by using the maximum-likelihood and maximum-parsimony algorithms. Sphingobium yanoikuyae NBRC 15102T was used as an outgroup. Bar, 0.005 substitutions per nucleotide position.
Strain SY-6T shared similar chemotaxonomic characteristics with members of the genus Sphingomonas sensu stricto in terms of DNA G+C content, polar lipids, major polyamine, predominant ubiquinone, major fatty acids and 16S rRNA gene signature nucleotides. However, strain SY6T could be distinguished from closely related species in some chemotaxonomic and phenotypic features, such as NaCl, temperature and pH ranges for growth, the presence of oxidase and the presence of a large amount of summed feature 4 (iso-C15 : 0 2-OH and/or C16 : 1v7c).
aroma; L. part. adj. vorans devouring; N.L. part. adj. oligoaromativorans degrading a few aromatic compounds).
Sphingomonas oligoaromativorans (o9li.go.a.ro.ma9ti.vo9rans. Gr. adj. oligos little, small; L. n. aroma, -atis spice,
Cells are Gram-negative, non-motile rods, 0.5–0.661.0– 1.3 mm. Colonies are circular, convex, entire, yellow and 1.0–2.0 mm in diameter on 1022 NB agar after 5 days of incubation at 28 uC. Growth is not observed on NB agar and occurs at 0–0.1 % (w/v) NaCl, with optimal growth at 0.01 % (w/v) NaCl. Grows at 20–35 uC (optimally at 28 uC) and pH 6.0–7.0 (optimally at pH 7.0). Catalase- and oxidase-positive. Aesculin is hydrolysed, but arginine, urea and gelatin are not. Nitrate is not reduced to nitrite. Indole is not produced. Acid is not produced from D-glucose. Utilizes D-xylose, cellobiose, sucrose and raffinose for growth, but not glycerol, erythritol, D- or L-arabinose, Dribose, L-xylose, adonitol, methyl b-D-xylopyranoside, Dgalactose, D-glucose, D-fructose, D-mannose, L-sorbose, rhamnose, dulcitol, inositol, D-mannitol, D-sorbitol, methyl a-D-mannopyranoside, methyl a-D-glucoside, Nacetylglucosamine, arbutin, amygdalin, aesculin, salicin, maltose, lactose, melibiose, trehalose, inulin, melezitose,
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Therefore, on the basis of the taxonomic evidence from our polyphasic study, we propose that the strain represents a novel species of the genus Sphingomonas, for which the name Sphingomonas oligoaromativorans sp. nov. is proposed. Description of Sphingomonas oligoaromativorans sp. nov.
Sphingomonas oligoaromativorans sp. nov.
starch, glycogen, xylitol, gentiobiose, turanose, D-lyxose, Dtagatose, D- or L-fucose, D- or L-arabitol, gluconate, 2ketogluconate or 5-ketogluconate. Degrades aromatic compounds, including benzoic acid, ferulic acid and pcoumaric acid. Enzyme activity is observed for esterase (C4), esterase lipase (C8), leucine arylamidase, acid phosphatase, naphthol-AS-BI-phosphohydrolase and aglucosidase, but not for alkaline phosphatase, lipase (C14), valine arylamidase, cystine arylamidase, trypsin, achymotrypsin, a- and b-galactosidase, b-glucuronidase, Nacetyl-b-glucosaminidase and a-mannosidase (API ZYM). Major fatty acids are C18 : 1v7c, summed feature 4 (isoC15 : 0 2-OH and/or C16 : 1v7c) and C16 : 0. The main hydroxy-fatty acid is C14 : 0 2-OH. Polar lipids are phosphatidylethanolamine, phosphatidylglycerol, sphingoglycolipid, diphosphatidylglycerol, phosphatidylcholine, an unknown glycolipid, unknown aminolipids and unidentified lipids. The major polyamine is sym-homospermidine. The predominant isoprenoid quinone is Q-10. The type strain, SY-6T (5KACC 12948T5NBRC 105508T), was isolated from a forest soil of Gyeryong Mountain National Park in Korea. The DNA G+C content of the type strain is 65.3 mol%.
Acknowledgements We are grateful to Dr Hans-Ju¨rgen Busse for analysis of the polyamine pattern and advice. This work was supported by a grant from the Regional SubGenBank Support Program of the Rural Development Administration, Republic of Korea.
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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. Yabuuchi, E., Kosako, Y., Naka, T., Suzuki, S. & Yano, I. (1999).
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DOI 10.1099/ijs.0.052894-0
Sphingomonas oligoaromativorans sp. nov., an oligotrophic bacterium isolated from a forest soil Song-Ih Han,1 Jae-Chan Lee,2 Hiroyuki Ohta3 and Kyung-Sook Whang1,2 Correspondence Kyung-Sook Whang [email protected]
1
Department of Biotechnology, Mokwon University, Doan-dong 800, Seo-gu, Daejeon 302-729, Republic of Korea
2
Institute of Microbial Ecology and Resources, Mokwon University, Doan-dong 800, Seo-gu, Daejeon 302-729, Republic of Korea
3
Department of Bioresource Science, College of Agriculture, Ibaraki University; Ami-machi, Ibaraki 300-0393, Japan
A halo- and organo-sensitive oligotrophic bacterium, designated strain SY-6T, was isolated from humus forest soil at Gyeryong mountain in Korea. Cells of the strain were Gram-negative, strictly aerobic, non-motile rods and the strain formed yellow-pigmented colonies on 100-fold-diluted nutrient broth. Strain SY-6T grew at pH 6.0–7.0 (optimal growth at pH 7.0), at 10–37 6C (optimal growth at 28 6C) and at salinities of 0–0.5 % (w/v) NaCl, growing optimally at 0.01 % (w/v) NaCl. On the basis of 16S rRNA gene sequence analysis, strain SY-6T was shown to belong to the genus Sphingomonas and showed the closest phylogenetic similarity to Sphingomonas polyaromaticivorans B2-7T (96.7 %). The major polar lipids were phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol, phosphatidylcholine and sphingoglycolipid. The predominant ubiquinone and polyamine were Q-10 and sym-homospermidine, respectively. The major fatty acids were C18 : 1v7c and C16 : 0. The DNA G+C content of the novel isolate was 65.3 mol%. On the basis of the evidence from this polyphasic study, strain SY-6T represents a novel species of the genus Sphingomonas, for which the name Sphingomonas oligoaromativorans sp. nov. is proposed. The type strain is SY-6T (5KACC 12948T5NBRC 105508T).
The genus Sphingomonas, belonging to the Alphaproteobacteria (Anzai et al., 2000; Lee et al., 2005), was described by Yabuuchi et al. (1990), with the type species Sphingomonas paucimobilis, and the description of the genus was subsequently emended by Takeuchi et al. (2001). At the time of writing, the genus Sphingomonas comprised 79 recognized species (Euze´by, 2013), including some recently described species (An et al., 2013; Chen et al., 2013; Niharika et al., 2012). Members of the genus Sphingomonas are rod-shaped, yellow, non-sporulating, aerobic, chemoheterotrophic, Gram-staining-negative and non-motile or motile by means of a single polar flagellum (Busse et al., 2003). They are characterized chemotaxonomically by the presence of ubiquinone 10 (Q-10) and 2hydroxy fatty acids and by the absence of 3-hydroxy fatty acids (Zhang et al., 2005), containing sym-homospermidine as the major polyamine and sphingoglycolipids as major components of their outer membrane (Kawahara et al., 1999). The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain SY-6T is FJ434127. Four supplementary figures and a supplementary table are available with the online version of this paper.
052894 G 2014 IUMS
Printed in Great Britain
Strain SY-6T was isolated from humus forest soil of Gyeryong Mountain National Park (36u 219 50.920 N 127u 119 34.230 E) in Korea during studies focused on the isolation of oligotrophic bacteria. It was isolated by diluting a soil sample in sterile water, plating on 100fold-diluted nutrient broth (1022 NB) containing 1.2 % (w/v) agar followed by aerobic incubation at 28 uC for 10 days. NB comprised 1 % (w/v) beef extract (Junsei), 1 % (w/v) polypeptone (Junsei) and 0.5 % (w/v) NaCl. The medium was adjusted to pH 7.0 with 1 M NaOH. The strain was subsequently purified three times by plating on 1022 NB agar at 28 uC for 5 days and maintained on the same medium. The strain was stored at 280 uC on this medium without agar and supplemented with 20 % (v/v) glycerol. In order to characterize strain SY-6T phenotypically, the isolate was routinely grown aerobically on 1022 NB agar for 5 days at 28 uC and pH 7.0, except where indicated otherwise. The morphology of the isolate was observed by Gram staining and scanning electron microscopy and motility was observed by phase-contrast microscopy (Nikon Eclipse 80i) using cells from exponentially growing cultures. Gram staining was performed by the Burke method (Murray et al., 1994). Catalase, oxidase, nitrate reduction, hydrolysis of 1679
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aesculin and production of indole were tested as recommended by Smibert & Krieg (1994) with 0.01 % (w/v) substrate. The substrate utilization profile was tested in minimal medium supplemented with carbon sources corresponding to the API 50CH gallery ingredients, including adipic acid, caproic acid, citric acid, malic acid and phenylacetic acid. All compounds were sterilized by filtration and then added to the medium. Cells were suspended in sterile distilled water. To test the profile of aromatic compound degradation, strain SY-6T was grown at 28 uC in 1022 NB supplemented with the following compounds (1 mM): benzoic acid, p-coumaric acid, ferulic acid, p-hydroxybenzoic acid, p-anistic acid, phenol and vanillic acid. Cultures were left for 10 days and degradation of the added compound was evaluated by the change in the UV absorption spectrum. Enzyme activities were tested using the API ZYM kit according to the instructions of the manufacturer (bioMe´rieux). To determine the optimal temperature and pH for growth of the strain, broth cultures were incubated in 1022 NB at 5–50 uC at intervals of 5 uC and at 28 and 37 uC and at pH 5–11 at intervals of 0.5 pH units. pH ,6, 6–9 and .9 was obtained by using sodium acetate/acetic acid, Tris/HCl and glycine/NaOH, respectively. Growth at 0, 0.01, 0.03. 0.05, 0.1, 0.5, 1.0, 3.0 and 5.0 % (w/v) NaCl was tested in 1022 NB medium at pH 7.0. Growth was monitored by turbidity at OD600 by using a spectroscopic method (model UV-1650PC; Shimadzu). Strain SY-6T showed a range of phenotypic properties typical of members of the genus Sphingomonas (Yabuuchi et al., 1990, 2002; Takeuchi et al., 2001; Busse et al., 2003). It was Gram-negative, non-motile and strictly aerobic and colonies were yellow, round and convex with entire margins when grown for 5 days at 28 uC on 1022 NB agar. Cells were rods, 0.5–0.6 mm wide and 1.0–1.3 mm long (Fig. S1, available in the online Supplementary Material). Strain SY-6T was able to grow at 10–37 uC, at pH 6.0–7.0 and in 0–0.5 % (w/v) NaCl. Optimal growth was observed at 28 uC, at pH 7.0 and in 0.01 % (w/v) NaCl. Cells were catalase- and oxidase-positive. It grew on R2A (Difco) and 1021 R2A agar. Growth was suppressed by fullstrength NB and was sensitive to 1021 NB. Strain SY-6T was a halo- and organo-sensitive bacterium, but a drastic change in the outer membrane structure was not observed with respect to salt sensitivity. Strain SY-6T was able to degrade benzoic acid, ferulic acid and p-coumaric acid. Other phenotypic features are included in the species description and characteristics that differentiate strain SY6T from its closest relative Sphingomonas polyaromaticivorans B2-7T are summarized in Table 1. Genomic DNA from strain SY-6T was prepared by using the method described by Tamaoka & Komagata (1984). The 16S rRNA gene was amplified by PCR with the forward primer Eubac 27F and reverse primer 1492R (DeLong, 1992). Direct sequence determination of the PCR-amplified DNA was carried out using an automated DNA sequencer (model ABI 3730XL; Applied Biosystems). 1680
The 16S rRNA gene sequence was aligned with the published sequences of closely related bacteria with the CLUSTAL W 2.0 software (Larkin et al., 2007). Gaps at the 59 and 39 ends of the alignment were omitted from further analyses. Phylogenetic trees were reconstructed by using three different methods, the neighbour-joining (Saitou & Nei, 1987), maximum-likelihood (Felsenstein, 1981) and maximum-parsimony (Fitch, 1971) algorithms, within the MEGA5 program (Tamura et al., 2011). Evolutionary distance matrices for the neighbour-joining method were calculated using the algorithm of Kimura’s two-parameter model (Kimura, 1980). To evaluate the stability of the phylogenetic tree, a bootstrap analysis (1000 replications) was performed (Felsenstein, 1985). The 16S rRNA gene sequences used for phylogenetic comparisons were obtained from GenBank and their strain designations and accession numbers are shown in Fig. 1. The almost-complete 16S rRNA gene sequence (1402 bp) of strain SY-6T was obtained and used for initial BLAST searches in GenBank and phylogenetic analysis. Identification of phylogenetic neighbours and calculation of pairwise 16S rRNA gene sequence similarity were achieved by using the EzTaxon-e server (http://www.eztaxon-e. ezcloud.net/; Kim et al., 2012). The neighbour-joining (Fig. 1), maximum-likelihood and maximum-parsimony methods (Figs S2 and S3) resulted in highly similar tree topologies, with strain SY-6T forming a distinct phyletic line with Sphingomonas polyaromaticivorans B2-7T with a bootstrap value of 100 %. DNA–DNA hybridization between strain SY-6T and its nearest phylogenetic neighbour, Sphingomonas polyaromaticivorans KCCM 42951T, was not attempted, as strains differing by .3 % in their 16S rRNA gene sequences are unlikely to exhibit .70 % relatedness at the whole-genome level (Stackebrandt & Goebel, 1994; Rossello´-Mora & Amann, 2001; Stackebrandt et al., 2002; Stackebrandt & Ebers, 2006). For analysis of fatty acids, strain SY-6T was cultured on 100-fold-diluted tryptic soy agar (1022 TSA; BD Difco) at 28 uC for 5 days and the closest type strain, Sphingomonas polyaromaticivorans KCCM 42951T, was cultured under the same conditions for 1 day. A different cultivation time had to be used because there was no single growth condition that allowed growth of both strains. Cells of the same physiological age were used in the comparison. Cellular fatty acids were extracted and analysed by GC (Agilent Technologies 6890N) according to the standard protocol of the Sherlock Microbial Identification System (version 4.5; MIDI database TSBA40 4.10). For analysis of quinones and polar lipids, cells were harvested in the late-exponential phase and freeze-dried. Isoprenoid quinones were extracted and analysed by HPLC (Shimadzu SPD-10AV), as described by Collins & Jones (1981). For polar lipid analysis, the cellular lipids were extracted twice, washed and hydrolysed with 0.5 M NaOH as described by Yabuuchi et al. (1990, 1999). Total lipids were examined by two-dimensional TLC and identified International Journal of Systematic and Evolutionary Microbiology 64
Sphingomonas oligoaromativorans sp. nov.
Table 1. Phenotypic characteristics for that differentiate strain SY-6T from the closest related type strain, Sphingomonas polyaromaticivorans KCCM 42951T Both strains are Gram-negative, non-motile rods and form yellow colonies. The two strains share the following characteristics: negative for nitrate reduction, catalase-positive, hydrolyse aesculin but not gelatin or arginine, assimilate sucrose and cellobiose but not D-adonitol, rhamnose, inositol, D-mannitol, D-sorbitol, N-acetylglucosamine, melibiose, xylitol, L-fucose, D-arabitol, D-gluconic acid, L-malate or phenylacetate. Both strains show optimum growth at pH 7.0. Both strains are positive for esterase (C4), esterase lipase (C8), leucine arylamidase, acid phosphatase, naphthol-AS-BIphosphohydrolase and a-glucosidase but negative for a-galactosidase, b-glucuronidase, N-acetyl-b-glucosaminidase, a-mannosidase and afucosidase. Data for Sphingomonas polyaromaticivorans B2-7T were taken from Luo et al. (2012) unless indicated. Characteristic NaCl concentration for growth (%, w/v) Range Optimum Growth temperature (uC) Range Optimum pH range for growth Oxidase activity Assimilation of: L-Arabinose D-Glucose Trehalose Raffinose Gentiobiose Malate API ZYM tests Alkaline phosphatase b-Glucosidase b-Galactosidase DNA G+C content (mol%)
SY-6T
Sphingomonas polyaromaticivorans KCCM 42951T
0–0.5 0.01
0.5–2.5 0.5
10–37 28 6.0–7.0 +
20–35 26 4.0–7.5 –
– – – + – –
+* +* +* –* +* +*
– – + 65.3
+* +* –* 61.8
*Data from this study.
using the method of Minnikin et al. (1984). To detect spots and their colour reactions, ethanolic molybdatophosphoric acid, Dittmer–Lester reagent, ninhydrin solution and 1-naphthol/sulfuric acid were used for total lipids, phospholipids, aminolipids and glycolipids, respectively. For polyamine analysis, strain SY-6T was grown aerobically in 6-fold-diluted PYE broth (DSM medium 655) at 30 uC and harvested in the late-exponential phase. Polyamines were extracted as described by Busse & Auling (1988) and analysed as described by Busse et al. (1997) using the HPLC equipment reported by Stolz et al. (2007). Isolation of DNA (Saito & Miura, 1963) and determination of the DNA G+C content were performed by HPLC (Shimadzu SPD-10AV), as described by Mesbah et al. (1989). Major fatty acids in strain SY-6T were C18 : 1v7c (56.9 %), C16 : 0 (10.1 %) and summed feature 4 (21.0 %; iso-C15 : 0 2OH and/or C16 : 1v7c). 2-Hydroxy fatty acids were present (C14 : 0 2-OH; 1.6 %), but 3-hydroxy fatty acid were not present. These are common characteristic features of members of the genus Sphingomonas. The presence of a large amount of summed feature 4 (iso-C15 : 0 2-OH and/or C16 : 1v7c) and the absence of C12 : 0 2-OH distinguished http://ijs.sgmjournals.org
strain SY-6T from the closely related Sphingomonas polyaromaticivorans B2-7T (Table S1). The major isoprenoid quinone of isolate SY-6T was ubiquinone 10 (Q-10), as in all known members of the Sphingomonadaceae. Polar lipids of SY-6T were phosphatidylethanolamine, phosphatidylglycerol, sphingoglycolipid, diphosphatidylglycerol, phosphatidylcholine, an unknown glycolipid, unknown aminolipids and unidentified lipids (Fig. S4). The presence of sphingoglycolipid in strain SY-6T indicates that the isolate is a member of the family Sphingomonadaceae. The polyamine pattern of strain SY-6T consisted predominantly of symhomospermidine [55.0 mmol (g dry weight)21], with spermidine [7.6 mmol (g dry weight)21] and spermine [2.1 mmol (g dry weight)21] present in smaller amounts, and putrescine [0.4 mmol (g dry weight)21], cadaverine [0.2 mmol (g dry weight)21] and 1,3 diaminopropane [0.1 mmol (g dry weight)21] as minor components. This polyamine pattern is in agreement with the assignment of strain SY-6T to the genus Sphingomonas sensu stricto (Busse et al., 1999, 2003; Geueke et al., 2007; Takeuchi et al., 2001). The G+C content of the DNA of strain SY-6T was 65.3 mol%, which is in the range (60.7–69.9 mol%) reported previously for members of the genus Sphingomonas. 1681
S.-I. Han and others
0.005
96
Sphingomonas sanguinis ISO 13937T (D84529) Sphingomonas paucimobilis ATCC 29837T (U37337) Sphingomonas pseudosanguinis G1-2T (AM412238)
99
Sphingomonas parapaucimobilis NBRC 15100T (D13724) 95
71
Sphingomonas roseiflava MK341T (D84520)
97 Sphingomonas yabuuchiae GTC 868T (AB071955) Sphingomonas ginsenosidimutnans Gsoil 1429T (HM204925) Sphingomonas yunnanensis YIM 003T (AY894691)
99 77
99
Sphingomonas phyllosphaerae FA2T (AY453855) 99 Sphingomonas endophytica YIM 65583T (HM629444) Sphingomonas japonica KC7T (AB428568) Sphingomonas xinjiangensis 10-1-84T (FJ754464)
71
Sphingomonas pituitosa EDIVT (AJ243751) 100 Sphingomonas trueperi LMG 2142T (X97776) 86 Sphingomonas azotifigens NBRC 15497T (AB033947)
68
Sphingomonas oligoaromativorans SY-6T (FJ434127) 100
Sphingomonas polyaromaticivorans B2-7T (EF467848) Sphingomonas ginsengisoli Gsoil 634T (AB245347)
94
Sphingomonas indica Dd16T (HQ449390) Sphingomonas starnbergensis 382T (JN591314)
81
Sphingomonas laterariae LNB2T (HM159118)
Sphingobium yanoikuyae NBRC 15102T (D13728)
Fig. 1. Rooted neighbour-joining tree based on 16S rRNA gene sequences of strain SY-6T and related bacteria in the class Alphaproteobacteria. Bootstrap values, expressed as percentages of 1000 replications, are given at branching points when .50 %. Filled circles at nodes indicate generic branches that were also recovered by using the maximum-likelihood and maximum-parsimony algorithms. Sphingobium yanoikuyae NBRC 15102T was used as an outgroup. Bar, 0.005 substitutions per nucleotide position.
Strain SY-6T shared similar chemotaxonomic characteristics with members of the genus Sphingomonas sensu stricto in terms of DNA G+C content, polar lipids, major polyamine, predominant ubiquinone, major fatty acids and 16S rRNA gene signature nucleotides. However, strain SY6T could be distinguished from closely related species in some chemotaxonomic and phenotypic features, such as NaCl, temperature and pH ranges for growth, the presence of oxidase and the presence of a large amount of summed feature 4 (iso-C15 : 0 2-OH and/or C16 : 1v7c).
aroma; L. part. adj. vorans devouring; N.L. part. adj. oligoaromativorans degrading a few aromatic compounds).
Sphingomonas oligoaromativorans (o9li.go.a.ro.ma9ti.vo9rans. Gr. adj. oligos little, small; L. n. aroma, -atis spice,
Cells are Gram-negative, non-motile rods, 0.5–0.661.0– 1.3 mm. Colonies are circular, convex, entire, yellow and 1.0–2.0 mm in diameter on 1022 NB agar after 5 days of incubation at 28 uC. Growth is not observed on NB agar and occurs at 0–0.1 % (w/v) NaCl, with optimal growth at 0.01 % (w/v) NaCl. Grows at 20–35 uC (optimally at 28 uC) and pH 6.0–7.0 (optimally at pH 7.0). Catalase- and oxidase-positive. Aesculin is hydrolysed, but arginine, urea and gelatin are not. Nitrate is not reduced to nitrite. Indole is not produced. Acid is not produced from D-glucose. Utilizes D-xylose, cellobiose, sucrose and raffinose for growth, but not glycerol, erythritol, D- or L-arabinose, Dribose, L-xylose, adonitol, methyl b-D-xylopyranoside, Dgalactose, D-glucose, D-fructose, D-mannose, L-sorbose, rhamnose, dulcitol, inositol, D-mannitol, D-sorbitol, methyl a-D-mannopyranoside, methyl a-D-glucoside, Nacetylglucosamine, arbutin, amygdalin, aesculin, salicin, maltose, lactose, melibiose, trehalose, inulin, melezitose,
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International Journal of Systematic and Evolutionary Microbiology 64
Therefore, on the basis of the taxonomic evidence from our polyphasic study, we propose that the strain represents a novel species of the genus Sphingomonas, for which the name Sphingomonas oligoaromativorans sp. nov. is proposed. Description of Sphingomonas oligoaromativorans sp. nov.
Sphingomonas oligoaromativorans sp. nov.
starch, glycogen, xylitol, gentiobiose, turanose, D-lyxose, Dtagatose, D- or L-fucose, D- or L-arabitol, gluconate, 2ketogluconate or 5-ketogluconate. Degrades aromatic compounds, including benzoic acid, ferulic acid and pcoumaric acid. Enzyme activity is observed for esterase (C4), esterase lipase (C8), leucine arylamidase, acid phosphatase, naphthol-AS-BI-phosphohydrolase and aglucosidase, but not for alkaline phosphatase, lipase (C14), valine arylamidase, cystine arylamidase, trypsin, achymotrypsin, a- and b-galactosidase, b-glucuronidase, Nacetyl-b-glucosaminidase and a-mannosidase (API ZYM). Major fatty acids are C18 : 1v7c, summed feature 4 (isoC15 : 0 2-OH and/or C16 : 1v7c) and C16 : 0. The main hydroxy-fatty acid is C14 : 0 2-OH. Polar lipids are phosphatidylethanolamine, phosphatidylglycerol, sphingoglycolipid, diphosphatidylglycerol, phosphatidylcholine, an unknown glycolipid, unknown aminolipids and unidentified lipids. The major polyamine is sym-homospermidine. The predominant isoprenoid quinone is Q-10. The type strain, SY-6T (5KACC 12948T5NBRC 105508T), was isolated from a forest soil of Gyeryong Mountain National Park in Korea. The DNA G+C content of the type strain is 65.3 mol%.
Acknowledgements We are grateful to Dr Hans-Ju¨rgen Busse for analysis of the polyamine pattern and advice. This work was supported by a grant from the Regional SubGenBank Support Program of the Rural Development Administration, Republic of Korea.
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