Introduction

The genus Actinopolyspora was firstly described by Gochnauer et al. [9], with the description of Actinopolyspora halophila as the type species. At the time of writing, the genus comprises 12 recognized species, all of them are halophilic actinomycetes, namely A. halophila [10], A. mortivallis [35], A. xinjiangensis [11], A. egyptensis [14], A. alba and A. erythraea [34], A. algeriensis [24], A. saharensis, A. righensis, and A. mzabensis [25–27], A. dayingensis [13], and A. lacussalsi [12].

Typically, members of the genus are characterized by fragmentation of both aerial and substrate mycelia into rods and ovoid elements, a chemotype IVA cell-wall (meso-diaminopimelic acid without glycine, and arabinose and galactose as diagnostic whole-cell sugars) [21], a phospholipid type PIII (phosphatidylcholine) pattern [22], the presence of MK-9(H4) and MK-10(H4) as the predominant menaquinones, the presence of anteiso-C17:0, iso-C15:0, and iso-C17:0 as the major fatty acids, and the absence of mycolic acids [10, 11, 34].

During an investigation on halophilic actinomycetes from Saharan soils in Biskra province (Northern Sahara), strain H254T was isolated and identified by a polyphasic approach. Based on phenotypic and genotypic characteristics, it is proposed that the strain H254T represents a novel species of the genus Actinopolyspora, for which the name Actinopolyspora biskrensis sp. nov. is proposed.

Materials and Methods

Isolation and Phenotypic Characterization

The strain H254T was isolated from a Saharan soil sample collected from Biskra (34°38′ North, 5°25′ East) in northeast of Algeria, by serial dilution method on complex medium (CM) agar [3] supplemented with 15 % (w/v) NaCl and 50 mg l−1 actidione. After 3–4 weeks incubation at 30 °C, the strain H254T was picked-up, purified, and preserved on CM agar medium at 4 °C. Strain H254T was deposited in the German Collection of Microorganisms and Cell Cultures (DSMZ) as strain DSM 46684T and in Spanish Type Culture Collection (CECT) as strain CECT 8576T.

The colors of substrate and aerial mycelia and any soluble pigments produced were determined by comparison with chips from the ISCC-NBS color charts [17]. Morphological characteristics were observed by light microscopy using 14 days old cultures grown on CM agar medium, International Streptomyces Project (ISP 4 and ISP 2) media, and nutrient agar. Growth was tested at pH 5.0–9.0 (at intervals of 1.0 pH units) and at 20, 30, 37, and 45 °C on nutrient agar. NaCl tolerance was studied on nutrient agar containing NaCl at final concentrations of 0–30 % (w/v) (at intervals of 5.0 %). Other physiological characteristics, including utilization of sole carbon and nitrogen sources, decarboxylation of nine organic acids, degradation of adenine, aesculin, arbutin, gelatin, guanine, hypoxanthine, starch, testosterone, Tween 80, tyrosine, and xanthine, and reduction of nitrate and sensibility to lysozyme, were assessed by the media and methods of Locci et al. [23].

Chemotaxonomic Characterization

Biomass for chemical and molecular studies was obtained by cultivation in shake flasks (250 rpm, 30 °C, 7 days) using complex medium broth (pH 7.0) supplemented with 15 % (w/v) NaCl. The whole-cell sugar pattern and the diagnostic isomers of diaminopimelic acid were analyzed according to the procedures developed by Becker et al. [1] and Lechevalier and Lechevalier [21]. Phospholipids were extracted and identified as described by Minnikin et al. [28]. The cellular fatty acid analysis was performed as described by Sasser [33] using the Microbial Identification System (MIDI). Menaquinones were extracted following the procedure of Minnikin et al. [30], and separated by HPLC [20]. Analysis of mycolic acids was performed using the method of Minnikin et al. [29].

16S rRNA Gene Sequence and Phylogenetic Analysis

The genomic DNA of strain H254T was extracted with DNA extraction kit (MasterPureTM Gram-Positive DNA Purification Kit, Epicentre® Biotechnologies, Madison). PCR amplification of the 16S rRNA gene was performed as described by Rainey et al. [31]. PCR products were purified with a PCR product purification kit (Qiagen, Germany). The primers used for sequencing were listed in Coenye et al. [4]. Multiple alignments with sequences of all species of the genus Actinopolyspora and calculations of levels of sequence similarity were carried out by the EzTaxon server [18]. Phylogenetic trees were constructed using the neighbor-joining method [32] with Jukes and Cantor model [16], maximum-likelihood [6] with Kimura’s two-parameter model [19], and maximum-parsimony methods [8]. The topology of the phylogenetic tree was evaluated using the bootstrap resampling method of Felsenstein [7] with 1,000 replicates.

DNA–DNA Hybridization

For DNA–DNA hybridization, cells were disrupted by a French pressure cell (Thermo Spectronic). DNA in the crude lysate was purified by chromatography on hydroxyapatite as described by Cashion et al. [2]. Genomic hybridization experiments between strain H254T and A. halophila DSM 43834T, A. saharensis DSM 45459T or A. algeriensis DSM 45476T were performed by the method described by De Ley et al. [5] under consideration of the modifications described by Huss et al. [15] 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). DNA–DNA hybridization experiments were done in duplicate in 2 × SSC in the presence of 10 % (w/v) formamide at 71 °C.

Results and Discussion

The strain H254T showed good growth on ISP 4 medium, moderate growth on nutrient agar and CM agar media, but no growth was observed on ISP 2 medium. The aerial mycelium was moderately produced with white to yellowish-white color on these media. The strain formed irregularly branched and fragmented substrate mycelium with dark brown color on ISP 4 medium, pinkish color on nutrient agar, and beige to reddish-orange color on CM agar medium. The diffusible pigments were produced on nutrient agar (pinkish) and ISP 4 (dark brown) media but not on CM agar medium. The aerial mycelium was irregularly branched and formed straight to flexuous chains of 5–15 spores (sometimes 20 spores) per chain. The spores were non-motile and rod-shaped. Strain H254T growth occurring in the presence of 10–30 % (w/v) NaCl (optimum 15–20 %), at pH 6–8 (optimum pH 7.0) and at 20–37 °C (optimum 30 °C). The detailed physiological features are indicated in Table 1 and in the species description. Cell-wall hydrolysate of strain H254T contained the meso-diaminopimelic acid isomer, but not glycine. Whole-cell sugars were arabinose, galactose, and ribose. This is typical of cell wall type IV and whole-cell sugar pattern type A [21]. The major menaquinones were MK-9(H4) (62.3 %) and MK-10(H4) (28.0 %), and minor amounts of MK-9(H2) (3.2 %), MK-9(H0) (2.1 %), MK-10(H6) (1.2 %), and MK-10(H2) (0.8 %) were also detected. The phospholipid profile contained diphosphatidylglycerol, phosphatidylglycerol, phosphatidylcholine, and three unknown glycolipids (Supplement Fig. 1). The fatty acid profiles were composed as follows: anteiso-C17:0 (32.8 %), iso-C15:0 (28.0 %), iso-C17:0 (12.3 %), 9-methyl C16:0 (8.6 %), anteiso-C15:0 (7.9 %), and iso-C16:0 (5.1 %).

Table 1 Differential characteristics of strain H254T compared with its closest relative recognized species of the genus Actinopolyspora
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Phylogenetic analysis of an almost complete 16S rRNA gene sequence (1,491 bp, GenBank accession number KJ574180) showed that strain H254T was related to members of the genus Actinopolyspora. Moreover, the 16S rRNA sequence exhibited highest 16S rRNA gene sequence similarity to A. saharensis DSM 45459T (99.2 %), A. halophila DSM 43834T (99.1 %), and A. algeriensis DSM 45476T (99.0 %). The phylogenetic relationship between strain H254T and the other Actinopolyspora species is seen in the neighbor-joining (Fig. 1), maximum-parsimony (Supplement Fig. 2), and maximum-likelihood (Supplement Fig. 3) dendrograms.

Fig. 1
figure 1

Phylogenetic tree for species of the genus Actinopolyspora calculated from almost complete 16S rRNA gene sequences using Jukes and Cantor [16] evolutionary distance methods and the neighbor-joining method of Saitou and Nei [32]. This illustrates the taxonomic position of strain H254T relative to the other species of the genus. Asterisks indicate branches that are conserved when the neighbor-joining, maximum-parsimony, and maximum-likelihood methods were used in constructing phylogenetic trees. Numbers at nodes are bootstrap values, expressed as percentages of 1,000 resamplings (only values >50 % are shown). Saccharopolyspora rosea IMMIB L-1070T was used as an outgroup. Bar 1 substitution per 100 nucleotides

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DNA–DNA relatedness between strain H254T and strains A. saharensis DSM 45459T, A. halophila DSM 43834T, and A. algeriensis DSM 45476T has respective mean values of 57.2, 68.4, and 45.6 %, respectively.

Strain H254T differs from these three closely related species of Actinopolyspora by several physiological characteristics, and also by some chemotaxonomical characteristics (Table 2). Strain H254T differs from A. halophila DSM 43834T, which is genetically the closest species (DNA–DNA relatedness of 68.4 %), by 17 physiological characteristics, by the percentage of fatty acid anteiso-C15:0 (only 7.9 % for H254T and 34.6 % for A. halophila DSM 43834T) and anteiso-C17:0 (32.8 % for H254T and only 10.2 % for A. halophila DSM 43834T), by the percentage of menaquinone MK-10(H4) (28.0 % for H254T and only 10.0 % for A. halophila DSM 43834T), by the presence of diphosphatidylglycerol (DPG), and by the absence of lyso-DPG.

Table 2 Cellular fatty acid, menaquinone, and phospholipid composition of Actinopolyspora biskrensis H254T in comparison with the closely related species A. halophila DSM 43834T, A. saharensis DSM 45459T, and A. algeriensis DSM 45476T
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It is evident from the phenotypic, chemotaxonomic, and genetic data that strain H254T represents a novel species in the genus Actinopolyspora, for which we propose the name Actinopolyspora biskrensis sp. nov.

Description of Actinopolyspora biskrensis sp. nov

Actinopolyspora biskrensis (bis.kren’sis, N.L. fem. adj. biskrensis pertaining to Biskra, where the type strain was isolated).

Gram-positive, aerobic, extremely halophilic actinomycete. Aerial mycelium is white to yellowish-white, and forms straight to flexuous chains of 5 to 15 (sometimes 20) rod-shaped and non-motile spores. The color of the substrate mycelium is pinkish on nutrient agar medium, dark brown on ISP 4 medium, and beige to reddish-orange on CM agar medium. The substrate mycelium is well developed and fragments with age into non-motile rods. The diffusible pigments are produced on nutrient agar (pinkish) and ISP 4 (dark brown) media, but not on CM agar medium. Temperature and pH ranges for growth are 20–37 °C (optimal at 30 °C) and pH 6.0–8.0 (optimal at pH 7.0). The NaCl concentration range for growth is 10–30 %, with optimal growth occurring at 15–20 %. Utilizes adonitol, l-arabinose, d-cellobiose, erythritol, d-fructose, d-galactose d-glucose, glycerol, d-lactose, maltose, d-mannose, d-raffinose, l-rhamnose, d-ribose, d-sorbitol, sucrose, d-trehalose, and d-xylose, as sole carbon sources, but not meso-inositol, mannitol, d-melezitose, and d-melibiose. Nitrates are not reduced. Aesculin, gelatin, hypoxanthine, starch, Tween 80, and xanthine are hydrolyzed; adenine, arbutin, casein, guanine, testosterone, and tyrosine are not decomposed. H2S is not formed. Citrate was decarboxylated, but not acetate benzoate, butyrate, oxalate, propionate, pyruvate, succinate, and tartrate. l-serine was used as source of nitrogen, but not l-alanine and l-proline. Moreover, the growth occurs in the presence of erythromycin (15 µg ml−1) and 0.005 % (w/v) lysozyme, but not in the presence of tetracycline (30 µg ml−1) and nalidixic acid (30 µg ml−1). Contains meso-diaminopimelic acid, as cell-wall diamino acid, arabinose, and galactose as major whole-cell sugars (chemotype IVA). The diagnostic phospholipid is phosphatidylcholine. Mycolic acids are absent. The major menaquinones are MK-9(H4) and MK-10(H4). The major fatty acids are anteiso-C17:0, iso-C15:0, and iso-C17:0.

The type strain is H254T (=DSM 46684T =CECT 8576T) isolated from a Saharan soil sample collected from Biskra region (northern Sahara).