Streptomyces aquilus sp. nov., a novel actinomycete isolated from a Chinese medicinal plant

1912 Streptomyces aquilus sp. nov., a novel actinomycete isolated from a Chinese medicinalplant KaiqinLi1†, YihuiGuo1†, JunzhenWang2, ZhiyongWang3, JiarongZhao1 and JianGao1,4,* TAXONOMIC DESCRIPTION Li etal., Int. J. Syst. Evol. Microbiol. 2020;70:1912–1917 DOI 10.1099/ijsem.0.003995 Author affiliations: 1School of Life Science, Hunan University of Science and technology, Xiangtan 411201, PR China; 2Xichang Institute of Agricultural Science, Liangshan 615000, PR China; 3Pengshui Branch of Chongqing Tobacco Company, Pengshui 409600, PR China; 4Key Laboratory of Ecological Remediation and Safe Utilization of Heavy MetalPolluted Soils, College of Hunan Province, Xiangtan 411201, PR China. *Correspondence: Jian Gao, xtgojian@ 126. com Keywords: Streptomyces aquilus sp. nov.; Xanthium sibiricum; Polyphasic taxonomy. Abbreviations: JCM, Japan correction of Microorganisms. 1. The GenBank accession numbers for 16S rRNA gene sequences of strain GGCR-6T is MH718844. 2. The GenBank accession numbers for whole genome sequences of strain GGCR-6T is CP034463. †These authors contributed equally to this work Five supplementary tables and three supplementary figures are available with the online version of this article. 003995 © 2020 The Authors Abstract The taxonomic position of a novel actinomycete isolate, designated strain GGCR-6T, isolated from the healthy leaves of Xanthium sibiricum collected from the botanic garden of Hunan University of Science and Technology in Hunan province, PR China, was determined by a polyphasic approach. GGCR-6T grew well on ISP series media and formed welldeveloped, branched substrate hyphae and aerial mycelium that differentiated into straight spore chains consisting of cylindrical spores with smooth surfaces. The diagnostic diamino acid was lldiaminopimelic acid. The major menaquinones were MK-9(H8), MK-9(H2), MK-9 and MK-9(H6). The polar lipids were diphosphatidylglycerol, phosphatidylethanolamine, phosphotidylinositol and phosphatidylinositol mannosides. The predominant fatty acids were C16 : 1ω9c, isoC16 : 0 and C16 : 0. The phenotypic characteristics of GGCR-6T indicated that it represented a member of the genus Streptomyces. Phylogenetic analysis based on the 16S rRNA gene sequence indicated that GGCR-6T was most closely related to Streptomyces cyaneus NRRL B2296T and Streptomyces griseoruber NRRL B1818T. However, the digital DNA–DNA hybridization, the average nucleotide identity and the multi locus sequence analysis evolutionary distance clearly separate GGCR-6T from the phylogenetically closely related species. Furthermore, the novel isolate was distinctly differentiated from S. cyaneus NRRL B2296T and S. griseoruber NRRL B1818T by morphological, physiological and biochemical characteristics. Based on these data, strain GGCR-6T should be designated as a representative of a novel species of the genus Streptomyces, for which the name Streptomyces aquilus sp. nov. is proposed. The type strain is strain GGCR-6T (=CICC 11055T=JCM 33584T). Antimicrobials, especially antibiotics, have played a crucial role in modern medicine. But in the past 25 years, their misuse and overuse has made them less effective as bacteria develop resistance [1, 2]. How can the challenges of resistance emergence be met? An important pathway is the development of new bioactive products, particularly those derived from microorganisms. Endophytes are microorganisms that exist inside plant tissues without having any negative effects on the host plant [3]. There is evidence that endophytic actinomycetes are found in almost all medicinal plants studied [4, 5], and possess the potential to produce unique secondary metabolites, which can be exploited in pharmaceutical, agricultural and other industries [6–9]. Thus, there is a growing interest by researchers in bioprospecting of endophytic actinobacteria communities inhabiting plants from various ecosystems. Recently, during our ongoing survey on diversity and biopotential of plantassociated endophytic actinobacteria, strain GGCR-6T, which exhibited weak antimicrobial activity against Staphylococcus aureus, was isolated from Xanthium sibiricum Patrin ex Widder. Phylogenetic analysis based on the 16S rRNA gene sequences indicated that GGCR-6T was most closely related to Streptomyces cyaneus NRRL B-2296T and Streptomyces griseoruber NRRL B-1818T. However, there were distinctly differences in morphological and cultural characteristics between GGCR-6 T and these strains. In the present work, the results of a polyphasic taxonomic study of a novel Streptomyces strain GGCR-6T, are presented. GGCR-6T was isolated from the healthy leaves of a medicinal plant X. sibiricum collected from the botanic garden of Hunan University of Science and Technology in Hunan province (27° 1913 Li etal., Int. J. Syst. Evol. Microbiol. 2020;70:1912–1917 54′ N, 112° 54′ E), PR China. The leaf segments were firstly washed in ultrapure water to remove adhered epiphytes and soil debris. Then, the tissue surfaces were sterilized according to a procedure described previously [10]. GGCR-6T was isolated and purified by the methods described by Mo et al. [11]. The purified isolate was maintained on Gauze’s synthetic agar [12] at 4 °C and stored in a 30 % (w/v) glycerol suspension at −80 °C. S. cyaneus CGMCC 4.1671T(=NRRL B-2296T) was purchased for reference from the China General Microbiological Culture Collection Centre (CGMCC). The reference strain was cultured under the same conditions for comparative testing. For chemical and molecular analyses, biomass was prepared by culturing in Gauze’s synthetic medium for 4–7 days at 28 °C in a rotary shaker (180 r.p.m.) and was then collected at the exponential phase of growth by centrifugation. The morphology of the spore chain and the spore surface ornamentation of GGCR-6T were observed by light microscope (BX41, Olympus) and scanning electron microscopy (JSM6610LV, JEOL) of 14dayold cultures on Gauze’s synthetic agar, respectively. The cultural properties of GGCR6T were evaluated according to the guidelines of the International Streptomyces Project (ISP) as described by Shirling and Gottlieb [13]. The colour of colonies and soluble pigments were determined according to the colour standard [14]. The optimum pH range, temperature and NaCl tolerance for cell growth were determined according to the methods of Verma et al. [7]. The utilization of carbon and nitrogen sources was determined by the methods of Shirling and Gottlieb [13]. Susceptibility to antimicrobial agents was examined by the disc diffusion method [15] using a set of antibioticimpregnated discs (Product code: S1100, Hangzhou Microbial Reagent). The other physiological and biochemical tests, such as aesculin hydrolysis, gelatin liquefaction, hydrogen sulfide production, nitrate reduction, starch hydrolysis and degradation tests for tweens (20, 40, 60 and 80), were carried out according to the methods described by Xu et al. [16]. Cellular fatty acids analysis was carried out by the China Centre of Industrial Culture Collection (CICC; Beijing, PR China) according to the protocol of the Sherlock Microbial ID System (http://www. midiinc. com/). Menaquinones were extracted according to the method of Collins et al. [17] and analyzed by HPLC [18]. The polar lipids analysis was performed as described by Komagata and Suzuki [19]. The isomer of diaminopimelic acid analysis and sugar analysis of wholecell hydrolysates were performed according to the procedures described by Hasegawa et al. [20] and Lechevailer and Lechevailer [21]. Extraction of genomic DNA was carried out by using the microwavebased method [22]. The 16S rRNA gene was amplified with the universal primers 27 f and 1492 r [23]. The PCR product was sequenced by Sangon Biotech (Shanghai, PR China). The obtained sequence was compared with available 16S rRNA gene sequences of species with validly published names from the EzBioCloud public databases (http://www. ezbiocloud. net/ eztaxon) in order to determine an approximate phylogenetic affiliation. The atpD, gyrB, recA, rpoB and trpB genes sequences were directly downloaded from GenBank or drawn from draft genome sequences, and concatenated head to tail inframe. Phylogenetic trees based on the 16S rRNA gene sequences and on the concatenated proteincoding sequences were reconstructed by using the neighborjoining (NJ) [24], maximum likelihood (ML) [25] and maximum parsimony (MP) [26] methods with 1000 bootstrap replications in mega 7.0 [27]. For the multilocus sequence analysis (MLSA) [28], the Kimura twoparameter model [29] was chosen to calculate the genetic distances. The 16S rRNA sequence has been deposited in the GenBank and assigned the accession number MH718844. Complete genome sequencing of GGCR-6T was finished by Beijing Novogene Bioinformatics (Beijing, PR China). The average nucleotide identity (ANI) and digital DNA– DNA hybridization (dDDH) values between the genomes of GGCR-6T and other relatives were calculated using the JSpeciesWS online service [30] and the genometogenome distance calculator [31], respectively. The G+C content of the genomic DNA of GGCR-6Twas calculated using ChunLab's online Average Nucleotide Identity (ANI) calculator [32]. The DNA–DNA hybridization experiment was performed using a method described by De Ley et al. [33]. The genome sequence Fig. 1. Optical micrograph (a) and scanning electron micrograph (b) of GGCR-6T grown on Gause’s synthetic medium at 28 °C after incubation for 14 days. 1914 Li etal., Int. J. Syst. Evol. Microbiol. 2020;70:1912–1917 obtained in this work was deposited in Genbank and assigned the accession number CP034463. GGCR-6T exhibited typical characteristics of members of the genus Streptomyces and formed welldeveloped, branched substrate mycelium and aerial hyphae. The colour of aerial hyphae and substrate mycelium was white and light yellow to brown on Gause’s agar, respectively. Aerial mycelia produced straight chains of cylindrical and smoothsurfaced spores (Fig.1). GGCR-6T grew well on ISP (27) media. No distinct soluble pigment was produced on all tested media. The detailed cultural characteristics are presented in Table S1 (available in the online version of this article). The growth temperature range of GGCR-6 T was 10–45 °C, with an optimal temperature at 30 °C. NaCl tolerance was up to 7 %. GGCR-6T could grow at between pH 5.0 and 12.0, with an optimum pH of 7.0. The detailed physiological and biochemical characteristics are presented in the species description. The cell wall of GGCR-6T contained alanine, asparagine, glutamate, glycine and lldiaminopimelic acid. Wholecell hydrolysates contained glucose, mannose and trace amounts of xylose. Cellular fatty acid compositions of strain GGCR-6T and reference strains are shown in Table S2. The predominant cellular fatty acids (>10 %) of strain GGCR-6T were C16 : 1ω9c (27.1%), isoC 16 : 0 (18.2%) and C 16 : 0 (14.1%); The fatty acids present in smaller amounts (>1 %) were anteisoC15 : 0 (8.5%), isoC 14 : 0 (7.1%), C 14 : 0 (5.1%), isoC 15 : 0 (3.7%), C 15 : 0 (3.4%), isoC16 : 1H (2.7%), anteisoC17 : 0 (1.6%), C15 : 1B (1.3%) and summed feature 6 (1.2 %). The polar lipids were diphosphatidylglycerol (DPG), phosphatidylethanolamine (PE), phosphotidylinositol (PI) and phosphatidylinositol mannosides (PIM) (Fig. S1). The predominant menaquinones were MK-9(H8) (36.3%), MK-9(H 2 ) (25.7%), MK-9 (17.3%) and MK-9(H 6 ) (15.7%). The G+C content of the DNA was 70.9 mol% from the whole genome sequence data. It is within the range (69–78 %) observed for the members of the genus Streptomyces [34]. All the data indicated that GGCR-6T represented a member of the genus Streptomyces. A sequence homology search based on the fulllength 16S rRNA gene sequence (1530 bp) of GGCR-6T indicated that it represented a member of the genus Streptomyces and exhibited 99.5, 99.1, 99.0, 99.0, 98.8, 98.8, 98.8, 98.8, 98.7 and 98.7 % similarities to S. cyaneus NRRL B-2296 T , S. shaanxiensis CCNWHQ 0031T, S. pseudovenezuelae DSM 40212T, S. caeruleatus NRRL B-24802T, S. curacoi DSM 40107T, S. canus Streptomyces longwoodensis DSM 41677T (KQ948572) Streptomyces bungoensis DSM 41781T (KQ948892) Streptomyces capoamus JCM 4734T (AB045877) Streptomyces corchorusii NBRC13032T (AB184267) SStreptomyces curacoi DSM 40107T (KQ948008) Streptomyces shaanxiensis CCNWHQ 0031QT (FJ465151) Streptomyces caeruleatus NRRL B24802T (KQ948975) Streptomyces lincolnensis NRRL 2936T (CP016438CP016438) Streptomyces aquilus GGCR-6T (MH718844) Streptomyces cyaneus NRRL B2296T (AF346475) Streptomyces griseoruber NRRL B1818T (LIQS01000280) Streptomyces mirabilis NBRC 13450T (AB184412) Streptomyces flavovariabilis NRRL B16367T (JNXD01000071) Streptomyces variegatus NRRL B16380T (JYJH01000110) Streptomyces canus DSM 40017T (KQ948708) Streptomyces pseudovenezuelae DSM 40212T (KQ948163) Streptomyces alboniger NRRL B1832T (LIQN01000245) Streptomyces resistomycificus NRRL ISP5133T (JOBA01000220) Streptomyces galilaeus JCM 4757T (AB045878) Streptomyces phaeoluteigriseus DSM 41896T (MPOH01000466) Streptomyces bobili JCM 4624T (AB045876) Streptomyces cinereoruber subsp. fructofermentans NBRC 15396T (AB184647) Streptomyces humiduspy NBRC 12877T (AB184213) Streptomyces rishiriensis NBRC 13407T (AB184383) Streptomyces phaeofaciens NBRC 13372T (AB184360) Mycobacterium tuberculosis H37Rv T (AL123456) 100 87 56 58 91 81 0.0100 * * 100 * * 86 * * Streptomyces galbus DSM 40089T (X79852) * Fig. 2. Neighborjoining phylogenetic tree based on 16S rRNA gene sequences showing the relationship between selected species of the genus Streptomyces. Mycobacterium tuberculosis H37RvT was used as an outgroup. Bootstrap percentages over 50 % derived from 1000 replications are shown at the nodes. Asterisks indicate branches also recovered in the maximumlikelihood and maximumparsimony trees. Bar, 0.01 nucleotide substitutions per site. 1915 Li etal., Int. J. Syst. Evol. Microbiol. 2020;70:1912–1917 DSM 40017T, S. griseoruber NRRL B-1818T, S. rishiriensis NBRC 13407T, S. resistomycificus NRRLISP 5133T and S. phaeoluteigriseus DSM 41896T, respectively, and ≤98.2 % similarities to the other species in the genus Streptomyces. A NJ tree based on the 16S rRNA gene sequences indicated that the phylogenetic neighbours of GGCR-6 T were S. cyaneus NRRL B-2296T and S. griseoruber NRRL B-1818T (Fig.2). The close association of GGCR-6T, S. cyaneus NRRL B-2296T and S. griseoruber NRRL B-1818 T was further supported by the ML and MP trees (Figs S2 and S3) based on the 16S rRNA gene sequences and the MLSA tree (Fig.3) based on the five housekeeping gene (atpD, gyrB, recA, rpoB and trpB) sequences (Table S3). However, the MLSA distances between this strain and the closely related species of the genus Streptomyces mentioned above were 0.041 and 0.039, respectively (Table S4), which was well above the species level threshold of 0.007 recommended by Rong and Huang [28], indicating that GGCR-6T represented a distinct species of the genus Streptomyces. Results of previous studies have indicated that the DNA–DNA reassociation experiments should be mandatory for testing the genomic uniqueness of a novel isolate which has more than or equal to 98.7 % 16S rRNA gene sequence similarities to the related species [35]. Thus, considering the higher 16S rRNA gene sequence similarities (≥98.7 %), DNA–DNA relatedness studies were carried out between GGCR-6T and the ten related type strains listed above. In the present work, the ANI and dDDH values were used for calculating the relatedness between their genome sequences. However, considering that the genome sequence data of S. shaanxiensis CCNWHQ 0031T is not available, DNA–DNA relatedness between it and strain GGCR-6T was determined using the DDH method [33]. Results indicated that the ANI and dDDH or DDH values between the wholegenome sequences of GGCR-6 T and the other strains were 86.5–87.2 and 24.5–31.3 % (or 33.8±0.9 %), which were well below the 95–96 and 70 % cutoff points recommended for delineating species (Table S5) [36, 37]. Furthermore, distinct differences in phenotypic traits between GGCR-6T and its phylogenetic neighbours, namely S. cyaneus NRRL B2296 T and S. griseoruber NRRL B1818T, also further indicated that GGCR-6T did not represent any known species of the genus Streptomyces phenotypically (Tables1, S1 and S5). Therefore, based on a combination of the genotypic and phenotypic data, Streptomyces rishiriensis NBRC 13407T Streptomyces humidus NBRC 12877T Streptomyces resistomycificus NRRL ISP5133T Streptomyces phaeofaciens NBRC 13372T Streptomyces phaeoluteigriseus DSM 41896T Streptomyces bobili JCM 4624T Streptomyces canuspy DSM 40017T Streptomyces pseudovenezuelae DSM 40212T Streptomyces lincolnensis NRRL 2936T Streptomyces cyaneuspyy NRRL B2296T Streptomyces aquiluspyq GGCR-6T Streptomyces griseoruberpyg NRRL B1818T Streptomyces shaanxiensis CCNWHQ 0031T Streptomyces caeruleatus NRRL B24802T Streptomyces curacoipyDSM 40107T Streptomyces flavovariabilis NRRL B16367T Streptomyces longwoodensis DSM 41677T Streptomyces galbus DSM 40089T Streptomyces bungoensis DSM 41781T Streptomyces capoamus JCM 4734T Streptomyces corchorusii NBRC13032T Streptomyces cinereoruber subsp. fructofermentans NBRC 15396 T Streptomyces mirabilis NBRC 13450T Streptomyces alboniger NRRL B1832T Streptomyces variegatus NRRL B16380T Streptomyces galilaeus JCM 4757T Mycobacterium tuberculosis H37Rv T 100 100 98 92 91 100 56 94 85 98 51 68 61 57 55 0.050 ⁎ ⁎ ⁎ ⁎ ⁎ ⁎ ⁎ ⁎ ⁎ ⁎ ⁎ ⁎ 91 ⁎ ⁎ ⁎ ⁎ Fig. 3. Neighborjoining tree based on fivegene concatenated sequences (atpD, gyrB, recA, rpoB and trpB, 2458 nt) showing the relationships between the related members of the genus Streptomyces. Mycobacterium tuberculosis H37RvT was used as an outgroup. Bootstrap percentages over 50 % derived from 1000 replications are shown at the nodes. Asterisks indicate branches that were also found using the maximumlikelihood method and the maximumparsimony method. Bar, 0.05 substitutions per site. 1916 Li etal., Int. J. Syst. Evol. Microbiol. 2020;70:1912–1917 GGCR-6 T represents a novel species of genus Streptomyces, for which the name Streptomyces aquilus sp. nov. is proposed. DESCRIPTION Of StreptomyceS aquiluS SP. NOv. Streptomyces aquilus sp. nov. (a' qui. lus. L. masc. adj. aquilus brown; referring to the ability of the organism to produce brown nutrient hyphae). Aerobic, Grampositive actinobacterium; forms welldeveloped branched aerial mycelium. Produces cylindrical and smoothsurfaced spores arranged in straight chains. The colour of the aerial mycelium is white and the colour of the substrate mycelium is light yellow to brown on ISP (2–7) media. No distinct soluble pigment is produced on tested media. Grows well on ISP (2–7) media. Growth occurs at pH 5.0–12.0, 10–45 °C and with 0–7% NaCl (w/v). Fructose, dgalactose, dglucose, mannitol, raffinose, ribose and sucrose can be used as sole carbon sources for growth. As a nitrogen source, it utilizes arginine, asparagine, lcysteine, lhistidine, lthreonine, methionine and tyrosine. Starch hydrolysis, H2S production, nitrate reduction, milk coagulation and milk peptonization, gelatin liquefaction and tweens (20, 40, 60 and 80) degradation are positive. Resistant to the following antibiotics: ampicillin, carbenicillin, cefoperazone, ceftazidime, clindamycin, norfloxacin, oxacillin, penicillin and vancomycin. The cell wall contains alanine, asparagine, glutamate, glycine and llDAP. Wholecell sugars are glucose, mannose and xylose. The predominant cellular fatty acids are C16 : 1ω9c, isoC16 : 0 and C16 : 0. The predominant menaquinones are MK-9(H8), MK-9(H2), MK-9 and MK-9(H6). The polar lipids contain phosphatidylethanolamine, diphosphatidylglycerol, phosphotidylinositol and phosphatidylinositol mannosides. The type strain is GGCR-6T (=CICC 11055T=JCM 33584T), which was isolated from the healthy leaves of a medicinal plant, Xanthium sibiricum, collected from the botanical garden of Hunan University of Science and Technology in Hunan province, PR China. The G+C content of the genomic DNA of the type strain is 70.9 mol%. The GenBank/EMBL/ DDBJ accession number for the 16S rRNA gene sequence of GGCR-6T is MH718844. The wholegenome shotgun project has been deposited at DDBJ/ENA/GenBank under the accession number CP034463. The version described in this paper is version CP034463.1. Funding information This research was supported by Postgraduate Research and Innovation Project of Hunan Province (CX2018B676) and Scientific Research Project of Hunan Province Department of Education (16K032). Acknowledgements The authors thank CICC (China Centre of Industrial Culture Collection) and Ms. Fu Zhuo (Analysis and Testing Centre, Xiangtan University) for providing excellent technical assistance. Conflicts of interest The authors declare that there are no conflicts of interest. References 1. Sharma VK, Johnson N, Cizmas L, McDonald TJ, Kim H. A review of the influence of treatment strategies on antibiotic resistant bacteria and antibiotic resistance genes. Chemosphere 2016;150:702–714. 2. Lathers CM. Role of veterinary medicine in public health: antibiotic use in food animals and humans and the effect on evolution of antibacterial resistance. J Clin Pharmacol 2001;41:595–599. 3. Schulz B, Boyle C. Microbial root endophytes. In: Sieber TN (editor). What Are Endophytes? 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