Nocardioides Silvaticus Sp. Nov., Isolated from Forest Soil

TAXONOMIC DESCRIPTION Li et al., Int J Syst Evol Microbiol 2018;68:1–6 DOI 10.1099/ijsem.0.003079

Nocardioides silvaticus sp. nov., isolated from forest soil

Chan Li, Kaixiang Shi, Yuxiao Zhang and Gejiao Wang*

Abstract A Gram-stain-positive, non-motile, rod-shaped bacterial strain S-34T was isolated from forest soil. According to 16S rRNA gene sequence analysis, strain S-34T was related to Nocardioides members and showed the highest similarities to Nocardioides thalensis NCCP-696T (97.3 %) and Nocardioides panacisoli Gsoil 346T (97.0 %), Nocardioides litorisoli X-2T (96.5 %) and Nocardioides immobilis FLL521T (96.4 %). Phylogenetic trees showed that strain S-34T fell within the cluster containing strain S-34T and N. immobilis FLL521T. The levels of DNA–DNA relatedness between strain S-34T and N. thalensis CCTCC AB 2016296T and between strain S-34T and N. panacisoli KCTC 19470T were 50.6 and 58.8 %, respectively. The genome orthoANI T T T value between strain S-34 and N. immobilis CCTCC AB 2017083 was 82.4 %. Strain S-34 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-C16 : 0,C17:1!8c,C17:1!6c,C17 : 0 and C17 : 0 10- T methyl (TBSA) as the major fatty acids. The genome length of strain S-34 was 4.53 Mb containing 52 contigs and with a DNA G+C content of 71.2 mol%. Strain S-34T 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- 34T represents a novel species of the genus Nocardioides, for which the name Nocardioides silvaticus sp. nov. is proposed. The type strain is S-34T (=KCTC 49137T=CCTCC AB 2018079T).

The genus Nocardioides was proposed by Prauser et al. in normal saline solution and serial dilutions were spread on 1976 with Nocardioides albus as the type species [1]. This Reasoner’s 2A (R2A; Sigma) agar plates and incubated at genus belongs to the phylum Actinobacteria, class Actino- 28 C for 7 days. Colonies were picked and repeatedly bacteria, order Actinomycetales and family Nocardiaceae. restreaked on R2A agar plates until purity was confirmed. To date, Nocardioides contains 101 species (www.bacterio. Strain S-34T was then purified after several sub-cultivation net/nocardioides.html) with strains isolated from diverse cycles and stored at À80 C with 25 % glycerol. environments, such as seawater [2], soil [3–6], desert [7] The 16S rRNA gene fragment of strain S-34T was amplified and glacier [8]. The members of Nocardioides are Gram- by PCR using the universal bacterial primer pair, 27F and stain-positive, aerobic, with high a DNA G+C content 1492R [10] according to Fan et al. [11]. After using T-A (64.9–74.0 mol%) [1–8]. Meanquinone-8(H4) is the major cloning with a pGEM-T Easy vector (Promega), the accurate respiratory quinone and LL-diaminopimelic acid is the diag- sequence of the PCR product was obtained. The 16S rRNA nostic amino acid of the cell- wall peptidoglycan. The fatty gene sequence of strain S-34T was compared with those acid profile includes both branched- and straight-chain fatty retrieved from GenBank and EzTaxon-e server databases acids, and iso-C is the dominant fatty acid [1–8]. The 16 : 0 (www.ezbiocloud.net/identify) [12]. Alignments of the major polar lipid is phosphatidylglycerol (PG) [1–9], some sequences were performed using MEGA version 6.0 [13]. strains have diphosphatidylglycerol (DPG) [1–6, 9] and/or Phylogenetic trees were reconstructed using the neighbour- phosphatidylinositol (PI) [2, 3, 5, 7]. In this study, a novel joining (NJ) [14], maximum-likelihood (ML) [15] and max- Nocardioides-like bacterial strain, designated strain S-34T, imum-parsimony (MP) [16] methods with bootstrap values was characterized using polyphasic taxonomic tools. determined based on 1000 replications [17]. For genome Strain S-34T was isolated from soil collected at Xinxiang analysis, the genome DNA of strain S-34T and Nocardioides Forest Ecology Park (35 18¢ 13.71† N, 113 55¢ 15.05† E) immobilis CCTCC AB 2017083T were extracted and frag- in Henan Province, PR China. The soil was suspended in mented by ultrasonication. Pair-end sequencing was carried

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, [email protected] Keywords: Nocardioides; Taxonomic Description; DNA hybridization; polar lipids; fatty acid. The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain S-34T is MH341589. The genome accession numbers of strain S-34T and Nocardioides immobilis CCTCC AB 2017083T are QGDD00000000 and QXGH00000000, respectively. Two tables and four supplementary figures are available with the online Supplementary materials

003079 ã 2018 IUMS

1 Li et al., Int J Syst Evol Microbiol 2018;68:1–6

Nocardioides albidus THG-S11.7T (KM073954) Nocardioides kongjuensis A2-4T (DQ218275) Nocardioides caeni MN8T (FJ423551) Nocardioides nitrophenolicus NSP41T (AF005024) 95 Nocardioides daeguensis 2C1-5 T (HQ246164) 0.01 Nocardioides aromaticivorans H-1T (AB087721) 66 62 Nocardioides simplex KCTC 9106 T (AF005009) 53 Nocardioides ginsengisoli Gsoil 1124T (AB245396) Nocardioides montaniterrae SR-1T (KF955611) Nocardioides humi DCY24 T (EF623863) Nocardioides ultimimeridianus RP-B26T (FM997998) 92 Nocardioides daecheongensis KLS 2-16T (KJ135310) 55 Nocardioides maradonensis RP-B30T (FM998000) 83 Nocardioides silvaticus S34T (MH341589) Nocardioides immobilis FLL521T (KY684256) 80 Nocardioides panacisoli Gsoil 346T (FJ666101) Nocardioides thalensis NCCP-696 T (LC193943) 72 97 99 Nocardioides litorisoli x-2T (KY287237) Nocardioides tritolerans MSL-14T (EF466107) Nocardioides sonneratiae BGMRC0092T (KY655023) 100 Nocardioides pyridinolyticus OS4T (U61298) Nocardioides aquiterrae GW-9T (AF529063) 59 Nocardioides deserti SC8A-24 T (KM816582) T 56 Nocardioides marinisabuli SBS-12 (AM422448) 100 Nocardioides lianchengensis CGMCC 4.6858 T (FMZM01000025) Nocardioides opuntiae OS1-21T (FN178400) Nocardioides albus KCTC 9186 T (AF004988) Terrabacter tumescens DSM 20308 T (X83812)

Fig. 1. An NJ tree based on 16S rRNA gene sequences showing the phylogenetic relationships of strain S-34T and other related strains. Terrabacter tumescens DSM 20308T 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.

out by Illumina Hiseq 2500 system by Wuhan Frasergen containing 52 contigs with an N50 length of 387 842 264Â Bioinformatics. sequencing depth of coverage, 4340 coding sequences, 3 rRNA operons and 49 tRNA operons. The genome length The nearly full-length 16S rRNA gene sequence of strain S- of N. immobilis CCTCC AB 2017083T was 6.52 Mb contain- 34T was determined to be 1448 bp and showed the highest ing 72 contigs with an N50 length of 166 910 840Â sequenc- sequence identities to those of Nocardioides thalensis ing depth of coverage, 6305 coding sequences, 3 rRNA NCCP-696T (97.3 %) and Nocardioides panacisoli Gsoil T T operons and 59 tRNA operons. The genome orthoANI 346 (97.0 %), following by Nocardioides litorisoli X-2 T T value between strain S-34 and N. immobilis CCTCC AB (96.5 %) and N. immobilis FLL521 (96.4 %). The NJ tree 2017083T was 82.4 %, which is below the maximum value T shows that strain S-34 is grouped with members of the for defining new species. Four closely related type strains genus Nocardioides and clustered with N. immobilis (N. thalensis CCTCC AB 2016296T, N. panacisoli KCTC T FLL521 (Fig. 1). Similar results were also obtained in the 19470T, N. litorisoli CCTCC AB 2016255T and N. immobilis ML and MP trees (Figs S1 and S2, available in the online CCTCC AB 2017083T) and the type species strain Supplementary Material). Thus, the genome of strain S-34T (Nocardioides albus KCTC 9186T), were obtained from the and N. immobilis CCTCC AB 2017083T were both collection centres and used as the reference strains in fol- sequenced. The genome length of strain S-34T was 4.53 Mb lowing analyses.

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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, phenyl- alanine deaminase, lipase (C14), trypsin, a-galactosidase, b-glucuronidase, N-acetyl-b-glucosaminidase, a-mannosidase and a-fucosidase. +, Posi- tive; À, negative; W, weak. The data of utilization of sole carbon sources are shown in Table S1.

Characteristic 1 2 3 4 5 6

Morphology Rod Rod Rod Rod Rod Hyphae Temperature 20–42 20–42* 10–42† 16–37‡ 16–32§ 18–37§ range ( C) pH range 7–9 5.5–9.0 5.5–8.5 6–9 6.5–9.0 7–10§ NaCl tolerance 5 4 2.5 1 0.5 11§ (%, w/v) Production ÀÀÀÀ + +

of H2S Hydrolysis of: Casein + + + + À + DNA + ÀÀÀÀÀ L-Arginine ÀÀÀÀ + + Starch ÀÀÀÀÀ + Tween 40 À + + + À + Tween 60 À + + + À + Tween 80 ÀÀ + ÀÀ + Urea À + À + À + Acid production: Cellobiose ÀÀ + + ÀÀ D-Galactose ÀÀ + ÀÀ + D-Ribose + + + + ÀÀ Fructose ÀÀÀ + ÀÀ Glucose À + + ÀÀ + Inositol ÀÀÀ + ÀÀ L-Arabinose + + W + À + L-Rhamnose À + ÀÀÀ + Maltose À + + + ÀÀ Mannitol À + À + + À Sucrose À + W + + À Xylose + + + + + À API ZYM results: a-Chymotrypsin ÀÀ + + ÀÀ b-Galactosidase ÀÀÀÀÀ + b-Glucuronidase + ÀÀÀÀ + Cysteine + + À + + + Esterase (C4) + + À + + + Esterase lipaes + + À + + + (C8) Valine arylamidase + + À + + + Polar lipids: APL ÀÀÀ + À DPG + ÀÀ + + L + ÀÀÀÀ PE ÀÀÀ + À PG + + + + + PI À + + À + ND PL + + À + +

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Table 1. cont.

Characteristic 1 2 3 4 5 6

DNA G+C content 71.2 71.6* 73.0† 71.1‡ 70.3§ 66.5§ (mol%)

*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-34T were detected strain S-34T occurred at a temperature range from 20 to by transmission electron microscopy (H-7650, Hitachi). 42 C (optimum, 37 C) and a pH range from 7 to 9 (opti- The Gram staining was performed using a Gram-staining mum, pH 7). It could tolerate salt up to 5 % NaCl (optimum kit (Jiancheng Biotech). Growth assays at different tempera- growth occurred in the absence of NaCl). The strain was tures (0, 4, 16, 20, 28, 37, 42, 45 and 47 C) and different pH positive for catalase activities but negative for oxidase, values (pH 4–9 at 1 pH unit intervals) were measured on indole production, H2S production, methyl red production R2A broth for 7 days. Citrate/phosphate buffer or Tris/HCl and Voges–Proskauer test. The strain could hydrolyse buffer solutions were used to adjust the pH. The NaCl toler- casein and DNA, but not gelatin, starch, cellulose, Tween ance tests for growth (0–6; in 1 % intervals, w/v) were 20, Tween 40, Tween 60 or Tween 80. Strain S-34T was observed in R2A medium for 7 days. Bacterial growth was resistant to oxacillin and furazolidone; susceptible to peni- determined by measuring the OD600. Anaerobic growth cillin G, ampicillin, carbenicillin, cephalexin, cefamezin, assay was tested in an anaerobic chamber (Mitsubishi) on cefuroxime, ceftriaxone, cefoperazone, midecamycinum, R2A agar for 2 weeks. The motility test was measured with norfloxacin, ofloxacin, ciprofloxacin, vancomycin and com- 0.3 % agar. pound sulphame–thoxazole. The differences between strain S-34T and the reference strains are summarized in Tables 1 Catalase activity was tested by observing the bubble and S1 (available in the online version of this article). The production with 3 % (v/v) H O and oxidase activity was 2 2 results of the susceptibilities of the antibiotics are listed in determined by testing the oxidation of 1 % (w/v) tetra- Table S2. Other characteristics of the novel strain are given methyl p-phenylenediamine [18]. The ability of degrading in the species description. gelatin, starch, Tweens (20, 40, 60 and 80) casein, cellulose and DNA were checked according to the methods of Cowan The genomic DNA G+C content of strain S-34T was calcu- and Steel [19]. Production of hydrogen sulfide and indole, lated based on the genomic sequence. Respiratory quinones and the methyl red and Voges–-Proskauer tests were carried were purified by TLC and identified by HPLC according to out according to the methods of Dong and Cai [20]. Phenyl- the method of Xie and Yokota [24]. Polar lipids were alanine dehydrogenase was tested using the method of Smi- extracted and examined by two-dimensional TLC [25]. The bert and Krieg [21]. Antibiotic susceptibility was assessed spray reagents molybdatophosphoric acid, ninhydrin, according to the conventional Kirby–Bauer method [22]. molybdenum blue, Dragendoff and 1-methyl naphthol were The following antibiotics were tested (g per disc unless used for the detect of all polar lipids, aminophospholipids, otherwise stated): penicillin G (10), oxacillin (1), ampicillin phospholipids, phosphatidylcholines and glycolipids, (10), carbenicillin (100), piperacillin (100), cephalexin (30), respectively. For analyses of the fatty acids, strain S-34T and cefamezin (30), cefradine (30), cefuroxime (30), ceftazidime the five reference strains were harvested at the mid-expo- (30), ceftriaxone (30), cefoperazone (75), midecamycinum nential phase. The fatty acids were analysed by gas chroma- (30), norfloxacin (10), ofloxacin (5), ciprofloxacin (5), van- tography according to the MIDI protocol (version 6.1 and comycin (30), polymyxin B (300), compound sulphame- TSBA library version 6.1) [26]. The diaminopimelic acid thoxazole (1.25) and furazolidone (300). Acid production isomers in the cell-wall peptidoglycan were determined by from various carbohydrates was measured according to TLC after being extracted, purified and then hydrolysed Hugh and Leifson [23]. Utilization of sole carbon sources with 6 M HCl at 100 C for 18 h [27]. DNA–DNA hybrid- was tested using API 32GN (bioMerieux) according to man- ization was performed by thermal denaturation and rena- ufacturer’s instructions and, if necessary, in combination turation method [28]. with traditional methods [20]. Additional biochemical and The DNA G+C content of strain S-34T was 71.2 mol%, enzyme activities were determined using the API 20NE and which lies within the range of DNA G+C for the genus ZYM systems (bioMerieux). Nocardioides. The main respiratory quinone detected in Strain S-34T was Gram-stain-positive, strictly aerobic, rod- strain S-34T was MK-8(H4) (93.7 %). The fatty acid compo- shaped (Fig. S3) and non-motile. Colonies grown on R2A sitions of strain S-34T and the five reference strains are pre- agar were cream-coloured, circular, smooth, raised and 1– sented in Table 2, the major components of cellular fatty T 2 mm in diameter after incubation for 3 days. Growth of acids (>5 %) of strain S-34 were iso-C16 : 0 (33.8 %), C17:1

4 Li et al., Int J Syst Evol Microbiol 2018;68:1–6

Table 2. Fatty acid compositions of strain S-34T and the phylogenetically related strains. All data are tested in this study 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. À, Not detected or less than 1 %. Summed features are groups of two or three fatty acids that cannot be separated by the MIDI system. Bold face is used to emphasize the major fatty acids (>5 %).

Fatty acid 1 2 3 4 5 6

Saturated:

C16 : 0 – 2.1 8.6 3.5 1.6 –

C17 : 0 11.0 9.1 6.2 2.4 2.0 –

C18 : 0 1.1 – 3.4 3.3 1.6 – Unsaturated:

C17:1!8c 22.8 13.9 5.6 1.9 3.7 2.2

C17:1!6c 8.2 11.6 3.6 9.3 1.9 8.8

C18:1!9c 3.6 8.7 19.2 14.4 11.9 1.9 Branched chain:

iso-C14 : 0 ––––– 1.7

iso-C15 : 0 –– 1.7 – 3.1 2.1

iso-C16 : 0 33.8 18.6 27 17.2 38.5 52.5

iso-C16 : 1 h 1.6 2.0 – 2.3 1.6 –

iso-C17 : 0 –– 1.2 – 12.1 2.4

ante-iso-C17 : 0 1.2 ––– 2.1 2.7

iso-C18 : 0 1.8 2.6 4.7 2.6 4.1 1.0

iso-C18 : 1 h – 1.1 1.2 2.3 ––

C17 : 0 10-methyl (TBSA) 5.1 10.9 2.8 4.8 4.6 7.7

C18 : 0 10-methyl (TBSA) – 4.6 9.6 29.1 6.2 – Hydroxy:

C17 : 0 2-OH 1.2 2.1 ––––

C16 : 0 2-OH – 1.1 1.2 2.3 –– Summed feature 3* – 1.7 1.5 1.5 – 1.4 Summed feature 6† 2.9 3.4 1.9 ––– Summed feature 8‡––– 1.1 –– Summed feature 9§ ––– 1.2 1.6 4.3

*Summed feature 3 contained C16:1!7c and/or C16:1!6c.

†Summed feature 6 contained iso-C19:1!9c and/or iso-C19:1!11c.

‡Summed feature 8 contained iso-C18:1!6c and/or iso-C18:1!7c.

§Summed feature 9 contained iso-C17:1!9c and/or 10-methyl C16 : 0.

!8c (22.8 %), C17 : 0 (11.0 %), C17:1!6c (8.2 %) and C17 : 0 10- DESCRIPTION OF NOCARDIOIDES SILVATICUS methyl (TBSA; 5.1 %), which were similar to the Nocardioides SP. NOV. strains but different in proportion. The polar lipids were ¢ DPG, PG, four unknown phospholipids (PL1–4) and one Nocardioides silvaticus sp. nov. (sil.va ti.cus. L. masc. adj. sil- unknown lipid (L) (Fig. S4) which were generally similar to vaticus belonging to a forest). the other Nocardioides strains, but with some differences Cells are Gram-stain-positive, strictly aerobic, rod-shaped, (Table 1). LL-Diaminopimelic acid was the diagnostic dia- approximately 0.8–1.3 µm long and 0.3–0.4 µm wide, and mino acid in the cell-wall peptidoglycan. The levels of non-motile. Colonies grown on R2A agar are cream- DNA–DNA relatedness between strain S-34T and N. thalen- coloured, circular, smooth, raised and 1–2 mm in diameter sis CCTCC AB 2016296T and between strain S-34T and N. after incubation for 3 days. The growth temperature and pH panacisoli KCTC 19470T were 50.6 and 58.8 %, respectively. range on R2A agar are 20–42 C (optimum, 37 C) and pH Strain S-34T could be distinguished from the other Nocar- 7–9 (optimum, pH 7). It can tolerate salt up to 5 % NaCl dioides members mainly based on the data of phylogenetic (optimum without addition of NaCl). Catalase is positive analyses, DNA–DNA hybridization, polar lipids and some but oxidase is negative. Negative for H2S production, indole biochemical differences. Therefore, strain S-34T is consid- production, methyl red production and Voges–Proskauer ered to represent a novel species of the genus Nocardioides, test. Hydrolyses casein and DNA, but not gelatin, starch, for which the name Nocardioides silvaticus sp. nov. is cellulose, Tween 20, Tween 40, Tween 60 and Tween 80. As proposed. sole carbon sources, sucrose, fructose, cysteine, propionate,

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