Introduction

The genus Micromonospora was described by Ørskov (1923) as a member of the family Micromonosporaceae. Micromonospora species were widely distributed in the environment, such as soil, insects, deep marine sediments and plants (Li and Hong 2016; Xiang et al. 2014; Veyisoglu et al. 2020; Kittiwongwattana et al. 2015). Members of the genus Micromonospora tended to feature in bioprospecting campaigns as they were a rich source of novel bioactive compounds of therapeutic value, such as aminoglycoside antibiotics (Kasai et al. 2000; Bérdy 2005; Genilloud 2012) and lupinacidins A and B with significant antitumour activity (Igarashi et al. 2007). All members of the genus produced a well-developed substrate mycelium that carried single spores either directly or on short sporophores. None of the strains formed aerial hyphae. They contained tetra- and hexa-hydrogenated menaquinones with either nine or ten isoprene units as predominant isoprenologues, meso-diaminopimelic and xylose. Saturated and unsaturated fatty acids, notably iso-C15:0 and iso-C16:0 have been discovered as the major fatty acids of the genus. Phosphatidylethanolamine is the diagnostic phospholipid (Carro et al. 2018). Currently, there were 105 validly named species (https://lpsn.dsmz.de/genus/micromonospora) including the latest described species Micromonospora orduensis (Veyisoglu et al. 2020), Micromonospora deserti (Saygin et al. 2020), Micromonospora craterilacus (Ay et al. 2020) and Micromonospora pelagivivens (Intra et al. 2020). In this study, we describe a novel species, strain NEAU-HG-1T, isolated from soil. Here we report on the taxonomic characterization and classification of the isolate and propose that strain NEAU-HG-1T represents a new species of the genus Micromonospora, for which the name Micromonospora rubida sp. nov. is proposed.

Materials and methods

Isolation and maintenance of the organism

Strain NEAU-HG-1T was isolated from soil collected from Harbin, Heilongjiang Province, Northeast China (45°36′ N, 127°41′ E). The soil sample was air-dried at room temperature for 14 days before isolation. For isolation, 5 g dried soil was diluted in sterile distilled water (45 ml), and then the soil suspension was incubated in a constant temperature shaker at 28 ºC and 250 g. for 30 min. Subsequently, the strain was isolated using the standard dilution plate method and grown on sodium succinate-asparagine agar (Piao et al. 2017) supplemented with cycloheximide (50 mg l−1) and nalidixic acid (20 mg l−1). After 3 weeks of aerobic incubation at 28 ºC, colonies were transferred and purified on oatmeal agar [International Streptomyces Project (ISP) medium 3] (Shirling and Gottlieb 1966) and maintained as glycerol suspensions (30%, v/v) at − 80 ºC. Reference type strains M. auratinigra DSM 44815T, Micromonospora olivasterospora JCM 7348T, Micromonospora pisi DSM 45175T, Micromonospora soli NBRC 110009T and Micromonospora kangleipakensis DSM 45612T were purchased and isolated from peat swamp forest soil (Thawai et al. 2004), soil of Hiroshima (Kawamoto et al. 1983), root nodules of Pisum sativum (Garcia et al. 2010), rice rhizosphere soil (Thawai et al. 2016) and sample of limestone quarry (Nimaichand et al. 2013), respectively. However, the isolation source of strain M. coerulea DSM 43143T had not been reported.

Phenotypic characterization

Morphological characteristics were observed by light microscopy (Nikon ECLIPSE E200) and scanning electron microscopy (Hitachi SU8010) using cultures grown on ISP 3 agar at 28 °C for 4 weeks (Jin et al. 2019). Samples for scanning electron microscopy were prepared by cutting a block from an agar plate and then fixing it in 2.5% glutaraldehyde buffer (pH 7.2) at 4 °C for approximately 1.5 h. After rinsing twice with phosphate buffer, samples were dehydrated through a graded series of ethanol, passed through tert-butanol and then critically point dried. The dried samples were placed onto a stub bearing adhesive and spatter-coated with gold under vacuum. (Guan et al. 2015). The cultural characteristics of strain NEAU-HG-1T were determined after 14 days at 28 ºC on various ISP (ISP 1–7) media (Shirling and Gottlieb 1966), Bennett’s agar (Waksman 1967) and nutrient agar (Jones 1949). ISCC-NBS colour charts (Kelly 1964) were used to determine colours of aerial and substrate mycelia. The cell motility was identified depending on turbidity development in a tube containing semisolid medium (Leifson 1960). Gram reaction was determined by using the KOH lysis test method (Cerny 1978). Growth at different temperatures (4, 10, 15, 18, 25, 28, 32, 35, 37, 40 and 42 °C) was determined on ISP 3 agar after incubation for 14 days. The pH range for growth (pH 3–12, at intervals of 1 pH units) was tested in glucose-yeast extract broth (GY) (Jia et al. 2013) using the buffer system: 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; pH 11.0–12.0, 0.2 M KH2PO4/0.1 M NaOH (Cao et al. 2020; Zhao et al. 2019). NaCl tolerance (0–12%, with an interval of 1%, w/v) for growth were tested after 14 days growth in GY broth at 28 °C. 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 (Gordon et al. 1974; Yokota et al. 1993). Hydrolysis of Tweens (20, 40 and 80) and production of urease were tested as described by Smibert and Krieg (1994). The related type strains were also included for comparison in all tests.

Chemotaxonomic analyses

For the chemotaxonomic analysis, freeze-dried biomass was prepared from cultures grown in GY medium on a rotary shaker (250 g) at 28 °C for 7 days. The isomer of diaminopimelic acid in the cell-wall hydrolysates was derivatized according to McKerrow et al. (2000) and analysed by the HPLC method described by Yu et al. (2013). The whole-cell sugars were performed according to the procedures developed by Lechevalier and Lechevalier (1980). The phospholipids in cell were separated by two-dimensional TLC and identified using the method of Minnikin et al. (1984). Menaquinones were extracted from freeze-dried biomass and purified according to Collins (1985). Extracts were analysed by a HPLC–UV method (Wu et al. 1989) using an Agilent Extend-C18 Column (150 × 4.6 mm, i.d. 5 µm), typically at 270 nm. The mobile phase was acetonitrile/propyl alcohol (60:40, v/v) (Song et al. 2019). The presence of mycolic acids was checked by the acid methanolysis method of Minnikin et al. (1980). To determine cellular fatty acid compositions, strain NEAU-HG-1T and its closely related strains were cultivated in GY medium in shake flasks at 28 °C for 7 days. Fatty acid methyl esters were extracted from the biomass as described by Gao et al. (2014) and analysed by GC–MS using the method of Xiang et al. (2011).

DNA preparation, amplification and determination of 16S rRNA and gyrB gene sequences

Extraction of chromosomal DNA and PCR amplification of the 16S rRNA gene sequence was carried out according to the procedure developed by Kim et al. (2000). The PCR product was purified and cloned into the vector pMD19-T (Takara) and sequenced using an Applied Biosystems DNA sequencer (model 3730XL). The almost full-length 16S rRNA gene sequence of strain NEAU-HG-1T, comprising 1509 bp, was obtained and compared with type strains available in the EzBioCloud server (https://www.ezbiocloud.net/) (Yoon et al. 2017a) and retrieved using NCBI BLAST (https://blast.ncbi.nlm.nih.gov/Blast.cgi;), and then submitted to the GenBank database. PCR amplification and gene sequencing of gyrB was carried out using primers GYF1 and GYR3B, as described by Garcia et al. (2010). The gyrB gene was proposed by Yamamoto and Harayama (1995) as a phylogenetic marker for bacterial identification and classification (Harayama and Yamamoto 1996; Yamamoto and Harayama 1996; Yamamoto et al. 1999). The gyrB-based methods would be more useful than 16S rDNA-based methods because the gyrB was a single-copy gene which encoded the ATPase domain of DNA gyrase, an enzyme essential for DNA replication (Huang 1996) in almost all of the bacteria examined (Watanabe et al. 1998, 1999). Sequences were multiple aligned in Molecular Evolutionary Genetics Analysis (MEGA) software version 7.0 using the Clustal W algorithm and trimmed manually where was necessary. Phylogenetic trees were constructed with maximum likelihood (Felsenstein 1981) and neighbour-joining (Saitou and Nei 1987) algorithms using MEGA 7.0 (Kumar et al. 2016). The stability of the topology of the phylogenetic tree was assessed using the bootstrap method with 1000 repetitions (Felsenstein 1985). A distance matrix was generated using Kimura’s two-parameter model (Kimura 1980). 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 (Yoon et al. 2017a).

Genomic analysis, DNA-DNA hybridization and DNA G + C content

The genomic DNA of strain NEAU-HG-1T was extracted by SDS method for genome sequencing and assembly. The harvested DNA was detected by the agarose gel electrophoresis and quantified by Qubit. Whole-genome sequencing was performed on the Illumina HiSeq PE150 platform. A-tailed, ligated to paired-end 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 low quality reads from the paired-end were filtered by the step of quality control using our own compling pipeline. All good quality paired reads were assembled using the SOAP denovo (Li et al. 2010, 2008) (http://soap.genomics.org.cn/soapdenovo.html) into a number of scaffolds. Then the filter reads were handled by the next step of the gap-closing. Whole-genome phylogeny was generated using TYGS server (http://tygs.dsmz.de) (Meier-Kolthoff and Göker 2019). “Antibiotics and secondary metabolite analysis shell” (antiSMASH) version 5.0 was employed to analyze the bioactive secondary metabolites (Blin et al. 2019). The DNA G + C content was calculated from the genome sequence.

Digital DNA-DNA hybridization (dDDH) and average nucleotide identity (ANI) values were employed to further distinguish strain NEAU-HG-1T from its phylogenetic relatives with available genome sequences (Yoon et al. 2017b; Meier-Kolthoff et al. 2013). In the present study, ANI and dDDH values were determined from the genomes of strain NEAU-HG-1T, M. auratinigra DSM 44815T, M. kangleipakensis DSM 45612T, M. olivasterospora JCM 7348T and M. pisi DSM 45175T using the ortho-ANIu algorithm from Ezbiocloud (Yoon et al. 2017a, b) and the genome-to-genome distance calculator (GGDC 2.1) (Meier-Kolthoff et al. 2013) at http://ggdc.dsmz.de. Because of lacking whole genome sequence of closely related strain M. coerulea DSM 43143T, DNA-DNA relatedness test between them was carried out by the thermal renaturation method described by De Ley et al. (1970) under consideration of the modifications described by Huss et al. (1983), using a model Cary 100 Bio UV/VIS-spectrophotometer equipped with a Peltier-thermostatted 6 × 6 multicell changer and a temperature controller with 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 (Thomas et al. 2000), then sheared using a JY92-II 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 mean of the three values.

Results and discussion

Phenotypic characteristics

Morphological observation of 4-week-old cultures of strain NEAU-HG-1T grown on ISP 3 medium revealed that it showed morphology consistent with the genus Micromonospora. Strain NEAU-HG-1T was observed to be an aerobic, Gram-stain positive actinobacterium and produced well-developed, branched and non-fragmented substrate mycelium, while the aerial mycelium was absent. Non-motile and oval spores (0.5–0.6 × 0.7–0.9 μm) developed singly on the substrate mycelium (Fig. 1). Strain NEAU-HG-1T was found to grow well on ISP 1, ISP 3 and Bennett’s agar; grow moderately on ISP 4 medium; grow poorly on ISP 2, ISP 6 and ISP 7 media; but no growth occurred on ISP 5 and nutrient agar media. The colour of the substrate hyphae was strong orange yellow to vivid reddish orange, but no diffusible pigment was observed on any of the media tested for the isolate. The summaries of cultural characteristics of strain NEAU-HG-1T were shown in Table S1. The strain was found to grow at a temperature range of 10–37 °C (optimum temperature 28 °C), pH 6–12 (optimum pH of 7) and in the presence of NaCl up to 3% (w/v). The physiological and biochemical properties of strain NEAU-HG-1T were shown in Table 1.

Fig. 1
figure 1

Scanning electron microscopy of strain NEAU-HG-1T grown on ISP 3 for 4 weeks at 28 °C. Bar, 1.0 μm

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Table 1 Differential characteristics of strain NEAU-HG-1T and its closely related species of the genus Micromonospora
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Chemotaxonomic characteristics

All the chemotaxonomic data were consistent with the assignment of strain NEAU-HG-1T to the genus Micromonospora. The isolate was determined to contain meso-diaminopimelic acid in the cell wall. Whole-cell sugars contained arabinose and xylose. The phospholipid profile consisted of diphosphatidylglycerol, phosphatidylethanolamine, glycolipid and phosphatidylinositol (Fig. S1). The menaquinones were MK-10(H6) (45.9%), MK-10(H4) (33.6%), MK-10(H2) (10.1%), MK-10(H8) (6.8%), MK-9(H4) (1.7%), MK-9(H6) (1.3%) and MK-9(H2) (0.6%). The cellular fatty acid profile was composed of C17:0 cycle (25.7%), iso-C15:0 (18.8%), iso-C16:0 (10.5%), C17:0 (7.9%), C18:1 (7.4%), iso-C18:0 (7.3%), 10-methyl C17:0 (6.9%), iso-C17:0 (4.4%), C19:1 (3.3%), C18:0 (2.4%), iso-C16:1 (1.4%), 10-methyl C18:0 (1.3%), anteiso-C17:0 (1.2%), iso-C17:1 (0.9%) and anteiso-C15:0 (0.6%). Mycolic acids were not found.

Molecular characteristics

EzBioCloud analysis based on 16S rRNA gene sequence (1509 bp; GenBank/EMBL/DDBJ accession number MG753996) indicated that strain NEAU-HG-1T should be affiliated to the genus Micromonospora and possessed high 16S rRNA gene sequence similarities to M. auratinigra DSM 44815T (98.9%) and M. coerulea DSM 43143T (98.7%). Phylogenetic analysis using the 16S rRNA gene sequences showed that the strain formed a stable clade with M. coerulea DSM 43143T and clustered with M. soli NBRC 110009T (98.1%) and M. kangleipakensis DSM 45612T (97.9%) in the neighbour-joining tree (Fig. 2). This relationship was also observed in the maximum-likelihood tree (Fig. S2). However, whole-genome phylogeny showed strain NEAU-HG-1T formed a stable phyletic line with M. olivasterospora JCM 7348T (98.1%) (Figure. S3). The partial gyrB gene sequence obtained for strain NEAU-HG-1T (1161 nt) has been deposited in the GenBank/EMBL/DDBJ databases as MW091032. The gyrB sequence analysis showed that strain NEAU-HG-1T was placed in the genus Micromonospora. Meanwhile, the phylogenetic analyses based on gyrB gene sequences showed that the strain NEAU-HG-1T formed a stable clade with M. pisi DSM 45175T (97.8%) in the neighbour-joining tree (Fig. S4), and the relationship was also supported by the corresponding maximum-likelihood (Fig. S5). Based on the 16S rRNA gene sequences similarities and phylogenic analysis, M. auratinigra DSM 44815T, M. coerulea DSM 43143T, M. olivasterospora JCM 7348T, M. pisi DSM 45175T, M. soli NBRC 110009T and M. kangleipakensis DSM 45612T were selected as the reference strains for comparative analysis.

Fig. 2
figure 2

Neighbour-joining tree showing the phylogenetic position of strain NEAU-HG-1T (1509 bp) and the 105 phylogenetically closely related representative species with validly-published names in the genus Micromonospora. The out-group used was Catellatospora citrea subsp. citrea strain DSM 43903T. Only bootstrap values above 50% (percentages of 1000 replications) are indicated. Asterisks indicate branches also recovered in the maximum-likelihood tree; Bar, 0.005 nucleotide substitutions per site

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DNA-DNA hybridization by the thermal renaturation method was carried out between strain NEAU-HG-1T and M. coerulea DSM 43143T to determine whether the isolate represented a novel species. Strain NEAU-HG-1T showed a DNA-DNA relatedness of 33.8 ± 2.2% with M. coerulea DSM 43143T. Digital DNA-DNA hybridization and ANI values were employed to further clarify the relatedness between strain NEAU-HG-1T and M. auratinigra DSM 44815T, M. kangleipakensis DSM 45612T, M. olivasterospora JCM 7348T and M. pisi DSM 45175T. The levels of digital DNA-DNA hybridization between them were 27.2% (24.8–29.7%), 29.2% (26.8–31.7%), 32.4% (29.9–34.9%) and 22.5% (20.2–24.9%), respectively. These values were below the threshold value of 70% recommended by Wayne et al. (1987) for assigning strains to the same genomic species. Similarly, the low ANI values between strain NEAU-HG-1T and its reference strains M. auratinigra DSM 44815T, M. kangleipakensis DSM 45612T, M. olivasterospora JCM 7348T and M. pisi DSM 45175T were found to be 83.7%, 84.8%, 86.4% and 78.6%, respectively, a result well below the threshold used to delineate prokaryote species (Richter and Rossello-Mora 2009; Chun and Rainey 2014).

The assembled genome sequence of strain NEAU-HG-1T was found to be 7,473,743 bp long and composed of 37 scaffolds with an N50 of 463,225 bp, a DNA G + C content of 72.8% and a coverage of 236 X. It was deposited in GenBank database under the accession number JAAALO000000000. NCBI Prokaryotic Genome Annotation Pipeline (PGAP) revealed four copies of the 5S rRNA genes, one copy of the 16S rRNA gene, one copy of the 23S rRNA gene, 51 tRNA genes, three copies of noncoding RNA genes and 6067 protein-coding genes (CDSs). Detailed genomic information and other general features of genome sequences were shown in Table S2. Among these CDSs, 5133 (84.6%) genes were classified into 24 clusters of orthologous groups of proteins. Most of the genes were associated with functions such as transcription (559), carbohydrate transport and metabolism (376), amino acid transport and metabolism (371), signal transduction mechanisms (309), lipid transport and metabolism (265), coenzyme transport and metabolism (264), cell wall/membrane/envelope biogenesis (263), translation, ribosomal structure and biogenesis (262), inorganic ion transport and metabolism (257), energy production and conversion (256), and secondary metabolites biosynthesis, transport and catabolism (227). These functions were essentials for nutritional/spatial competition and antagonism against microorganisms to compete in various ecosystems (Liu et al. 2019). Genome mining analysis using antiSMASH 5.0 led to the identification of 24 putative gene clusters in the genome of strain NEAU-HG-1T, including 20 gene clusters that showed very low similarity to the known gene clusters of hopene, kirromycin, nystatin-like Pseudonocardia polyene, divergolide A, nostopeptolide A2, enduracidin, formicamycins A-M, gentamicin and so on. Hence, it had great potential to biosynthesize various secondary metabolite and produce novel bioactive compounds. One cluster showed 83% similarity to the reported biosynthetic gene cluster of desferrioxamine B in Streptomyces coelicolor A3(2) (Becerril et al. 2018). The other three putative gene clusters with more than 50% similarity to the known clusters were related to the production of catenulipeptin (Wang and van der Donk 2012), alkyl-O-dihydrogeranyl-methoxyhydroquinones (Awakawa et al. 2011) and avilamycin A (Weitnauer et al. 2001). The strain NEAU-HG-1T contained genes predicted to code for indole-3-glycerol phosphate synthase, an intermediate in the tryptophan synthetic pathway associated with the production of indol-acetic acid (IAA) which stimulated plant growth (Ouyang et al. 2000). Genome analysis also showed that strain NEAU-HG-1T contained 115 glycoside hydrolases (GHs), 103 glycosyl transferases (GTs), 15 carbohydrate esterases (CEs), 5 auxiliary activities (AAs) and 2 polysaccharide lyases (PLs) genes. To sum up, it was speculated that strain NEAU-HG-1T had immense potential to be a rich source for producing various bioactive compounds and display striking functions.

Beside of the genotypic evidence above, the strain NEAU-HG-1T also could differentiate from its closely related strains by phenotypic characteristics (Table 1). Differential cultural characteristics contained: pH range of strain NEAU-HG-1T was up to 12, which is higher than that of other reference strains. Other phenotypic differences included: temperature range and NaCl tolerance for growth, hydrolysis of starch and aesculin, decomposition of cellulose, liquefaction of gelatin, production of H2S, coagulation and peptonization of milk and utilization of inositol, d-mannitol, d-raffinose, d-fructose, lactose, l-rhamnose, d-xylose, l-arabinose, d-ribose, l-alanine, creatine, l-glutamic acid, l-glutamine, l-threonine, l-tyrosine, glycine, l-proline and l-serine. Therefore, based on a combination of chemotaxonomic, morphological, molecular and physiological data, strain NEAU-HG-1T represents a novel species of the genus Micromonospora, for which the name Micromonospora rubida sp. nov. is proposed.

Description of Micromonospora rubida sp. nov.

Micromonospora rubida (ru'bi.da. L. fem. adj. rubida reddish)

Aerobic, Gram-stain-positive actinobacterium that forms single oval spores on well-developed branched substrate hyphae. Aerial mycelium is not produced and the spores are 0.5–0.6 × 0.7–0.9 μm in size. Good growth on ISP 1, ISP 3 and Bennett’s agar media; moderate growth on ISP 4 medium; poor growth on ISP 2, ISP 6 and ISP 7 media; but no growth on ISP 5 and nutrient agar media. The colour of the substrate hyphae is strong orange yellow to vivid reddish orange, and no soluble pigment is produced on all used media. The growth temperature range is 10–37 °C, with an optimum at 28 °C. Growth is occurred at pH 6–12 (optimum pH 7.0). The maximum NaCl concentration for growth is 3.0% (w/v). Weakly positive for hydrolysis of aesculin, but negative for hydrolysis of Tweens (20,40 and 80) and starch, liquefaction of gelatin, production of urease, coagulation and peptonization of milk, decomposition of cellulose, production of H2S, and reduction of nitrate. Utilize raffinose, d-mannitol, d-galactose, d-glucose, lactose, maltose, l-rhamnose, d-fructose and sucrose as the sole carbon source for growth, while the following substrates are not used as carbon sources: inositol, l-arabinose, d-mannose, d-ribose, d-sorbitol and d-xylose. l-alanine, l-arginine, l-aspartic acid, l-asparagine, l-serine and creatine are utilized as the sole nitrogen source, but not l-glutamic acid, l-glutamine, l-proline, l-threonine, l-tyrosine or glycine. Cell walls contain meso-diaminopimelic acid as the diagnostic diamino acid and the whole-cell sugars are arabinose and xylose. The phospholipid profile is identified to contain diphosphatidylglycerol, phosphatidylethanolamine, glycolipid and phosphatidylinositol. The predominant menaquinones (> 10%) are MK-10(H2), MK-10(H4) and MK-10(H6). The major fatty acids (> 10%) are C17:0 cycle, iso-C15:0, and iso-C16:0. The DNA G + C content of the type strain is 72.8%.

The type strain is NEAU-HG-1T (= CGMCC 4.7479T = JCM 32386T), isolated from soil collected from Harbin, Heilongjiang Province, Northeast China. The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain NEAU-HG-1T is MG753996. The GenBank/EMBL/DDBJ accession number for the partial gyrB gene sequence of strain NEAU-HG-1T is MW091032. The Whole Genome Shotgun project has been deposited at DDBJ/ENA/GenBank under the accession JAAALO000000000. The version described in this paper is version JAAALO000000000.1.