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Article

Characterization of Streptomyces sporangiiformans sp. nov., a Novel Soil Actinomycete with Antibacterial Activity against Ralstonia solanacearum

1
Key Laboratory of Agricultural Microbiology of Heilongjiang Province, Northeast Agricultural University, No. 59 Mucai Street, Xiangfang District, Harbin 150030, China
2
State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing 100193, China
*
Authors to whom correspondence should be addressed.
These authors contributed equally to this work.
Submission received: 27 August 2019 / Revised: 13 September 2019 / Accepted: 13 September 2019 / Published: 17 September 2019

Abstract

:
Ralstonia solanacearum is a major phytopathogenic bacterium that attacks many crops and other plants around the world. In this study, a novel actinomycete, designated strain NEAU-SSA 1T, which exhibited antibacterial activity against Ralstonia solanacearum, was isolated from soil collected from Mount Song and characterized using a polyphasic approach. Morphological and chemotaxonomic characteristics of the strain coincided with those of the genus Streptomyces. The 16S rRNA gene sequence analysis showed that the isolate was most closely related to Streptomyces aureoverticillatus JCM 4347T (97.9%). Phylogenetic analysis based on 16S rRNA gene sequences indicated that the strain formed a cluster with Streptomyces vastus JCM4524T (97.4%), S. cinereus DSM43033T (97.2%), S. xiangluensis NEAU-LA29T (97.1%) and S. flaveus JCM3035T (97.1%). The cell wall contained LL-diaminopimelic acid and the whole-cell hydrolysates were ribose, mannose and galactose. The polar lipids were diphosphatidylglycerol (DPG), phosphatidylethanolamine (PE), hydroxy-phosphatidylethanolamine (OH-PE), phosphatidylinositol (PI), two phosphatidylinositol mannosides (PIMs) and an unidentified phospholipid (PL). The menaquinones were MK-9(H4), MK-9(H6), and MK-9(H8). The major fatty acids were iso-C17:0, C16:0 and C17:1 ω9c. The DNA G+C content was 69.9 mol %. However, multilocus sequence analysis (MLSA) based on five other house-keeping genes (atpD, gyrB, recA, rpoB, and trpB), DNA–DNA relatedness, and physiological and biochemical data showed that the strain could be distinguished from its closest relatives. Therefore, it is proposed that strain NEAU-SSA 1T should be classified as representatives of a novel species of the genus Streptomyces, for which the name Streptomyces sporangiiformans sp. nov. is proposed. The type strain is NEAU-SSA 1T (=CCTCC AA 2017028T = DSM 105692T).

1. Introduction

Ralstonia solanacearum is the causal agent of bacterial wilt, one of the most devastating plant pathogenic bacteria around the world [1], which has an unusually wide host range, infecting over 200 plant species [2], including many important agricultural crops such as potato, tomato, banana and pepper. Even though different approaches have been developed to control bacterial wilt, we still lack an efficient and environmentally friendly control measure for most of the host crops [3]. Therefore, the search and discovery of novel, environmentally friendly, commercially significant, naturally bioactive compounds are in demand to control this disease at present.
The actinobacteria are known to produce biologically active secondary metabolites, including antibiotics, enzymes, enzyme inhibitors, antitumour agents and antibacterial compounds [4,5,6]. The genus Streptomyces, within the family Streptomycetaceae, is the largest genus of the phylum Actinobacteria, first proposed by Waksman and Henrici (1943) [7] and currently encompasses more than 800 species with valid published names (http://www.bacterio.net/streptomyces.html), which are widely distributed in soils throughout the world. Therefore, members of novel Streptomyces species are in demand as sources of novel, environmentally friendly, commercially significant, naturally bioactive compounds [8,9]. During our search for antagonistic actinobacteria from soil in Mount Song, an aerobic actinomycete, strain NEAU-SSA 1T with inhibitory activity against phytopathogenic bacterium Ralstonia solanacearum was isolated and subjected to the polyphasic taxonomy analysis. Results demonstrated that the strain represents a novel species of the genus Streptomyces, for which the name Streptomyces sporangiiformans sp. nov. is proposed.

2. Materials and Methods

2.1. Isolation of Actinomycete Strain

Strain NEAU-SSA 1T was isolated from soil collected from Mount Song (34°29′ N, 113°2′ E), Dengfeng, Henan Province, China. The soil sample was air-dried at room temperature for 14 days before isolation for actinomycetes. After drying, the soil sample was ground into powder and then suspended in sterile distilled water, followed by a standard serial dilution technique. The diluted soil suspension was spread on humic acid-vitamin agar (HV) [10] supplemented with cycloheximide (50 mg L−1) and nalidixic acid (20 mg L−1). After 28 days of aerobic incubation at 28 °C, colonies were transferred and purified on the International Streptomyces Project (ISP) medium 3 [11], and maintained as glycerol suspensions (20%, v/v) at −80 °C for long-term preservation.

2.2. Morphological and Physiological and Biochemical Characteristics of NEAU-SSA 1T

Gram staining was carried out by using the standard Gram stain, and morphological characteristics were observed using light microscopy (Nikon ECLIPSE E200, Nikon Corporation, Tokyo, Japan) and scanning electron microscopy (Hitachi SU8010, Hitachi Co., Tokyo, Japan) using cultures grown on ISP 3 agar at 28 °C for 6 weeks. Samples for scanning electron microscopy were prepared as described by Jin et al. [12]. Cultural characteristics were determined on the ISP 1 agar [11], ISP media 2–7 [8], Czapek’s agar [13], Bennett’s agar [14], and Nutrient agar [15] after 14 days at 28 °C. Color determination was done with color chips from the ISCC-NBS (Inter-Society Color Council-National Bureau of Standards) color charts [16]. Growth at different temperatures (10, 15, 20, 25, 28, 32, 35, 40, 45, and 50 °C) was determined on ISP 3 medium after incubation for 14 days. Growth tests for pH range (pH 4.0–12.0, at intervals of 1.0 pH unit) and NaCl tolerance (0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, and 20%, w/v) were tested in GY (Glucose-Yeast extract) medium [17] at 28 °C for 14 days on a rotary shaker. The buffer systems were: pH 4.0–5.0, 0.1 M citric acid/0.1 M sodium citrate; pH 6.0–8.0, 0.1 M KH2PO4/0.1 M NaOH; pH 9.0–10.0, 0.1 M NaHCO3/0.1 M Na2CO3; and pH 11.0–12.0, 0.2 M KH2PO4/0.1 M NaOH. Hydrolysis of Tweens (20, 40, and 80) and production of urease were tested as described by Smibert and Krieg [18]. The utilization of sole carbon and nitrogen sources, decomposition of cellulose, hydrolysis of starch and aesculin, reduction of nitrate, coagulation and peptonization of milk, liquefaction of gelatin, and production of H2S were examined as described previously [19,20].

2.3. Chemotaxonomic Analysis of NEAU-SSA 1T

Biomass for chemotaxonomic studies was prepared by growing the organisms in GY medium in shake flasks at 28 °C for 5 days. Cells were harvested using centrifugation, washed with distilled water, and freeze-dried. The isomer of diaminopimelic acid (DPA) in the cell wall hydrolysates was derivatized and analyzed using an HPLC (High Performance Liquid Chromatography) method [21] with an Agilent TC-C18 Column (250 × 4.6 mm i.d. 5 µm; Agilent Technologies, Santa Clara, CA, USA) that had 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 (Agilent Technologies, Santa Clara, CA, USA) with a 365 nm excitation and 455 nm longpass emission filters. The whole-cell sugars were analyzed according to the procedures developed by Lechevalier and Lechevalier [22]. The polar lipids were examined using two-dimensional TLC (Thin-Layer Chromatography) and identified using the method of Minnikin et al. [23]. Menaquinones were extracted from the freeze-dried biomass and purified according to Collins [24]. Extracts were analyzed using a HPLC-UV method [25] with an Agilent Extend-C18 Column (150 × 4.6 mm, i.d. 5 µm; Agilent Technologies, Santa Clara, CA, USA) at 270 nm. The mobile phase was acetonitrile-iso-propyl alcohol (60:40, v/v). To determine cellular fatty acid compositions, the strain NEAU-SSA 1T was cultivated in GY medium in shake flasks at 28 °C for 4 days. Fatty acid methyl esters were extracted from the biomass as described by Gao et al. [26] and analyzed using GC-MS according to the method of Xiang et al. [27].

2.4. Phylogenetic Analysis of NEAU-SSA 1T

For DNA extraction, strain NEAU-SSA 1T was cultured in GY medium for 3 days to the early stationary phase and harvested using centrifugation. The chromosomal DNA was extracted according to the method of sodium dodecyl sulfate (SDS)-based DNA extraction [28]. PCR amplification of the 16S rRNA gene sequence was carried out using the universal bacterial primers 27F (5′-AGAGTTTGATCCTGGCTCAG-3´) and 1541R (5´-AAGGAGGTGATCCAGCC-3´) under conditions described previously [29,30]. The PCR product was purified and cloned into the vector pMD19-T (Takara) and sequenced using an Applied Biosystems DNA sequencer (model 3730XL, Applied Biosystems Inc., Foster City, California, USA). The almost complete 16S rRNA gene sequence of strain NEAU-SSA 1T (1412bp) was obtained and compared with type strains available at the EzBioCloud server (https://www.ezbiocloud.net/), retrieved using NCBI BLAST (National Center for Biotechnology Information, Basic Local Alignment Search Tool; https://blast.ncbi.nlm.nih.gov/Blast.cgi;) and then submitted to the GenBank database. Phylogenetic trees were constructed based on the 16S rRNA gene sequences of strain NEAU-SSA 1T and related reference species. Sequences were multiply aligned in Molecular Evolutionary Genetics Analysis (MEGA) software version 7.0 using the Clustal W algorithm and trimmed manually where necessary. Phylogenetic trees were constructed with neighbor-joining [31] and maximum likelihood [32] algorithms using MEGA [33]. The stability of the topology of the phylogenetic tree was assessed using the bootstrap method with 1000 repetitions [34]. A distance matrix was generated using Kimura’s two-parameter model [35]. All positions containing gaps and missing data were eliminated from the dataset (complete deletion option). 16S rRNA gene sequence similarities between strains were calculated on the basis of pairwise alignment using the EzBioCloud server [36]. To further clarify the affiliation of strain NEAU-SSA 1T to its closely related strains, phylogenetic relationships of the strain NEAU-SSA 1T were also confirmed using sequences of five individual housekeeping genes (atpD, gyrB, recA, rpoB, and trpB) for core-genome analysis. The sequences of NEAU-SSA 1T and its related strains were obtained from the genomes or GenBank/EMBL/DDBJ (European Molecular Biology Laboratory/DNA Data Bank of Japan). GenBank accession numbers of the sequences used are given in Table 1. The sequences of each locus were aligned using MEGA 7.0 software and trimmed manually at the same position before being used for further analysis. Trimmed sequences of the five housekeeping genes were concatenated head-to-tail in-frame in the order atpD-gyrB-recA-rpoB-trpB. Phylogenetic analysis was performed as described above. Genome mining for bioactive secondary metabolites was performed using “antibiotics and secondary metabolite analysis shell” (antiSMASH) version 4.0 [37].

2.5. Draft Genome Sequencing and Assembly of NEAU-SSA 1T

For draft genome sequencing and assembly, the genomic DNA of strain NEAU-SSA 1T was extracted using the method of SDS-based DNA extraction [28]. The harvested DNA was detected using agarose gel electrophoresis and quantified using Qubit® 2.0 Fluorometer (Thermo Scientific). Whole-genome sequencing was performed on the Illumina HiSeq PE150 (Illumina, San Diego, CA, USA) platform. A-tailed, ligated to paired-end adaptors, and PCR amplified samples with a 350 bp insert were used for the library construction at the Beijing Novogene Bioinformatics Technology Co., Ltd. Illumina PCR adapter reads and low-quality reads from the paired-end were filtered using a quality control step using our own compling pipeline. All good-quality paired reads were assembled using the SOAP (Short Oligonucleotide Alignment Program) denovo [38,39] (https://github.com/aquaskyline) into a number of scaffolds. Then, the filter reads were handled by the next step of the gap-closing.

2.6. DNA–DNA Relatedness Tests

Because of a lacking number of genome sequences of Streptomyces aureoverticillatus JCM4347T, Streptomyces vastus JCM4524T, S. cinereus DSM43033T, and S. xiangluensis NEAU-LA29T, DNA–DNA relatedness tests between strain NEAU-SSA 1T and those strains were carried out as described by De Ley et al. [40] under consideration of the modifications described by Huss et al. [41], using a model Cary 100 Bio UV/VIS-spectrophotometer (Hitachi U-3900, Hitachi Co., Tokyo, Japan) equipped with a Peltier-thermostatted 6 × 6 multicell changer and a temperature controller with in situ temperature probe (Varian). The genomic DNAs of strain NEAU-SSA 1T and its closely related species—S. aureoverticillatus JCM4347T, S. vastus JCM4524T, S. cinereus DSM43033T, and S. xiangluensis NEAU-LA29T—were extracted using the method of SDS-based DNA extraction [28]. The concentration and purity of these DNA samples were determined by measuring the optical density (OD) 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 JY92-II ultrasonic cell disruptor (ultrasonic time 3 s, interval time 4 s, 90 times; Ningbo Scientz Biotechnology Co., Ltd, Ningbo, China). 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 a mean of the three values. Several genomic metrics are now available to distinguish between orthologous genes of closely related prokaryotes, including the calculation of average nucleotide identity (ANI) and digital DNA–DNA hybridization (dDDH) values [42,43]. In the present study, ANI and dDDH values were determined from the genomes of strain NEAU-SSA 1T and S. flaveus JCM3035T (JOCU00000000) using the ortho-ANIu algorithm from Ezbiotaxon and the genome-to-genome distance calculator (GGDC 2.0) at http://ggdc.dsmz.de.

2.7. In Vitro Antibacterial Activity Test

The antibacterial activity of strain NEAU-SSA 1T against two pathogenic bacteria (Micrococcus luteus and Ralstonia solanacearum) was evaluated using the agar well diffusion method [44] with the cultures growth on ISP 3 medium at 28 °C for four weeks as follows: All the spores and mycelia were collected from one ISP 3 plate (diameter, 9mm) and then extracted using 1 mL methanol with an ultrasonic step (300 W, 30–60 min). Afterwards, 200 µL methanol extract or methanol was added to the agar well, and methanol was used as the control. To further investigate the antibacterial components produced by NEAU-SSA 1T, the strain was cultured in tryptone-glucose-soluble starch-yeast extract medium (tryptone 0.2%, glucose 1%, soluble starch 0.5%, yeast extract 0.2%, NaCl 0.4%, K2HPO4 0.05%, MgSO4.7H2O 0.05%, CaCO3 0.2%, w/v, pH 7.0–7.4), and the inhibitory activity was tested. Briefly, strain NEAU-SSA 1T was inoculated into MB medium and incubated at 28 °C for seven days in a rotary shaker. The supernatant (100 mL for this study) was obtained via centrifugation at 8000 rpm and 4 °C for 10 min and subsequently extracted by using an equal volume of ethyl acetate. Then, the extract was dried in a rotary evaporator at 40 °C and eluted with proper volume methanol (1 mL used in this study). The cell precipitate was extracted with an equal volume of methanol and also condensed as above. After that, the antibacterial activity was evaluated using the agar well diffusion method, and each well contained 200 µL of the methanol extract. To examine the effect of temperature on antibacterial activity, the ten-fold dilution methanol extract was placed in a water bath at 40, 60, 80, and 100 °C for 30 min, and then cooled to room temperature. The antibacterial activity was evaluated using the agar well diffusion method.

3. Result and Discussion

3.1. Polyphasic Taxonomic Characterization of NEAU-SSA 1T

The morphological characteristics of strain NEAU-SSA 1T showed that the strain had the typical characteristics of the genus Streptomyces. Observation of 6-week cultures of strain NEAU-SSA 1T grown on ISP 3 medium revealed that it formed well-developed, branched substrate hyphae and aerial mycelia. Sporangia consisted of cylindrical, and rough-surfaced spores (0.6–0.8 μm × 0.9–1.6 μm) were produced on aerial mycelia, but spore chains were not observed (Figure 1). Strain NEAU-SSA 1T exhibited good growth on ISP 3, ISP 4, ISP 7, and Nutrient agar media; moderate growth on ISP 1, ISP 2, ISP 5, ISP 6, and Czapek’s agar media; and poor growth on Bennett’s agar medium. The cultural characteristics of strain NEAU-SSA 1T is shown in Table S1. Strain NEAU-SSA 1T grew well between pH 6.0 and 11.0, with an optimum pH of 7.0. The range of temperature of the strain was determined to be 15–45 °C, with the optimum growth temperature being 28 °C. The strain grew in the presence of 0–6% NaCl (w/v) with an optimal level of 0–1% (w/v). Detailed physiological characteristics are presented in the species description (Table 2 and Table S1).
Chemotaxonomic analyses revealed that strain NEAU-SSA 1T exhibited characteristics that are typical of representatives of the genus Streptomyces. The strain was found to contain LL-diaminopimelic acid as diamino acid. The whole-cell hydrolysates of the strain were determined to contain ribose, mannose, and galactose. The menaquinones of strain NEAU-SSA 1T were MK-9(H4) (29.5%), MK-9(H6) (41.2%), and MK-9(H8) (29.4%). The cellular fatty acid profile of strain NEAU-SSA 1T was composed of iso-C17:0 (30.9%), C16:0 (26.4%), C17:1ω9c (19.9%), C15:0 (7.8%), C17:0 (4.4%), C14:0 (3.3%), iso-C16:0 (1.7%), anteiso-C15:0 (1.7%), C18:1ω9c (1.7%), C16:0 1-OH (1.2%), and iso-C18:0 (1.1%). The polar lipids of the strain consisted of diphosphatidylglycerol (DPG), phosphatidylethanolamine (PE), hydroxy-phosphatidylethanolamine (OH-PE), phosphatidylinositol (PI), two phosphatidylinositol mannosides (PIMs), and an unidentified phospholipid (PL) (Supplementary Figure S1). All the chemotaxonomic data are consistent with the assignment of strain NEAU-SSA 1T to the genus Streptomyces.
Sequence analysis of the 16S rRNA gene showed that strain NEAU-SSA 1T were affiliated with the genus Streptomyces and most closely related to S. aureoverticillatus JCM 4347T (97.9%). Phylogenetic analysis based on 16S rRNA gene sequences indicated that the strain formed a cluster with S. vastus JCM4524T (97.4%), S. cinereus DSM43033T (97.2%), S. xiangluensis NEAU-LA29T (97.1%), and S. flaveus JCM3035T (97.1%) in the neighbor-joining tree (Figure 2), a relationship also recovered by the maximum-likelihood algorithm (Figure S2). Phylogenetic trees based on the neighbor-joining and maximum-likelihood algorithms were constructed from the concatenated sequence alignment of the five housekeeping genes (Figure 3 and Figure S3), and had the same topology as the 16S rRNA gene tree. Moreover, pairwise distances calculated for NEAU-SSA 1T and the related species using the concatenated sequences of atpD-gyrB-recA-rpoB-trpB were well above 0.007 (Table S2), which is considered to be the threshold for species determination by Rong et al. [46]. DNA–DNA hybridization was employed to further clarify the relatedness between the strain and S. aureoverticillatus JCM 4347T, S. vastus JCM4524T, S. cinereus DSM 43033T, and S. xiangluensis NEAU-LA29T. Results showed that strain NEAU-SSA 1T shared DNA–DNA relatedness of 37.1 ± 3.4% with S. aureoverticillatus JCM 4347T, 35.4 ± 4.3% with S. vastus JCM 4524T, 33.1 ± 4.1% with S. cinereus DSM 43033T, and 29.0 ± 4.9% with S. xiangluensis NEAU-LA29T. Digital DNA–DNA hybridization was employed to clarify the relatedness between strain NEAU-SSA 1T and S. flaveus JCM 3035T. The level of digital DNA–DNA hybridization between them was 24.9 ± 2.4%. These five values are all below the threshold value of 70% recommended by Wayne et al. [47] for assigning strains to the same genomic species. Similarly, a low ANI value of 80.99% was found between strain NEAU-SSA 1T and S. flaveus JCM 3035T, a result well below the threshold used to delineate prokaryote species [48,49].
The assembled genome sequence of strain NEAU-SSA 1T was found to be 10,364,704 bp long and composed of 352 contigs with an N50 of 59,982 bp, a DNA G+C content of 69.9 mol % and a coverage of 200x. It was deposited into GenBank under the accession number VCHX00000000. The 16S rRNA gene sequence from the whole genome sequence shared a 100% similarity with that from PCR sequencing, suggesting that the genome sequence was not contaminated. Detailed genomic information is presented in the Table S3.
Comparison of phenotypic characteristics between strain NEAU-SSA 1T and its closely related species—S. aureoverticillatus JCM 4347T, S. vastus JCM4524T, S. cinereus DSM 43033T, S. xiangluensis NEAU-LA29T, and S. flaveus JCM 3035T—was performed to differentiate these strains (Table 2). Differential cultural characteristics included: NaCl tolerance of the strain was up to 5.0%, which is lower than that of S. aureoverticillatus JCM 4347T (15%) and S. flaveus JCM 3035T (7%); and the strain could grow at pH 11.0, while S. vastus JCM4524T, S. cinereus DSM 43033T, and S. xiangluensis NEAU-LA29T could not. Other phenotypic differences included the production of H2S; decomposition of cellulose; liquefaction of gelatin; growth temperature; hydrolysis of Tweens (20, 40, and 80); and utilization of L-arabinose, D-galactose, D-fructose, D-maltose, lactose, L-rhamnose, D-ribose, D-sorbitol, D-mannose, raffinose, D-xylose, myo-inositol, L-glutamine, glycine, L-threonine, L-tyrosine, L-serine, L-proline, L-asparagine, and L-arginine.
On the basis of morphological, physiological, chemotaxonomic, and phylogenetic results, strain NEAU-SSA 1T is considered to represent a novel species within the genus Streptomyces, for which the name Streptomyces sporangiiformans is proposed.

3.2. Description of Streptomyces sporangiiformans sp. nov.

Streptomyces sporangiiformans (spo.ran.gi.i.for’mans. N.L. neut. n. sporangium; L. pres. part. formans forming; N.L. part. adj. sporangiiformans forming sporangia).
Gram-stain-positive, aerobic actinomycete that formed well-developed, branched substrate hyphae and aerial mycelia. Sporangia consisted of cylindrical and rough surfaced spores (0.6–0.8 μm × 0.9–1.6 μm) were produced on aerial mycelia, but spore chains were not observed. Good growth on ISP 3, ISP 4, ISP 7, and Nutrient agar media; moderate growth on ISP 1, ISP 2, ISP 5, ISP 6, and Czapek’s agar media; and poor growth on Bennett’s agar medium. Growth occurred at pH values between 6.0 and 11.0, the optimum being pH 7.0. Tolerates up to 6.0% NaCl and grows optimally in 0–1% (w/v) NaCl. Growth was observed at temperatures between 15 and 45 °C, with an optimum temperature of 28 °C. Positive for decomposition of Tweens (40 and 80) and cellulose, hydrolysis of aesculin and starch, liquefaction of gelatin and production of urease; and negative for coagulation and peptonization of milk, hydrolysis of Tween 20, production of H2S, and reduction of nitrate. D-fructose, D-galactose, D-glucose, inositol, lactose, D-maltose, D-mannose, D-raffinose, L-rhamnose, and D-sucrose were utilized as sole carbon sources, but not L-arabinose, dulcitol, D-ribose, D-sorbitol, or D-xylose. L-alanine, L-arginine, L-asparagine, L-aspartic acid, creatine, L-glutamic acid, L-glutamine, L-proline, L-serine, and L-threonine were utilized as sole nitrogen sources, but not glycine or L-tyrosine. Cell wall contained LL-diaminopimelic acid and the whole-cell hydrolysates were ribose, mannose, and galactose. The polar lipids contained diphosphatidylglycerol (DPG), phosphatidylethanolamine (PE), hydroxy-phosphatidylethanolamine (OH-PE), phosphatidylinositol (PI), two phosphatidylinositol mannosides (PIMs), and an unidentified phospholipid (PL). The menaquinones were MK-9(H4), MK-9(H6), and MK-9(H8). Major fatty acids were iso-C17:0, C16:0, and C17:1ω9c.
The type strain was NEAU-SSA 1T (=CCTCC AA 2017028T = DSM 105692T), isolated from soil collected from Mount Song, Dengfeng, Henan Province, China. The DNA G+C content of the type strain was 69.9 mol %, calculated from the assembly for the draft genome sequence. The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain NEAU-SSA 1T is MH842151. This Whole Genome Shotgun project has been deposited at DDBJ/ENA/GenBank under the accession VCHX00000000. The version described in this paper is version VCHX00000000.2.

3.3. Antibacterial Activity of NEAU-SSA 1T against Ralstonia solanacearum

Strain NEAU-SSA 1T exhibited antibacterial activity against Ralstonia solanacearum with inhibitory zone diameters of 23 mm (Figure 4a). However, no inhibitory effect on the growth of Micrococcus luteus (Figure 4b) was observed. Comparison of the antibacterial activity of the extract of the supernatant with that of the cell pellet suggested that the antibacterial substances of strain NEAU-SSA 1T were in both the supernatant and cell pellet since the extracts all showed inhibition of the growth of Ralstonia solanacearum with the inhibitory zone diameters of 31.5 and 26.4 mm, respectively (Figure 5a,b). The antibacterial substances in the supernatant were stable after they were placed in a water bath at 40 and 60 °C for 30 min, while they did not show antibacterial activity after 80 and 100 °C bath (Figure 6a), which indicated that they were sensitive to temperature. In contrast, the antibacterial substances in the cell pellet were insensitive to temperature (Figure 6b), which demonstrated that the antibacterial substances in the supernatant and cell pellet were different. The antiSMASH analysis led to the identification of 49 gene clusters, including 24 gene clusters that showed very low similarity to the known gene clusters of mediomycin A, cremimycin, primycin, ibomycin, naphthomycin, lasalocid, informatipeptin, polyoxypeptin, kutznerides, anisomycin, paulomycin, himastatin, desotamide, nystatin, tiacumicin B, oxazolomycin, and 4-Z-annimycin. Therefore, the relationships between the corresponding secondary metabolites produced by NEAU-SSA 1T and the antibacterial activity are still ambiguous. Streptomyces are well known as important biological resources for their biologically active secondary metabolites, which play important roles in protecting plants against pathogens [50]. Strain NEAU-SSA 1T, which shows a stronger antibacterial activity against Ralstonia solanacearum, is a novel species of the genus Streptomyces, and possesses 24 lower similarity gene clusters. Therefore, it is interesting and significant to isolate and identify the secondary metabolites of the strain in further studies.

4. Conclusions

A novel strain NEAU-SSA 1T that exhibited antibacterial activity against Ralstonia solanacearum was isolated from a soil sample. Morphological features, phylogenetic analysis based on 16S rRNA gene sequences, and multilocus sequence analysis based on five other house-keeping genes (atpD, gyrB, recA, rpoB, and trpB) suggested that strain NEAU-SSA 1T belonged to the genus Streptomyces. Physiology and biochemical characteristics, together with DDH relatedness values and ANI values, clearly indicated that strain NEAU-SSA 1T could be differentiated from the closely related strains S. aureoverticillatus JCM 4347T, S. vastus JCM 4524T, S. cinereus DSM 43033T, S. xiangluensis NEAU-LA29T, and S. flaveus JCM 3035T. Based on the polyphasic analysis, it is proposed that strain NEAU-SSA 1T should be classified as representatives of a novel species of the genus Streptomyces, for which the name Streptomyces sporangiiformans sp. nov. is proposed. The type strain is NEAU-SSA 1T (=CCTCC AA 2017028T = DSM 105692T).

Supplementary Materials

The following are available online at https://0-www-mdpi-com.brum.beds.ac.uk/2076-2607/7/9/360/s1, Figure S1: Maximum-likelihood tree showing the phylogenetic position of strain NEAU-SSA 1T (1412 bp) and the related species based on 16S rRNA gene sequences. The out-group used was Kitasatospora setae LM-6054T. Only bootstrap values above 50% (percentages of 1000 replications) are indicated. Bar, 0.01 nucleotide substitutions per site. Figure S2: Maximum-likelihood tree based on MLSA analysis of the concatenated partial sequences from five housekeeping genes (atpD, gyrB, recA, rpoB, and trpB) of isolate NEAU-SSA 1T and related taxa. Only bootstrap values above 50% (percentages of 1000 replications) are indicated. Kitasatospora setae LM-6054T was used as an out-group. Bar, 0.05 nucleotide substitutions per site. Table S1. Growth and cultural characteristics of strain NEAU-SSA 1T. Table S2: MLAS distance values for selected strains in this study. Table S3: General features of the genome sequence of the type strain NEAU-SSA 1T.

Author Contributions

J.Z. and L.H. performed the isolation and morphological and biochemical characterization of strain NEAU-SSA 1T. M.Y. performed the antifungal test. P.C. analyzed DNA sequencing data and genomic sequencing data. D.L. performed chemotaxonomic analysis and phylogenetic analysis. X.G. prepared the figures and tables. Y.L. performed the morphological observation by transmission electron microscopy. X.W. and W.X. designed the experiments and edited the manuscript.

Funding

This work was supported in part by grants from the National Key Research and Development Program of China (No. 2017YFD0201606), the National Natural Youth Science Foundation of China (No. 31701858), the China Postdoctoral Science Foundation (2018M631907), the Heilongjiang Postdoctoral Fund (LBH-Z17015), the University Nursing Program for Young Scholars with Creative Talents in Heilongjiang Province (UNPYSCT-2017017), and the “Young Talents” Project of Northeast Agricultural University (17QC14).

Acknowledgments

The authors would like to thank Aharon Oren (Department of Plant and Environmental Sciences, the Alexander Silberman Institute of Life Sciences, the Hebrew University of Jerusalem) for helpful advice on the specific epithet.

Conflicts of Interest

The authors declare that there are no conflict of interest.

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Figure 1. Scanning electron micrograph of strain NEAU-SSA 1T grown on ISP 3 agar for 6 weeks at 28 °C; Scale bar represents 1 μm.
Figure 1. Scanning electron micrograph of strain NEAU-SSA 1T grown on ISP 3 agar for 6 weeks at 28 °C; Scale bar represents 1 μm.
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Figure 2. Neighbor-joining tree showing the phylogenetic position of strain NEAU-SSA 1T (1412 bp) and the related species of the genus Streptomyces based on 16S rRNA gene sequences. The out-group used was Kitasatospora setae LM-6054T. Only bootstrap values above 50% (percentages of 1000 replications) are indicated. Asterisks indicate branches also recovered in the maximum-likelihood tree. Scale bar represents 0.005 nucleotide substitutions per site.
Figure 2. Neighbor-joining tree showing the phylogenetic position of strain NEAU-SSA 1T (1412 bp) and the related species of the genus Streptomyces based on 16S rRNA gene sequences. The out-group used was Kitasatospora setae LM-6054T. Only bootstrap values above 50% (percentages of 1000 replications) are indicated. Asterisks indicate branches also recovered in the maximum-likelihood tree. Scale bar represents 0.005 nucleotide substitutions per site.
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Figure 3. Neighbor-joining tree based on MLSA analysis of the concatenated partial sequences from five housekeeping genes (atpD, gyrB, recA, rpoB, and trpB) of isolate NEAU-SSA 1T (in bold) and related taxa. Only bootstrap values above 50% (percentages of 1000 replications) are indicated. Kitasatospora setae LM-6054T was used as an out-group. Asterisks indicate branches also recovered in the maximum-likelihood tree. Scale bar represents 0.02 nucleotide substitutions per site.
Figure 3. Neighbor-joining tree based on MLSA analysis of the concatenated partial sequences from five housekeeping genes (atpD, gyrB, recA, rpoB, and trpB) of isolate NEAU-SSA 1T (in bold) and related taxa. Only bootstrap values above 50% (percentages of 1000 replications) are indicated. Kitasatospora setae LM-6054T was used as an out-group. Asterisks indicate branches also recovered in the maximum-likelihood tree. Scale bar represents 0.02 nucleotide substitutions per site.
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Figure 4. The antibacterial activity of strain NEAU-SSA 1T against Ralstonia solanacearum (a) and Micrococcus luteus (b).
Figure 4. The antibacterial activity of strain NEAU-SSA 1T against Ralstonia solanacearum (a) and Micrococcus luteus (b).
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Figure 5. The antibacterial activity of the extract of the supernatant (a) and cell pellet of strain NEAU-SSA 1T (b) against Ralstonia solanacearum.
Figure 5. The antibacterial activity of the extract of the supernatant (a) and cell pellet of strain NEAU-SSA 1T (b) against Ralstonia solanacearum.
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Figure 6. The effect of temperature on the antibacterial activity of the extract of the supernatant (a) and cell pellet of strain NEAU-SSA 1T (b) against Ralstonia solanacearum.
Figure 6. The effect of temperature on the antibacterial activity of the extract of the supernatant (a) and cell pellet of strain NEAU-SSA 1T (b) against Ralstonia solanacearum.
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Table 1. GenBank Accession Numbers of the Sequences Used in MLSA.
Table 1. GenBank Accession Numbers of the Sequences Used in MLSA.
StrainType StrainWhole GenomeatpDgyrBrecArpoBtrpB
Streptomyces sporangiiformansNEAU-SSA 1TVCHX00000000
Streptomyces coelescensDSM 40421TGU383344AY508508KT385220GU383768KT389192
Streptomyces violaceolatusDSM 40438TGU383347AY508509 KT385451GU383771KT389418
Streptomyces anthocyanicusNBRC 14892TKT384465KT384814KT385162KT388784KT389134
Streptomyces humiferusDSM 43030TKT384598 KT384947KT385296KT388918KT389267
Streptomyces violaceoruberNBRC 12826TCP020570KT384751KT385099KT385453KT389071 KT389420
Streptomyces rubrogriseusLMG 20318TBEWD00000000KT384715KT385065KT385416KT389036KT389384
Streptomyces tendaeATCC 19812TKT384733KT385082KT385434KT389053KT389402
Streptomyces violaceorubidusLMG 20319TJODM00000000
Streptomyces lienomyciniLMG 20091TKT384622KT384971KT385321KT388942KT389291
Streptomyces diastaticus subsp. ardesiacusNRRL B-1773TBEWC00000000KT384534KT384883KT385231KT388853KT389203
Streptomyces albaduncusJCM 4715TKT384449KT384798KT385146KJ996741KT389118
Streptomyces matensisNBRC 12889TKT384637KT384986KT385337KT388957KT389306
Streptomyces althioticusNRRL B-3981TKT384460KT384809KT385157KT388779KT389129
Streptomyces davaonensisJCM 4913THE971709
Streptomyces canusDSM 40017TLMWO00000000KT384500KT384849KT385197KT388819KT389169
Streptomyces lincolnensisNRRL 2936TCP016438
Streptomyces pseudovenezuelaeDSM 40212TLMWM00000000KT384695KT385045KT385396KT389016KT389364
Streptomyces xiangluensisNEAU-LA29TMH291276MH345670MH291277MH291275MH291278
Streptomyces vastusNBRC 13094TKU323834KT385093KU975607KT389065KT389414
Streptomyces cinereusNBRC 12247T KT384513KT384862KT385210KJ996667KT389182
Streptomyces flaveusNRRL B-16074TJOCU00000000KT384551KT384900KT385249KT388870KT389220
Streptomyces chilikensisRC 1830TLWCC00000000
Streptomyces coeruleorubidusISP 5145TKT384528KT384877KT385225KT388847KT389197
Streptomyces misionensisDSM 40306TFNTD00000000KT384647KT384996KT385347KT388967KT389316
Streptomyces phaeoluteichromatogenesNRRL 5799TKT384680KT385030KT385381KT389001KT389350
Streptomyces tricolorNBRC 15461TMUMF00000000KT384741KT385089KT385443KT389061KT389410
Streptomyces achromogenes subsp. achromogenes NBRC 12735TJODT00000000
Streptomyces eurythermusATCC 14975TKT384544KT384893KT385242KT388863KT389213
Streptomyces nogalaterJCM 4799TKT384664KT385014KT385365KT388984KT389333
Streptomyces jietaisiensisFXJ46TFNAX00000000KT384605KT384954KT385304KT388925KT389274
Streptomyces griseoaurantiacusNBRC 15440TAEYX00000000
Streptomyces lavenduligriseusNRRL ISP-5487TJOBD00000000KT384620AB072859KT385319KT388940KT389289
Streptomyces uncialisDCA2648TLFBV00000000
Streptomyces albonigerNRRL B-1832TKT384455KT384804KT385152KT388774KT389124
Streptomyces alfalfaeXY25TCP015588
Streptomyces lasiicapitis3H-HV17(2)TMH651782KY229066MH651785MH651788MH651791
Streptomyces aureocirculatusNRRL ISP-5386TJOAP00000000KT384476KT384825KT385173KT388795KT389145
Streptomyces aureoverticillatusNRRL B-3326TKT384478KT384827KT385175KT388797KT389147
Streptomyces alboflavusNRRL B-2373TCP021748
Streptomyces rutgersensisNBRC 12819TKT384716KT385066KT385417KT389037KT389385
Streptomyces intermediusNBRC 13049TKT384602KT384951KT385301KT388922KT389271
Streptomyces gougerotiiNBRC 3198TKT384572KT384921KT385270KT388891KT389241
Streptomyces diastaticus subsp. diastaticusNBRC 3714TKT384535KT384884KT385232KT388854KT389204
Kitasatospora setaeLM-6054TAP010968
Table 2. Differential characteristics of strain NEAU-SSA 1T, S. aureoverticillatus JCM 4347T, S. vastus JCM4524T, S. cinereus DSM43033T, S. xiangluensis NEAU-LA29T, and S. flaveus JCM3035T.
Table 2. Differential characteristics of strain NEAU-SSA 1T, S. aureoverticillatus JCM 4347T, S. vastus JCM4524T, S. cinereus DSM43033T, S. xiangluensis NEAU-LA29T, and S. flaveus JCM3035T.
Characteristic123456a
Decomposition of cellulose++ND
Production of H2S+
Tween 20+ND
Tween 40+++ND
Tween 80+++ND
Liquefaction of gelatin+ND
Growth temperature (℃)15–4510–4520–4020–4020–4010–37
pH range for growth6–115–126–106–96–9ND
NaCl tolerance range (w/v, %)0–60–150–5 0–50–60–7
Carbon source utilization
D-fructose++++
D-galactose++++
Lactose++++
D-maltose+++ND
L-rhamnose+++++
D-ribose+
D-sorbitol++ND
D-mannose+++++
Raffinose+++++
L-arabinose+
D-xylose+
Myo-inositol+++++
Nitrogen source utilization
L-glutamine+++ND
Glycine+++ND
L-threonine+++
L-tyrosine++ND
L-arginine+++++
L-asparagine++++ND
L-serine+++++
L-proline++++
Strains: 1—NEAU-SSA 1T; 2—S. aureoverticillatus JCM 4347T; 3—S. vastus JCM4524T; 4—S. cinereus DSM43033T; 5—S. xiangluensis NEAU-LA29T; 6—S. flaveus JCM3035T. Abbreviation: +, positive; –, negative. All data are from this study except where marked. a Data from Michael Goodfellow et al. [45].

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Zhao, J.; Han, L.; Yu, M.; Cao, P.; Li, D.; Guo, X.; Liu, Y.; Wang, X.; Xiang, W. Characterization of Streptomyces sporangiiformans sp. nov., a Novel Soil Actinomycete with Antibacterial Activity against Ralstonia solanacearum. Microorganisms 2019, 7, 360. https://0-doi-org.brum.beds.ac.uk/10.3390/microorganisms7090360

AMA Style

Zhao J, Han L, Yu M, Cao P, Li D, Guo X, Liu Y, Wang X, Xiang W. Characterization of Streptomyces sporangiiformans sp. nov., a Novel Soil Actinomycete with Antibacterial Activity against Ralstonia solanacearum. Microorganisms. 2019; 7(9):360. https://0-doi-org.brum.beds.ac.uk/10.3390/microorganisms7090360

Chicago/Turabian Style

Zhao, Junwei, Liyuan Han, Mingying Yu, Peng Cao, Dongmei Li, Xiaowei Guo, Yongqiang Liu, Xiangjing Wang, and Wensheng Xiang. 2019. "Characterization of Streptomyces sporangiiformans sp. nov., a Novel Soil Actinomycete with Antibacterial Activity against Ralstonia solanacearum" Microorganisms 7, no. 9: 360. https://0-doi-org.brum.beds.ac.uk/10.3390/microorganisms7090360

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