Introduction

The genus Streptomyces was first described by Waksman and Henrici [1]. Members of the genus Streptomyces are aerobic, Gram-positive filamentous bacteria which can form branched substrate and aerial mycelia. They have LL-diaminopimelic acid with no characteristic sugars in the cell wall [2] and have high genomic DNA G+C contents [3, 4]. Streptomyces strains are an important source of a broad range of bioactive secondary metabolites, which are widely used in the fields of food, agriculture, and pharmaceutical industries [5, 6].

Streptomyces can live in a range of environments, including both fertile and barren soils [7,8,9]. When we investigated the diversity of actinobacteria in the west of China, a novel actinobacterium producing antibiotic activity against Methicillin-resistant Staphylococcus aureus (MRSA) was isolated from sand collected from the Qaidam Basin, China. MRSA is a major cause of hospital-acquired infections, and has acquired resistance to many current frontline antibiotic classes, and consequently it is becoming increasingly difficult to combat MRSA infections [10]. The strain S10T may produce new bioactive compounds with anti-MRSA activity. Consequently, it is of considerable interest for further research.

Materials and methods

Bacterial strains and isolation

Strain S10T was isolated from a sand sample collected in the Qaidam Basin in Qinghai province, China, by using Gause’s synthetic agar medium (20.0 g soluble starch, 1.0 g KNO3, 0.5 g K2HPO4.3H2O, 0.5 g MgSO4.7H2O, 0.001 g FeSO4, 0.5 g NaCl and 20.0 g agar in 1.0 l tap water, pH 7.2), supplemented with nalidixic acid (25 μg ml−1) incubated for 7 days at 28 °C. The strain was stored at −86 °C in the presence of 20% (v/v) glycerol. The reference strains were Streptomyces chartreusis NBRC 12753T, Streptomyces variegatus NRRL B-16380T, Streptomyces flavovariabilis NRRL B-16367T, and Streptomyces kunmingensis NBRC 14463T.

Morphological, physiological, and biochemical tests

The morphology of spore-chains and hyphae were determined by light microscopy (BH-2; Olympus) and scanning electron microscopy (SU8010, Hitachi; JSM-5600, JEOL) using cultures grown on Gause’s synthetic agar medium at 30 °C for 20 days. Cultural characteristics was examined after growth on standard media ISP 2–7 [11], Czapek’s agar [12] and nutrient agar after incubation at 30 °C for 14 days. The utilization of sole carbon and nitrogen sources, and metabolism of starch and cellulose, were examined as described previously [13, 14]. Growth at various temperatures (4, 10, 15, 20, 30, 37, 40, 45, and 50 °C) and NaCl concentrations (0–10%) was examined on yeast extract-malt extract (ISP 2). The pH range and the optimum pH were determined by incubating at 28 °C in ISP 2 broth with the pH adjusted to between 4 and 12 by addition of KH2PO4/HCl, KH2PO4/K2HPO4, and K2HPO4/NaOH (at intervals of 1.0 pH unit). The antibacterial activity of strain S10T was determined using a cylinder plug antibacterial bioassay using a clinical methicillin-resistant Staphylococcus aureus isolate, a gift of Jodi Lindsay, St George’s Hospital, London, as the indicator strain [15]. This indicator strain, EMRSA-8, was resistant to a range of beta-lactam antibiotics, ciprofloxacin, erythromycin, rifampicin, tetracycline, and trimethoprim, but sensitive to vancomycin.

Chemotaxonomy

Hyphae for chemotaxonomic studies were prepared by growing the strain in TSB medium in shake flasks for 10 days at 30 °C. The hyphae were harvested by centrifugation and washed twice with distilled water. Then the cells were recentrifuged and freeze-dried. The diaminopimelic acid isomers in the cell wall and whole-cell sugars were analyzed with the method described by Lechevalier and Lechevalier [2] and Staneck and Roberts [16], respectively. The menaquinones were analyzed by the method of Collins et al. [17] and analysed by HPLC [18]. The polar lipids were examined using two-dimensional TLC and identified according to method of Minnikin et al. [19]. The cellular fatty acids methyl esters were extracted by the method of Sasser [20] and analysis by according to the standard protocol of the Sherlock Microbial identification (MIDI) system [21].

Molecular analysis

The genomic DNA of strain S10T was extracted and the 16S rRNA gene was amplified as described by Harunari et al. [22]. Closely related 16S rRNA gene sequences to that of strain S10T were identified using the EzTaxon-e server [23]. A phylogenetic tree was generated using the neighbor-joining [24], maximum-parsimony [25], and maximum-likelihood [25] algorithms in MEGA5.0 [26]. Evolutionary distances were calculated using the model of Jukes and Cantor [27]. Topologies of the resultant tree were evaluated by bootstrap analyses [28] based on 1000 resamplings. The G+C content of the DNA was examined by HPLC according to the method of Tamaoka and Komagata [29]. The genomic DNA of strain S10T was also used to obtain a draft genome sequence using Illumina sequencing; the draft genome consisted of 1 contigs with an estimated genome size of 8.66 Mb(CP015098). The whole-genome average nucleotide identity (ANI) value were calculated by Goris et al. [30]. A maximum-composite likelihood tree was performed using PhyloPhlAn and the method of Segata et al. [31]. Initially, all protein sequences from all annotated Streptomyces genomes (.faa files) were retrieved autonomously from the GenBank FTP site (last accessed November 2016) using the term “Streptomyces” as a query. Ortholog identification and alignment was performed in Phylophlan using the “-u” command. A maximum likelihood phylogeny was reconstructed from the concatenated alignments in FastTree MP implemented by the Cipres Science Gateway Server [32]. The tree was drawn using the JTT+CAT model with 20 discrete categories (-cat 20). Topology refinement was performed using the follow parameters: -nni 10 -spr 2 -sprlength 10. Nodal support was inferred from 1000 bootstrap pseudoreplicates.

Nucleotide sequence accession number

The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain S10T are MH257693- MH257698. The GenBank accession number for the genome of S10T is CP015098.1.

Results and discussion

Morphological, cultural, and physiological characteristics

Strain S10T formed extensively branched substrate hyphae, and the aerial hyphae formed straight spore chains (Fig. 1). The spores were cylindrical with a rough-textured surface (Fig. 1a). The growth characteristics of strain S10T cultured on different growth media were compared to those of the reference strains Streptomyces chartreusis NBRC 12753T, Streptomyces variegatus NRRL B-16380T, Streptomyces flavovariabilis NRRL B-16367T, and Streptomyces kunmingensis NBRC 14463T (Table S1). Strain S10T grew well on ISP medium 2–7, Czapek agar and nutrient agar. Sporulation was poor on ISP 5 and nutrient agar, and no sporulation or growth of aerial hyphae was observed for cultures grown on ISP 6 medium. Black soluble pigments were produced on ISP 6 and brown pigments were produced on nutrient agar. Prior to sporulation, aerial mycelia produced on all media except ISP 6 were white. The morphological features of isolate S10T were consistent with its classification in the genus Streptomyces [33] and were distinct from those of the reference strains.

Fig. 1
figure 1

Fig.1 Scanning electron micrograph showed cylindrical spores with a rough-textured surface (a) and straight spore chains (b) of strain S10T growing on Gause’s synthetic agar medium at 30 °C for 20 days

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The physiological properties of strain S10T, Streptomyces chartreusis NBRC 12753T, Streptomyces variegatus NRRL B-16380T, Streptomyces flavovariabilis NRRL B-16367T, and Streptomyces kunmingensis NBRC13368T were compared and found to be different (Table 1). Strain S10T could utilize L-myo-inositol, L-arabinose, D-fructose, D-lactose, D-mannitol, D-raffinose, L-rhamnose or D-xylose as sole carbon sources. It also utilized L-alanine, L-asparagine or L-histidine as sole nitrogen sources, but not L-leucine and L-cysteine (Table 1). The strain S10T could degrade starch, cellulose, gelatin, tween 20, tween 80 or urea. The temperature range for growth of strain S10T was 20–40 °C (optimum temperature 30 °C). The pH range for growth was 6–11 (optimum pH 8.0). The maximum NaCl concentration for growth was 10% (w/v) (optimum 1%).

Table 1 Phenotypic properties of strain S10T and related type species
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Cylinder plugs of strain S10T grown for 10 days on ISP 4 medium produced clearing zones of 10 (+/−2) mm, compared to zones of 13 mm (+/−2) mm produced when 10 μl of a 50 mg/ml solution of vancomycin was applied to a 10 mm diameter sterile filter disc on the lawn of an MRSA strain bacteria, indicating that the strain S10T produces antibiotic activity against MRSA.

Chemotaxonomic characteristics

Cell wall analysis indicated that strain S10T contained LL-diaminopimelic acid, as is characteristic for the genus Streptomyces. The whole-cell hydrolysate contained galactose and ribose. The predominant isoprenoid quinone compound were MK-9(H6) (61.3%) and MK-9(H8) (21.5%). The polar lipids detected were diphosphatidyl glycerol, phosphatidyl ethanolamine, phosphatidyl choline, three unknown phospholipids, an unknown aminophospholipid and an unknown phosphatidyl glycolipid (Figure S1). The major fatty acids were anteiso-C15:0 (31.3%), anteiso-C17:0 (17.5%), iso-C15:0 (12.8%), and iso-C16:0 (11.7%); the fatty acid composition was different to Streptomyces chartreusis NBRC 12753T, Streptomyces variegatus NRRL B-16380T and Streptomyces flavovariabilis NRRL B-16367T (Table 2). The DNA G+C content of strain S10T was 71.3 mol%.

Table 2 Cellular fatty acid composition of strain S10T and related type species
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Molecular analysis

There were six copies of 16S rDNA in S10T which were not same exactly. There were high sequence similarities between strain S10T and Streptomyces chartreusis NBRC 12753T (99.31%), Streptomyces phaeoluteigriseus DSM 41896T (99.24%), variegatus NRRL B-16380T (99.17%), and Streptomyces flavovariabilis NRRL B-16367T (99.17%) comparing with MH257693, MH257695, MH257696, MH257697, and MH257698. Similarities with Streptomyces kunmingensis NBRC14463T (98.82%), Streptomyces bungoensis DSM 41781T(98.76%), S. chartreusis NBRC 12753T (98.69%), and S. phaeoluteigriseus DSM 41896T (98.62%) with MH257694. Based on the 16S rRNA gene sequence, phylogenetic analysis also confirmed that strain S10T represented a member of the genus Streptomyces (Fig. 2). The whole-genome ANI value between strain S10T and S. chartreusis NBRC 12753T, S. phaeoluteigriseus DSM 41896T, S. variegatus NRRL B-16380T, S. flavovariabilis NRRL B-16367T, S. kunmingensis NBRC 14463T, S. bungoensis DSM 41781T were 83.63%, 82.89%, 92.55%, 92.51%, 79.29% and 82.87%, respectively. These values were below the species demarcation threshold of 95–96% ANI suggested for prokaryotic species [34], indicating that S10T was a new Streptomyces species. A phylogenetic tree based on 16S rRNA sequences alone was found to be unstable. As a consequence, a maximum-composite likelihood tree based on analysis of 400 protein sequences from 336 sequenced Streptomyces genomes was constructed. This analysis indicated strain S10T shared highest similarity with S. variegatus NRRL B-16380T and S. flavovariabilis NRRL B-16367T (Figure S2).

Fig. 2
figure 2

Neighbor-joining phylogenetic tree, based on nearly complete 16S rRNA gene sequences, showing the relationships between strain S10Tand strains of related species of the genus Streptomyces. Numbers at nodes are bootstrap values based on 1000 re-samplings (only values above 50% are shown).Asterisks (*, #) indicate that the clades are recovered in maximum-likelihood and maximum-parsimony trees, respectively

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Based on its phenotypic, phylogenetic, and chemotaxonomic characteristics, strain S10T represents a novel species within the genus Streptomyces, for which the name Streptomyces qaidamensis sp. nov. is proposed.

Description of Streptomyces qaidamensis sp. nov

Streptomyces qaidamensis (qai.dam.en’sis. N.L. masc. adj. qaidamensis pertaining to Qaidam, China, where the type strain was isolated).

Cells are aerobic and Gram-stain-positive. The substrate hyphae are branched and aerial mycelia formed straight spore chains. The spores are cylindrical with a rough-textured surface. It grow well on ISP medium 2–7, Czapek agar and nutrient agar. Sporulation is poor on ISP 5 and nutrient agar, and not detected on ISP 6. Aerial hyphae are white on all media except ISP 6. Production of black soluble pigments is observed on ISP 6 and brown pigments are produced on nutrient agar. Growth occur between 20–40 °C (optimum temperature 30 °C), at pH 6–11 (optimum pH 8.0) and with 0–10% (w/v) NaCl. It utilize myo-inositol, L-arabinose, D-fructose, D-lactose, D-mannitol, D-raffinose, L-rhamnose or D-xylose as sole carbon sources. It also utilize L-alanine, L-asparagine or L-histidine as sole nitrogen sources, but not L-leucine and L-cysteine. The strain S10T degrade starch, cellulose, gelatin, tween 20, tween 80 or urea. The cell wall contain LL-diaminopimelic acid, galactose and ribose. The predominant isoprenoid quinine compounds are MK-9(H6) and MK-9(H8). The polar lipids detected are diphosphatidyl glycerol, phosphatidyl ethanolamine, phosphatidyl choline, three unknown phospholipids, an unknown aminophospholipid, and an unknown phosphatidyl glycolipid. The major fatty acids are anteiso-C15:0, anteiso-C17:0, iso-C15:0 and iso-C16:0. The DNA G+C content of strain S10T is 71.3 mol%.

The type strain, S10T (=JCM 31184T =CGMCC 4.7315T) is isolated from a sand sample collected from the Qaidam Basin, China.