International Journal of Systematic and Evolutionary Microbiology (2014), 64, 1030–1035
DOI 10.1099/ijs.0.055954-0
Sphingomonas yantingensis sp. nov., a mineralweathering bacterium isolated from purplish paddy soil Jing Huang, Zhi Huang, Zhen-Dong Zhang, Lin-Yan He and Xia-Fang Sheng Correspondence Xia-Fang Sheng
Key Laboratory of Agricultural Environment Microbiology, Ministry of Agriculture, College of Life Sciences, Nanjing Agricultural University, Nanjing 210095, PR China
[email protected]
A novel type of mineral-weathering bacterium was isolated from purplish soils collected from Yanting (Sichuan, south-western China). Cells of strain 1007T were Gram-stain-negative and rod-shaped, motile and yellow-pigmented. The isolate was strictly aerobic, catalase- and oxidasepositive, and grew optimally at 28-30 6C and pH 6.0-7.0. The genomic DNA G+C content of strain 1007T was 67±0.7 mol%. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain 1007T belonged to the genus Sphingomonas and was most closely related to Sphingomonas pruni IFO 15498T (97.3 %), Sphingomonas mali IFO 15500T (97.2 %), Sphingomonas japonica KC7T (97.2 %) and Sphingomonas koreensis JSS26T (97.0 %). This affiliation of strain 1007T to the genus Sphingomonas was confirmed by the presence of Q-10 as the major ubiquinone, sphingoglycolipid, C14 : 0 2-OH and by the absence of 3-hydroxy fatty acids. The major polyamine was homospermidine. The main cellular fatty acids included summed feature 8 (comprising C18 : 1v7c and/or C18 : 1v6c) and C16 : 0. Based on the low level of DNA–DNA relatedness (ranging from 26.1 % to 58.7 %) to these type strains of species of the genus Sphingomonas and unique phenotypic characteristics, strain 1007T represents a novel species of the genus Sphingomonas, for which the name Sphingomonas yantingensis sp. nov. is proposed. The type strain is 1007T (5DSM 27244T5JCM 19201T5CCTCC AB 2013146T).
The genus Sphingomonas was first proposed by Yabuuchi et al. (1990) and emended by Takeuchi et al. (1993), Yabuuchi et al. (1999, 2002), Takeuchi et al. (2001) and Busse et al. (2003). Members of the genus Sphingomonas are yellow-pigmented, nonfermentative, Gram-negative, non-motile or motile rods with a single polar flagellum and are characterized by the presence of a large amount of a unique sphingolipid with the long-chain base dihydrosphingosin, by the presence of 2-hydroxymyristic acid (C14 : 0 2-OH) and ubiquinone 10 (Q-10), and by the absence of 3-hydroxy fatty acids. Homospermidine is the major polyamine component (Busse et al., 1999; Takeuchi et al., 2001). Species of the genus Sphingomonas have been isolated from differing environments such as soil, aquatic habitats, clinical material and marine animals. In this study, polyphasic characterization of a yellowpigmented bacterium, strain 1007T, was performed. Phylogenetic analysis based on 16S rRNA gene sequences confirmed The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain 1007T is JX566547. Five supplementary figures are available with the online version of this paper.
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that strain 1007T belonged to the genus Sphingomonas and may represent a novel species of this genus. Strain 1007T was isolated from a purple soil sample collected from Yanting, a county in the south-west of China (105u 279 E 31u 169 N, with altitude of 420 m). Strain 1007T was able to weather biotite (a silicate mineral). It could grow aerobically on LB agar, R2A agar, tryptic soy agar (TSA), nutrient agar (NA) and marine agar 2216 (all from Difco) at 30 uC. It was cultured on NA at 30 uC and maintained as 30 % glycerol (v/v) suspensions at 280 uC. The characteristics of strain 1007T were investigated using routine cultivation on NA at 30 uC. For analysis of the 16S rRNA gene sequence, bacterial genomic DNA was extracted using a Qiagen Genomic DNA kit (Qiagen). PCR-mediated amplification of the 16S rRNA gene and purification of PCR products were carried out according to the method of Zhao et al. (2013). The PCR products were sequenced by Invitrogen (Shanghai, China). Full sequences of the 16S rRNA gene were compiled using Seqman software (DNASTAR) and identified using the EzTaxon server (Kim et al., 2012). The 16S rRNA gene sequences of related taxa were obtained from the GenBank database. Multiple alignments were made using the 055954 G 2014 IUMS Printed in Great Britain
Sphingomonas yantingensis sp. nov.
program (Thompson et al., 1997) and gaps were edited in the BioEdit program (Hall, 1999). Evolutionary distances were calculated using the Kimura two-parameter model (Kimura, 1983). Phylogenetic trees were reconstructed with the neighbour-joining, minimum-evolution and maximum-likelihood methods within the MEGA 5.0 program (Tamura et al., 2011). Bootstrap values were determined based on 1000 replications. DNA–DNA hybridization was carried out as described by De Ley et al. (1970) using a UV/VIS spectrophotometer (UV1201; Beijing Rayleigh Analytical Instrument Corporation), in which strain 1007T was used to generate the labeled DNA probe for hybridization studies.
CLUSTALX
The almost-complete 16S rRNA gene sequence of strain 1007T (1425 bp) was determined and subjected to comparative analysis. A sequence similarity search of the EzTaxon server revealed that strain 1007T belonged to the genus Sphingomonas. The highest sequence similarities were found with sequences from recognized members of the genus Sphingomonas, including Sphingomonas pruni IFO 15498T (97.3 %), Sphingomonas mali IFO 15500T (97.2 %), Sphingomonas japonica KC7T (97.2 %) and Sphingomonas koreensis JSS26T (97.0 %). Phylogenetic analysis based on 16S rRNA gene sequence indicated that strain 1007T clustered with S. japonica KC7T in a branch (Fig. 1). These findings were confirmed by analysis based on the minimum-evolution and
maximum-likelihood algorithms (Figs S1 and S2, available in the online Supplementary Material). Motility was observed by the hanging drop method. Cell morphology was investigated by light microscopy (CX21; Olympus) of exponential phase cells grown in NA liquid medium. Growth in NA liquid medium at different temperatures (4, 15, 25, 30, 37 and 42 uC) and at various pH values (pH 5.0–10.0, at intervals of 1 pH unit) were assessed after incubation for 5 days. The buffers that were used to adjust the pH of the NA medium (each at a final concentration of 100 mM) were acetate (for pH 4.0–5.0), phosphate (for pH 6.0–8.0) and Tris (for pH 9.0–10.0). Salt tolerance was tested on NA supplemented with 0.5– 10 % (w/v) NaCl, after incubation for 5 days. The Gramreaction was performed using the standard Gram staining method. Oxidase activity and catalase activity were determined according to the method of An et al. (2013). 3Ketolactose production, indole production, nitrate reduction, H2S production and hydrolysis of gelatin were tested (Halt et al., 1994). Carbon-source utilization and enzyme activities were tested by using the API 20NE, API CH50 B/E and API ZYM test kits (bioMe´rieux) according to the manufacturer’s instructions. The API ZYM tests were read after 4 h of incubation at 37 uC, the other API tests after 2 days at 30 uC.
T 98 Sphingomonas mali IFO 15500 (Y09638) 81 Sphingomonas pruni IFO 15498T (Y09637) Sphingomonas asaccharolytica IFO 15499T (Y09639) Sphingomonas soli T5-04T (AB166883) Sphingomonas koreensis JSS26T (AF131296)
0.01
Sphingomonas adhaesiva GIFU 11458T (D16146) Sphingomonas panni C52T (AJ575818) Sphingomonas aquatilis JSS7T (AF131295) Sphingomonas mucosissima CP173-2T (AM229669) Sphingomonas molluscorum KMM 3882T (AB248285) Sphingomonas yantingensis 1007T (JX566547) Sphingomonas japonica KC7T (AB428568) Sphingomonas trueperi LMG 2142T (X97776) 100 Sphingomonas pituitosa EDIVT (AJ243751) Sphingomonas paucimobilis ATCC 29837T (U20776) Sphingomonas roseiflava MK341T (D84520)
74 99 98
66
71 88
Sphingomonas sanguinis IFO 13937T (D13726) Sphingomonas parapaucimobilis IFO 15100T (D13724) Sphingomonas wittichii DSM 6014T (AB021492) Sphingopyxis terrae IFO 15098T (D13727)
100
Novosphingobium capsulatum IFO 12533T (D16147) Novosphingobium rosa IAM 14222T (D13945) Zymomonas mobilis ATCC 10988T (AF281031)
Fig. 1. Neighbour-joining phylogenetic tree based on 16S rRNA gene sequences showing the relationships between strain 1007T and the type strains of related bacteria. Bootstrap values (expressed as percentages of 1000 replications) over 50 % are shown at branch points. Zymomonas mobilis ATCC 10988T was used as an outgroup. Bar, 0.01 substitutions per nucleotide position. http://ijs.sgmjournals.org
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J. Huang and others
Table 1. Differential phenotypic and physiological characteristics of strain 1007T and closely related type strains of the genus Sphingomonas Strains: 1, 1007T; 2, S. pruni IFO 15498T (DSM 10566T) (Takeuchi et al., 1995); 3, S. mali IFO 15500T (DSM 10565T) (Takeuchi et al., 1995); 4, S. japonica KC7T (DSM 22753T) (Romanenko et al., 2009); 5, S. koreensis JSS26T (DSM 15582T) (Lee et al., 2001). All data are from this study except those for the DNA G+C content of the reference strains, taken from the corresponding public documents. All strains are positive for arginine dihydrolase, urease, aesculin hydrolysis, assimilation of D-glucose, maltose, cellobiose and lactose and activity of alkaline phosphatase, acid phosphatase, esterase (C4), esterase lipase (C8), leucine arylamidase, cystine arylamidase, naphthol-AS-BI-phosphohydrolase and a-glucosidase. All strains are negative for Gram-staining, methyl red and Voges Proskauer tests, nitrate reduction, indole production, fermentation from glucose, utilization of D-mannitol, capric acid, trisodium citrate, phenylacetic acid, glycerol, erythritol, D-arabinose, D-ribose, L-xylose, D-adonitol, L-sorbose, dulcitol, inositol, D-sorbitol, methyl a-D-mannopyranoside, inulin, glycogen, xylitol, D-lyxose, D-tagatose, D- and L-arabitol, potassium 2-ketogluconate and potassium 5-ketogluconate, and activity of a-galactosidase, a-mannosidase and a-fucosidase. All strains are positive for H2S production, catalase and oxidase. PY, pale yellow; Y, yellow; YO, yellow–orange. +, Positive reaction; –, negative reaction; W, weakly positive reaction; NR, not reported. Characteristic
1
2
3
4
5
Colony colour Hydrolysis of gelatin 3-Ketolactose production Oxidase Utilization of: D-Xylose Methyl b-Dxylopyranoside D-Galactose D-Fructose L-Rhamnose Methyl a-Dglucopyranoside N-Acetylglucosamine Amygdalin Arbutin Salicin Melibiose Sucrose Trehalose Melezitose Raffinose Starch Gentiobiose Turanose D-Fucose L-Fucose Assimilation of (API 20 NE): L-Arabinose D-Mannose
Y – + W
PY – + +
PY – + +
YO + + +
Y – – +
+ –
– –
– –
+ +
+ –
+ – + +
– –
– – + –
+ – – –
– + +
–
+ –
– – – – – – + – – – + – + +
– – – – + – – – – + – – + –
– – – + + – + – + – + + – –
+ +
– +
– +
– +
– –
1032
+ + + + – + + + + – + – W
W
– + W W W
– – + – – + + W
W
Table 1. cont. Characteristic
1
2
3
4
5
N-Acetylglucosamine Potassium gluconate Adipic acid Malic acid API ZYM tests Lipase Valine arylamidase Trypsin a-Chymotrypsin b-Galactosidase b-Glucuronidase b-Glucosidase N-Acetyl-bglucosaminidase DNA G+C content (mol%)
+ – – +
+
– – – –
– – +
+ – – +
W
W
+ – – +
– + – – – – – –
– – +
W
+ – – + – + +
65
66
NR
66
2 + + W
– + + + 67
W W W
W W
W
W
– – + +
For the measurement of DNA G+C content of strain 1007T, genomic DNA was extracted, purified and enzymically degraded into nucleosides. The DNA G+C content was then determined as described by Mesbah et al. (1989), using a reversed-phase HPLC (Agilent 1100, Agilent Technologies, Palo Alto, CA). The DNA G+C content was calculated using Escherichia coli K-12 as the reference strain. For polar lipid and fatty acid analysis, strain 1007T was cultivated on NA at 30 uC for 3 days and lipids were extracted using the chloroform/methanol extraction method (Minnikin et al., 1984). Polar lipids were analysed as described by Denner et al. (2001). Fatty acid methyl esters were obtained by acid methanolysis as described by Rowe et al. (2000) and identified by using the Microbial Identification software package (Sasser , 1990). Respiratory quinones were analysed according to the method of Komagata & Suzuki (1987). Polyamines were extracted and analysed according to the protocol of Tala` et al. (2013). Cells of strain 1007T were Gram-stain-negative, aerobic, yellow-pigmented, motile rods with single polar flagella (Fig. S3). Strain 1007T grew strongly on many media such as NA, R2A agar, TSA and LB agar. Colonies of strain 1007T grown on NA were circular, convex, opaque and yellow and became sticky after 48 h of incubation at 30 uC. On NA medium, strain 1007T was able to grow at 15–30 uC but not at 4 uC or 42 uC, and grew weakly at 37 uC. The pH range for growth was pH 5.0–10.0 (optimum, pH 6.0–7.0). Optimal growth occurred in the absence of NaCl; growth was inhibited by the presence of .2 % (w/v) NaCl. Strain 1007T was positive for H2S production, oxidase, catalase and Tween 80 hydrolysis, but negative for indole production, nitrate reduction, gelatin hydrolysis, oxidation/fermentation test and gas/acid production test with glucose. Other physiological, biochemical and chemotaxonomic characteristics that differentiated strain 1007T from the closest phylogenetic relatives are listed in Table 1. The major respiratory quinone of strain 1007T was ubiquinone Q-10 (see Fig. S4). The polar lipid profile of International Journal of Systematic and Evolutionary Microbiology 64
Sphingomonas yantingensis sp. nov.
strain 1007T contained phosphatidylglycerol, phosphatidylethanolamine, glycosylsphingolipid, phosphatidylcholine, sphingolipid, two unidentified phospholipids and three unidentified lipids (Fig. S5). Major fatty acids were summed feature 8 (comprising C18 : 1v7c or C18 : 1v6c) (60.4 %) and C16 : 0 (15.8 %). C14 : 0 2-OH (3.9 %) was the major 2-hydroxy fatty acid (Table 2) and 3-hydroxy fatty acid was absent. The presence of 2-hydroxy fatty acid and the lack of 3-hydroxy fatty acid are important characteristics of members of the family Sphingomonadaceae (Busse et al., 2003; Takeuchi et al., 2001; Takeuchi & Hiraishi, 2001). The fatty acid profile of strain 1007T was in agreement with those of close representatives of the genus and consistent with the assignment of strain 1007T to the genus Sphingomonas. The DNA G+C content of strain 1007T was 67 mol%. The major polyamine of strain 1007T was homospermidine, which was in accordance with the members of the genus Sphingomonas sensu stricto (Takeuchi et al., 2001; Maruyama et al., 2006). Despite the similarities, DNA–DNA hybridization studies showed low relatedness between strain 1007T and S. pruni IFO 15498T (36.0 %), S. mali IFO 15500T (51.0 %), S. japonica KC7T (58.7 %) and S. koreensis JSS26T (26.1 %). On the basis of phenotypic, chemotaxonomic and phylogenetic data and the results of DNA–DNA hybridizations, strain 1007T is proposed to be assigned to the genus Sphingomonas as a novel species, for which the name Sphingomonas yantingensis sp. nov. is proposed. Description of Sphingomonas yantingensis sp. nov. Sphingomonas yantingensis (yan.ting.en9sis. N.L. fem. adj. yantingensis pertaining to Yanting County in China, where the type strain was isolated). Cells are Gram-negative-staining, strictly aerobic, yellowpigmented, motile short rods with single polar flagella and do not form spores. Cells are 0.5–0.8 mm in width and 1.0– 1.6 mm in length. On NA, forms yellow, circular, dry colonies (0.8–1.2 mm in diameter) within 48 h. Grows at 15– 37 uC (optimum, 28–30 uC) and pH 5.0–10.0 (optimum, pH 6.0–7.0). Growth is inhibited in the presence of .2 % (w/ v) NaCl. Does not reduce nitrate to nitrite or nitrite to N2. Positive for H2S production, Tween 80 hydrolysis, aesculin hydrolysis, catalase and oxidase. Negative for methyl red test, Voges–Proskauer test, indole production and hydrolysis of gelatin. Positive for arginine dihydrolase, urease, valine arylamidase, trypsin, a-chymotrypsin (weakly), b-glucuronidase, a- and b-glucosidase, N-acetyl-b-glucosaminidase, alkaline- and acid- phosphatase, esterase (C4), esterase lipase (C8), leucine arylamidase, cystine arylamidase and naphtholAS-BI-phosphohydrolase activities. Negative for a-galactosidase, a-mannosidase, a-fucosidase, lipase and b-galactosidase activities. D-Glucose, maltose, cellobiose, lactose, D-xylose, D-galactose, L-rhamnose, methyl a-D-glucopyranoside, Nacetylglucosamine, amygdalin, arbutin, salicin, sucrose, trehalose, melezitose, raffinose, gentiobiose, D-fucose (weakly), http://ijs.sgmjournals.org
Table 2. Cellular fatty acid profiles of strain 1007T and type strains of related species of the genus Sphingomonas Strains: 1, 1007T; 2, S. pruni IFO 15498T (DSM 10566T); 3, S. mali IFO 15500T (DSM 10565T); 4, S. japonica KC7T (DSM 22753T); 5, S. koreensis JSS26T (DSM 15582T). All data were generated in this study. Values shown are percentages of the total fatty acids. –, Not detected; TR, trace amount (,0.1 %). The major cellular fatty acids of the five strains tested were C16 : 0 and summed feature 8, and the major hydroxyl fatty acid was C14 : 0 2-OH. 3-Hydroxy fatty acid was not detected. Fatty acid C12 : 0 C14 : 0 C16 : 0 C17 : 0 C18 : 0 C12 : 0 2-OH C13 : 0 2-OH C14 : 0 2-OH C15 : 0 2-OH anteiso-C15 : 0 iso-C15 : 0 C16 : 0 2-OH iso-C16 : 0 C16 : 1v5c anteiso-C17 : 0 C17 : 1v6c C17 : 1v8c C18 : 1v5c C18 : 1v9c C19 : 0 cyclo v8c 11-Methyl C18 : 1v7c 10-Methyl C19 : 0 Summed feature 2 Summed feature 3 Summed feature 7 Summed feature 8
1
2
3
4
5
– 0.4 15.8 0.2 0.7 – – 3.9 – – – – – 2.2 – 1.3 0.3 1.4 1.2 – 3.8 – – 8.2 – 60.4
– 0.3 11.1 2.8 1.1 –
0.2 0.4 12.6 0.4 1.2
– 0.9 12.4 0.1 0.1 – – 5.0 0.1 – – – – 1.8 – 2.4 – 0.8 0.1 – 3.0 – 0.2 29.4 – 43.4
– 0.5 13.2 – 2.0 – – 5.5
TR
4.3 3.1 0.6 0.6 0.6 TR
0.2 0.3 16.7 3.4 2.4 1.1 – 5.8 – – 0.6 0.7 44.2
TR
– 6.7 0.5 0.1 0.2 0.4 – 0.3 0.1 3.0 0.7 1.6 1.4 – 5.8 TR
– 1.8 – 62.2
TR
– – 0.7 – 1.3 – 0.9 – 2.5 1.2 0.4 10.0 – – 2.6 – 59.2
*Summed feature 2, unknown fatty acids; summed feature 3 contains C16 : 1v6c and/or C16 : 1v7c; summed feature 7 contains C19 : 1v6c and/ or C19 : 0 cyclov8c; summed feature 8 contains C18 : 1v7c and/or C18 : 1v6c.
L-arabinose, D-mannose
and malic acid are utilized as sole carbon and energy sources, but D-mannitol, capric acid, trisodium citrate, phenylacetic acid, glycerol, erythritol, Darabinose, D-ribose, L-xylose, D-adonitol, L-sorbose, dulcitol, inositol, D-sorbitol, methyl a-D-mannopyranoside, inulin, glycogen, xylitol, D-lyxose, D-tagatose, D- and L-arabitol, potassium gluconate, potassium 2-ketogluconate, potassium 5-ketogluconate, methyl b-D-xylopyranoside, D-fructose, melibiose, starch, turanose, L-fucose and adipic acid are not. Ubiquinone Q-10 is the predominant respiratory quinone. The major polyamine is homospermidine. The major fatty acids are summed feature 8 (comprising C18 : 1v7c and/or C18 : 1v6c) and C16 : 0. C14 : 0-2-OH is the major 2-hydroxyl 1033
J. Huang and others
fatty acid. The polar lipid profile comprises phosphatidylglycerol, phosphatidylethanolamine, glycosylsphingolipid, phosphatidylcholine, sphingolipid, two unidentified phospholipids and three unidentified lipids. The type strain is 1007T (5DSM 27244T5JCM 19201T5 CCTCC AB 2013146T), which was isolated from a purplish soil sample collected in Yanting (Sichuan, China). The DNA G+C content of the type strain is (67±0.7) mol%.
Acknowledgements This research was supported by National Science Foundation of China (project no. 41071173).
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Yabuuchi, E., Kosako, Y., Fujiwara, N., Naka, T., Matsunaga, I., Ogura, H. & Kobayashi, K. (2002). Emendation of the genus
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International Journal of Systematic and Evolutionary Microbiology 64
Sphingomonas yantingensis sp. nov. Sphingomonas Yabuuchi et al. 1990 and junior objective synonymy of the species of three genera, Sphingobium, Novosphingobium and Sphingopyxis, in conjunction with Blastomonas ursincola. Int J Syst Evol Microbiol 52, 1485–1496.
http://ijs.sgmjournals.org
Zhao, F., Guo, X. Q., Wang, P., He, L. Y., Huang, Z. & Sheng, X. F. (2013). Dyella jiangningensis sp. nov., a c-proteobacterium isolated
from the surface of potassium-bearing rock. Int J Syst Evol Microbiol 63, 3154–3157.
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DOI 10.1099/ijs.0.055954-0
Sphingomonas yantingensis sp. nov., a mineralweathering bacterium isolated from purplish paddy soil Jing Huang, Zhi Huang, Zhen-Dong Zhang, Lin-Yan He and Xia-Fang Sheng Correspondence Xia-Fang Sheng
Key Laboratory of Agricultural Environment Microbiology, Ministry of Agriculture, College of Life Sciences, Nanjing Agricultural University, Nanjing 210095, PR China
[email protected]
A novel type of mineral-weathering bacterium was isolated from purplish soils collected from Yanting (Sichuan, south-western China). Cells of strain 1007T were Gram-stain-negative and rod-shaped, motile and yellow-pigmented. The isolate was strictly aerobic, catalase- and oxidasepositive, and grew optimally at 28-30 6C and pH 6.0-7.0. The genomic DNA G+C content of strain 1007T was 67±0.7 mol%. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain 1007T belonged to the genus Sphingomonas and was most closely related to Sphingomonas pruni IFO 15498T (97.3 %), Sphingomonas mali IFO 15500T (97.2 %), Sphingomonas japonica KC7T (97.2 %) and Sphingomonas koreensis JSS26T (97.0 %). This affiliation of strain 1007T to the genus Sphingomonas was confirmed by the presence of Q-10 as the major ubiquinone, sphingoglycolipid, C14 : 0 2-OH and by the absence of 3-hydroxy fatty acids. The major polyamine was homospermidine. The main cellular fatty acids included summed feature 8 (comprising C18 : 1v7c and/or C18 : 1v6c) and C16 : 0. Based on the low level of DNA–DNA relatedness (ranging from 26.1 % to 58.7 %) to these type strains of species of the genus Sphingomonas and unique phenotypic characteristics, strain 1007T represents a novel species of the genus Sphingomonas, for which the name Sphingomonas yantingensis sp. nov. is proposed. The type strain is 1007T (5DSM 27244T5JCM 19201T5CCTCC AB 2013146T).
The genus Sphingomonas was first proposed by Yabuuchi et al. (1990) and emended by Takeuchi et al. (1993), Yabuuchi et al. (1999, 2002), Takeuchi et al. (2001) and Busse et al. (2003). Members of the genus Sphingomonas are yellow-pigmented, nonfermentative, Gram-negative, non-motile or motile rods with a single polar flagellum and are characterized by the presence of a large amount of a unique sphingolipid with the long-chain base dihydrosphingosin, by the presence of 2-hydroxymyristic acid (C14 : 0 2-OH) and ubiquinone 10 (Q-10), and by the absence of 3-hydroxy fatty acids. Homospermidine is the major polyamine component (Busse et al., 1999; Takeuchi et al., 2001). Species of the genus Sphingomonas have been isolated from differing environments such as soil, aquatic habitats, clinical material and marine animals. In this study, polyphasic characterization of a yellowpigmented bacterium, strain 1007T, was performed. Phylogenetic analysis based on 16S rRNA gene sequences confirmed The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain 1007T is JX566547. Five supplementary figures are available with the online version of this paper.
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that strain 1007T belonged to the genus Sphingomonas and may represent a novel species of this genus. Strain 1007T was isolated from a purple soil sample collected from Yanting, a county in the south-west of China (105u 279 E 31u 169 N, with altitude of 420 m). Strain 1007T was able to weather biotite (a silicate mineral). It could grow aerobically on LB agar, R2A agar, tryptic soy agar (TSA), nutrient agar (NA) and marine agar 2216 (all from Difco) at 30 uC. It was cultured on NA at 30 uC and maintained as 30 % glycerol (v/v) suspensions at 280 uC. The characteristics of strain 1007T were investigated using routine cultivation on NA at 30 uC. For analysis of the 16S rRNA gene sequence, bacterial genomic DNA was extracted using a Qiagen Genomic DNA kit (Qiagen). PCR-mediated amplification of the 16S rRNA gene and purification of PCR products were carried out according to the method of Zhao et al. (2013). The PCR products were sequenced by Invitrogen (Shanghai, China). Full sequences of the 16S rRNA gene were compiled using Seqman software (DNASTAR) and identified using the EzTaxon server (Kim et al., 2012). The 16S rRNA gene sequences of related taxa were obtained from the GenBank database. Multiple alignments were made using the 055954 G 2014 IUMS Printed in Great Britain
Sphingomonas yantingensis sp. nov.
program (Thompson et al., 1997) and gaps were edited in the BioEdit program (Hall, 1999). Evolutionary distances were calculated using the Kimura two-parameter model (Kimura, 1983). Phylogenetic trees were reconstructed with the neighbour-joining, minimum-evolution and maximum-likelihood methods within the MEGA 5.0 program (Tamura et al., 2011). Bootstrap values were determined based on 1000 replications. DNA–DNA hybridization was carried out as described by De Ley et al. (1970) using a UV/VIS spectrophotometer (UV1201; Beijing Rayleigh Analytical Instrument Corporation), in which strain 1007T was used to generate the labeled DNA probe for hybridization studies.
CLUSTALX
The almost-complete 16S rRNA gene sequence of strain 1007T (1425 bp) was determined and subjected to comparative analysis. A sequence similarity search of the EzTaxon server revealed that strain 1007T belonged to the genus Sphingomonas. The highest sequence similarities were found with sequences from recognized members of the genus Sphingomonas, including Sphingomonas pruni IFO 15498T (97.3 %), Sphingomonas mali IFO 15500T (97.2 %), Sphingomonas japonica KC7T (97.2 %) and Sphingomonas koreensis JSS26T (97.0 %). Phylogenetic analysis based on 16S rRNA gene sequence indicated that strain 1007T clustered with S. japonica KC7T in a branch (Fig. 1). These findings were confirmed by analysis based on the minimum-evolution and
maximum-likelihood algorithms (Figs S1 and S2, available in the online Supplementary Material). Motility was observed by the hanging drop method. Cell morphology was investigated by light microscopy (CX21; Olympus) of exponential phase cells grown in NA liquid medium. Growth in NA liquid medium at different temperatures (4, 15, 25, 30, 37 and 42 uC) and at various pH values (pH 5.0–10.0, at intervals of 1 pH unit) were assessed after incubation for 5 days. The buffers that were used to adjust the pH of the NA medium (each at a final concentration of 100 mM) were acetate (for pH 4.0–5.0), phosphate (for pH 6.0–8.0) and Tris (for pH 9.0–10.0). Salt tolerance was tested on NA supplemented with 0.5– 10 % (w/v) NaCl, after incubation for 5 days. The Gramreaction was performed using the standard Gram staining method. Oxidase activity and catalase activity were determined according to the method of An et al. (2013). 3Ketolactose production, indole production, nitrate reduction, H2S production and hydrolysis of gelatin were tested (Halt et al., 1994). Carbon-source utilization and enzyme activities were tested by using the API 20NE, API CH50 B/E and API ZYM test kits (bioMe´rieux) according to the manufacturer’s instructions. The API ZYM tests were read after 4 h of incubation at 37 uC, the other API tests after 2 days at 30 uC.
T 98 Sphingomonas mali IFO 15500 (Y09638) 81 Sphingomonas pruni IFO 15498T (Y09637) Sphingomonas asaccharolytica IFO 15499T (Y09639) Sphingomonas soli T5-04T (AB166883) Sphingomonas koreensis JSS26T (AF131296)
0.01
Sphingomonas adhaesiva GIFU 11458T (D16146) Sphingomonas panni C52T (AJ575818) Sphingomonas aquatilis JSS7T (AF131295) Sphingomonas mucosissima CP173-2T (AM229669) Sphingomonas molluscorum KMM 3882T (AB248285) Sphingomonas yantingensis 1007T (JX566547) Sphingomonas japonica KC7T (AB428568) Sphingomonas trueperi LMG 2142T (X97776) 100 Sphingomonas pituitosa EDIVT (AJ243751) Sphingomonas paucimobilis ATCC 29837T (U20776) Sphingomonas roseiflava MK341T (D84520)
74 99 98
66
71 88
Sphingomonas sanguinis IFO 13937T (D13726) Sphingomonas parapaucimobilis IFO 15100T (D13724) Sphingomonas wittichii DSM 6014T (AB021492) Sphingopyxis terrae IFO 15098T (D13727)
100
Novosphingobium capsulatum IFO 12533T (D16147) Novosphingobium rosa IAM 14222T (D13945) Zymomonas mobilis ATCC 10988T (AF281031)
Fig. 1. Neighbour-joining phylogenetic tree based on 16S rRNA gene sequences showing the relationships between strain 1007T and the type strains of related bacteria. Bootstrap values (expressed as percentages of 1000 replications) over 50 % are shown at branch points. Zymomonas mobilis ATCC 10988T was used as an outgroup. Bar, 0.01 substitutions per nucleotide position. http://ijs.sgmjournals.org
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J. Huang and others
Table 1. Differential phenotypic and physiological characteristics of strain 1007T and closely related type strains of the genus Sphingomonas Strains: 1, 1007T; 2, S. pruni IFO 15498T (DSM 10566T) (Takeuchi et al., 1995); 3, S. mali IFO 15500T (DSM 10565T) (Takeuchi et al., 1995); 4, S. japonica KC7T (DSM 22753T) (Romanenko et al., 2009); 5, S. koreensis JSS26T (DSM 15582T) (Lee et al., 2001). All data are from this study except those for the DNA G+C content of the reference strains, taken from the corresponding public documents. All strains are positive for arginine dihydrolase, urease, aesculin hydrolysis, assimilation of D-glucose, maltose, cellobiose and lactose and activity of alkaline phosphatase, acid phosphatase, esterase (C4), esterase lipase (C8), leucine arylamidase, cystine arylamidase, naphthol-AS-BI-phosphohydrolase and a-glucosidase. All strains are negative for Gram-staining, methyl red and Voges Proskauer tests, nitrate reduction, indole production, fermentation from glucose, utilization of D-mannitol, capric acid, trisodium citrate, phenylacetic acid, glycerol, erythritol, D-arabinose, D-ribose, L-xylose, D-adonitol, L-sorbose, dulcitol, inositol, D-sorbitol, methyl a-D-mannopyranoside, inulin, glycogen, xylitol, D-lyxose, D-tagatose, D- and L-arabitol, potassium 2-ketogluconate and potassium 5-ketogluconate, and activity of a-galactosidase, a-mannosidase and a-fucosidase. All strains are positive for H2S production, catalase and oxidase. PY, pale yellow; Y, yellow; YO, yellow–orange. +, Positive reaction; –, negative reaction; W, weakly positive reaction; NR, not reported. Characteristic
1
2
3
4
5
Colony colour Hydrolysis of gelatin 3-Ketolactose production Oxidase Utilization of: D-Xylose Methyl b-Dxylopyranoside D-Galactose D-Fructose L-Rhamnose Methyl a-Dglucopyranoside N-Acetylglucosamine Amygdalin Arbutin Salicin Melibiose Sucrose Trehalose Melezitose Raffinose Starch Gentiobiose Turanose D-Fucose L-Fucose Assimilation of (API 20 NE): L-Arabinose D-Mannose
Y – + W
PY – + +
PY – + +
YO + + +
Y – – +
+ –
– –
– –
+ +
+ –
+ – + +
– –
– – + –
+ – – –
– + +
–
+ –
– – – – – – + – – – + – + +
– – – – + – – – – + – – + –
– – – + + – + – + – + + – –
+ +
– +
– +
– +
– –
1032
+ + + + – + + + + – + – W
W
– + W W W
– – + – – + + W
W
Table 1. cont. Characteristic
1
2
3
4
5
N-Acetylglucosamine Potassium gluconate Adipic acid Malic acid API ZYM tests Lipase Valine arylamidase Trypsin a-Chymotrypsin b-Galactosidase b-Glucuronidase b-Glucosidase N-Acetyl-bglucosaminidase DNA G+C content (mol%)
+ – – +
+
– – – –
– – +
+ – – +
W
W
+ – – +
– + – – – – – –
– – +
W
+ – – + – + +
65
66
NR
66
2 + + W
– + + + 67
W W W
W W
W
W
– – + +
For the measurement of DNA G+C content of strain 1007T, genomic DNA was extracted, purified and enzymically degraded into nucleosides. The DNA G+C content was then determined as described by Mesbah et al. (1989), using a reversed-phase HPLC (Agilent 1100, Agilent Technologies, Palo Alto, CA). The DNA G+C content was calculated using Escherichia coli K-12 as the reference strain. For polar lipid and fatty acid analysis, strain 1007T was cultivated on NA at 30 uC for 3 days and lipids were extracted using the chloroform/methanol extraction method (Minnikin et al., 1984). Polar lipids were analysed as described by Denner et al. (2001). Fatty acid methyl esters were obtained by acid methanolysis as described by Rowe et al. (2000) and identified by using the Microbial Identification software package (Sasser , 1990). Respiratory quinones were analysed according to the method of Komagata & Suzuki (1987). Polyamines were extracted and analysed according to the protocol of Tala` et al. (2013). Cells of strain 1007T were Gram-stain-negative, aerobic, yellow-pigmented, motile rods with single polar flagella (Fig. S3). Strain 1007T grew strongly on many media such as NA, R2A agar, TSA and LB agar. Colonies of strain 1007T grown on NA were circular, convex, opaque and yellow and became sticky after 48 h of incubation at 30 uC. On NA medium, strain 1007T was able to grow at 15–30 uC but not at 4 uC or 42 uC, and grew weakly at 37 uC. The pH range for growth was pH 5.0–10.0 (optimum, pH 6.0–7.0). Optimal growth occurred in the absence of NaCl; growth was inhibited by the presence of .2 % (w/v) NaCl. Strain 1007T was positive for H2S production, oxidase, catalase and Tween 80 hydrolysis, but negative for indole production, nitrate reduction, gelatin hydrolysis, oxidation/fermentation test and gas/acid production test with glucose. Other physiological, biochemical and chemotaxonomic characteristics that differentiated strain 1007T from the closest phylogenetic relatives are listed in Table 1. The major respiratory quinone of strain 1007T was ubiquinone Q-10 (see Fig. S4). The polar lipid profile of International Journal of Systematic and Evolutionary Microbiology 64
Sphingomonas yantingensis sp. nov.
strain 1007T contained phosphatidylglycerol, phosphatidylethanolamine, glycosylsphingolipid, phosphatidylcholine, sphingolipid, two unidentified phospholipids and three unidentified lipids (Fig. S5). Major fatty acids were summed feature 8 (comprising C18 : 1v7c or C18 : 1v6c) (60.4 %) and C16 : 0 (15.8 %). C14 : 0 2-OH (3.9 %) was the major 2-hydroxy fatty acid (Table 2) and 3-hydroxy fatty acid was absent. The presence of 2-hydroxy fatty acid and the lack of 3-hydroxy fatty acid are important characteristics of members of the family Sphingomonadaceae (Busse et al., 2003; Takeuchi et al., 2001; Takeuchi & Hiraishi, 2001). The fatty acid profile of strain 1007T was in agreement with those of close representatives of the genus and consistent with the assignment of strain 1007T to the genus Sphingomonas. The DNA G+C content of strain 1007T was 67 mol%. The major polyamine of strain 1007T was homospermidine, which was in accordance with the members of the genus Sphingomonas sensu stricto (Takeuchi et al., 2001; Maruyama et al., 2006). Despite the similarities, DNA–DNA hybridization studies showed low relatedness between strain 1007T and S. pruni IFO 15498T (36.0 %), S. mali IFO 15500T (51.0 %), S. japonica KC7T (58.7 %) and S. koreensis JSS26T (26.1 %). On the basis of phenotypic, chemotaxonomic and phylogenetic data and the results of DNA–DNA hybridizations, strain 1007T is proposed to be assigned to the genus Sphingomonas as a novel species, for which the name Sphingomonas yantingensis sp. nov. is proposed. Description of Sphingomonas yantingensis sp. nov. Sphingomonas yantingensis (yan.ting.en9sis. N.L. fem. adj. yantingensis pertaining to Yanting County in China, where the type strain was isolated). Cells are Gram-negative-staining, strictly aerobic, yellowpigmented, motile short rods with single polar flagella and do not form spores. Cells are 0.5–0.8 mm in width and 1.0– 1.6 mm in length. On NA, forms yellow, circular, dry colonies (0.8–1.2 mm in diameter) within 48 h. Grows at 15– 37 uC (optimum, 28–30 uC) and pH 5.0–10.0 (optimum, pH 6.0–7.0). Growth is inhibited in the presence of .2 % (w/ v) NaCl. Does not reduce nitrate to nitrite or nitrite to N2. Positive for H2S production, Tween 80 hydrolysis, aesculin hydrolysis, catalase and oxidase. Negative for methyl red test, Voges–Proskauer test, indole production and hydrolysis of gelatin. Positive for arginine dihydrolase, urease, valine arylamidase, trypsin, a-chymotrypsin (weakly), b-glucuronidase, a- and b-glucosidase, N-acetyl-b-glucosaminidase, alkaline- and acid- phosphatase, esterase (C4), esterase lipase (C8), leucine arylamidase, cystine arylamidase and naphtholAS-BI-phosphohydrolase activities. Negative for a-galactosidase, a-mannosidase, a-fucosidase, lipase and b-galactosidase activities. D-Glucose, maltose, cellobiose, lactose, D-xylose, D-galactose, L-rhamnose, methyl a-D-glucopyranoside, Nacetylglucosamine, amygdalin, arbutin, salicin, sucrose, trehalose, melezitose, raffinose, gentiobiose, D-fucose (weakly), http://ijs.sgmjournals.org
Table 2. Cellular fatty acid profiles of strain 1007T and type strains of related species of the genus Sphingomonas Strains: 1, 1007T; 2, S. pruni IFO 15498T (DSM 10566T); 3, S. mali IFO 15500T (DSM 10565T); 4, S. japonica KC7T (DSM 22753T); 5, S. koreensis JSS26T (DSM 15582T). All data were generated in this study. Values shown are percentages of the total fatty acids. –, Not detected; TR, trace amount (,0.1 %). The major cellular fatty acids of the five strains tested were C16 : 0 and summed feature 8, and the major hydroxyl fatty acid was C14 : 0 2-OH. 3-Hydroxy fatty acid was not detected. Fatty acid C12 : 0 C14 : 0 C16 : 0 C17 : 0 C18 : 0 C12 : 0 2-OH C13 : 0 2-OH C14 : 0 2-OH C15 : 0 2-OH anteiso-C15 : 0 iso-C15 : 0 C16 : 0 2-OH iso-C16 : 0 C16 : 1v5c anteiso-C17 : 0 C17 : 1v6c C17 : 1v8c C18 : 1v5c C18 : 1v9c C19 : 0 cyclo v8c 11-Methyl C18 : 1v7c 10-Methyl C19 : 0 Summed feature 2 Summed feature 3 Summed feature 7 Summed feature 8
1
2
3
4
5
– 0.4 15.8 0.2 0.7 – – 3.9 – – – – – 2.2 – 1.3 0.3 1.4 1.2 – 3.8 – – 8.2 – 60.4
– 0.3 11.1 2.8 1.1 –
0.2 0.4 12.6 0.4 1.2
– 0.9 12.4 0.1 0.1 – – 5.0 0.1 – – – – 1.8 – 2.4 – 0.8 0.1 – 3.0 – 0.2 29.4 – 43.4
– 0.5 13.2 – 2.0 – – 5.5
TR
4.3 3.1 0.6 0.6 0.6 TR
0.2 0.3 16.7 3.4 2.4 1.1 – 5.8 – – 0.6 0.7 44.2
TR
– 6.7 0.5 0.1 0.2 0.4 – 0.3 0.1 3.0 0.7 1.6 1.4 – 5.8 TR
– 1.8 – 62.2
TR
– – 0.7 – 1.3 – 0.9 – 2.5 1.2 0.4 10.0 – – 2.6 – 59.2
*Summed feature 2, unknown fatty acids; summed feature 3 contains C16 : 1v6c and/or C16 : 1v7c; summed feature 7 contains C19 : 1v6c and/ or C19 : 0 cyclov8c; summed feature 8 contains C18 : 1v7c and/or C18 : 1v6c.
L-arabinose, D-mannose
and malic acid are utilized as sole carbon and energy sources, but D-mannitol, capric acid, trisodium citrate, phenylacetic acid, glycerol, erythritol, Darabinose, D-ribose, L-xylose, D-adonitol, L-sorbose, dulcitol, inositol, D-sorbitol, methyl a-D-mannopyranoside, inulin, glycogen, xylitol, D-lyxose, D-tagatose, D- and L-arabitol, potassium gluconate, potassium 2-ketogluconate, potassium 5-ketogluconate, methyl b-D-xylopyranoside, D-fructose, melibiose, starch, turanose, L-fucose and adipic acid are not. Ubiquinone Q-10 is the predominant respiratory quinone. The major polyamine is homospermidine. The major fatty acids are summed feature 8 (comprising C18 : 1v7c and/or C18 : 1v6c) and C16 : 0. C14 : 0-2-OH is the major 2-hydroxyl 1033
J. Huang and others
fatty acid. The polar lipid profile comprises phosphatidylglycerol, phosphatidylethanolamine, glycosylsphingolipid, phosphatidylcholine, sphingolipid, two unidentified phospholipids and three unidentified lipids. The type strain is 1007T (5DSM 27244T5JCM 19201T5 CCTCC AB 2013146T), which was isolated from a purplish soil sample collected in Yanting (Sichuan, China). The DNA G+C content of the type strain is (67±0.7) mol%.
Acknowledgements This research was supported by National Science Foundation of China (project no. 41071173).
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