International Journal of Systematic and Evolutionary Microbiology (2014), 64, 4109–4114
DOI 10.1099/ijs.0.063610-0
Nocardioides daecheongensis sp. nov., isolated from soil Jun-Muk Lim,1,2 Soo-Jin Kim,1 Moriyuki Hamada,3 Jae-Hyung Ahn,1 Hang-Yeon Weon,1 Ken-ichiro Suzuki,3 Tae-Young Ahn2 and Soon-Wo Kwon1 Correspondence Soon-Wo Kwon [email protected]
1
Agricultural Microbiology Division, National Academy of Agricultural Science, Rural Development Administration, Suwon, Republic of Korea
2
Department of Microbiology, Dankook University, Anseo-dong, Cheonan 330-714, Republic of Korea
3
NITE Biological Resource Center (NBRC), National Institute of Technology and Evaluation, 2-5-8 Kazusakamatari, Kisarazu, Chiba 292-0818, Japan
Strain KIS2-16T was isolated from a soil sample collected from Daecheong Island of Incheon region, South Korea. KIS2-16T was Gram-staining-positive, aerobic, non-spore-forming, nonmotile, catalase-positive, oxidase-negative and mesophilic. On the basis of 16S rRNA gene sequence analysis, strain KIS2-16T represented a member of the genus Nocardioides, being most closely related to the type strains of species of the genus Nocardioides, Nocardioides maradonensis RP-B30T (97.8 % sequence similarity) and Nocardioides ultimimeridianus RPB26T (97.0 %). The fatty acid profile of KIS2-16T was dominated by C18 : 1v9c, C17 : 1v8c, C16 : 0, C18 : 0 10-methyl (TBSA), C16 : 0 2-OH and C17 : 0 2-OH. The major isoprenoid quinone was MK8(H4), and the major polar lipids were diphosphatidylglycerol, phosphatidylglycerol and phosphatidylinositol. The peptidoglycan structure was A3c-type with LL-diaminopimelic acid. The genomic DNA G+C content of KIS2-16T was 64.9 mol%. Strain KIS2-16T showed DNA–DNA hybridization values of less than 70 % with the closely related species of the genus Nocardioides. Based on phenotypic, genotypic and phylogenetic data, the isolate represents a novel species of the genus Nocardioides, for which the name Nocardioides daecheongensis sp. nov. (type strain KIS2-16T5DSM 27136T5KACC 17297T5NBRC 109597T) is proposed.
The genus Nocardioides was proposed by Prauser (1976) with a single species. Since then, many additional species have been classified as members of the genus Nocardioides, and at the time of writing 65 species were included in the List of Prokaryotic Names with Standing in Nomenclature (http://www.bacterio.net/). Members of the genus contain LL-2,6-diaminopimelic acid in the cell-wall peptidoglycan and MK-8(H4) as the major menaquinone (Prauser, 1976; O’Donnell et al., 1982; Urzı` et al., 2000). The major fatty acids of the species of the genus Nocardioides are diverse, with many species containing iso-C16 : 0, C17 : 1v8c or C18 : 1v9c and some species having C16 : 0, C17 : 0, C15 : 0, iso-C15 : 0 or iso-C17 : 0 (Cui et al., 2009; Kim et al., 2009; Lee et al., 2011; Liu et al., 2013; Yamamura et al., 2011; Zhang et al., 2009, 2012). Members of the genus have been isolated from diverse environments such as soil, sand, tidal flat The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain KIS2-16T is KJ135310. Two supplementary figures are available with the online Supplementary Material.
063610 G 2014 IUMS
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sediment, fresh water, seawater, wastewater, walls, cryoconite, plants, sludge, plankton, lichen, glaciers and oil shale. Strain KIS2-16T was isolated from a forest soil sample collected from Daecheong Island of Incheon region, South Korea. The serial dilution technique using R2A (BD) agar at the incubation temperature of 28 uC for a week was used for the isolation. The isolate was routinely cultivated on R2A at 28 uC and stored as aqueous glycerol suspensions (20 %, v/v) at 270 uC. Biochemical and physiological tests were performed at 28 uC for KIS2-16T and the reference strains Nocardioides maradonensis KACC 17350T and Nocardioides ultimimeridianus KACC 17349T. The Gram reaction was determined with a Difco Gram staining kit. Cell morphology was observed using transmission electron microscopy (model 912AB; LEO) after incubation at 28 uC for 48 h in R2A agar. Catalase activity was determined using bubble production in 3 % (v/v) H2O2 solution, and oxidase activity was tested using 1 % (w/v) N,N,N9,N9-tetramethyl p-phenylenediamine reagent. Growth under anaerobic 4109
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conditions was determined after incubation in the BBL GasPak Anaerobic System (Difco) for 14 days at 28 uC. Biochemical tests were carried out using API 20NE, API ID 32GN and API ZYM test kits according to the protocols of the manufacturer (bioMe´rieux). Casein, CM-cellulose, hypoxanthine, starch, Tween 80, tyrosine and xanthine hydrolyses were examined on R2A plates containing milk powder [5 % (w/v)], CM-cellulose [1 % (w/v)], hypoxanthine [0.5 % (w/v)], starch [1 % (w/v)], Tween 80 [1 % (w/v)], tyrosine [0.1 % (w/v)] and xanthine [0.5 % (w/v)], respectively. DNase activity was determined with DNase test agar (Difco). Growth at 4, 10, 15, 18, 20, 25, 28, 30, 35, 37 and 40 uC and at pH 3.0–12.0 (at intervals of 1.0 pH unit) was assessed after 5 days of incubation in R2A broth. Tolerance to NaCl concentrations was tested on R2A broth from 0 % additional NaCl to 5 % additional NaCl (w/v) in R2A broth at 1 % intervals. Growth was tested on the following media (all from BD) at 28 uC: R2A, ISP 2, nutrient agar (NA) and trypticase soy agar (TSA).
KIS2-16T were grown in shake flasks containing liquid ISP 2 medium on a rotary shaker for 4 days at 30 uC. Amino acids and the isomers in cell-wall hydrolysates were analysed as described by Hamada et al. (2012). The DNA G+C content was determined by the fluorometric method (Gonzalez & Saiz-Jimenez, 2002) using SYBR Green 1 and a real-time PCR thermocycler (Bio-Rad). Genomic DNA samples from Bacillus amyloliquefaciens subsp. plantarum DSM 23117T, Pseudomonas stutzeri ATCC 17588T and Micrococcus luteus ATCC 4698T were used as calibration references.
Chromosomal DNA was isolated with a Wizard Genomic DNA Purification kit (Promega). The gene encoding the 16S rRNA was amplified by PCR with the bacterial 16S rRNA gene-specific primers 9F and 1512R (Weisburg et al., 1991). The 16S rRNA gene sequence was determined at Genotec (Daejeon, South Korea) using sequencing primers 27F, 1492R (Weisburg et al., 1991), 518R (59-GTATTACCGCGGCTGCTGG-39) and 785F (59-GGATTAGATACCCTGGTA-39). The sequence determined for strain KIS2-16T was 1450 bp in length. The 16S rRNA gene sequence of strain KIS2-16T was compared with those retrieved from EzTaxon (Kim et al., 2012) and the GenBank database (http://blast.ncbi.nlm.nih.gov/Blast.cgi.). 16S rRNA gene sequence similarities were calculated using the EzTaxon server. Alignment of the sequence data were performed with the SILVA Incremental Aligner (Pruesse et al., 2012). Phylogenetic trees were reconstructed using MEGA version 5.0 (Tamura et al., 2011) on the basis of the neighbourjoining (Saitou & Nei, 1987), maximum-parsimony (Kluge & Farris 1969) and maximum-likelihood (Felsenstein, 1981) algorithms. In each case bootstrap values were calculated based on 1000 replications.
Cells of strain KIS2-16T were Gram-staining-positive, aerobic, curved or straight rod-shaped, non-spore-forming, nonflagellated, catalase-positive and oxidase-negative, which was similar to the phenotypes of two closely related species of the genus Nocardioides (Table 1) (Fig. S1 available in the online Supplementary Material). However, strain KIS2-16T could also be differentiated from these two closely related species of the genus Nocardioides on the basis of various physiological and biochemical properties such as cell morphology, temperature and NaCl ranges for growth, nitrate reduction, hydrolyses of several substances, assimilation of various substances and enzymic activities (Table 1).
For cellular fatty acid analysis, strain KIS2-16T and reference strains were grown on R2A at 28 uC for 2– 4 days. Fatty acid extraction was performed using cells in the late exponential phase of growth (Sasser, 1990). The fatty acids were analysed by GC (6890; Hewlett Packard) according to the standard protocol of the Sherlock Microbial Identification System (Sherlock version 6.10, database TSBA 6; MIDI). Menaquinones and phospholipids were extracted and purified according to the protocol of Minnikin et al. (1984). Polar lipids were examined using two-dimensional TLC. The phospholipid pattern was determined as described by Collins et al. (1980) using molybdophosphoric acid (for detection of all lipids), ninhydrin (lipids containing free amino groups), Zinzadze (phosphorus-containing lipids) and a-naphthol reagents (glycolipids). For peptidoglycan analysis, cells of strain 4110
DNA–DNA hybridization was carried out according to the method described by Seldin & Dubnau (1985). Probe labelling was conducted using the nonradioactive DIG High Prime DNA Labelling and Detection Starter Kit II (Roche Molecular Biochemicals). Each of strain KIS2-16T and Nocardioides maradonensis KACC 17350T was labelled. The hybridized DNA was visualized using the DIG luminescent detection kit (Roche Molecular Biochemicals). DNA–DNA relatedness was quantified with a densitometer (Bio-Rad).
According to results of the 16S rRNA gene sequence analysis, strain KIS2-16T showed the highest pairwise sequence similarity with N. maradonensis RP-B30T (97.8 %) and N. ultimimeridianus RP-B26T (97.0 %), revealing low sequence similarity values below 96.6 % with the other species of the genus Nocardioides. The neighbour-joining tree (Fig. 1) indicated that strain KIS2-16T was a representative of a member of the genus Nocardioides and formed a subcluster with N. maradonensis RP-B30T and N. ultimimeridianus RPB26T with 86 % bootstrap value. The clustering of strain KIS2-16T in the neighbour-joining tree was also observed in the maximum-parsimony and maximum-likelihood trees (Data not shown). The cellular fatty acid profiles of strain KIS2-16T and closely related members of the genus Nocardioides are shown in Table 2. The major fatty acids of strain KIS2-16T were C18 : 1v9c, C17 : 1v8c, C16 : 0, C18 : 0 10-methyl (TBSA), C16 : 0 2-OH and C17 : 0 2-OH. The fatty acid profile of strain KIS2-16T was similar to those of N. maradonensis KACC 17350T and N. ultimimeridianus KACC 17349T with relatively large amounts of C16 : 0, C16 : 0 2-OH, C17 : 0, C17 : 0 2-OH, C17 : 1v8c and C18 : 1v9c although the minor fatty acids were variable among those strains. The major isoprenoid quinone of strain KIS2-16T was MK-8(H4) in agreement with previous results for the other members of International Journal of Systematic and Evolutionary Microbiology 64
Nocardioides daecheongensis sp. nov.
Table 1. Phenotypic comparisons among strain KIS2-16T and the closely related members of the genus Nocardioides Strain: 1, KIS2-16T; 2, N. maradonensis KACC 17350T; 3, N. ultimimeridianus KACC 17349T. All strains are non-motile, catalase-positive and oxidase-negative. All strains are positive for aesculin hydrolysis and assimilation of D-glucose, malic acid, sodium acetate, L-alanine, L-serine, propionic acid, valeric acid and 3-hydroxybutyric acid, but negative for indole production, glucose fermentation, arginine dihydrolase, urease and assimilation of D-mannose, D-mannitol, N-acetylglucosamine, capric acid, adipic acid, phenylacetic acid, D-ribose, inositol, itaconic acid, sodium malonate, lactic acid, potassium 5-ketogluconate, melibiose, L-fucose, D-sorbitol and potassium 2-ketogluconate. All strains showed positive activities for esterase (C4), esterase lipase (C8), leucine arylamidase, acid phosphatase and naphthol-AS-BI-phosphohydrolase, but were negative for alkaline phosphatase, valine arylamidase, cystine arylamidase, trypsin, a-chymotrypsin, a-galactosidase, b-galactosidase, b-glucuronidase, b-glucosidase, N-acetyl-b-glucosaminidase, a-mannosidase and a-fucosidase. +, Positive; W, weakly positive; 2, negative. Characteristic Isolation source Cell morphology Cell size (mm) Colony colour Temperature range for growth (uC) NaCl range for growth (%) Nitrate reduction Hydrolysis of: Casein Gelatin Hypoxanthine Tyrosine Assimilation of: L-Arabinose Maltose Potassium gluconate Trisodium citrate L-Rhamnose Sucrose Suberic acid Glycogen 3-Hydroxybenzoic acid Salicin L-Histidine 4-Hydroxybenzoic acid L-Proline Enzymic activity of: Lipase (C14) a-Glucosidase DNA G+C content (mol%)
1
2
3
Soil Rods 0.5–0.761.2–2.2 White 10–35 0–3 +
Rhizosphere soil* Short rods* 0.6–1.061.1–1.5* Light yellow* 20–37* 0–1 2
Rhizosphere soil* Short rods* 0.6–1.061.0–1.6* Moderately yellow* 10–42* 0–1 2
+ 2 2 +
2* 2 +* 2
+* + 2* 2
+ 2 2 + + 2 2
2 + + 2 2 + + 2 2 + 2 W
2 + + 2 2 + + + 2 2 + 2 +
2 + 71.7*
2 + 73.0*
W
+ 2 + + 2 + 2 65
W
*Data from Lee et al. (2011).
the genus Nocardioides (Busse & Schumann 1999). Strain KIS2-16T contained diphosphatidylglycerol, phosphatidylglycerol and phosphatidylinositol as the major polar lipids, which have also been observed in other species of the genus Nocardioides such as Nocardioides perillae, Nocardioides iriomotensis and Nocardioides hwasunensis (Fig. S2; Du et al., 2013; Lee et al., 2008; Yamamura et al., 2011). However, this polar lipid pattern was different from those of the two close relatives of strain KIS2-16T, N. ultimimeridianus and N. maradonensis, both of which had phosphatidylcholine and phosphatidylethanolamine as additional major polar lipids (Lee et al., 2011). The peptidoglycan structure of strain http://ijs.sgmjournals.org
KIS2-16T was A3c-type with LL-diaminopimelic acid, which is common in the species of the genus Nocardioides. The genomic DNA G+C content of strain KIS2-16T was 64.9 mol%. According to the results of DNA–DNA hybridization, strain KIS2-16T showed less than 70 % hybridization with N. maradonensis KACC 17350T (37±4 %; reciprocal 31±4 %) and N. ultimimeridianus KACC 17349T (32±3 %). In conclusion, based on the phenotypic, chemotaxonomic, phylogenetic and DNA–DNA hybridization data presented, strain KIS2-16T would appear to represent a novel species 4111
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Nocardioides simplex KCTC 9106T (AF005009) Nocardioides aromaticivorans H-1T (AB087721) Nocardioides kongjuensis A2-4T (DQ218275) Nocardioides caeni MN8T (FJ423551) 58 Nocardioides humi DCY24T (EF623863) Nocardioides daecheongensis KIS2-16T (KJ135310) 77 Nocardioides ultimimeridianus RP-B26T (FM997998) 86 91 Nocardioides maradonensis RP-B30T (FM998000) Nocardioides panacisoli GSoil 346T (FJ666101) 100 Nocardioides oleivorans DSM 16090T (AJ698724) Nocardioides furvisabuli SBS-26T (DQ411542) Nocardioides albertanoniae CD40127T (HE801966) Nocardioides aestuarii JC2056T (AY423719) Nocardioides marinus CL-DD14T (DQ401093) Nocardioides terrae VA15T (FJ423762) 59 Nocardioides panacihumi Gsoil 616T (AB271053) 100 Nocardioides salarius CL-Z59T (DQ401092) Nocardioides basaltis J112T (EU143365) Nocardioides marinquilinus CL-GY44T (JX164255) 98 Nocardioides ginsengagri BX5-10T (GQ339904) Nocardioides fonticola NAA-13T (EF626689) Nocardioides dilutus MSL-11T (EF466121) Nocardioides pyridinolyticus OS4T (U61298) 66 82 Nocardioides hankookensis DS-30T (EF555584) Nocardioides perillae I10A-01402T (JN869461) Nocardioides daphniae D287T (AM398438) Terrabacter tumescens KCTC 9133T (AF005023) 66 100
0.01
57
60
Fig. 1. Phylogenetic analysis of strain KIS2-16T and type strains of other related species of the genus Nocardioides based on 16S rRNA gene sequences. Distances and clustering were performed using the neighbour-joining method with the software package MEGA version 5.0. Bootstrap values (.50 %) based on 1000 replications are listed as percentages at the branching points. Filled circles indicate that the corresponding nodes were also recovered in trees generated with the maximum-likelihood and maximum-parsimony algorithms. Bar, 0.01 substitutions per nucleotide position.
of the genus Nocardioides, for which the name Nocardioides daecheongensis sp. nov. is proposed.
Cells are Gram-staining-positive, aerobic, non-spore-forming, non-flagellated and curved or straight rods (0.5– 0.761.2–2.2 mm). Catalase-positive and oxidase-negative. Colonies are whitish, irregular and flat on R2A medium. Growth occurs at 10–35 uC (optimum, 28–30 uC) and at pH 4.0–10.0 (optimum, 7.0). Growth occurs in the presence of 0–3.0 % (w/v) added NaCl. Hydrolyses casein, tyrosine and Tween 80, but does not hydrolyse chitin, CMcellulose, DNA, hypoxanthine, starch or xanthine. Positive activities for nitrate reduction and aesculin hydrolysis, but negative for indole production, glucose fermentation, arginine dihydrolase, urease and gelatin hydrolysis (API 20NE test strips). Assimilates D-glucose, L-arabinose, malic
acid, trisodium citrate, L-rhamnose, sodium acetate, Lalanine, 3-hydroxybenzoic acid, L-serine, propionic acid, valeric acid, L-histidine, 3-hydroxybutyric acid and 4hydroxybenzoic acid, weakly assimilates glycogen, but does not assimilate D-mannose, D-mannitol, N-acetylglucosamine, maltose, potassium gluconate, capric acid, adipic acid, phenylacetic acid, D-ribose, inositol, sucrose, itaconic acid, suberic acid, sodium malonate, lactic acid, potassium 5-ketogluconate, salicin, melibiose, L-fucose, D-sorbitol, potassium 2-ketogluconate or L-proline (API 20NE and API ID 32GN test strips). Positive activities for esterase (C4), esterase lipase (C8), lipase (C14), leucine arylamidase, acid phosphatase and naphthol-AS-BI-phosphohydrolase, but negative for alkaline phosphatase, valine arylamidase, cystine arylamidase, trypsin, a-chymotrypsin, a-galactosidase, b-galactosidase, b-glucuronidase, a-glucosidase, b-glucosidase, N-acetyl-b-glucosaminidase, a-mannosidase and a-fucosidase (API ZYM test strip). The major fatty acids (.5 %) are C18 : 1v9c, C17 : 1v8c, C16 : 0, C18 : 0 10-methyl (TBSA), C16 : 0 2-OH and C17 : 0 2-OH. The predominant menaquinone is MK-8(H4). The main polar lipids consist of diphosphatidylglycerol, phosphatidylglycerol
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Description of Nocardioides daecheongensis sp. nov. Nocardioides daecheongensis (dae.che.ong.en9sis, N.L. masc. adj. daecheongensis referring to the Daecheong Island, from where the type strain was isolated).
Nocardioides daecheongensis sp. nov.
Table 2. Fatty acids compositions of strain KIS2-16T and type strains of the closely related species of the genus Nocardioides Strain: 1, KIS2-16T; 2, N. maradonensis KACC 17350T; 3, N. ultimimeridianus KACC 17349T. –, Not detected or ,0.5 % of total fatty acids.
Cui, Y. S., Lee, S.-T. & Im, W.-T. (2009). Nocardioides ginsengisoli sp.
nov., isolated from soil of a ginseng field. Int J Syst Evol Microbiol 59, 3045–3050. Du, H.-J., Wei, Y.-Z., Su, J., Liu, H.-Y., Ma, B.-P., Guo, B.-L., Zhang, Y.-Q. & Yu, L.-Y. (2013). Nocardioides perillae sp. nov., isolated from
surface-sterilized roots of Perilla frutescens. Int J Syst Evol Microbiol 63, 1068–1072. Felsenstein, J. (1981). Evolutionary trees from DNA sequences: a
Fatty acid C14 : 0 C15 : 0 2-OH C15 : 1v6c C16 : 0 C16 : 0 2-OH C16 : 1v9c iso-C16 : 0 C17 : 0 C17 : 0 2-OH C17 : 0 10-methyl C17 : 1v6c C17 : 1v8c C18 : 0 C18 : 0 10-methyl (TBSA) C18 : 1v9c iso-C18 : 0 C19 : 0 cyclo v8c Summed feature* 3 6
1
2
3
– 1.0 – 9.2 7.4 1.0 1.5 8.5 6.7 1.3 1.3 9.8 2.5 7.8 31.8 2.6 0.5
0.7 1.2 0.5 8.2 7.5 – 3.9 6.0 5.0 4.9 2.9 17.9 0.7 4.6 19.7 5.9 2.0
0.9 0.9 – 10.3 5.1 1.9 – 10.3 5.7 – – 14.8 2.7 1.5 35.6 – –
2.9 1.7
5.0 1.2
2.4 4.4
*Summed features are groups of two or three fatty acids that cannot be separated by GLC with the MIDI system: summed feature 3, C16 : 1v6c and/or C16 : 1v7c; summed feature 6, C19 : 1v9c and/or C19 : 1v11c.
and phosphatidylinositol. The peptidoglycan contains LLdiaminopimelic acid in the cell wall and has A3c-type peptidoglycan. The type strain, KIS2-16T (5DSM 27136T5KACC 17297T5 NBRC 109597T), was isolated from a soil sample collected from Daecheong Island of Incheon region, South Korea. The DNA G+C content of the type strain is 64.9 mol%.
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terium isolated from the rhizosphere of a mangrove. Int J Syst Evol Microbiol 62, 1731–1735. Kim, M. K., Srinivasan, S., Park, M.-J., Sathiyaraj, G., Kim, Y.-J. & Yang, D.-C. (2009). Nocardioides humi sp. nov., a b-glucosidase-
producing bacterium isolated from soil of a ginseng field. Int J Syst Evol Microbiol 59, 2724–2728. Kim, O. S., Cho, Y. J., Lee, K., Yoon, S. H., Kim, M., Na, H., Park, S. C., Jeon, Y. S., Lee, J. H. & other authors (2012). Introducing EzTaxon-e:
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Acknowledgements This study was carried out with the support of the National Academy of Agricultural Science, Rural Development Administration, Republic of Korea (project no. PJ008666).
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DOI 10.1099/ijs.0.063610-0
Nocardioides daecheongensis sp. nov., isolated from soil Jun-Muk Lim,1,2 Soo-Jin Kim,1 Moriyuki Hamada,3 Jae-Hyung Ahn,1 Hang-Yeon Weon,1 Ken-ichiro Suzuki,3 Tae-Young Ahn2 and Soon-Wo Kwon1 Correspondence Soon-Wo Kwon [email protected]
1
Agricultural Microbiology Division, National Academy of Agricultural Science, Rural Development Administration, Suwon, Republic of Korea
2
Department of Microbiology, Dankook University, Anseo-dong, Cheonan 330-714, Republic of Korea
3
NITE Biological Resource Center (NBRC), National Institute of Technology and Evaluation, 2-5-8 Kazusakamatari, Kisarazu, Chiba 292-0818, Japan
Strain KIS2-16T was isolated from a soil sample collected from Daecheong Island of Incheon region, South Korea. KIS2-16T was Gram-staining-positive, aerobic, non-spore-forming, nonmotile, catalase-positive, oxidase-negative and mesophilic. On the basis of 16S rRNA gene sequence analysis, strain KIS2-16T represented a member of the genus Nocardioides, being most closely related to the type strains of species of the genus Nocardioides, Nocardioides maradonensis RP-B30T (97.8 % sequence similarity) and Nocardioides ultimimeridianus RPB26T (97.0 %). The fatty acid profile of KIS2-16T was dominated by C18 : 1v9c, C17 : 1v8c, C16 : 0, C18 : 0 10-methyl (TBSA), C16 : 0 2-OH and C17 : 0 2-OH. The major isoprenoid quinone was MK8(H4), and the major polar lipids were diphosphatidylglycerol, phosphatidylglycerol and phosphatidylinositol. The peptidoglycan structure was A3c-type with LL-diaminopimelic acid. The genomic DNA G+C content of KIS2-16T was 64.9 mol%. Strain KIS2-16T showed DNA–DNA hybridization values of less than 70 % with the closely related species of the genus Nocardioides. Based on phenotypic, genotypic and phylogenetic data, the isolate represents a novel species of the genus Nocardioides, for which the name Nocardioides daecheongensis sp. nov. (type strain KIS2-16T5DSM 27136T5KACC 17297T5NBRC 109597T) is proposed.
The genus Nocardioides was proposed by Prauser (1976) with a single species. Since then, many additional species have been classified as members of the genus Nocardioides, and at the time of writing 65 species were included in the List of Prokaryotic Names with Standing in Nomenclature (http://www.bacterio.net/). Members of the genus contain LL-2,6-diaminopimelic acid in the cell-wall peptidoglycan and MK-8(H4) as the major menaquinone (Prauser, 1976; O’Donnell et al., 1982; Urzı` et al., 2000). The major fatty acids of the species of the genus Nocardioides are diverse, with many species containing iso-C16 : 0, C17 : 1v8c or C18 : 1v9c and some species having C16 : 0, C17 : 0, C15 : 0, iso-C15 : 0 or iso-C17 : 0 (Cui et al., 2009; Kim et al., 2009; Lee et al., 2011; Liu et al., 2013; Yamamura et al., 2011; Zhang et al., 2009, 2012). Members of the genus have been isolated from diverse environments such as soil, sand, tidal flat The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain KIS2-16T is KJ135310. Two supplementary figures are available with the online Supplementary Material.
063610 G 2014 IUMS
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sediment, fresh water, seawater, wastewater, walls, cryoconite, plants, sludge, plankton, lichen, glaciers and oil shale. Strain KIS2-16T was isolated from a forest soil sample collected from Daecheong Island of Incheon region, South Korea. The serial dilution technique using R2A (BD) agar at the incubation temperature of 28 uC for a week was used for the isolation. The isolate was routinely cultivated on R2A at 28 uC and stored as aqueous glycerol suspensions (20 %, v/v) at 270 uC. Biochemical and physiological tests were performed at 28 uC for KIS2-16T and the reference strains Nocardioides maradonensis KACC 17350T and Nocardioides ultimimeridianus KACC 17349T. The Gram reaction was determined with a Difco Gram staining kit. Cell morphology was observed using transmission electron microscopy (model 912AB; LEO) after incubation at 28 uC for 48 h in R2A agar. Catalase activity was determined using bubble production in 3 % (v/v) H2O2 solution, and oxidase activity was tested using 1 % (w/v) N,N,N9,N9-tetramethyl p-phenylenediamine reagent. Growth under anaerobic 4109
J.-M. Lim and others
conditions was determined after incubation in the BBL GasPak Anaerobic System (Difco) for 14 days at 28 uC. Biochemical tests were carried out using API 20NE, API ID 32GN and API ZYM test kits according to the protocols of the manufacturer (bioMe´rieux). Casein, CM-cellulose, hypoxanthine, starch, Tween 80, tyrosine and xanthine hydrolyses were examined on R2A plates containing milk powder [5 % (w/v)], CM-cellulose [1 % (w/v)], hypoxanthine [0.5 % (w/v)], starch [1 % (w/v)], Tween 80 [1 % (w/v)], tyrosine [0.1 % (w/v)] and xanthine [0.5 % (w/v)], respectively. DNase activity was determined with DNase test agar (Difco). Growth at 4, 10, 15, 18, 20, 25, 28, 30, 35, 37 and 40 uC and at pH 3.0–12.0 (at intervals of 1.0 pH unit) was assessed after 5 days of incubation in R2A broth. Tolerance to NaCl concentrations was tested on R2A broth from 0 % additional NaCl to 5 % additional NaCl (w/v) in R2A broth at 1 % intervals. Growth was tested on the following media (all from BD) at 28 uC: R2A, ISP 2, nutrient agar (NA) and trypticase soy agar (TSA).
KIS2-16T were grown in shake flasks containing liquid ISP 2 medium on a rotary shaker for 4 days at 30 uC. Amino acids and the isomers in cell-wall hydrolysates were analysed as described by Hamada et al. (2012). The DNA G+C content was determined by the fluorometric method (Gonzalez & Saiz-Jimenez, 2002) using SYBR Green 1 and a real-time PCR thermocycler (Bio-Rad). Genomic DNA samples from Bacillus amyloliquefaciens subsp. plantarum DSM 23117T, Pseudomonas stutzeri ATCC 17588T and Micrococcus luteus ATCC 4698T were used as calibration references.
Chromosomal DNA was isolated with a Wizard Genomic DNA Purification kit (Promega). The gene encoding the 16S rRNA was amplified by PCR with the bacterial 16S rRNA gene-specific primers 9F and 1512R (Weisburg et al., 1991). The 16S rRNA gene sequence was determined at Genotec (Daejeon, South Korea) using sequencing primers 27F, 1492R (Weisburg et al., 1991), 518R (59-GTATTACCGCGGCTGCTGG-39) and 785F (59-GGATTAGATACCCTGGTA-39). The sequence determined for strain KIS2-16T was 1450 bp in length. The 16S rRNA gene sequence of strain KIS2-16T was compared with those retrieved from EzTaxon (Kim et al., 2012) and the GenBank database (http://blast.ncbi.nlm.nih.gov/Blast.cgi.). 16S rRNA gene sequence similarities were calculated using the EzTaxon server. Alignment of the sequence data were performed with the SILVA Incremental Aligner (Pruesse et al., 2012). Phylogenetic trees were reconstructed using MEGA version 5.0 (Tamura et al., 2011) on the basis of the neighbourjoining (Saitou & Nei, 1987), maximum-parsimony (Kluge & Farris 1969) and maximum-likelihood (Felsenstein, 1981) algorithms. In each case bootstrap values were calculated based on 1000 replications.
Cells of strain KIS2-16T were Gram-staining-positive, aerobic, curved or straight rod-shaped, non-spore-forming, nonflagellated, catalase-positive and oxidase-negative, which was similar to the phenotypes of two closely related species of the genus Nocardioides (Table 1) (Fig. S1 available in the online Supplementary Material). However, strain KIS2-16T could also be differentiated from these two closely related species of the genus Nocardioides on the basis of various physiological and biochemical properties such as cell morphology, temperature and NaCl ranges for growth, nitrate reduction, hydrolyses of several substances, assimilation of various substances and enzymic activities (Table 1).
For cellular fatty acid analysis, strain KIS2-16T and reference strains were grown on R2A at 28 uC for 2– 4 days. Fatty acid extraction was performed using cells in the late exponential phase of growth (Sasser, 1990). The fatty acids were analysed by GC (6890; Hewlett Packard) according to the standard protocol of the Sherlock Microbial Identification System (Sherlock version 6.10, database TSBA 6; MIDI). Menaquinones and phospholipids were extracted and purified according to the protocol of Minnikin et al. (1984). Polar lipids were examined using two-dimensional TLC. The phospholipid pattern was determined as described by Collins et al. (1980) using molybdophosphoric acid (for detection of all lipids), ninhydrin (lipids containing free amino groups), Zinzadze (phosphorus-containing lipids) and a-naphthol reagents (glycolipids). For peptidoglycan analysis, cells of strain 4110
DNA–DNA hybridization was carried out according to the method described by Seldin & Dubnau (1985). Probe labelling was conducted using the nonradioactive DIG High Prime DNA Labelling and Detection Starter Kit II (Roche Molecular Biochemicals). Each of strain KIS2-16T and Nocardioides maradonensis KACC 17350T was labelled. The hybridized DNA was visualized using the DIG luminescent detection kit (Roche Molecular Biochemicals). DNA–DNA relatedness was quantified with a densitometer (Bio-Rad).
According to results of the 16S rRNA gene sequence analysis, strain KIS2-16T showed the highest pairwise sequence similarity with N. maradonensis RP-B30T (97.8 %) and N. ultimimeridianus RP-B26T (97.0 %), revealing low sequence similarity values below 96.6 % with the other species of the genus Nocardioides. The neighbour-joining tree (Fig. 1) indicated that strain KIS2-16T was a representative of a member of the genus Nocardioides and formed a subcluster with N. maradonensis RP-B30T and N. ultimimeridianus RPB26T with 86 % bootstrap value. The clustering of strain KIS2-16T in the neighbour-joining tree was also observed in the maximum-parsimony and maximum-likelihood trees (Data not shown). The cellular fatty acid profiles of strain KIS2-16T and closely related members of the genus Nocardioides are shown in Table 2. The major fatty acids of strain KIS2-16T were C18 : 1v9c, C17 : 1v8c, C16 : 0, C18 : 0 10-methyl (TBSA), C16 : 0 2-OH and C17 : 0 2-OH. The fatty acid profile of strain KIS2-16T was similar to those of N. maradonensis KACC 17350T and N. ultimimeridianus KACC 17349T with relatively large amounts of C16 : 0, C16 : 0 2-OH, C17 : 0, C17 : 0 2-OH, C17 : 1v8c and C18 : 1v9c although the minor fatty acids were variable among those strains. The major isoprenoid quinone of strain KIS2-16T was MK-8(H4) in agreement with previous results for the other members of International Journal of Systematic and Evolutionary Microbiology 64
Nocardioides daecheongensis sp. nov.
Table 1. Phenotypic comparisons among strain KIS2-16T and the closely related members of the genus Nocardioides Strain: 1, KIS2-16T; 2, N. maradonensis KACC 17350T; 3, N. ultimimeridianus KACC 17349T. All strains are non-motile, catalase-positive and oxidase-negative. All strains are positive for aesculin hydrolysis and assimilation of D-glucose, malic acid, sodium acetate, L-alanine, L-serine, propionic acid, valeric acid and 3-hydroxybutyric acid, but negative for indole production, glucose fermentation, arginine dihydrolase, urease and assimilation of D-mannose, D-mannitol, N-acetylglucosamine, capric acid, adipic acid, phenylacetic acid, D-ribose, inositol, itaconic acid, sodium malonate, lactic acid, potassium 5-ketogluconate, melibiose, L-fucose, D-sorbitol and potassium 2-ketogluconate. All strains showed positive activities for esterase (C4), esterase lipase (C8), leucine arylamidase, acid phosphatase and naphthol-AS-BI-phosphohydrolase, but were negative for alkaline phosphatase, valine arylamidase, cystine arylamidase, trypsin, a-chymotrypsin, a-galactosidase, b-galactosidase, b-glucuronidase, b-glucosidase, N-acetyl-b-glucosaminidase, a-mannosidase and a-fucosidase. +, Positive; W, weakly positive; 2, negative. Characteristic Isolation source Cell morphology Cell size (mm) Colony colour Temperature range for growth (uC) NaCl range for growth (%) Nitrate reduction Hydrolysis of: Casein Gelatin Hypoxanthine Tyrosine Assimilation of: L-Arabinose Maltose Potassium gluconate Trisodium citrate L-Rhamnose Sucrose Suberic acid Glycogen 3-Hydroxybenzoic acid Salicin L-Histidine 4-Hydroxybenzoic acid L-Proline Enzymic activity of: Lipase (C14) a-Glucosidase DNA G+C content (mol%)
1
2
3
Soil Rods 0.5–0.761.2–2.2 White 10–35 0–3 +
Rhizosphere soil* Short rods* 0.6–1.061.1–1.5* Light yellow* 20–37* 0–1 2
Rhizosphere soil* Short rods* 0.6–1.061.0–1.6* Moderately yellow* 10–42* 0–1 2
+ 2 2 +
2* 2 +* 2
+* + 2* 2
+ 2 2 + + 2 2
2 + + 2 2 + + 2 2 + 2 W
2 + + 2 2 + + + 2 2 + 2 +
2 + 71.7*
2 + 73.0*
W
+ 2 + + 2 + 2 65
W
*Data from Lee et al. (2011).
the genus Nocardioides (Busse & Schumann 1999). Strain KIS2-16T contained diphosphatidylglycerol, phosphatidylglycerol and phosphatidylinositol as the major polar lipids, which have also been observed in other species of the genus Nocardioides such as Nocardioides perillae, Nocardioides iriomotensis and Nocardioides hwasunensis (Fig. S2; Du et al., 2013; Lee et al., 2008; Yamamura et al., 2011). However, this polar lipid pattern was different from those of the two close relatives of strain KIS2-16T, N. ultimimeridianus and N. maradonensis, both of which had phosphatidylcholine and phosphatidylethanolamine as additional major polar lipids (Lee et al., 2011). The peptidoglycan structure of strain http://ijs.sgmjournals.org
KIS2-16T was A3c-type with LL-diaminopimelic acid, which is common in the species of the genus Nocardioides. The genomic DNA G+C content of strain KIS2-16T was 64.9 mol%. According to the results of DNA–DNA hybridization, strain KIS2-16T showed less than 70 % hybridization with N. maradonensis KACC 17350T (37±4 %; reciprocal 31±4 %) and N. ultimimeridianus KACC 17349T (32±3 %). In conclusion, based on the phenotypic, chemotaxonomic, phylogenetic and DNA–DNA hybridization data presented, strain KIS2-16T would appear to represent a novel species 4111
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Nocardioides simplex KCTC 9106T (AF005009) Nocardioides aromaticivorans H-1T (AB087721) Nocardioides kongjuensis A2-4T (DQ218275) Nocardioides caeni MN8T (FJ423551) 58 Nocardioides humi DCY24T (EF623863) Nocardioides daecheongensis KIS2-16T (KJ135310) 77 Nocardioides ultimimeridianus RP-B26T (FM997998) 86 91 Nocardioides maradonensis RP-B30T (FM998000) Nocardioides panacisoli GSoil 346T (FJ666101) 100 Nocardioides oleivorans DSM 16090T (AJ698724) Nocardioides furvisabuli SBS-26T (DQ411542) Nocardioides albertanoniae CD40127T (HE801966) Nocardioides aestuarii JC2056T (AY423719) Nocardioides marinus CL-DD14T (DQ401093) Nocardioides terrae VA15T (FJ423762) 59 Nocardioides panacihumi Gsoil 616T (AB271053) 100 Nocardioides salarius CL-Z59T (DQ401092) Nocardioides basaltis J112T (EU143365) Nocardioides marinquilinus CL-GY44T (JX164255) 98 Nocardioides ginsengagri BX5-10T (GQ339904) Nocardioides fonticola NAA-13T (EF626689) Nocardioides dilutus MSL-11T (EF466121) Nocardioides pyridinolyticus OS4T (U61298) 66 82 Nocardioides hankookensis DS-30T (EF555584) Nocardioides perillae I10A-01402T (JN869461) Nocardioides daphniae D287T (AM398438) Terrabacter tumescens KCTC 9133T (AF005023) 66 100
0.01
57
60
Fig. 1. Phylogenetic analysis of strain KIS2-16T and type strains of other related species of the genus Nocardioides based on 16S rRNA gene sequences. Distances and clustering were performed using the neighbour-joining method with the software package MEGA version 5.0. Bootstrap values (.50 %) based on 1000 replications are listed as percentages at the branching points. Filled circles indicate that the corresponding nodes were also recovered in trees generated with the maximum-likelihood and maximum-parsimony algorithms. Bar, 0.01 substitutions per nucleotide position.
of the genus Nocardioides, for which the name Nocardioides daecheongensis sp. nov. is proposed.
Cells are Gram-staining-positive, aerobic, non-spore-forming, non-flagellated and curved or straight rods (0.5– 0.761.2–2.2 mm). Catalase-positive and oxidase-negative. Colonies are whitish, irregular and flat on R2A medium. Growth occurs at 10–35 uC (optimum, 28–30 uC) and at pH 4.0–10.0 (optimum, 7.0). Growth occurs in the presence of 0–3.0 % (w/v) added NaCl. Hydrolyses casein, tyrosine and Tween 80, but does not hydrolyse chitin, CMcellulose, DNA, hypoxanthine, starch or xanthine. Positive activities for nitrate reduction and aesculin hydrolysis, but negative for indole production, glucose fermentation, arginine dihydrolase, urease and gelatin hydrolysis (API 20NE test strips). Assimilates D-glucose, L-arabinose, malic
acid, trisodium citrate, L-rhamnose, sodium acetate, Lalanine, 3-hydroxybenzoic acid, L-serine, propionic acid, valeric acid, L-histidine, 3-hydroxybutyric acid and 4hydroxybenzoic acid, weakly assimilates glycogen, but does not assimilate D-mannose, D-mannitol, N-acetylglucosamine, maltose, potassium gluconate, capric acid, adipic acid, phenylacetic acid, D-ribose, inositol, sucrose, itaconic acid, suberic acid, sodium malonate, lactic acid, potassium 5-ketogluconate, salicin, melibiose, L-fucose, D-sorbitol, potassium 2-ketogluconate or L-proline (API 20NE and API ID 32GN test strips). Positive activities for esterase (C4), esterase lipase (C8), lipase (C14), leucine arylamidase, acid phosphatase and naphthol-AS-BI-phosphohydrolase, but negative for alkaline phosphatase, valine arylamidase, cystine arylamidase, trypsin, a-chymotrypsin, a-galactosidase, b-galactosidase, b-glucuronidase, a-glucosidase, b-glucosidase, N-acetyl-b-glucosaminidase, a-mannosidase and a-fucosidase (API ZYM test strip). The major fatty acids (.5 %) are C18 : 1v9c, C17 : 1v8c, C16 : 0, C18 : 0 10-methyl (TBSA), C16 : 0 2-OH and C17 : 0 2-OH. The predominant menaquinone is MK-8(H4). The main polar lipids consist of diphosphatidylglycerol, phosphatidylglycerol
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Description of Nocardioides daecheongensis sp. nov. Nocardioides daecheongensis (dae.che.ong.en9sis, N.L. masc. adj. daecheongensis referring to the Daecheong Island, from where the type strain was isolated).
Nocardioides daecheongensis sp. nov.
Table 2. Fatty acids compositions of strain KIS2-16T and type strains of the closely related species of the genus Nocardioides Strain: 1, KIS2-16T; 2, N. maradonensis KACC 17350T; 3, N. ultimimeridianus KACC 17349T. –, Not detected or ,0.5 % of total fatty acids.
Cui, Y. S., Lee, S.-T. & Im, W.-T. (2009). Nocardioides ginsengisoli sp.
nov., isolated from soil of a ginseng field. Int J Syst Evol Microbiol 59, 3045–3050. Du, H.-J., Wei, Y.-Z., Su, J., Liu, H.-Y., Ma, B.-P., Guo, B.-L., Zhang, Y.-Q. & Yu, L.-Y. (2013). Nocardioides perillae sp. nov., isolated from
surface-sterilized roots of Perilla frutescens. Int J Syst Evol Microbiol 63, 1068–1072. Felsenstein, J. (1981). Evolutionary trees from DNA sequences: a
Fatty acid C14 : 0 C15 : 0 2-OH C15 : 1v6c C16 : 0 C16 : 0 2-OH C16 : 1v9c iso-C16 : 0 C17 : 0 C17 : 0 2-OH C17 : 0 10-methyl C17 : 1v6c C17 : 1v8c C18 : 0 C18 : 0 10-methyl (TBSA) C18 : 1v9c iso-C18 : 0 C19 : 0 cyclo v8c Summed feature* 3 6
1
2
3
– 1.0 – 9.2 7.4 1.0 1.5 8.5 6.7 1.3 1.3 9.8 2.5 7.8 31.8 2.6 0.5
0.7 1.2 0.5 8.2 7.5 – 3.9 6.0 5.0 4.9 2.9 17.9 0.7 4.6 19.7 5.9 2.0
0.9 0.9 – 10.3 5.1 1.9 – 10.3 5.7 – – 14.8 2.7 1.5 35.6 – –
2.9 1.7
5.0 1.2
2.4 4.4
*Summed features are groups of two or three fatty acids that cannot be separated by GLC with the MIDI system: summed feature 3, C16 : 1v6c and/or C16 : 1v7c; summed feature 6, C19 : 1v9c and/or C19 : 1v11c.
and phosphatidylinositol. The peptidoglycan contains LLdiaminopimelic acid in the cell wall and has A3c-type peptidoglycan. The type strain, KIS2-16T (5DSM 27136T5KACC 17297T5 NBRC 109597T), was isolated from a soil sample collected from Daecheong Island of Incheon region, South Korea. The DNA G+C content of the type strain is 64.9 mol%.
maximum likelihood approach. J Mol Evol 17, 368–376. Gonzalez, J. M. & Saiz-Jimenez, C. (2002). A fluorimetric method for
the estimation of G+C mol% content in microorganisms by thermal denaturation temperature. Environ Microbiol 4, 770–773. Hamada, M., Tamura, T., Yamamura, H., Suzuki, K. & Hayakawa, M. (2012). Lysinimicrobium mangrovi gen. nov., sp. nov., an actinobac-
terium isolated from the rhizosphere of a mangrove. Int J Syst Evol Microbiol 62, 1731–1735. Kim, M. K., Srinivasan, S., Park, M.-J., Sathiyaraj, G., Kim, Y.-J. & Yang, D.-C. (2009). Nocardioides humi sp. nov., a b-glucosidase-
producing bacterium isolated from soil of a ginseng field. Int J Syst Evol Microbiol 59, 2724–2728. Kim, O. S., Cho, Y. J., Lee, K., Yoon, S. H., Kim, M., Na, H., Park, S. C., Jeon, Y. S., Lee, J. H. & other authors (2012). Introducing EzTaxon-e:
a prokaryotic 16S rRNA gene sequence database with phylotypes that represent uncultured species. Int J Syst Evol Microbiol 62, 716–721. Kluge, A. G. & Farris, J. S. (1969). Quantitative phyletics and the
evolution of anurans. Syst Zool 18, 1–32. Lee, S. D., Lee, D. W. & Kim, J. S. (2008). Nocardioides hwasunensis sp.
nov. Int J Syst Evol Microbiol 58, 278–281. Lee, D. W., Lee, S.-Y., Yoon, J.-H. & Lee, S. D. (2011). Nocardioides
ultimimeridianus sp. nov. and Nocardioides maradonensis sp. nov., isolated from rhizosphere soil. Int J Syst Evol Microbiol 61, 1933–1937. Liu, Q., Xin, Y.-H., Liu, H.-C., Zhou, Y.-G. & Wen, Y. (2013).
Nocardioides szechwanensis sp. nov. and Nocardioides psychrotolerans sp. nov., isolated from a glacier. Int J Syst Evol Microbiol 63, 129–133. Minnikin, D. E., O’Donnell, A. G., Goodfellow, M., Alderson, G., Athalye, M., Schaal, A. & Parlett, J. H. (1984). An integrated
procedure for the extraction of bacterial isoprenoid quinones and polar lipids. J Microbiol Methods 2, 233–241. O’Donnell, A. G., Goodfellow, M. & Minnikin, D. E. (1982). Lipids in
the classification of Nocardioides: reclassification of Arthrobacter simplex (Jensen) Lochhead in the genus Nocardioides (Prauser) emend. O’Donnell et al. as Nocardioides simplex comb. nov. Arch Microbiol 133, 323–329. Prauser, H. (1976). Nocardioides, a new genus of the order Actinomycetales. Int J Syst Bacteriol 26, 58–65. Pruesse, E., Peplies, J. & Glo¨ckner, F. O. (2012). SINA: accurate high-
Acknowledgements This study was carried out with the support of the National Academy of Agricultural Science, Rural Development Administration, Republic of Korea (project no. PJ008666).
throughput multiple sequence alignment of ribosomal RNA genes. Bioinformatics 28, 1823–1829. Saitou, N. & Nei, M. (1987). The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 4, 406– 425. Sasser, M. (1990). Identification of bacteria by gas chromatography of
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