DOI: 10.15468/3g8a6j
title: Nocardioides silvaticus sp. nov., isolated from forest soil

description: This dataset contains the digitized treatments in Plazi based on the original journal article Li, Chan, Shi, Kaixiang, Zhang, Yuxiao, Wang, Gejiao (2019): Nocardioides silvaticus sp. nov., isolated from forest soil. International Journal of Systematic and Evolutionary Microbiology 69 (1): 68-73, DOI: 10.1099/ijsem.0.003079, URL: http://dx.doi.org/10.1099/ijsem.0.003079AbstractA Gram-stain-positive, non-motile, rod-shaped bacterial strain S-34 T was isolated from forest soil. According to 16S rRNA gene sequence analysis, strain S-34 T was related to Nocardioides members and showed the highest similarities to Nocardioides thalensis NCCP-696 T (97.3 %) and Nocardioides panacisoli Gsoil 346 T (97.0 %), Nocardioides litorisoli X-2 T (96.5 %) and Nocardioides immobilis FLL 521 T (96.4 %). Phylogenetic trees showed that strain S-34 T fell within the cluster containing strain S-34 T and N. immobilis FLL 521 T. The levels of DNA–DNA relatedness between strain S-34 T and N. thalensis CCTCC AB 2016296 T and between strain S-34 T and N. panacisoli KCTC 19470 T were 50.6 and 58.8 %, respectively. The genome orthoANI value between strain S-34 T and N. immobilis CCTCC AB 2017083 T was 82.4 %. Strain S-34 T had LL- diaminopimelic acid in the cell-wall peptidoglycan, diphosphatidylglycerol, phosphatidylglycerol, four unknown phospholipids and one unknown lipid as the polar lipids, meanquinone-8(H4) as the only respiratory quinone and iso-C 16: 0, C 17: 1! 8 c, C 17:1! 6 c, C 17: 0 and C 17: 0 10- methyl (TBSA) as the major fatty acids. The genome length of strain S-34 T was 4.53 Mb containing 52 contigs and with a DNA G+C content of 71.2 mol%. Strain S-34 T could be distinguished from the other Nocardioides members mainly based on the data of phylogenetic analyses, DNA–DNA hybridization, polar lipids and some biochemical differences. Therefore, strain S-34 T represents a novel species of the genus Nocardioides, for which the name Nocardioides silvaticus sp. nov. is proposed. The type strain is S-34 T (= KCTC 49137 T = CCTCC AB 2018079 T).The genus Nocardioides was proposed by Prauser et al. in 1976 with Nocardioides albus as the type species [1]. This genus belongs to the phylum Actinobacteria, class Actinobacteria, order Actinomycetales and family Nocardiaceae. To date, Nocardioides contains 101 species (www.bacterio. net/nocardioides.html) with strains isolated from diverse environments, such as seawater [2], soil [3 – 6], desert [7] and glacier [8]. The members of Nocardioides are Gramstain-positive, aerobic, with high a DNA G+C content (64.9–74.0 mol%) [1 – 8]. Meanquinone-8(H4) is the major respiratory quinone and LL- diaminopimelic acid is the diagnostic amino acid of the cell-wall peptidoglycan. The fatty acid profile includes both branched- and straightchain fatty acids, and iso-C 16: 0 is the dominant fatty acid [1 – 8]. The major polar lipid is phosphatidylglycerol (PG) [1 – 9], some strains have diphosphatidylglycerol (DPG) [1 – 6, 9] and/or phosphatidylinositol (PI) [2, 3, 5, 7]. In this study, a novel Nocardioides -like bacterial strain, designated strain S-34 T, was characterized using polyphasic taxonomic tools.Strain S-34 T was isolated from soil collected at Xinxiang Forest Ecology Park (35 Ǫ 18′ 13.71″ N, 113 Ǫ 55′ 15.05″ E) in Henan Province, PR China. The soil was suspended in normal saline solution and serial dilutions were spread on Reasoner’ s 2A (R 2A; Sigma) agar plates and incubated at 28 Ǫ C for 7 days. Colonies were picked and repeatedly restreaked on R 2A agar plates until purity was confirmed. Strain S-34 T was then purified after several sub-cultivation cycles and stored at ‒80 Ǫ C with 25 % glycerol.The 16S rRNA gene fragment of strain S-34 T was amplified by PCR using the universal bacterial primer pair, 27F and 1492 R [10] according to Fan et al. [11]. After using T-A cloning with a pGEM - T Easy vector (Promega), the accurate sequence of the PCR product was obtained. The 16S rRNA gene sequence of strain S-34 T was compared with those retrieved from GenBank and EzTaxon-e server databases (www.ezbiocloud.net/identify) [12]. Alignments of the sequences were performed using MEGA version 6.0 [13]. Phylogenetic trees were reconstructed using the neighbourjoining (NJ) [14], maximum-likelihood (ML) [15] and Author affiliation: State Key Laboratory of Agricultural Microbiology, College of Life Science and Technology, Huazhong Agricultural University, Wuhan, Hubei 430070, PR China.*Correspondence: Gejiao Wang, gejiao@mail.hzau.edu.cnKeywords: Nocardioides; Taxonomic Description; DNA hybridization; polar lipids; fatty acid.Abbreviations: PG, phosphatidylglycerol; DPG, diphosphatidylglycerol; PI, phosphatidylinositol; PL, phospholipid; L, lipid; APL, unknown amino phospholipid; PE, phosphatidylethanolamine; NJ, neighbor-joining; ML, maximum-likelihood; MP, maximum-parsimony; H 2 S, hydrogen sulfide; HPLC, high performance chromatography; TLC, thin layer chromatography.The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain S-34 T is MH 341589. The genome accession numbers of strain S-34 T and Nocardioides immobilis CCTCC AB 2017083 T are QGDD 00000000 and QXGH00000000, respectively.Two supplementary tables and four supplementary figures are available with the online version of this article. maximum-parsimony (MP) [16] methods with bootstrap values determined based on 1000 replications [17]. For genome analysis, the genome DNA of strain S-34 T and Nocardioides immobilis CCTCC AB 2017083 T were extracted and fragmented by ultrasonication. Pair-end sequencing was carried out by Illumina Hiseq 2500 system by Wuhan Frasergen Bioinformatics.Fig. 1. An NJ tree based on 16S rRNA gene sequences showing the phylogenetic relationships of strain S-34 T and other related strains. Terrabacter tumescens DSM 20308 T was used as an outgroup member. Filled circles indicate that the corresponding nodes were also recovered in trees reconstructed with the MP tree and ML tree. Numbers at nodes indicate levels of bootstrap support based on 1000 resampled datasets. Values below 50 % are not shown. Bar, 0.01 substitutions per nucleotide position.The nearly full-length 16S rRNA gene sequence of strain S-34 T was determined to be 1448 bp and showed the highest sequence identities to those of Nocardioides thalensis NCCP-696 T (97.3 %) and Nocardioides panacisoli Gsoil 346 T (97.0 %), following by Nocardioides litorisoli X-2 T (96.5 %) and N. immobilis FLL 521 T (96.4 %). The NJ tree shows that strain S-34 T is grouped with members of the genus Nocardioides and clustered with N. immobilis FLL 521 T (Fig. 1). Similar results were also obtained in the ML and MP trees (Figs S1 and S2, available in the online version of this article). Thus, the genome of strain S-34 T and N. immobilis CCTCC AB 2017083 T were both sequenced. The genome length of strain S-34 T was 4.53 Mb containing 52 contigs with an N50 length of 387 842 264× sequencing depth of coverage, 4340 coding sequences, 3 rRNA operons and 49 tRNA operons. The genome length of N. immobilis CCTCC AB 2017083 T was 6.52 Mb containing 72 contigs with an N50 length of 166 910 840× sequencing depth of coverage, 6305 coding sequences, 3 rRNA operons and 59 tRNA operons. The genome orthoANI value between strain S-34 T and N. immobilis CCTCC AB 2017083 T was 82.4 %, which is below the maximum value for defining new species. Four closely related type strains (N. thalensis CCTCC AB 2016296 T, N. panacisoli KCTC 19470 T, N. litorisoli CCTCC AB 2016255 T and N. immobilis CCTCC AB 2017083 T) and the type species strain (Nocardioides albus KCTC 9186 T), were obtained from the collection centres and used as the reference strains in following analyses.Table 1. Differential phenotypic characteristics among strain S-34T and the phylogenetically related strains and the Nocardioides type species strain Strains: 1, N. silvaticus S-34T; 2, N. thalensis CCTCC AB 2016296T; 3, N. panacisoli KCTC 19470T; 4, N. litorisoli CCTCC AB 2016255T; 5, N. immobilis CCTCC AB 2017083T; 6, N. albus KCTC 9186T. All data are tested in this study except the DNA G+C contents of reference strains. All strains are positive for the activity of catalase; hydrolysis of aesculin; and for the activity of alkaline phosphatase, leucine arylamidase, acid phosphatase, naphthol-AS-BI-phosphohydrolase, 4-nitrophenyl-b- D- galactopyranoside and a- galactosidase. All strains are Gram-stain-positive and negative for indole production, methyl red production and Voges–Proskauer test, Tween 20, gelatin and CM-cellulose hydrolyses; and for the activity of oxidase, phenylalanine deaminase, lipase (C14), trypsin, a- galactosidase, b- glucuronidase, N -acetyl-b- glucosaminidase, a- mannosidase and a- fucosidase. +, Positive; ‒, negative; W, weak. The data of utilization of sole carbon sources are shown in Table S1.Characteristic 1 2 3 4 5 6 Morphology Temperature range (Ǫ C) Rod 20–42 Rod 20–42 * Rod 10–42 † Rod 16–37 ‡ Rod 16–32 § Hyphae 18–37 § pH range 7–9 5.5–9.0 5.5–8.5 6–9 6.5–9.0 7–10 § NaCl tolerance (%, w/v) 5 4 2.5 1 0.5 11§ Production ‒ ‒ ‒ ‒ + + of H2S Hydrolysis of: Casein + + + + ‒ + DNA + ‒ ‒ ‒ ‒ ‒ L- Arginine ‒ ‒ ‒ ‒ + + Starch ‒ ‒ ‒ ‒ ‒ + Tween 40 ‒ + + + ‒ + Tween 60 ‒ + + + ‒ + Tween 80 ‒ ‒ + ‒ ‒ + Urea ‒ + ‒ + ‒ + Acid production: Cellobiose ‒ ‒ + + ‒ ‒ D- Galactose ‒ ‒ + ‒ ‒ + D- Ribose + + + + ‒ ‒ Fructose ‒ ‒ ‒ + ‒ ‒ Glucose ‒ + + ‒ ‒ + Inositol ‒ ‒ ‒ + ‒ ‒ L- Arabinose + + W + ‒ + L- Rhamnose ‒ + ‒ ‒ ‒ + Maltose ‒ + + + ‒ ‒ Mannitol ‒ + ‒ + + ‒ Sucrose ‒ + W + + ‒ Xylose + + + + + ‒ API ZYM results: a - Chymotrypsin ‒ ‒ + + ‒ ‒ b- Galactosidase ‒ ‒ ‒ ‒ ‒ + b- Glucuronidase + ‒ ‒ ‒ ‒ + Cysteine + + ‒ + + + Esterase (C4) + + ‒ + + + Esterase lipaes (C8) + + ‒ + + + Valine arylamidase + + ‒ + + + Polar lipids: APL ‒ ‒ ‒ + ‒ DPG + ‒ ‒ + + L + ‒ ‒ ‒ ‒ PE ‒ ‒ ‒ + ‒ PG + + + + + PI ‒ + + ‒ + ND PL + + ‒ + +Table 1. cont.Characteristic 1 2 3 4 5 6 DNA G+C content (mol%) 71.2 71.6* 73.0† 71.1‡ 70.3§ 66.5§*Data from Khan et al. [7]. †Data from Cho et al. [6]. ‡Data from Wang et al. [4]. §Data from Lu et al. [5].Morphological characteristics of strain S-34 T were detected by transmission electron microscopy (H-7650, Hitachi). The Gram staining was performed using a Gram-staining kit (Jiancheng Biotech). Growth assays at different temperatures (0, 4, 16, 20, 28, 37, 42, 45 and 47 Ǫ C) and different pH values (pH 4–9 at 1 pH unit intervals) were measured on R 2A broth for 7 days. Citrate/phosphate buffer or Tris/HCl buffer solutions were used to adjust the pH. The NaCl tolerance tests for growth (0–6; in 1 % intervals, w/v) were observed in R 2A medium for 7 days. Bacterial growth was determined by measuring the OD 600. Anaerobic growth assay was tested in an anaerobic chamber (Mitsubishi) on R 2A agar for 2 weeks. The motility test was measured with 0.3 % agar.Catalase activity was tested by observing the bubble production with 3 % (v/v) H 2 O 2 and oxidase activity was determined by testing the oxidation of 1 % (w/v) tetramethyl p-phenylenediamine [18]. The ability of degrading gelatin, starch, Tweens (20, 40, 60 and 80) casein, cellulose and DNA were checked according to the methods of Cowan and Steel [19]. Production of hydrogen sulfide and indole, and the methyl red and Voges–Proskauer tests were carried out according to the methods of Dong and Cai [20]. Phenylalanine dehydrogenase was tested using the method of Smibert and Krieg [21]. Antibiotic susceptibility was assessed according to the conventional Kirby–Bauer method [22]. The following antibiotics were tested (g per disc unless otherwise stated): penicillin G (10), oxacillin (1), ampicillin (10), carbenicillin (100), piperacillin (100), cephalexin (30), cefamezin (30), cefradine (30), cefuroxime (30), ceftazidime (30), ceftriaxone (30), cefoperazone (75), midecamycinum (30), norfloxacin (10), ofloxacin (5), ciprofloxacin (5), vancomycin (30), polymyxin B (300), compound sulphamethoxazole (1.25) and furazolidone (300). Acid production from various carbohydrates was measured according to Hugh and Leifson [23]. Utilization of sole carbon sources was tested using API 32GN (bioḾerieux) according to manufacturer’ s instructions and, if necessary, in combination with traditional methods [20]. Additional biochemical and enzyme activities were determined using the API 20NE and ZYM systems (bioḾerieux).Strain S-34 T was Gram-stain-positive, strictly aerobic, rodshaped (Fig. S3) and non-motile. Colonies grown on R 2A agar were cream-coloured, circular, smooth, raised and 1– 2 mm in diameter after incubation for 3 days. Growth of strain S-34 T occurred at a temperature range from 20 to 42 Ǫ C (optimum, 37 Ǫ C) and a pH range from 7 to 9 (optimum, pH 7). It could tolerate salt up to 5 % NaCl (optimum growth occurred in the absence of NaCl). The strain was positive for catalase activities but negative for oxidase, indole production, H 2 S production, methyl red production and Voges–Proskauer test. The strain could hydrolyse casein and DNA, but not gelatin, starch, cellulose, Tween 20, Tween 40, Tween 60 or Tween 80. Strain S-34 T was resistant to oxacillin and furazolidone; susceptible to penicillin G, ampicillin, carbenicillin, cephalexin, cefamezin, cefuroxime, ceftriaxone, cefoperazone, midecamycinum, norfloxacin, ofloxacin, ciprofloxacin, vancomycin and compound sulphame–thoxazole. The differences between strain S-34 T and the reference strains are summarized in Tables 1 and S1 (available in the online version of this article). The results of the susceptibilities of the antibiotics are listed in Table S2. Other characteristics of the novel strain are given in the species description.The genomic DNA G+C content of strain S-34 T was calculated based on the genomic sequence. Respiratory quinones were purified by TLC and identified by HPLC according to the method of Xie and Yokota [24]. Polar lipids were extracted and examined by two-dimensional TLC [25]. The spray reagents molybdatophosphoric acid, ninhydrin, molybdenum blue, Dragendoff and 1-methyl naphthol were used for the detect of all polar lipids, aminophospholipids, phospholipids, phosphatidylcholines and glycolipids, respectively. For analyses of the fatty acids, strain S-34 T and the five reference strains were harvested at the mid-exponential phase. The fatty acids were analysed by gas chromatography according to the MIDI protocol (version 6.1 and TSBA library version 6.1) [26]. The diaminopimelic acid isomers in the cell-wall peptidoglycan were determined by TLC after being extracted, purified and then hydrolysed with 6 M HCl at 100 Ǫ C for 18 h [27]. DNA–DNA hybridization was performed by thermal denaturation and renaturation method [28].The DNA G+C content of strain S-34 T was 71.2 mol%, which lies within the range of DNA G+C for the genus Nocardioides. The main respiratory quinone detected in strain S-34 T was MK-8(H4) (93.7 %). The fatty acid compositions of strain S-34 T and the five reference strains are presented in Table 2, the major components of cellular fatty acids (&gt;5 %) of strain S-34 T were iso-C 16: 0 (33.8 %), C 17:1! 8 c (22.8 %), C 17: 0 (11.0 %), C 17:1! 6 c (8.2 %) and C 17: 0 10- methyl (TBSA; 5.1 %), which were similar to the Nocardioides strains but different in proportion. The polar lipids were DPG, PG, four unknown phospholipids (PL1–4) and one unknown lipid (L) (Fig. S4) which were generally similar to the other Nocardioides strains, but with some differences (Table 1). LL- Diaminopimelic acid was the diagnostic diamino acid in the cell-wall peptidoglycan. The levels of DNA–DNA relatedness between strain S-34 T and N. thalensis CCTCC AB 2016296 T and between strain S-34 T and N. panacisoli KCTC 19470 T were 50.6 and 58.8 %, respectively. Strain S-34 T could be distinguished from the other Nocardioides members mainly based on the data of phylogenetic analyses, DNA–DNA hybridization, polar lipids and some biochemical differences. Therefore, strain S-34 T is considered to represent a novel species of the genus Nocardioides, for which the name Nocardioides silvaticus sp. nov. is proposed.