Microbispora clausenae sp. nov., an endophytic actinobacterium isolated from the surface-sterilized stem of a Thai medicinal plant, Clausena excavala Burm. f.

An endophytic actinobacterium, strain CLES2T, was discovered from the surface-sterilized stem of a Thai medicinal plant, Clausena excavala Burm. f., collected from the Phujong-Nayoa National Park, Ubon Ratchathani Province, Thailand. The results of a polyphasic taxonomic study identified this strain as a member of the genus Microbispora and a Gram-stain-positive, aerobic actinobacterium. It had well-developed substrate mycelia, which were non-motile and possessed paired spores. A phylogenetic evaluation based on 16S rRNA gene sequence analysis placed this strain in the family Streptosporangiaceae , being most closely related to Microbispora bryophytorum NEAU-TX2-2T (99.4 %), Microbispora camponoti 2C-HV3T (99.2 %), Microbispora catharanthi CR1-09T (99.2 %) and Microbispora amethystogenes JCM 3021T and Microbispora fusca NEAU-HEGS1-5T (both at 99.1 %). The major cellular fatty acid of this strain was iso-C16 : 0 and major menaquinone was MK-9(H4). The polar lipid profile of strain CLES2T contained diphosphatidylglycerol, phosphatidylmethylethanolamine, phosphatidylinositol and phosphatidylinositol dimannosides. These chemotaxonomic data confirmed the affiliation of strain CLES2T to the genus Microbispora . The DNA G+C content of this strain was 70 mol%. Digital DNA–DNA hybridization and average nucleotide identity blast values between strain CLES2T and M. catharanthi CR1-09T were 62.4 and 94.0 %, respectively. The results of the polyphasic study allowed the genotypic and phenotypic differentiation of strain CLES2T from its closest species with valid names. The name proposed for the new species is Microbispora clausenae sp. nov. The type strain is CLES2T (=DSM 101759T=NRRL B-65340T).


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Strain CLES2 T was isolated from the stem sample of a Thai medicinal plant (Clausena excavala Burm. f.) collected from the Phujong-Nayoa National Park, Ubon Ratchathani Province, Thailand (14.438954° N 105.344589° E), and processed within 4 h of collection [14]. Surface-sterilized stem tissue was placed onto VL70 medium containing a defined amino acid mixture and solidified with 0.8 % gellan gum [14,15]. Strain CLES2 T emerged as a small colony from the stem tissue after incubation for 2 weeks at 27 °C. Polyphasic taxonomy showed that this strain represents a novel species of the genus Microbispora, for which the name Microbispora clausenae sp. nov. is proposed.
Genomic DNA of strain CLES2 T was extracted and used for 16S rRNA gene amplification and sequencing as described previously [14]. The 16S rRNA gene sequence of CLES2 T was analysed using the EzTaxon-e server ( www. ezbiocloud. net) [16]. The 16S rRNA gene sequences of representatives of all valid strains of the genus Microbispora available from GenBank/EMBL were subsequently aligned with strain CLES2 T using clustal_x [17] with Nonomuraea cavernae SYSU K10005 T as the outgroup. The phylogenetic trees were reconstructed based on the maximum-likelihood and neighbour-joining algorithms using the software package mega version X [18]. The Tamura-Nei model [19] was applied to the maximum-likelihood analysis using the Subtree-Pruning-Regrafting-Extensive (SPR level 5) program. The neighbour-joining algorithm [20] was used according to Kimura's two-parameter model [21]. The topology of the tree  Genomic DNA for whole genome sequencing of strain CLES2 T was extracted using GenElute (Sigma) and a short insert size library was prepared. The genome was sequenced by the Hiseq X-ten platform (Illumina; 2×150 bp paired-end reads) at the Beijing Genome Institute (BGI; Hong Kong). De novo assembly of the reads was achieved by using Unicycler (version 0.4.8; without long reads) [23].
The draft assembly of the genome of strain CLES2 T was submitted to GenBank with the accession number JACBWX000000000. The phylogenetic tree of the genomes of strain CLES2 T and its related taxa was reconstructed using the Type (strain) Genome Server (TYGS) [24,25]. The tree inferred with FastME version 2.1.6.1 [26] from genome blast distance phylogeny (GBDP) and distances were calculated from genome sequences. The branch lengths were scaled in terms of GBDP distance formula d4.
The average nucleotide identity (ANI) values between strain CLES2 T and four related species were evaluated with pairwise genome alignment by using the ANI-blast (ANIb) and ANI-MUMmer (ANIm) algorithms [27]. Correlation indexes of tetra-nucleotide signature (Tetra) were applied within the JSpecies Web Server [27,28]. Digital DNA-DNA hybridization (dDDH) values were calculated by applying the Genometo-Genome Distance calculator (GGDC 2.1; blast+ method) in which formula 2 (identities/HSP length) was applied to the incomplete draft genome [24].
The draft genome sequence of strain CLES2 T was 7.25 Mb with a DNA G+C content of 70 mol%. The genome analysis resulted in the following ANIb and ANIm values between the draft genome of strain CLES2 T and its related species: . According to the report of Richter and Rosselló-Móra [28], the ANI species delineation cutoff is 95-96 %. However, the ANIm value between strain CLES2 T and M. catharanthi CR1-09 T was 95.6 % ( Table 1). An investigation by Kim et al. [29] revealed that some strains were identified as novel species having an ANI value higher than 96 %. Therefore, the differential characteristics between strain CLES2 T and this type strain should be considered thoroughly. The Tetra values between strain CLES2 T and M. catharanthi CR1-09 T , M. bryophytorum NEAU-TX2-2 T and M. fusca NBRC 13915 T were 0.9982, 0.9980 and 0.9942, respectively, which were well below the cut off value of ≥0.999 for the same species [28].
The phylogenetic tree based on the TYGS revealed the relationship between strain CLES2 T and the related type strains (Fig. 2).
The result clearly showed that strain CLES2 T was positioned in a different node with its closest strains, M. bryophytorum NEAU-TX2-2 T and M. catharanthi CR1-09 T . Also, the phylogenetic tree of the genome showed that strain CLES2 T was placed in a different species cluster from these two type strains (Fig. 2).
Whole-cell sugar was analysed by the TLC method of Hasegawa et al. [31] and diaminopimelic acid (DAP) was identified by TLC using the method of Bousfield et al. [32].
The meso-isomer of DAP was detected from strain CLES2 T and the whole-cell sugar contained galactose, glucose, mannose and madurose, while the whole-cell sugars of the closest type strains, M. bryophytorum NEAU-TX2-2 T , were glucose and madurose [8] and M. catharanthi CR1-09 T contained galactose, glucose, madurose and a small amount of xylose [9].
Isoprenoid quinones were extracted and purified using the method of Minnikin et al. [34] and analysed by reverse phase LC-MS employing UV detection and electrospray mass spectrometry (ESI) according to Kaewkla and Franco [36]. For the analysis of whole-cell fatty acids, strain CLES2 T and its three closest type strains were grown for 7 days at 27 °C in tryptic soya broth (Oxoid) in an Erlenmeyer flask at 150 r.p.m. and harvested by centrifugation. Washed cells (100 mg) were saponified, methylated and extracted, and then the fatty acid methyl esters (FAMEs) were determined by following the protocols described by Microbial Identification Inc. (midi) [37]. The sactin6 method and Sherlock version 6.3 were used for analysis.
The results of our chemotaxonomic study showed that strain CLES2 T was clearly different from M. catharanthi CR1-09 T .  [39]. Colour determination was based on the Methuen Handbook of Colour [40]. Strain CLES2 T showed morphology belonging to the genus Microbispora, with a substrate mycelium that was well developed and an aerial mycelium formed well in some media. Cultural characteristics on different media are demonstrated in Table S1. Electron microscopy revealed that it formed paired spores (approximately 1×0.8 microns) with smooth surfaces (Fig. S3).
The physiological and biochemical characteristics of strain CLES2 T and its four closest type strains were studied. Acid production from 23 carbohydrates and decomposition of l-tyrosine, urea and aesculin were evaluated according to the methods of Gordon et al. [41]. Hydrolysis of starch, catalase production, assimilation of seven organic acids and utilization of four phenolic compounds as sole carbon source were described by Kurup and Schmitt [42]. Growth at different temperatures (4,15,27,37,45 and 55 °C), NaCl concentrations (1, 3, 5, 10, 15 and 20 %, w/v) and pH between pH 4 and 10 (in 1 pH unit intervals) were evaluated after incubation at 37 °C for 7-14 days on ISP 2 medium [42].
Strain CLES2 T could produce acid from fucose, maltose, myo-inositol and methyl d-glucopyranoside, but the closest type strain, M. bryophytorum NEAU-TX2-2 T , could not. In contrast, the closest type strain could produce acid from Table 3. Differential characteristics between strain CLES2 T and related species of Microbispora Strain: 1, CLES2 T ; 2, Microbispora catharanthi CR1-09 T ; 3, Microbispora bryophytorum NEAU-TX2-2 T ; 4, Microbispora camponoti 2C-HV3 T ; 5, Microbispora amethystogenes JCM 3021 T ; +, Positive or present; w, weakly positive; −, negative or absent; nd, not done. Catalase was positive for all strains. All strains could produce acid from arabinose, fructose, galactose, glucose, mannose, mannitol, sucrose and xylose but not from sorbitol. All strains could assimilate acetate but not tartrate. They could not use phenol and benzene as sole carbon sources. All strains could hydrolyse aesculin. They could grow at 1 % NaCl (w/v) but not at 15, and 20 % (w/v) NaCl. All strains could grow at between pH 6 and 10 and between 27 and 37 °C but could not grow at 4 and 55 °C and at pH 4. meso-erythritol but strain CLES2 T could not. Also, strain CLES2 T could decompose l-tyrosine, assimilate malate and propionate, grow at 45 °C and use pyridine and toluene as sole carbon sources but the closest type strain could not. On the other hand, the closest type strain could decompose urea, grow at 5 and 10 % NaCl (w/v) but strain CLES2 T could not.
Based on the data of ANIb and ANIm including dDDH, strain CLES2 T shared the highest values with M. catharanthi CR1-09 T . The physiology and biochemical properties of these two strains were compared. The result showed that strain CLES2 T differed significantly from this reference strain. Strain CLES2 T could not produce soluble pigment, but the reference strain could. The spore colour of strain CLES2 T was reddish white on ISP 2 and ISP 7, but that of the reference strain was pinkish white. In addition, strain CLES2 T could hydrolyse starch and skimmed milk, while the reference strain could not. Also, strain CLES2 T grew weakly at pH 5 and 15 °C, but the reference strain could not. They differed in terms of acid production and organic assimilation. Strain CLES2 T produced acid from raffinose, rhamnose and trehalose and assimilated propionate and malate, but the reference strain could not. Also, strain CLES2 T produced acid from myo-inositol and maltose, but the reference strain could only do so weakly. Also, strain CLES2 T could use pyridine and toluene as sole carbon sources, but the reference strain could not.
Based on the results of this polyphasic study, strain CLES2 T is proposed to represent a novel species of the genus Microbispora, named Microbispora clausenae sp. nov.
Aerobic and catalase-positive. Grows between 15 and 45 °C, but best growth occurs between 27 and 45 °C. Grows well between pH 6.0 and 10.0 and in the presence of 3 % (w/v) NaCl. Colonies are wrinkled with a dry surface. Substrate mycelium develops well on most media and aerial mycelium forms well on some media. Diffusible pigments are observed on ISP 2. The mycelium is extensively branched and forms paired spores. Paired rod-shaped spores (0.8×1.0 µm) are observed. Produces acid from arabinose, cellobiose, fucose, fructose, galactose, glucose, mannose, mannitol, maltose, myo-inositol, methyl d-glucopyranoside, sucrose, trehalose, rhamnose, ribose, salicin, trehalose and xylose, but not from ducitol, meso-erythritol or sorbitol. Assimilates acetate, citrate, lactate, malate and propionate, but not tartrate. Decomposes l-tyrosine, starch and skimmed milk, but not urea. Uses pyridine and toluene, but not phenol and benzene as a sole carbon source.
The type strain, CLES2 T (=DSM 101759 T =NRRL B-65340 T ), is an endophytic actinobacterium isolated from the stem of a Thai medicinal plant, Clausena excavala Burm. f., which grows in Phujong-Nayoa National Park, Ubon Ratchathani Province, Thailand. The GenBank/EMBL/ DDBJ accession number for the 16S rRNA gene sequence of strain CR1-09T is KX394342. The Whole Genome Shotgun project of strain CLES2 T has been deposited at DDBJ/ENA/GenBank under the accession JACBWX000000000. The version described in this paper is version JACBWX000000000.

Funding information
This work has been granted by the Office of Higher Education Commission and Thailand Research Fund, contract number MRG5580168.