DOI: 10.15468/3haqp7
title: Nocardioides sambongensis sp. nov., isolated from Dokdo Islands soil

description: This dataset contains the digitized treatments in Plazi based on the original journal article Hwang, Ye-Ji, Son, Jin-Soo, Lee, Soo-Yeong, He, Yuxi, Jo, YoungJae, Shin, Jae-Ho, Ghim, Sa-Youl (2020): Nocardioides sambongensis sp. nov., isolated from Dokdo Islands soil. International Journal of Systematic and Evolutionary Microbiology 70 (1): 16-22, DOI: 10.1099/ijsem.0.003689, URL: http://dx.doi.org/10.1099/ijsem.0.003689AbstractStrain KUDC 5002 T was isolated from soil sampled on the Dokdo Islands, Republic of Korea. This bacterial strain was Gram stain-positive, non-motile, rod-shaped, capable of growing at 25 – 37 Ǫ C and pH 5.0 – 12.0, and showed optimal growth at 30 Ǫ C and pH 7.0 – 8.0. Strain KUDC 5002 T could be grown in tryptic soy broth containing less than 7.0 % NaCl (w/v). The cell width ranged from 0.5 to 0.6 µm and length ranged from 0.8 to 1.0 µm. Strain KUDC 5002 T was catalase- and oxidase-positive. Its genomic G+C content was 72.2 mol%. Its major fatty acids were C 18: 1! 9 c (17.3 %), iso-C 16: 0 (16.0 %) and iso-C 17: 0 (11.4 %). Phylogenetic analysis, based on 16S rRNA gene sequences, showed that strain KUDC 5002 T belongs to the genus Nocardioides and is most closely related to strain Nocardioides humi DCY 24 T (97.0 %). Based on its phenotypic, phylogenetic, genetic and chemotaxonomic features, strain KUDC 5002 T should be considered a novel species in the genus Nocardioides, for which we have proposed the name Nocardioides sambongensis sp. nov. The type strain is KUDC 5002 T (= KCTC 39855 T = DSM 106604 T).The genus Nocardioides belongs to the family Nocardiaceae [1], with Nocardioides albus [2] as the type strain. At the time of writing, according to LPSN (www.bacterio.net/ index.html; [3]), this genus contains more than 104 recognised species. Members of this genus are Gram-stain-positive and mesophilic, and have LL- diaminopimelic acid cell walls and a high DNA G+C content (68–74 mol%) [4]. The major respiratory quinone is menaquinone MK-8(H 4) and the fatty acid profile contains both branched and straightchain fatty acids [5]. Nocardioides species have been isolated from industrial wastewater [6], groundwater [7], alkaline soil [8 – 10], marine sediment [11 – 16] and desert soil [17].The Dokdo Islands are a group of volcanic islands located in the middle of the East Sea, east of the Republic of Korea (37 Ǫ 14′ 24.2″ N, 131 Ǫ 52′ 12.2″ E; Uljin-gun, Gyeongsangbuk-do). Dokdo is characterized by barren environmental conditions, including shallow soil depth, steep inclinations, drought, high soil salinity, and high uric acid and low organic matter content in soil [18]. From 2005 to the present day, 57 novel bacterial species have been isolated from the Dokdo Islands. Furthermore, these include several Nocardioides type strains (Nocardioides insulae [19], Nocardioides terrigena [20], Nocardioides hankookensis [21], Nocardioides dokdonensis [16] and Nocardioides paucivorans [22]).Here, we report the taxonomic analysis of a novel bacterial strain (KUDC 5002 T) isolated from dry soil sampled on these islands and propose the name Nocardioides sambongensis sp. nov.In November 2011, soil samples were collected from the rhizosphere of plants distributed across Dong-do, one of the two Dokdo Islands (Ulleung-gun, Gyeongsangbuk-Do). The samples were collected and stored as described previously [23]. They were suspended in a saline solution (0.85 %, w/v, NaCl) and serial dilutions (10 ‒4 –10 ‒6) were prepared. A 100 µl aliquot of each dilution was plated onto tryptic soy agar (TSA; Difco) and incubated at 25 Ǫ C (under ambient conditions) for 7 days [24]. Morphologically different colonies were picked up and individual colonies were further purified by repeated streaking on TSA. Strain KUDC 5002 T was cultivated on TSA and maintained at ‒70 Ǫ C in a 0.85 % NaCl solution supplemented with 15 % glycerol (v/v). The strains used in this study were subcultured on Reasoner’ s 2A (R 2A) agar at 30 Ǫ C.Author affiliations: 1 School of Life Sciences, Research Institute for Dok-do & Ulleung-do Island, Kyungpook National University, 80 Daehakro, Bukgu, Daegu 41566, Republic of Korea; 2 School of Life Sciences, BK21 Plus KNU Creative BioResearch Group, Kyungpook National University, 80 Daehakro, Bukgu, Daegu 41566, Republic of Korea; 3 School of Applied Biosciences, College of Agriculture and Life Sciences, Kyungpook National University, Daegu 41566, Republic of Korea, 80 Daehakro, Bukgu, Daegu 41566, Republic of Korea.*Correspondence: Sa-Youl Ghim, ghimsa@knu.ac.krKeywords: Nocardioides; Dokdo Islands; Soil bacteria.Abbreviations: ANI, average nucleotide identity; dDDH, digital DNA – DNA hybridization; R 2A, Reasoner ’ s 2A; TSA, tryptic soy agar; TSB, tryptic soy broth.The GenBank/ EMBL / DDBJ accession numbers for 16S rRNA gene sequence and the whole genome sequence of strain KUDC 5002 T are KX858534 and CP041091, respectively.Five supplementary figures are available with the online version of this article.The phylogenetic position of the isolate was determined based on comparative analysis of the 16S rRNA gene. The 16S rRNA gene was amplified and PCR products were purified as described previously [25]. Universal primer sets 27F/ 1492 R [26] and 518F/800 R [27] were used. Direct sequencing of the 16S rRNA gene was carried out by Macrogen with sequencing primers (27F, 1492 R, 518F and 800 R) and an ABI 3730xl automated sequencer (Applied Biosystems). The EzTaxon-e server (http://eztaxon-e.ezbiocloud.net/) [28] was used to identify the closest phylogenetic neighbours and calculate pairwise 16S rRNA gene sequence similarity values with respect to the novel strain.The complete genome of strain KUDC 5002 T and Nocardioides humi DCY 24 T were sequenced using the PacBio RSII sequencing system (Pacific Biosciences) by Macrogen. The reads were assembled de novo using Hierarchical Genome Assembly Process version 3.0 (HGAP3.0) in SMRT analysis version 2.3.0 [29]. The complete genome sequence was annotated using the combined results from the automatic NCBI Prokaryotic Genomes Annotation Pipeline (PGAP).The 16S rRNA gene sequence of strain KUDC 5002 T (1462 bp) was determined, as described previously [30]. This strain showed the highest 16S rRNA gene sequence similarity (96.96 %) to N. humi DCY 24 T, followed by Nocardioides daecheongensis KIS 2-16 T (96.95 %), Nocardioides panacisoli GSoil 346 T (96.74 %), Nocardioides kongjuensis A2-4 T (96.60 %) and Nocardioides nitrophenolicus NSP 41 T (96.60 %). A comparison of the preliminary 16S rRNA gene sequences showed that strain KUDC 5002 T was related to members of the genus Nocardioides.The complete genomes determined in this study have been deposited in the NCBI GenBank database under the accession numbers CP041091 and CP041146. The complete genome of strain KUDC 5002 T consists of a circular 4 434 294 bp chromosome. Strain KUDC 5002 T contained 71.7 mol% G+C in its DNA. This value was similar to the value of 72.6 mol% for N. humi DCY 24 T.The average nucleotide identity (ANI) values based on the BLAST+ algorithm (ANIb) and the MUMmer ultra-rapid aligning tool (ANIm) were calculated through the website of JSpeciesWS (http://jspecies.ribohost.com/jspeciesws) [30]. The average amino acid identity (AAI) values were obtained through the Kostas lab website (http://enve-omics.ce.gatech. edu/). The digital DNA–DNA hybridization (dDDH) values between strain KUDC 5002 T and N. humi DCY 24 T were calculated using the server-based Genome-to-Genome Distance Calculator version 2.1 (http://ggdc.dsmz.de/distcalc2. php) [31]. The dDDH results were based on recommended formula 2 (identities/HSP length).The ANIb and ANIm values of strain KUDC 5002 T between the most closely related strain, N. humi DCY 24 T, were 76.5 and 85.1 %. Also, the in dDDH value of strain KUDC 5002 T compared with N. humi DCY 24 T was 22.00 %. The values were clearly lower than their recommended thresholds (ANI,~95 %; dDDH, 70 %) [32, 33]. The AAI value between strain KUDC 5002 T and N. humi DCY 24 T was 69.1 %. Based on these genomic data, it is clearly indicated that strain KUDC 5002 T represents a novel species of the genus Nocardioides.Multiple sequence alignment and gap removal were carried out using CLUSTAL_W [34] and the sequence data were analysed with the software package BioEdit [35]. Gaps at the 5′ and 3′ ends of the alignment were omitted from further analyses. The Jukes and Cantor model [36] was used to generate an evolutionary distance matrix for the neighbourjoining algorithm. Phylogenetic trees were reconstructed using the neighbour-joining [37] algorithm in PHYLIP version 3.696 [38]. The SEQBOOT and CONSENSE programs in the PHYLIP package was used to evaluate the resulting tree topologies by bootstrap analysis with 1000 replications [39]. Phylogenetic trees were inferred using the maximumparsimony [40] and maximum-likelihood [41] tree-making algorithms in the MEGA7 software [42] with 1000 bootstrap replicates. Maximum-likelihood phylogeny was generated by using Kimura’ s two-parameter model [43]. Kribbella flavida was used as an outgroup. Trees were rooted and drawn using DRAWGRAM in PHYLIP, MEGA 7 in the Newick format.In the neighbour-joining phylogenetic (Fig. 1) and maximum-likelihood trees (Fig. S1, available in the online version of this article), strain KUDC 5002 T was grouped with N. humi DCY 24 T, whereas N. daecheongensis KIS 2-16 T and other related strains were located in another cluster. In the maximum-parsimony tree (Fig. S2), strain KUDC 5002 T was located within a group containing N. humi DCY 24 T, N. daecheongensis KIS 2-16 T, N. kongjuensis A2-4 T, N. nitrophenolicus NSP 41 T and ten other Nocardioides species. Furthermore, strain KUDC 5002 T exhibited high 16S rRNA gene similarity values and a distinct phylogenetic lineage, corresponding to a novel species in the genus Nocardioides (Figs 1 S1, S2).Cells morphology was observed under a stereomicroscope equipped with a Zentech digital camera (Sw 804255, Samwon Optics and Seige), a scanning electron microscope (SU 8220, Hitachi) and a transmission electron microscope (H-7100, Hitachi) after incubation on R 2A agar at 30 Ǫ C for 2 days. Gram-staining was performed using a Gram-stain kit (bioMerieux), according to the manufacturer’ s instructions. Growth of this strain at different temperatures (4, 10, 18, 25, 30, 37, 40 and 45 Ǫ C) was assessed on TSA, and the pH tolerance was tested in tryptic soy broth (TSB; Difco) by varying the pH (pH 4–11, in 0.5 pH unit increments). The pH values were adjusted as described by K€ ampfer et al. [44]. The NaCl tolerance and growth in the absence of NaCl were tested at 0–8 % NaCl concentration (w/v; in 0.5 % increments) in TSB. For phenotypic characterization and comparative purposes, three reference strains, N. humi DCY 24 T, N. daecheongensis KIS 2-16 T and N. panacisoli GSoil 346 T were used.Catalase activity was determined in a solution of 3 % hydrogen peroxide (v/v) and oxidase activity was tested using BBL Oxidase Reagent (Becton Dickinson). To assess anaerobic growth, strains were cultured on R 2A agar for 7 days at 30 Ǫ C under anaerobic conditions in a GasPak EZ Standard Incubation Container (Becton Dickinson). Motility was tested by observing cell growth on motility test medium agar with triphenyltetrazolium chloride (Carolina Biological Supply Company). Hydrolysis of casein, starch, Tween 20, Tween 40, Tween 60 and Tween 80 was determined as described by Cowan and Steel [45]. Hydrolysis of hypoxanthine, tyrosine and xanthine, at the substrate concentrations specified by Cowan and Steel [45], were assessed with an incubation in TSA at 30 Ǫ C. Other physiological and biochemical tests were carried out using API 50 CH and API 20 NE kits (bioMerieux) after incubation at 30 Ǫ C for 2 days. Enzyme activity was tested using an API ZYM kit (bio-Merieux) after incubation at 25 Ǫ C for 10 h. To detect endospore formation, strain KUDC 5002 T was grown on R 2A for 2 days and spores were visualised using a scanning electron microscope (SU 8220, Hitachi) and a transmission electron microscope (H-7100, Hitachi).19: 0The cells of strain KUDC 5002 T were Gram-positive nonendospore forming rods (Figs S3 and S4) that proliferated well on TSA and R 2A agar plates. Single cells were 0.5– 0.6×0.8–1.0 µm. Flagella and endospores were not observed by transmission or scanning electron microscopy. The cells grew at 25–37 Ǫ C (optimally at 30 Ǫ C) in the presence of 0– 7.0% NaCl (w/v; optimally in 0.5–2.0 %) and at pH 5–12 (optimally at pH 7–8) under aerobic conditions on TSA and R 2A agar. Cells were positive for catalase and oxidase activity. The characteristics of strain KUDC 5002 T are listed in the species description and phenotypic differences between this strain and the reference strain are presented in Table 1. Strain KUDC 5002 T is non-motile and does not possess flagella in contrast to the motile flagella of N. humi DCY 24 T. Strain KUDC 5002 T and other closely related strains differed considerably with respect to utilization of different substrates as sole carbon sources via hydrolysis and other enzymatic activities. Strain KUDC 5002 T can grow in culture media containing more than 3 % NaCl.Strain KUDC 5002 T was further characterized using chemotaxonomic analysis. Strain KUDC 5002 T and the closely related Nocardioides type strains were collected from R 2A plates after culturing at 30 Ǫ C for 2 days for analysing fatty acid methyl esters. Fatty acid methyl esters were saponified, methylated and extracted using the standard MIDI protocol (Sherlock Microbial Identification System, version 4.0). The fatty acids were analysed by gas chromatography (6890, Hewlett Packard) and identified using the Microbial Identification System TSBA 40 database [46]. Polar lipids were examined by two-dimensional TLC and identified using previously described methods [47]. For analysing cell-wall peptidoglycans, all strains were cultured in TSB at 37 Ǫ C for 48 h until OD 600 was 0.8 (the mid-exponential phase). Peptidoglycans were hydrolysed according to a previously described method [48]. Amino acids and isomers in the cell-wall hydrolysates were analysed as described by Hamada et al. [49]. Isoprenoid quinones were extracted using a previously described method [50] and analysed by reversedphase HPLC on a YMC ODS-A (250× 4.6 mm) column [51].MK-8(H 4) was the only predominant isoprenoid quinone in strain KUDC 5002 T cells. This is consistent with the previous finding that MK-8(H 4) is the major quinone in the majority of the Nocardioides species [48]. The cellular fatty acid profiles of strain KUDC 5002 T and the most closely related type strains are presented in Table 2. The major fatty acids in the novel strain were C 18: 1! 9 c (17.3 %), iso-C 16: 0 (16.0 %) and iso-C 17: 0 (11.4 %). Strain KUDC 5002 T contained C, C 17: 0 2-OH and C 19: 1! 9 c and/or C 19: 1! 11 c fatty acid fractions and a high abundance of saturated C 17: 0 fatty acid fractions, but did not contain C 16: 0 10-methyl, C 16: 0 2-OH, C 16: 1! 6 c or C 16: 1! 7 c, which was different from the fatty acid compositions of N. humi DCY 24 T and N. panacisoli GSoil 346 T. Strain KUDC 5002 T contained the unsaturated fatty acid C 16: 1! 11 c (1.8 %). The polar lipids of strain KUDC 5002 T were diphosphatidylglycerol, phosphatidylglycerol, two unidentified aminophospholipids, three unidentified aminolipids, one unidentified phospholipid and and one unidentified lipid. (Fig. S5). The cell-wall peptidoglycan contained LL-2,6-diaminopimelic acid as the diagnostic amino acid, which is in agreement with the other Nocardioides strains [52]. These features are common among members of Nocardioides [53].Based on the phenotypic, chemotaxonomic, phylogenetic and genetic data presented here, strain KUDC 5002 T should be considered a novel species affiliated with the genus Nocardioides for which we propose the name Nocardioides sambongensis sp. nov.