Introduction

Members of the phylum Actinobacteria are widely distributed in nature, and one important characteristic of this phylum is its role in degradation of organic matter (Lechevalier and Lechevalier 1967; Goodfellow and Williams 1983) and as source of antibiotics and other bioactive molecules (Chun et al. 1997; Labeda et al. 1997; Iwai and Takahashi 1992). Members of the genus Streptomyces constitute the largest actinomycete group and have been extensively studied over the past several decades for their potential agricultural, pharmaceutical or industrial applications (Watve et al. 2001; Saadoun and Gharaibeh 2002; Okoro et al. 2009; Goodfellow and Fiedler 2010; Santhanam et al. 2012; Mohammadipanah and Wink 2015; Nithya et al. 2015). Finding new actinobacterial species will presumably lead to the discovery of potentially new structural and beneficial secondary metabolites (Antony-Babu and Goodfellow 2008; Thumar et al. 2010). One way to explore new species is mining of underexplored habitats such as hyper-arid soils (Okoro et al. 2009; Santhanam et al. 2012; Mohammadipanah and Wink 2015). This paper is based on the polyphasic characterization of a novel Streptomyces sp. strain LPA192T, isolated from a soil sample collected from Lop Nur, a dried salt lake in Xinjiang Uygur Autonomous Region, Northwest China. Having dried for more than 60 years, Lop Nur is now primarily a salt flats with a potential salt deposit of about 1–2 m thick (Wood 2004). Recent studies have indicated that the source is unique to several new bacterial species (Li et al. 2015a; Liu et al. 2013, 2015; Zhang et al. 2012; Zheng et al. 2014)

Materials and methods

Isolation

Strain LPA192T was isolated from a soil sample collected from Lop Nur (90°52′E, 39°58′N) during October 2010. Sample (1 g) was suspended in a flask containing 10 ml sterile water and several glass beads. The suspension was kept in an orbital shaker (28 °C, 180 rpm) for 1 h. One milliliter of the solution was serially diluted 10- and 100-folds, and 100 µl of the 10−3 dilution plated on Reasoner’s 2A (R2A) agar medium (for composition see DSMZ 830). The plates were incubated at 28 °C for 14 days. The isolates obtained were subcultured in the same medium to obtain pure cultures. Based on the 16S rRNA gene phylogenetic profiles, strain LPA192T was selected among other strains for further characterization using polyphasic taxonomy. Pure cultures of strain LPA192T were maintained on R2A at 28 °C and also stored as glycerol suspensions (20 %, v/v) at −80 °C. Physiological tests were performed by cultivating the strain in R2A broth at 28 °C for 5 days as the basal growth condition, unless otherwise stated. Streptomyces tanashiensis CGMCC 4.1924T, S. nashvillensis CGMCC 4.1741T, S. roseolus CGMCC 4.2005T and S. filamentosus CGMCC 4.1565T were obtained from the China General Microbiological Culture Collection Center (CGMCC, Beijing, China), and S. gulbargensis CCTCC AA 206001T from the China Center for Type Culture Collection (CCTCC, Wuhan, China). All reference strains were grown under similar culture condition for subsequent comparative analysis.

Phenotypic characteristics

Gram reaction was determined by using the standard Gram’s staining method (Beveridge 2001) and confirmed by KOH lysis test (Cerny 1978). Morphology was observed using light (Philips XL 30) and scanning electron microscopy (ESEM-TMP). Cultural characteristics were examined on Gause’s synthetic agar, T5 (Ming et al. 2014), International Streptomyces Project (ISP; Shirling and Gottlieb 1966) media, Czapek’s agar (Waksman 1967), R2A agar and tryptic soy agar (TSA Difco). Color determination was carried out by using color chips from the ISCC-NBS color charts (Kelly 1964). Growth at different temperatures (4, 10, 15, 20, 28, 30, 37, 40, 45, 50, 55 and 60 °C) and NaCl tolerance (0–10.0 % w/v, with interval of 0.5 % unit) were tested on R2A medium. The pH range (4.0–13.0, at intervals of 1.0 pH unit) for growth was determined in R2A broth (28 °C, 14–21 days) using the buffer system as described by Xu et al. (2005). Catalase activity was detected by assessing production of bubbles on addition of a drop of 3 % (v/v) H2O2. Oxidase activity was identified by the oxidation of tetramethyl-p-phenylenediamine (Kovacs 1956). Antimicrobial susceptibility was tested according to Masadeh et al. (2011). Other physiological and biochemical characteristics were assessed by using the media and methods described by Gordon et al. (1974) and Williams et al. (1989). Carbon source utilization was evaluated according to the methods of Shirling and Gottlieb (1966) and Locci (1989). Nitrogen source utilization was determined as described by Williams et al. (1989).

Chemotaxonomy

Whole-cell hydrolysates were extracted, purified and analyzed by the method of Hasegawa et al. (1983) and Tang et al. (2009). Polar lipids were extracted and separated by two-dimensional thin-layer chromatography (TLC) (Minnikin et al. 1979) and analyzed as described by Collins and Jones (1980). Menaquinones were extracted according to the method of Collins et al. (1977) and identified by HPLC (Kroppenstedt 1982). Biomass for cellular fatty acid analysis of strain LPA192T and the reference strains was harvested from cultures grown in TSB (28 °C, 2 days). Cellular fatty acid analysis was performed by using Microbial Identification System (Sherlock version 6.1; MIDI database: TSBA6) (Sasser 1990).

Molecular analysis and DNA–DNA hybridizations

Genomic DNA extraction and PCR amplification of the 16S rRNA gene were performed as described by Li et al. (2007). Almost full sequence of 16S rRNA gene of strain LPA192T was compared with sequences of cultured species present in EzTaxon database (Kim et al. 2012). Alignments with sequences of the most closely related taxa and calculations of levels of sequence similarity were carried out using CLUSTALX program (Thompson et al. 1997). Phylogenetic analyses were performed by using three tree-making algorithms, neighbor-joining (Saitou and Nei 1987), maximum-likelihood (Felsenstein 1981) and maximum-parsimony (Fitch 1971) methods. Phylogenetic dendrograms were generated by using MEGA software package version 5.0 (Tamura et al. 2011). Evolutionary distance matrices of phylogenetic trees were calculated according to Kimura’s two-parameter model (Kimura 1980). Bootstrap analysis was performed with 1000 replications (Felsenstein 1985). G + C content of the genomic DNA was determined by HPLC (Mesbah et al. 1989) using Escherichia coli JM-109 as the reference strain. DNA–DNA relatedness was carried out by the fluorometric microwell method (Ezaki et al. 1989; Li et al. 2015b) at the optimal hybridization temperature (50 °C). The experiments were set with eight replications between strain LPA192T and its closest phylogenetic neighbors as indicated above.

Results and discussion

Phenotypic characteristics

Strain LPA192T was observed to be Gram-positive, aerobic and non-motile. Strain LPA192T grew well on R2A, yeast extract-malt extract agar (ISP 2), oatmeal agar (ISP 3), inorganic salts-starch agar (ISP 4), glycerol-asparagine agar (ISP 5), T5, Czapek’s agar and TSA, while moderately on Gause’s synthetic agar. Smooth, oval spores were borne in long, straight-to-flexuous chains (Fig. S1). Aerial mycelium was observed to be either white or gray on Gause’s synthetic agar, ISP 2, ISP 3, ISP 4, ISP 5, T5, Czapek’s agar, R2A and TSA. Substrate mycelium was observed to be orange yellow in majority of the tested media, but white on ISP 4, gray on TSA and vivid greenish blue on Czapek’s agar media. The strain did not produce diffusible pigment (Table S1). Growth of strain LPA192T was observed at 10–40 °C (optimum 28–37 °C), pH 5.0–8.0 (optimum pH 7.0) and 0–7 % (w/v) NaCl (optimum 0–2.5 %). The strain showed positive results for catalase, milk peptonization, milk coagulation and hydrolysis of gelatin, starch, oxidase and tweens (20, 40, 60 and 80) tests, but negative for hydrolysis of cellulose, nitrate reduction, urease and hydrogen sulfide production tests. The differential characteristics between LPA192T and the related type strains in the genus Streptomyces are shown in Table 1. The detailed physiological characteristics of strain LPA192T are given in the species description.

Table 1 Differential characteristics between strain LPA192T and its closely related type strains in the genus Streptomyces
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Chemotaxonomy

The diagnostic diamino acid of strain LPA192T was LL-diaminopimelic acid, while major sugar in whole-cell hydrolysates was mannose. Polar lipid profile contained phosphatidylethanolamine, diphosphatidylglycerol, phosphatidylinositol, an unidentified aminophospholipid, an unidentified aminolipid, two unidentified phospholipids and four unidentified polar lipids (Fig. S2). The predominant respiratory menaquinones of strain LPA192T were identified as MK-9 (H6) (48.4 %) and MK-10 (H4) (42.3 %). Fatty acid profile (>10 %) consisted of C16:0 (18.9 %), anteiso-C15:0 (18.7 %) and C18:1 ω9c (14.0 %). Detailed fatty acid profiles (>0.5 %) of strain LPA192T and its most closely related reference type strains are shown in Table 2.

Table 2 Fatty acid profiles (>0.5 %) of strain LPA192T and its closely related type strains in the genus Streptomyces
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Phylogenetic analysis and DNA–DNA relatedness

Sequence analysis of almost complete 16S rRNA gene of strain LPA192T (1535 bp; GenBank accession number KU301049) using EzTaxon-e server showed the strain is closely related to Streptomyces tanashiensis LMG 20274T (99.3 % pairwise sequence similarity), S. gulbargensis DAS131T (99.3 %), S. nashvillensis NBRC 13064T (99.3 %), S. roseolus NBRC 12816T (99.2 %) and S. filamentosus NBRC 12767T (99.1 %) while showing below 98.5 % sequencing similarities with other validly published Streptomyces species. Strain LPA192T also formed a clade with these closely related type strains in all the three phylogenetic dendrograms (Fig. 1, Figs. S3 and S4). DNA–DNA relatedness values between strain LPA192T and type strains S. tanashiensis CGMCC 4.1924T, S. gulbargensis CCTCC AA206001T, S. nashvillensis CGMCC 4.1741T, S. roseolus CGMCC 4.2005T and S. filamentosus CGMCC 4.1565T were 21.8 ± 3.5, 11.7 ± 0.7, 33.3 ± 1.7, 15.5 ± 2.2 and 33.9 ± 2.2 %, respectively, which is significantly lower than 70 % threshold value used for recognition of genomic species (Stackebrandt and Goebel 1994). The genomic DNA G + C content of strain LPA192T was 69.3 mol %.

Fig. 1
figure 1

Unrooted neighbor-joining phylogenetic tree based on 16S rRNA gene sequences of strain LPA192T and its closely related strains. Bootstrap values (expressed as percentages of 1000 replications) of above 50 % are shown at the branch points. Asterisks indicate that the corresponding nodes were also recovered in trees generated with the maximum-parsimony and maximum-likelihood methods. Bar, 0.005, nucleotide substitutions per site

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On the basis of low DNA–DNA relatedness values, phylogenetic analysis, morphological, physiological and chemotaxonomic data, the strain LPA192T is considered to represent a new species of the genus Streptomyces, for which the name Streptomyces xinjiangensis sp. nov. is proposed.

Description of Streptomyces xinjiangensis sp. nov.

Streptomyces xinjiangensis (xin.ji.ang.en’sis. N.L. adj. xinjiangensis pertaining to Xinjiang, a province of China from where the sample was collected).

Aerobic, non-motile, Gram-staining positive. Long, smooth, oval-shaped spores are arranged in straight chains. Aerial mycelium is white or gray without fragmentation. Substrate mycelium is orange yellow. Diffusible or melanoid pigments are not produced. Growth occurs at 10–40 °C, pH 5.0–8.0 and with 0–7 % (w/v) NaCl. Positive for catalase, milk peptonization, milk coagulation, gelatin, starch, oxidase and tweens (20, 40, 60 and 80) hydrolysis tests, but negative for cellulose hydrolysis, nitrate reduction, urease and hydrogen sulfide production tests. Utilizes D-arabinose, d -fructose, D(+)-galactose, d -mannose, D(+)-xylose, inositol, lactose, L-rhamnose, maltose as sole carbon sources, but not D(+)-trehalose or raffinose. Utilizes l -aspartic acid, l -arginine, l -asparagine, L-cystine, l -glutamic acid, l -histidine, l -isoleucine, l -lysine, l -methionine, l -phenylalanine, l -serine, l -threonine, l -tyrosine and l -valine as sole nitrogen sources, but not l -tryptophan. Cell-wall diamino acid is LL-diaminopimelic acid. Whole-cell hydrolysates contain mannose. Major polar lipids are phosphatidylethanolamine, diphosphatidylglycerol and phosphatidylinositol. The predominant menaquinones are MK-9 (H6) and MK-10(H4). Major fatty acids (>10 %) are C16:0, anteiso-C15:0 and C18:1 ω9c. The DNA G + C content of strain LPA192T is 69.3 mol  %. The type strain LPA192T (=KCTC 39601T = CGMCC 4.7288T) was isolated from a soil sample collected from Lop Nur in Xinjiang province in Northwest China. The GenBank accession number for the 16S rRNA gene sequence of strain LPA192T is KU301049.