Introduction

The genus Nocardiopsis, belonging to the family Nocardiopsaceae [1] of the order Streptosporangiales [2], was initially described by Meyer in 1976 [3]. At the time of writing, this taxon includes over 60 species with validly published and correct names (http://www.bacterio.net/nocardiopsis.html). Members of the genus Nocardiopsis are distributed in various environments such as saline soil [4, 5], marine sediments [6], alkaline environment [7, 8], desert soil [9], rhizosphere soil [10, 11], mangrove sediment [12] and host plants [13]. Although previous studies have suggested that Nocardiopsis is opportunistic pathogens [14, 15], there is recent evidence that some species of this genus is a potential source of bioactive compounds [16]. Thus, it is very important for microbiologists to explore the natural environment with the aim of discovering novel species in this genus. During the course of screening for novel Nocardiopsis species from roots of Eucommia ulmoides Oliv., a strain designated as Mg02T, was obtained. Sequence analysis of 16S rRNA gene indicated that the strain was related to the genus of Nocardiopsis. This study aimed to determine their taxonomic positions by using a polyphasic taxonomic approach.

Materials and methods

Isolation, maintenance and cultivation of strain

Strain Mg02T was isolated from roots of Eucommia ulmoides Oliv. collected from Changde City, Hunan Province, China (29°03'6.48” N, 111°40'00.10” E). The samples were processed as described by Qin et al. [13] with some modifications. After being thoroughly dried under sterile conditions, the samples were subjected to drying at 37 °C for 5 days. Then, the roots sample were crushed and evenly distributed on modified Gause’s synthetic medium [17]. The purified isolates were prepared for short-term preservation on Gause’s synthetic medium [18] at 4 °C and suspended in sterile 30% (w/v) glycerol solution for long-term conservation at −80 °C. Strain Nocardiopsis flavescens CGMCC 4.5723T [obtained from China General Microbiological Culture Collection Center (CGMCC), Beijing, China] was used as reference species for comparative studies.

Phenotypic tests, morphological and cultural properties

The morphological property of strain Mg02T were observed by light microscope (NE620, Ningbo Yongxin Optics CO., LTD, China) and scanning electron microscope (FEI-Quanta 450, America) after incubation on Gause’s synthetic medium for about 30 days at 28 °C. The cultural characteristics of strain Mg02T and N. flavescens CGMCC 4.5723T were observed on Gause’s synthetic medium and ISP serial media [19]. The color of aerial mycelium, substrate mycelium and diffusible pigmentation were determined by Color Standards and Color Nomenclature [20]. The growth pH values (2, 3, 4, 5, 6, 7, 8, 9, 10 and 11) were assayed in ISP 2 broth. The effect of NaCl concentration on growth was determined on the ISP 2 agar plates supplemented with 0 to 14.0% (w/v) NaCl. Growth at different temperature (4–45 °C) was also observed on the ISP 2 agar plates. The utilization of carbon sources was determined using the methods previously described by [19]. The following tests, including urease, liquefaction of gelatin, hydrolysis of starch and aesculin, reduction of nitrate and degradation tests for Tweens (20, 40, 60 and 80) were carried out using methods described in the literatures [21, 22]. The experiments were carried out in triplicate.

Chemotaxonomic characteristics

Cellular fatty acids composition was analyzed using cells of strain Mg02T and N. flavescens CGMCC 4.5723T cultured using trypticase soy broth (TSB) medium at 28 °C for 7 days on a rotary shaker. The cellular fatty acids were extracted, methylated and analysed according to the protocol of the Sherlock Microbial ID System (http://www.midi-inc.com/) [23], which was carried out by the MCCC (Marine Culture Collection of China). The isomer of diaminopimelic acid and sugar analyses of whole-cell hydrolysates were determined according to the procedures described by Hasegawa et al. [24] and Lechevalier MP and Lechevalier H [25]. Menaquinones were extracted according to the method of Collins et al. [26] and analyzed by HPLC [27]. The polar lipids were extracted and separated by two dimensional TLC [28] and analysed on Merck silica gel 60 F254 aluminum-backed thin-layer plates [29].

Phylogeny and genomic DNA-DNA correlation analysis

Genomic DNA for PCR amplification was prepared as followed: firstly, amount of biomass was transferred from solid medium to a sterile mortar frozen for 48 h in advance. Second, added proper sterile sand and 1.5 ml 2× CTAB (2× Hexadecyl trimethyl ammonium Bromide), and grind rapidly within 10 s. Third, quickly moved into two 1.5 ml Eppendorf tubes, and bathed in water at 65 °C for 40 min, and gently mixed every 10 min. Then, added the same volume of chloroform: isopentyl alcohol mixture (24:1, V/V), fully mixed, and centrifuge at 4 °C 11000 r/min for 12 min. Followed, transferred the 0.6 ml supernatant to a new 1.5 ml Eppendorf tube. Finally, the DNA solution was purified using a MagExtractor®-PCR&Gel Clean up kit (toyobo) according to the manufacturers’ instructions. The 16S rRNA gene of strain Mg02T was amplified using universal primer set, 27 F (5′-AGA GTT TGA TCC TGG CTC AG-3′) and 1492 R (5ʹ-GGT TAC CTT GTT ACG ACT T-3ʹ) [30]. PCR reactions were performed in Proflex PCR System (Applied Biosystems, USA) in a total volume of 50 µl reaction mixture containing 2 µl templates DNA, 21 µl ddH2O, 25 µl Quick TaqTM HS Dye Mix and 1.0 µl 25 µM of each primer. The thermal cycling conditions were programmed as follows: initial denaturation at 94 °C for 5 min; followed by 30 cycles of 94 °C for 1 min, 65 °C for 1 min and 72 °C for 1.5 min, and final extension at 72 °C for 10 min. The amplified products were determined by 1.0% (w/v) agarose gel electrophoresis. The PCR products were sequenced directly using an automated DNA sequencing system (ABI 3730XL; Applied Biosystems) by Sangon Biotech (Shanghai, China). The 16S rRNA gene sequence of strain Mg02T was compared with public databases and EzBioCloud database (https://www.ezbiocloud.net/), [31]. Based on the EzBioCloud results, closely related reference strains were used for constructing phylogenetic trees, using the neighbor-joining (NJ) [32], maximum-likelihood (ML) [33], maximum-parsimony (MP) [34] algorithms within the Molecular Evolutionary Genetics Analysis (MEGA7.0) software [35]. The confidence values of resultant trees were evaluated with bootstrap analysis based on 1000 replicates [36]. The genome of strain Mg02T was sequenced by using a Nanopore PromethION sequencing system performed at Wuhan Benagen Technology Co., Ltd (Hubei, China). The sequenced reads was assembled using SOAPnuke (Version: 2.1.2). The gene prediction analysis and functional annotation of the genome of strain Mg02T was performed by Rapid Annotation by using Subsystems Technology (RAST, http://rast.nmpdr.org/), specifically the RAST Toolkit (RASTtk) option [37] and visualized with the SEED viewer [38]. Based on results from 16S rRNA gene sequence analysis, the genome sequences of strain Mg02T and related type species were selected for reconstructing phylogenomic tree using the Type (Strain) Genome Server [39]. The average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) values between the genomes of strain Mg02T and N. flavescens CGMCC 4.5723T were calculated using the JSpeciesWS online service [40] and Formula 2 of the Genome- to-Genome Distance Calculator 2.1 [41] online platforms, respectively. The DNA G + C content of strain Mg02T was from the genomic data using the JSpecies WS online service.

Results and discussion

Morphological and physio-biochemical characteristics

Strain Mg02T was observed to form white aerial mycelium and yellow substrate mycelium on Gause’s synthetic agar medium at 28 °C for 30 days, which produced straight to slightly flexuous spore chains consisting of rod-shaped spores with smooth surfaces (Fig. 1). Strain Mg02T grew well on all media tested at 28 °C after incubation for 21 days. No diffusible pigment and melanin were observed on all tested media (Fig. S2). Growth is observed between 20–45 °C (optimum, 28 °C), from pH 6.0–11.0 (optimum, pH 7.0), and in the presence of 0–8.0% (w/v) NaCl (optimum, 1.0%). Cell-wall hydrolysates of strain Mg02T contained meso-diaminopimelic acid and no diagnostic sugars. The polar lipid profile of strain Mg02T consisted of diphosphatidylglycerol (DPG), phosphatidylethanolamine (PE), phosphatidylinositol mannosides (PIM), phosphatidyl methyl ethanolamine (PME), phospholipidscholine (PC), phosphatidyl glycerol (PG), phosphotidyl inositol (PI), phosphoglycolipid (PGL) and unidentified phospholipids (L1, L2, L3) (Fig. S1). The major cellular fatty acids of strain Mg02T (>5.0%) were iso-C16:0 (27.4%), C18:1ω9c (15.4%), anteiso-C17:0 (9.1%), anteiso-C15:0 (6.8%) and C17:1ω8c (6.3%) and other trace components (Table S1). The menaquinones of this strain were identified as MK-10(H4) (38.4%), MK-10(H2) (32.4%), MK-10(H8) (18.4%), MK-10 (5.4%) and MK-10(H6) (2.3%). The morphological and chemical characteristics indicated that strain Mg02T belonged to the genus Nocardiopsis.

Fig. 1
figure 1

Optical micrograph (a) and scanning electron micrograph (b) of strain Mg02T grown on Gause’s synthetic agar medium at 28 °C after incubation about 30 days

Full size image

Phylogenomic analysis

The assembled genome sequence was found to be 7,194,689 bp with a G + C content of 73.5% and composed of 4 contigs. A total of 6639 genes (6447 coding genes, 83 RNA genes and 109 pseudo genes), 6556 CDSs (6447 CDSs with protein and 109 CDSs without protein) were predicted and annotated. The antiSMASH secondary metabolite biosynthetic gene clusters prediction revealed 21 gene clusters in strain Mg02T. The five main biosynthetic gene clusters were type 1 polyketide synthase (T1PKS), terpene, Linear azol(in)e-containing peptides (LAP), non-ribosomal peptide synthetase (NRPS) and thiopeptide. Strain Mg02T had 306 subsystems that could be classified into 24 categories, and the subsystem coverage was 17%, according to the annotation using the RAST server, The represented subsystem features identified were “Carbohydrate” (307 CDSs), “Amino Acids and Derivatives” (296 CDSs), “Cofactors, Vitamins, Prosthetic Groups, Pigments” (194 CDSs), “Protein Metabolism” (203 CDSs), “Fatty Acids, Lipids, and Isoprenoids” (99 CDSs), “Nucleosides and Nucleotides” (99 CDSs), “DNA Metabolism” (95 CDSs), “Respiration” (86 CDSs), “RNA Metabolism” (49 CDSs), “Membrane Transport” (48 CDSs), “Virulence, Disease and Defense” (46 CDSs), “Stress Response” (41 CDSs), “Metabolism of Aromatic Compounds” (36 CDSs), “Cell Wall and Capsule” (28 CDSs), “Miscellaneous” (25 CDSs), “Nitrogen Metabolism” (25 CDSs), “Phosphorus Metabolism” (22 CDSs), “Regulation and Cell signaling” (19 CDSs), “Sulfur Metabolism” (7 CDSs), “Secondary Metabolism” (6 CDSs), “Iron acquisition and metabolism” (6 CDSs), “Potassium metabolism” (4 CDSs),“Phages, Prophages, Transposable elements, Plasmids” (2 CDSs) and “Dormancy and Sporulation” (1 CDSs). The length of the complete 16S rRNA gene sequence of strain Mg02T was 1532 bp. Sequence analysis of 16S rRNA gene indicated that strain Mg02T is affiliated to the genus Nocardiopsis and had 99.1% sequence similarity to N. flavescens CGMCC 4.5723T and <98.7% to other species of the genus Nocardiopsis with validly published names. The phylogenetic tree based on 16S rRNA gene sequence indicated that strain Mg02T was clustered with members of genus Nocardiopsis and formed a distinct cluster distinguishable from other species of the genus in the ML tree (Fig. 2). The similar results were also observed in the NJ and MP trees (Figs. S3, S4). Given that the phylogenomic analysis exhibited better resolution than the phylogenetic analysis based on 16S rRNA gene sequence [42], so, in the present work, the phylogenomic analysis was carried out in order to further clarify the taxonomic status of strain Mg02T. It was shown in Fig. 3, strain Mg02T was closely related to N. flavescens CGMCC 4.5723T and distinct from the latter according to the clustering patterns. The ANIm and dDDH values between them were 88.5% and 36.7%, respectively, below the 95–96% and 70% cutoff points recommended for delineating species, further supporting that strain Mg02T represented a novel species of the genus Nocardiopsis [43, 44]. In addition, the cultural, physiological and biochemical characteristics were dissimilar enough to distinguish strain Mg02T from N. flavescens CGMCC 4.5723T (Table 1, Table S1, Table S2 and Fig. S3). For example, strain Mg02T formed orange-citrine aerial mycelium and substrate mycelium on ISP 2 medium, while N. flavescens CGMCC 4.5723T formed white aerial mycelium and yellow substrate mycelium. The major cellular fatty acids of strain Mg02T (>5.0%) were iso-C16:0 (27.4%), C18:1ω9c (15.4%), anteiso-C17:0 (9.1%), anteiso-C15:0 (6.8%) and C17:1ω8c (6.3%). While N. flavescens CGMCC 4.5723T were iso-C16:0 (15.2%), C18:1ω9c (5.2%), anteiso-C17:0 (7.0%), anteiso-C15:0 (22.4%), iso-C14:0 (9.6%) and C16:0 (5.2%). These results illustrated that strain Mg02T should be recognized as a novel Nocardiopsis species.

Fig. 2
figure 2

Maximum-likelihood phylogenetic tree based on 16S rRNA gene sequences (1390 bp) showing the relationship between selected species of the genus Nocardiopsis. Actinomadura madurae DSM 43067T was used as an outgroup. Bootstrap percentages over 50% derived from 1000 replications are showed at the node. Bar represents 0.0100 changes per nucleotide position

Full size image
Fig. 3
figure 3

Phylogenetic tree based on whole-genome sequences of strain Mg02T and related reference strains. Tree inferred with FastME 2.1.6.1 [45] from GBDP distances calculated from genome sequences. The branch lengths are scaled in terms of GBDP distance formula d5. The numbers above branches are GBDP pseudo-bootstrap support values >60% from 100 replications, with an average branch support of 96.0%. The tree was rooted at the midpoint [46]

Full size image
Table 1 Differential physio-biochemical characteristics between strain Mg02T and N. flavescens CGMCC 4.5723T
Full size table

Conclusions

According to the phylogenetic, phenotypic and chemotaxonomic data, strain Mg02T was considered to be a member of the genus Nocardiopsis. The ANI and dDDH values confirmed that strain Mg02T represented a novel species of the genus Nocardiopsis, for which the name Nocardiopsis changdeensis sp. nov. is proposed, with Mg02T (=MCCC 1K06174T = JCM 34709T) as the type strain.

Description of Nocardiopsis changdeensis sp. nov

Nocardiopsis changdeensis (chang.de.en’sis.N.L. fem. adj. changdeensis, pertaining to changde, a city of Hunan Province in China).

Grow well on all tested media at 28 °C after incubation for 21 days. No diffusible pigment and melanin were observed on all tested media. Growth occurs at 20–45 °C (optimum, 28 °C), pH 6.0–11.0 (optimum, 7.0), and 0–8.0% NaCl (optimum, 1.0%). Nitrate is reduced. Positive in tests for hydrolyses starch, gelatin liquefaction, Tween 40 and urease, negative for Tweens (20, 60 and 80) decomposition. Utilizes acetate, cellobiose, d-fructose, d-galactose, d-mannitol, d-mannose, d-ribose, d-xylose, glucose, glycerol, l-arabinose, l-rhamnose, melibiose, raffinose and sucrose as sole carbon sources for growth, but not lactose or myo-inositol. The whole-cell hydrolysates contain meso-diaminopimelicacid, but no diagnostic sugars. The menaquinones are MK-10(H4), MK-10(H2), MK-10(H8), MK-10 and MK-10(H6). The major cellular fatty acids (>5.0%) are iso-C16:0, C18:1 ω9c, anteiso-C17:0, anteiso-C15:0 and C17:1 ω8c. The polar lipid profile consists of diphosphatidylglycerol (DPG), phosphatidylethanolamine (PE), phosphatidylinositol mannosides (PIM), phosphatidyl methyl ethanolamine (PME), phospholipidscholine (PC), phosphatidyl glycerol (PG), phosphotidyl inositol (PI), phosphoglycolipid (PGL) and unidentified phospholipids (L1, L2, L3).

The GenBank/EMBL/DDBJ accession numbers for the full-length 16S rRNA gene sequence and genome sequence of strain Mg02T are OM368593 and CP074133, respectively.