Actinomadura harenae sp. nov., a novel actinomycete isolated from sea sand in Sanya

766 Actinomadura harenae sp. nov., a novel actinomycete isolated from sea sand inSanya JiangmeihuiHu1, ChuanyuHan1, BingYu1, JunweiZhao1, XiaoweiGuo1, YueShen1, XiangjingWang1,* and WenshengXiang1,2,* TAXONOMIC DESCRIPTION Hu etal., Int. J. Syst. Evol. Microbiol. 2020;70:766–772 DOI 10.1099/ijsem.0.003819 Author affiliations: 1Key Laboratory of Agricultural Microbiology of Heilongjiang Province, Northeast Agricultural University, No. 59 Mucai Street, Xiangfang District, Harbin 150030, PR China; 2State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing, PR China. *Correspondence: Xiangjing Wang, wangneau2013@ 163. com; Wensheng Xiang, xiangwensheng@ neau. edu. cn Keywords: Actinomadura harenae sp. nov.; polyphasic taxonomy; 16S rRNA gene. Abbreviations: ANI, average nucleotide identity; BA, Bennett’s agar; CA, Czapek’s agar; dDDH, digital DNA–DNA hybridization; ISP, International Streptomyces Project; NA, nutrient agar. The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain NEAUHt49T is MK203829. This Whole Genome Shotgun project has been deposited at DDBJ/ENA/GenBank under the accession RFFG00000000. The version described in this paper is version RFFG01000000.1. Three supplementary figures and three supplementary tables are available with the online version of this article. 003819 © 2020 The Authors Abstract A novel actinomycete, designated strain NEAUHt49T, was isolated from sea sand sampled in Sanya and characterized by using a polyphasic approach. The 16S rRNA gene sequence analysis showed that strain NEAUHt49T was most closely related to Actinomadura rhizosphaerae SDA37T (98.8 %), Actinomadura logoneensis NEAUG17T (98.6 %), Actinomadura oligospora ATCC 43269T (98.6 %) and Actinomadura gamaensis NEAUGz5T (98.6 %). The results of phylogenetic analysis based on the 16S rRNA gene sequences indicated that strain NEAUHt49T formed a cluster with A. rhizosphaerae SDA37T, A. logoneensis NEAUG17T, A. oligospora ATCC 43269T, A. gamaensis NEAUGz5T and Actinomadura rupiterrae CS5AC15T (96.4 %). Mesodiaminopimelic acid was detected in its cell walls and glucose, madurose, mannose and ribose were detected in wholecell hydrolysate. The polar lipids were found to consist of diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol, phosphatidylinositolmannoside and two unidentified lipids. The majoy menaquinone was MK-10(H6) and the minor menaquinones were MK-9(H4) and MK-9(H8). The major fatty acids were C16 : 0, C18 : 1ω9c, 10methyl C18 : 0 and isoC16 : 0. Moreover, morphological and chemotaxonomic characteristics of properties of strain NEAUHt49T also confirmed the affiliation of the isolate to the genus Actinomadura. However, DNA–DNA relatedness, physiological and biochemical data showed that strain NEAUHt49T could be distinguished from its closest relatives. Therefore, strain NEAUHt49T represents a novel species of the genus Actinomadura, for which the name Actinomadura harenae sp. nov. is proposed, with strain NEAUHt49T (=CGMCC 4.7499T=JCM 32659T) as the type strain. The genus Actinomadura, a member of the family Thermomonosporaceae, was first established by Lechevalier and Lechevalier [1] with Actinomadura madurae as type species and its description has been emended by Zhang et al. [2, 3], Miyadoh and Miyara [4], and Zhao et al. [5]. Actinomadura strains are mostly distributed in terrestrial soils [6, 7] but many strains are found in other ecosystems such as plant tissues [8, 9] and marine sources [10, 11]. Morphologically, the genus is characterized by the production of welldeveloped, nonfragmenting vegetative hyphae and aerial mycelium, on the tips of which spore chains of various lengths are arranged in straight, hooked or spiral form. The smooth, spiny or wartysurfaced spores are nonmotile, oval or rodshaped. Single spherical spore vesicles may be produced. The members of the genus Actinomadura are also characterised by a number of chemical properties, including mesodiaminopimelic acid as the diagnostic diamino acid in the cellwall peptidoglycan and madurose as the characteristic sugar in wholecell hydrolysates; hexahydrogenated menaquinones with nine isoprene units [MK-9(H6)] as the predominant isoprenologue; diphosphatidylglycerol, phosphatidylinositol and phosphatidylinositolmannoside as major phospholipids; complex mixtures of fatty acids with major amounts of hexadecanoic (C16 : 0), 14methylpentadecanoic (isoC16 : 0) and 10methyloctadecanoic acid (tuberculostearic acid) [12]. The G+C contents of the genomic DNA range from 65.0 to 73.0 mol%. During an investigation of novel rare actinomycetes from sand collected from Wuzhizhou island in Sanya, 767 Hu etal., Int. J. Syst. Evol. Microbiol. 2020;70:766–772 an Actinomaduralike strain, NEAUHt49T, was isolated. In this study, we performed polyphasic taxonomy on the isolate including morphological, physiological, chemotaxonomic and phylogenetic characteristics and proposed that it should be classified as a new species of the genus Actinomadura. Strain NEAUHt49 T was isolated from sea sand collected from Wuzhizhou island in Sanya, Hainan Province, PR China (18° 32′ N, 109° 77′ E). The sand sample was airdried at room temperature for 14 days before isolation for actinomycetes. After drying, 5 g sand sample was mixed with 45 ml distilled water and followed shaking on a rotary shaker at 250 r.p.m., 28 °C for 30 min. Then a 200 µl sample of the suspension was spread on a plate of Gause’s synthetic agar no. 1 [13] supplemented with cycloheximide (50 mg l −1 ) and nalidixic acid (20 mg l−1). After 21 days of aerobic incubation at 28 °C, colonies were transferred and purified on oatmeal agar [International Streptomyces Project (ISP) medium 3] [14] and maintained as glycerol suspensions (20 %, v/v) at −80 °C. The reference strains Actinomadura rhizosphaerae SDA37T was purchased from the Japan NITE Biological Resource Centre (NBRC), Actinomadura oligospora ATCC 43269T was purchased from the Japan Collection of Microorganisms (JCM) and Actinomadura rupiterrae CS5AC15T was purchased from the Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSM). The strains Actinomadura logoneensis NEAUG17T and Actinomadura gamaensis NEAUGz5T were isolated and stored in our laboratory. All strains were cultured under the same conditions for comparative analyses. Morphological characteristics were observed by light microscopy (eclipse E200, Nikon) and scanning electron microscopy (SU8010, Hitachi) using cultures grown on ISP 3 agar at 28 °C for 7 and 14 days, respectively. Samples for scanning electron microscopy were prepared as described by Jin et al. [15] Cultural characteristics were determined on ISP 1–7 agars [14], Bennett’s agar (BA) [16], nutrient agar (NA) [17] and Czapek’s agar (CA) [17] after 14 days at 28 °C. Colour determination was done by using colour chips from the ISCCNBS colour charts standard samples No. 2106 [18]. Growth at different temperatures (4, 10, 15, 20, 28, 35, 37, 40, 45 and 50 °C) was determined on ISP 3 medium after incubation for 14 days. Growth tests for pH range (pH 3.0–12.0, at intervals of 1.0 pH unit) and NaCl tolerance (0, 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10 %, w/v) were tested in Glucoseyeast extract powder (GY) broth [19] using the buffer system described by Xu et al. [20] at 28 °C for 14 days on a rotary shaker. The utilization of sole carbon and nitrogen sources (0.5 % w/v), decomposition of cellulose, hydrolysis of starch and aesculin, reduction of nitrate, coagulation and peptonization of milk, liquefaction of gelatin, and production of H 2 S were examined as described previously [21, 22]. Production of catalase, hydrolysis of Tweens (20, 40 and 80) and production of urease were tested as described by Gordon et al. [21] and Williams et al. [23] Biomass for chemotaxonomic studies was prepared by growing these strains in GY broth in shake flasks (250 r.p.m.) at 28 °C for 5 days. Cells were harvested by centrifugation, washed with distilled water and freezedried. The isomer of mesodiaminopimelic acid in the cellwall peptidoglycan was derivatised and analysed by an HPLC method [24] using an Agilent TCC18 column (250×4.6 mm i.d. 5 µm) with a mobile phase consisting of acetonitrile: 0.05 mol l−1 phosphate buffer, pH 7.2 (15 : 85, v/v), at a flow rate of 0.5 ml min−1. The peak detection used an Agilent G1321A fluorescence detector with a 365 nm excitation and 455 nm longpass emission filters. The wholecell sugars were analysed according to the procedures developed by Lechevalier and Lechevalier [25]. Polar lipids in cells were extracted and identified by the method of Minnikin et al. [26]. Menaquinones were extracted from freezedried biomass and purified according to Collins [27]. Extracts were analysed by an HPLCUV method [28] using an Agilent ExtendC18 column (150×4.6 mm, i.d. 5 µm), typically at 270 nm. The mobile phase was acetonitrile–propyl alcohol (60 : 40, v/v). To determine cellular fatty acid compositions, strain NEAUHt49 T and its closely related species were cultivated in GY broth in shake flasks at 28 ℃ for 5 days. Fatty acid methyl esters were extracted from the biomass as described by Gao et al. [29] and analysed by GCMS using the method of Xiang et al. [30]. Extraction of chromosomal DNA and PCR amplification of the 16S rRNA gene sequence were carried out according to the procedure developed by Kim et al. [31]. The PCR product was purified and cloned into the vector pMD19T (Takara) and sequenced using an Applied Biosystems DNA sequencer (model 3730XL). The almost fulllength 16S rRNA gene sequence of strain NEAUHt49T (1519 bp) was obtained and multiply aligned in mega using the clustal_w algorithm and trimmed manually where necessary. The phylogenetic dendrograms of the isolate and its closely related strains were reconstructed with the maximumlikelihood [32] and neighbourjoining [33] algorithms using mega software version 7.0 [34]. The stability of the topology of each phylogenetic tree was assessed by using the bootstrap method with 1000 rtitions [35]. A distance matrix was generated using Kimura’s twoparameter model [36]. All positions containing gaps and missing data were eliminated from the dataset (complete deletion option). The 16S rRNA gene sequence similarities between the strains were calculated on the basis of pairwise alignment using the EzBioCloud server [37]. For draft genome sequencing and assembly, the genomic DNA of strain NEAUHt49T was extracted with the SDS method. The harvested DNA was detected by agarose gel electrophoresis and quantified by Qubit. Wholegenome sequencing was performed on the Illumina HiSeq PE150 platform. Atailed, ligated to pairedend adaptors and PCR amplified with a 350 bp insert was used for the library construction at the Beijing Novogene Bioinformatics Technology Co., Ltd. Illumina PCR adapter reads and lowquality reads from the pairedend were filtered by the step of quality control using our own compiling pipeline. All goodquality paired reads were assembled by using SOAPdenovo [38, 39] (https:// github. com/ aquaskyline) into a number of scaffolds. Then the filter reads were handled by the next step of the gapclosing. Several genomic metrics are now available to 768 Hu etal., Int. J. Syst. Evol. Microbiol. 2020;70:766–772 distinguish between orthologous genes of closely related prokaryotes, including the calculation of average nucleotide identity (ANI) and digital DNA–DNA hybridization (dDDH) values [40, 41]. In the present study, ANI and dDDH values were determined from the genomes of strain NEAUHt49T, A. logoneensis NEAUG17T (QURH00000000) and A. oligospora ATCC 43269T (JADG00000000) using the orthoANIu algorithm from Ezbiocloud [37, 40] and the GenometoGenome Distance Calculator (GGDC 2.0) at http:// ggdc. dsmz. de. Because of the lack of whole genome sequences of strains A. rhizosphaerae NBRC SDA37T, A. gamaensis NEAUGz5T and A. rupiterrae CS5AC15T, DNA–DNA relatedness tests between strain NEAUHt49T and these three related type strains were carried out as described by De Ley et al. [42], under consideration of the modifications described by Huss et al. [43], using a model Cary 100 Bio UV/VISspectrophotometer equipped with a Peltierthermostatted 6×6 multicell changer and a temperature controller with an in situ temperature probe (Varian). The concentration and purity of DNA samples were determined by measuring the optical density at 260, 280 and 230 nm. The DNA samples used for hybridization were diluted to OD260 around 1.0 using 0.1×SSC (saline sodium citrate buffer), then sheared using a JY92II ultrasonic cell disruptor (ultrasonic time 3 s, interval time 4 s, 90 times). The DNA renaturation rates were determined in 2×SSC at 70 °C. The experiments were performed with three replications and the DNA–DNA relatedness value was expressed as the mean of the three values. Morphological observation of a 2 week old culture of strain NEAUHt49T grown on ISP 3 medium revealed that it had the typical characteristics of members of the genus Actinomadura. The cells of strain NEAUHt49T were found to be Grampositive and aerobic. White aerial mycelium was produced abundantly and differentiated into flexuous or straight spore chains consisting of cylindrical spores (0.4–0.6×0.8–1.0 µm); the spore surface was rough (Fig.1). Strain NEAUHt49T exhibited good growth on ISP 1 agar, ISP 2 agar, ISP 3 agar, ISP 6 agar, ISP 7 agar, NA and BA media; moderate growth on ISP 4; and poor growth on ISP 5 and CA media. The colony colours varied from brilliant yellow to greygreen yellow. Moderate olive brown soluble pigment was observed on ISP 7 medium. A summary of cultural characteristics of the isolate are shown in Tables S1 and Fig. S1 (available in the online version of this article). Strain NEAUHt49T grew at a temperature range of 10–45 °C (optimum, 28 °C), pH 5–10 (pH 7) and NaCl tolerance of 0–3 % (0 %). The physiological and biochemical properties of strain NEAUHt49T are given in Table1 and the species description. Chemotaxonomic analyses revealed that strain NEAUHt49T exhibited characteristics which are typical of members of the genus Actinomadura, such as the presence of mesodiaminopimelic acid as the cellwall diamino acid and glucose, madurose, mannose and ribose as wholecell sugars. The polar lipids of strain NEAUHt49T consisted of diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol, phosphatidylinositolmannoside and two unidentified lipids (Fig. S2). Menaquinones of strain NEAUHt49 T were identified as MK-9(H 6 ) (61.5 %), MK-9(H4) (21.5 %) and MK-9(H8) (17.0 %). The cellular fatty acid profile was found to be composed of C16 : 0 (26.4 %), C18 : 1ω9c (25.3 %) and 10methly C18 : 0 (22.7 %), isoC16 : 0 (8.5 %), C 16 : 1 ω7c (5.0 %), C 18 : 0 (4.8 %), C 14 : 0 (2.5 %), C 17 : 1 ω7c (2.4 %), C 17 : 0 (1.7 %), anteisoC 17 : 0 (0.4 %) and isoC 14 : 0 (0.3 %) (Table S2). The DNA G+C content was 72.1 mol%. All the data of the morphological and chemotaxonomic analyses are consistent with the assignment of strain NEAUHt49T to the genus Actinomadura. The 16S rRNA gene sequence (1519 bp) indicated that strain NEAUHt49T should be assigned to the genus Actinomadura using the EzBioClould analysis and was most closely related to A. rhizosphaerae SDA37T (98.8 %), A. logoneensis NEAUG17T (98.6 %), A. oligospora ATCC 43269T (98.6 %) and A. gamaensis NEAUGz5T (98.6 %). The phylogenetic tree based on 16S rRNA gene sequences showed that strain NEAUHt49T formed a stable cluster with A. rhizosphaerae SDA37T, A. logoneensis NEAUG17T, A. oligospora ATCC 43269T, A. gamaensis NEAUGz5T and A. rupiterrae CS5AC15T (96.4 %) in the neighbourjoining tree (Fig.2), a relation - ship also recovered by the maximumlikelihood algorithm (Fig. S3). The assembled genome sequence of strain NEAUHt49T was found to be 9 356 375 bp long and composed of 283 contigs with an N50 value of 75 833 bp, a DNA G+C content of 72.1 mol% and a coverage of 177×. It was deposited in GenBank under the accession number RFFG00000000. The ncbi Prokaryotic Genome Annotation Pipeline revealed seven copies of the 5S rRNA genes, one copy of the 16S rRNA genes, three copies of the 23S rRNA genes, 65 tRNA genes and three copies of noncoding RNA genes. The 16S rRNA gene sequence from whole genome sequence shared 100 % similarity to that from PCR sequencing suggesting that the genome sequence was not contaminated. Other general features of the genome sequence are presented in Table S3. Fig. 1. Scanning electron microscope image of strain NEAUHt49T grown on ISP 3 medium for 2 weeks at 28 °C. Bar 1 µm 769 Hu etal., Int. J. Syst. Evol. Microbiol. 2020;70:766–772 DNA–DNA hybridization was carried out between strain NEAUHt49T and A. rhizosphaerae SDA37T, A. gamaensis NEAUGz5T and A. rupiterrae CS5AC15T to determine whether the isolate represents a novel species. Strain NEAUHt49T displayed a DNA–DNA relatedness of 50.5±3.6 % with A. rhizosphaerae SDA37T, 34.9±3.4 % with A. gamaensis NEAUGz5T and 25.5±4.1 % with A. rupiterrae CSAC15T. dDDH and ANI values were employed to further clarify the relatedness between strain NEAUHt49T and A. logoneensis NEAUG17T and A. oligospora ATCC 43269T. The levels of Table 1. Differential physiological characteristics of strain NEAUHt49T and its closely related type strains Strains: 1, NEAUHt49T; 2, Actinomadura rhizosphaerae SDA37T; 3, Actinomadura logoneensis NEAUG17T; 4, Actinomaduraoligospora ATCC 43269T; 5, Actinomadura gamaensis NEAUGz5T; 6, Actinomadura rupiterrae CS5AC15T. All data are from this study. +, Positive; −, negative. Characteristic 1 2 3 4 5 6 Utilization as sole carbon source: lArabinose −++−+− Dulcitol ++−+−− dFructose +++−+− dGalactose −−+−+− mesoInositol +−−−−+ Lactose −++−−+ Maltose +++++− dMannitol +−−−−+ Raffinose −−−−+− lRhamnose +−+−−− dRibose −+−+−− dSorbitol −+−+−− Sucrose +−−−+− dXylose −+−−−− Utilization as sole nitrogen source: lAsparagine ++−−−− Creatine −++−−− lGlutamic acid ++−+−+ lSerine +++−−− Growth temperature range (°C) 10–45 20–40 25–42 25–42 15–40 20–42 Growth pH range 5–10 6–8 5–10 6–10 6–8 5–11 NaCl tolerance range (w/v, %) 0–3 0–4 0–4 0–2 0–3 0–2 Aesculin hydrolysis + + − + − + Cellulose decomposition + − − − + − Gelatin liquefaction − + − + − + Nitrate reduction − + − − − − Production of H2S −−−−−+ Production of urease + − − − − − Tween hydrolysis: 40 ++−+++ 80 ++−+++ 770 Hu etal., Int. J. Syst. Evol. Microbiol. 2020;70:766–772 Actinomadura verrucosospora NBRC 14100T (U49011) Actinomadura luteofluorescens IFO 13057T (U49008) Actinomadura citrea IFO 14678T (AJ420139) Actinomadura mexicana A290T (AF277195) Actinomadura formosensis JCM 7474T (AJ293703) Actinomadura macra NBRC 14102T (U49009) Actinomadura madurae DSM 43067T (X97889) Actinomadura geliboluensis A8036T (HQ157187) Actinomadura napierensis B60T (AY568292) Actinomadura latina JCM 10674T (AY035998) Actinomadura meyerae DSM 44715T (AY273787) Actinomadura bangladeshensis 3-46-b3T (AB331652) Actinomadura chokoriensis 3-45-a/11T (AB331730) Actinomadura xylanilytica BK147T (FR692101) Actinomadura yumaensis JCM 3369T (AF163122) Actinomadura livida JCM 3387T (AJ293706) Actinomadura sputi IMMIB L-889T (FM957483) Actinomadura adrarensis ACD12T (KU356942) Actinomadura rugatobispora IFO 14382T (U49010) Actinomadura vinacea JCM 3325T (AF134070) Actinomadura viridis IFO 15238T (AJ420141) Actinomadura sporangiiformans NEAU-Jh2-5T (KM000834 ) Actinomadura jiaoheensis NEAU-Jh1-3T (KM000835) Actinomadura apis IM17-1T (AB557596) Actinomadura deserti BMP B8004T (MF972517) Actinomadura fibrosa ATCC 49459T (AJ293702) Actinomadura barringtoniae GKU 128T (KF667497) Actinomadura nitritigenes DSM 44137T (AY035999) Actinomadura fulvescens IFO 14347T (AJ420137) Actinomadura montaniterrae CYP1-1BT (LC126428) Spirillospora albida IFO12248T (D85498) Actinomadura rudentiformis HMC1T (DQ285420) Actinomadura flavalba YIM 61435T (FJ157185) Actinomadura rayongensis RY35-68T (AB889544) Actinomadura keratinilytica WCC-2265T (EU637009) Actinomadura miaoliensis BC 44T-5T (EF116925) Actinomadura rubrobrunea NBRC 15275T (EU637008) Actinomadura viridilutea IFO14480T (D86943) Actinomadura oligospora ATCC 43269T (AF163118) Actinomadura rupiterrae CS5-AC15T (FM210337) Actinomadura gamaensis NEAU-Gz5T (KT989505) Actinomadura logoneensis NEAU-G17T (MG189967) Actinomadura harenae NEAU-Ht49T (MK203829) Actinomadura rhizosphaerae SDA37T (LC369500) Actinomadura hibisca NBRC 15177T (AJ293705) Actinomadura kijaniata NBRC 14229T (X97890) Actinomadura namibiensis DSM 44197T (AJ420134) Actinocorallia lasiicapitis 3H-GS17T (KR261652) Actinocorallia glomerate JCM 9376T (AF134068) Actinocorallia aurea IFO 14752T (AB006177) Actinocorallia herbida DSM 44254T (RJKE01000001) Streptomyces glauciniger FXJ14 T (AY314782) 100 95 94 98 100 61 68 97 100 90 87 59 100 80 77 67 68 83 84 72 83 51 67 59 76 55 68 57 98 52 0.01 * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * Fig. 2. Neighbourjoining tree based on 16S rRNA gene sequences (1371 bp) showing the relationships between strain NEAUHt49T (in bold) and related species of top 50 16S rRNA gene sequence. Asterisks indicate branches that were also recovered using the maximumlikelihood methods. Bootstrap values (expressed as percentages of 1000 replications) of above 50 % are shown at branch points. Streptomyces glauciniger FXJ14T (AY314782) was used as the outgroup. Bar, 0.01 substitutions per nucleotide position. 771 Hu etal., Int. J. Syst. Evol. Microbiol. 2020;70:766–772 dDDH between them were 32.9±2.5 and 37.1±2.5 %, respectively. These values were below the threshold value of 70 % recommended by Wayne et al. [44] for assigning strains to the same genomic species. Similarly, low ANI values of 86.8 and 89.0 % were found between strain NEAUHt49T and its reference strains respectively for A. logoneensis NEAUG17T and A. oligospora ATCC 43269T, a result well below the threshold used to delineate prokaryote species [45, 46]. Besides the genotypic evidence above, the novel strain showed remarkable difference to the other closely related type strains by several phenotypic characteristics. For instance, their different colony colours on ISP 2, ISP 3, ISP 7 and Bennett’s agar media at 28 °C for 14 days (Fig. S3). Strain NEAUHt49T could grow at 45 °C, while A. rhizosphaerae NBRC SDA37T, A. logoneensis NEAUG17 T , A. oligospora ATCC 43269 T , A. gamaensis NEAUGz5T and A. rupiterrae CS5AC15T could not. The novel strain could utilize dmannitol and mesoinositol as sole carbon source, while its reference strains, A. rhizosphaerae NBRC SDA37T, A. logoneensis NEAUG17T, A. oligospora ATCC 43269T and A. gamaensis NEAUGz5T, could not; lasparagine was utilized as a sole nitrogen source for strain NEAUHt49T, while in A. logoneensis NEAUG17T, A.oligospora ATCC 43269 T , A. gamaensis NEAUGz5 T and A. rupiterrae CS5AC15T it was not. Other phenotypic differences included NaCl tolerance, pH range of growth, decomposition of cellulose, hydrolysis of aesculin and Tweens (40 and 80), liquefaction of gelatin, nitrate reduction, production of H2S and urease, and utilization of larabinose, dulcitol, dfructose, dgalactose, lactose, maltose, raffinose, lrhamnose, dribose, dsorbitol, sucrose, dxylose, creatine, lglutamic acid and lserine (Table1). In conclusion, it is evident from the genotypic, phenotypic and chemotaxonomic data that strain NEAUHt49 T represents a novel species of the genus Actinomadura, for which the name Actinomadura harenae sp. nov. is proposed. DESCRIPTION Of ActinomAdurA hArenAe SP. NOv. Actinomadura harenae ( ha. re′nae. L. gen. n. harenae of sand). The cells of the strain are found to be Grampositive and aerobic. White aerial mycelium is produced abundantly and differentiates into flexuous or straight spore chains consisted of cylindrical spores (0.4–0.6×0.8–1.0 µm), the spore surface is rough on ISP 3 medium. Good growth on ISP 1 agar, ISP 2 agar, ISP 3 agar, ISP 6 agar, ISP 7 agar, NA and BA media; moderate growth on ISP 4 agar; and poor growth on ISP 5 agar and CA media. The colony colours vary from brilliant yellow to greygreen yellow on agar media and the strain produces moderate olive brown soluble pigment on ISP 7 medium. Strain NEAUHt49T grows at 10–45 °C (optimum, 28 °C), pH 5–10 (pH 7) and NaCl tolerance of 0–3 % (0 %). Positive for decomposition of cellulose, hydrolysis of aesculin and Tweens (40 and 80) and production of urease, but negative for hydrolysis of starch and Tween 20, liquefaction of gelatin, peptonization and coagulation of milk, production of H 2 S, and reduction of nitrate. Dulcitol, dfructose, dglucose, mesoinositol, maltose, dmannitol, dmannose, lrhamnose and sucrose are utilized as sole carbon sources, but not larabinose, dgalactose, lactose, raffinose, dribose, dsorbitol or dxylose. lAlanine, larginine, lasparagine, laspartic acid, lglutamic acid, lglutamine, glycine, lproline, lserine, lthreonine and ltyrosine are utilized as sole nitrogen sources, but not creatine. The diagnostic diamino acid of the cell wall is mesodiaminopimelic acid. Wholecell sugars contain glucose, madurose, mannose and ribose. The polar lipid profile consists of diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol, phosphatidylinositolmannoside and two unidentified lipids. The menaquinones are MK-9(H6), MK-9(H4) and MK-9(H8). Major fatty acids are C16 : 0, C18 : 1ω9c, 10methyl C18 : 0 and isoC16 : 0 (>8 %). The G+C content of the DNA of the type strain is 72.1 mol%. The type strain is NEAUHt49T (=CGMCC 4.7499T=JCM 32659T), isolated from sea sand collected from Wuzhizhou island in Sanya, Hainan Province, PR China. The GenBank/ EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain NEAUHt49 T is MK203829. This Whole Genome Shotgun project has been deposited at DDBJ/ENA/ GenBank under the accession number RFFG00000000. The version described in this paper is version RFFG01000000.1. Funding information This work was supported in part by grants from the National Natural Science Foundation of China (No. 31572070) and the Research Science Foundation in Technology Innovation of Harbin (2017RAQXJ022). Acknowledgements We are grateful to Professor Aharon Oren for helpful advice on the specific epithet. Conflicts of interest The authors declare that there are no conflicts of interest. References 1. Lechevalier HA, Lechevalier MP. A critical evaluation of the genera of aerobic actinomycetes. In: Prauser H (editor). The Actinomycetales. Jena: VEB Gustav Fischer Verlag; 1968. pp. 393–405. 2. Zhang Z, Wang Y, Ruan J. Reclassification of Thermomonospora and Microtetraspora. Int J Syst Bacteriol 1998;48:411–422. 3. Zhang Z, Kudo T, Nakajima Y, Wang Y. Clarification of the relationship between the members of the family Thermomonosporaceae on the basis of 16S rDNA, 16S23S rRNA internal transcribed spacer and 23S rDNA sequences and chemotaxonomic analyses. 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