Introduction

The genus Saccharopolyspora was first described by Lacey and Goodfellow1 for actinomycetes isolated from spontaneously heated sugarcane bagasse, and was assigned to the family Pseudonocardiaceae.2 Nevertheless, most species of this genus were isolated from soil samples. At the time of writing, the genus comprises 23 recognized species, with Saccharopolyspora hirsuta as the type species.1 Most species of the genus Saccharopolyspora were non-halophilic, and some of them were halotolerant. However, only these species of this genus were reported to be extremely halophilic: S. halophila,3 S. qijiaojingensis4 and S. lacisalsi.5

Members of the genus Saccharopolyspora were characterized by extensively branched substrate mycelium that may fragment into rod-shaped and non-motile elements, and aerial mycelium that may segment into bead-like chains of spores. The strains of this genus were characterized chemotaxonomically by the presence of meso-diaminopimelic acid in the cell wall, arabinose and galactose as diagnostic sugars in whole-cell hydrolysates (wall chemotype IVA6), iso and anteiso-branched chain fatty acids, major amounts of phosphatidylcholine (phospholipid pattern PIII7), and MK-9(H4) as the predominant menaquinone, but lack mycolic acids.8 The DNA base compositions of members of this genus fall within the range of 66–77 mol% G+C.9 Several strains and species belonging to the genus Saccharopolyspora produce different bioactive secondary metabolites, such as the enzyme inhibitor CL307-2410 and the antimicrobial compounds erythromycin11 and spinosyns.12

During our investigation of actinomycetes from Saharan soils, the strain H53T was isolated. This strain showed a good antibacterial and antifungal activity, especially against Bacillus subtilis (ATCC 6633), Micrococcus luteus (ATCC 9314) and Mucor ramannianus (NRRL 1829). In this paper we describe the characterization of new species in the genus Saccharopolyspora.

Materials and methods

Isolation of actinomycete strain

During our investigations on extremophilic actinomycetes diversity in Algerian Saharan soils, the halophilic strain H53T was isolated from a non-saline soil sample (electrical conductivity=0.15 mS cm−1) collected from Chaâbet Ntissa (32°27′52′′N, 3°40′19′′E), Béni-isguen (Mzab), Ghardaïa province (South of Algeria), by a dilution-plate agar method using humic acid–vitamin agar medium13 supplemented with actidione (50 mg l−1) and 20% (w/v) NaCl at 30 °C for 45 days. The strain, which formed a white colony, was purified and maintained at 4 °C on complex medium (CM) agar14 containing 20% (w/v) NaCl. Strain H53T was deposited in the German Collection of Microorganisms and Cell Cultures as strain DSM 45606T, in the Culture Collection, University of Göteborg, as strain CCUG 63370T, and in the Spanish Type Culture Collection as strain CECT 8304T.

Cultural and micro-morphological characteristics

Cultural characteristics of strain H53T were investigated after 7, 14 and 21 days of incubation at 30 °C using the media of the International Streptomyces Project, ISP 2 and ISP 4,15 CM agar14 and nutrient agar (bacteriological peptone, 5 g; meat extract, 1 g; yeast extract, 2 g; NaCl, 5 g; agar, 18 g; in 1000 ml deionized water; pH 7.2). The colors of the substrate, and aerial mycelia and any soluble pigments produced were determined by comparison with ISCC–NBS color charts.16 The morphological characteristics of strain H53T, including spore size, spore-chain morphology and surface ornamentation, were examined by light microscope (Motic, B1 Series, Hong Kong) and scanning electron microscope (model S450; Hitachi, Japan) after two weeks growth on ISP 2 medium at 30 °C. All media used for morphological characteristics contained 15% (w/v) NaCl.

Physiological characterization

Physiological tests were used to characterize the actinomycete strain H53T in comparison with its closest relative halophilic species (S. lacisalsi TRM 40133T and S. qijiaojingensis YIM 91168T). Growth at different temperatures (10, 15, 20, 25, 28, 30, 32, 35, 40, 45 and 47 °C), various pH values (5, 6, 7, 8 and 9) and NaCl concentrations (0, 7, 10, 15, 20, 25, 28, 30, 32 and 35%; w/v), and also in the presence of antibiotics, were determined by using nutrient agar medium, with the cultures incubated for 21 days at 30 °C. All media used for physiological tests contained 15% (w/v) NaCl (except for the NaCl concentration test). Production of acid from carbohydrates, and decarboxylation of organic acids were studied using the method of Gordon et al.17 Degradation of different other organic compounds was evaluated as described by Goodfellow.18 Lysozyme sensitivity and production of nitrate reductase were determined according to the methods of Gordon and Barnett19 and Marchal et al.,20 respectively. The production of melanoid pigments was evaluated on ISP 6 and ISP 7 media as recommended by Shirling and Gottlieb.15

Chemotaxonomic characterization

For the chemotaxonomic analyses, strain H53T was grown in CM broth containing 15% (w/v) NaCl at 30 °C for 10 days on a rotary shaker (250 r.p.m.). Biomass was harvested by centrifugation at 3500 r.p.m. and washed several times with distilled water. The isomeric form of diaminopimelic acid and the presence (or absence) of glycine in the cell wall were realized as described by Becker et al.21 The composition of whole-cell sugars was determined as described by Lechevalier and Lechevalier.6 Polar lipids were determined according to the method described by Minnikin et al.22 and separated by two dimensional TLC. The menaquinones were extracted according to the procedure of Minnikin et al.23 and were analyzed by HPLC.24, 25 For extraction and analysis of cellular fatty acids, the physiological age of the strain was standardized by consistently choosing the same factor (the last quadrant streaked) on (CM+YE medium, see DSMZ web pages, https://www.dsmz.de/catalogues/catalogue-microorganisms/culture-technology/list-of-media-for-microorganisms.html) incubated at 28 °C for 10 days. The analysis was conducted using the Microbial Identification System (MIDI) Sherlock software version 4.5 (method TSBA40, TSBA6 database) as described by Sasser.26 The analysis of mycolic acids was performed using the method of Minnikin et al.27

Determination of 16S ribosomal RNA gene sequence and phylogenetic analyses

For DNA analysis, the strain H53T was grown on the CM broth supplemented with 15% (w/v) NaCl. The genomic DNA was extracted with DNA extraction kit (JetFlex, Hannover, Germany) according to the method of Liu et al.28 PCR-mediated amplification of the 16S ribosomal RNA (rRNA) gene and sequencing of the purified PCR products were carried out as described by Rainey et al.29 PCR products were purified with a PCR product purification kit (Qiagen, Hilden, Germany). The primers used for sequencing are listed in Coenye et al.30 The 16S rRNA sequence has been deposited in the GenBank data library and assigned the accession number KC427277. The sequences obtained were compared with sequences present in the public sequence databases as well as with the EzTaxon-e server (http://eztaxon-e.ezbiocloud.net/;31), a web-based tool for the identification of prokaryotes based on 16S rRNA gene sequences from type strains. Phylogenetic analyses were conducted using MEGA version 5.32 The 16S rRNA sequence of strain H53T was aligned using the CLUSTAL W33 against corresponding nucleotide sequences retrieved from GenBank. Phylogenetic trees were constructed by using the neighbor joining34 with Jukes and Cantor35 model, maximum likelihood36 with Kimura two-parameter37 model and maximum-parsimony38 methods. The topology of the tree was evaluated by bootstrap analysis based on 1000 replicates.39

Determination of G+C content of DNA

The G+C content of the chromosomal DNA was determined by HPLC according to Mesbah et al.40

Results and Discussion

Morphological characteristics

Strain H53T was observed to grow well on ISP 2, nutrient agar and CM agar media, and the aerial mycelium was moderately abundant with white color on these media. However, poor growth was observed on ISP 4 medium. Substrate mycelium was dark reddish orange color on ISP 2 and nutrient agar media, and was light yellow on CM agar and ISP 4 media. Melanoid pigments and other diffusible pigments were not produced on tested media (ISP 6, ISP 7 and other media). The substrate mycelium was well developed and fragmented into non-motile cocci. It exhibited an abundant fragmentation on both solid and liquid media. Aerial mycelia form long chains of spores that are non-motile, smooth surfaced, oval or spherical in shape (Figure 1). No morphological forms or structures resembling sporangia, sclerotia or synnemata were observed.

Figure 1
figure 1

Scanning electron micrograph of strain H53T showing long spore chains of smooth surface spores after 15 days growth at 30 °C on yeast extract–malt extract agar (ISP 2) containing 15% (w/v) NaCl. Bar=5 μm.

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Chemotaxonomic characteristics

Strain H53T was determined to contain meso-diaminopimelic acid (but not glycine) in its cell wall. Whole-cell hydrolysates were found to contain arabinose and galactose (chemotype pattern IVA of Lechevalier and Lechevalier).6 Furthermore, the presence of ribose was also confirmed. Mycolic acids were not detected. The phospholipids detected were phosphatidylcholine (in very small amount), diphosphatidylglycerol, phosphatidylglycerol and phosphatidylinositol, as well as several glycolipids and unknown phospholipid (Supplementary Figures S1a and b). This polar lipid pattern corresponds to phospholipid type III.7 The predominant menaquinones were determined to be MK-9 (H4) (88.9%) and MK-9 (H6) (4.6%), and minor amounts of MK-9 (H2), MK-9 (H8), as well as unidentified menaquinones were also detected. The fatty acid’s profile was composed as follows: anteiso-C17:0 (27.8%), iso-C15:0 (26.2%), iso-C17:0 (14.0%), cis9 iso-C17:1 (11.7%), iso-C16:0 (5.6%) and anteiso-C15:0 (3.5%) (Supplementary Table S1). The morphological and chemical characteristics described above clearly support the placement of strain H53T within the genus Saccharopolyspora.

Physiological characteristics

The strain H53T grew in a wide range of NaCl concentrations (7–32% w/v on nutrient agar medium) and was strictly halophilic. It used the majority of sugars and other organic compounds for its growth. The organism is resistant to kanamycin (5 μg ml−1), erythromycin (10 μg ml−1), streptomycin (10 μg ml−1), penicillin (25 μg ml−1) and lysozyme (0.005% w/v), but sensitive to chloramphenicol (25 μg ml−1). The complete physiological characteristics of strain H53T are given in the species description.

Phylogenetic analysis based on 16S rRNA gene sequence comparison and G+C content of DNA

Phylogenetic analysis of an almost complete 16S rRNA gene sequence (1491 bp, GenBank accession number KC427277) showed that strain H53T was related to members of the genus Saccharopolyspora. Moreover, the 16S rRNA sequence similarities between strain H53T and other species of the genus Saccharopolyspora ranged from 92.1 to 94.3%. The phylogenetic relationship between strain H53T and the other Saccharopolyspora species is seen in the neighbor-joining dendrogram (Figure 2). Maximum parsimony and maximum-likelihood calculation resulted in a similar tree topology.

Figure 2
figure 2

Phylogenetic tree for species of the genus Saccharopolyspora calculated from almost complete 16S rRNA gene sequences using Jukes and Cantor35 evolutionary distance methods and the neighbor-joining method of Saitou and Nei.34 This illustrates the taxonomic position of strain H53T 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 the nodes are bootstrap values, expressed as a percentage of 1000 resamplings (only values >50% are shown). Bar=0.01 nucleotide substitutions per site.

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The strain H53T has a DNA G+C content of 72.6 mol%.

Conclusion

The results of the morphological and chemotaxonomic investigations and phylogenetic analysis supported the affiliation of the strain H53T to the genus Saccharopolyspora. The comparison with S. lacisalsi TRM 40133T showed differences in the color of substrate mycelium, the G+C content, the decomposition of adenine, hypoxanthine, gelatin, starch and tyrosine, the growth at 45 °C, and the growth with 28, 30, 32 and 35% NaCl (w/v) on nutrient agar medium as shown in Table 1. Furthermore, the differences in the production of acids from the utilization of L-arabinose, D-fructose, D-lactose, D-mannitol and D-xylose were observed. In addition, a comparison with the S. qijiaojingensis YIM 91168T showed differences in the color of substrate mycelium, the G+C content, the decomposition of adenine, gelatin, starch, Tween 80, tyrosine and xanthine, the production of nitrate reductase, the growth at 45 °C, and the growth with 25, 28, 30, 32 and 35% NaCl (w/v) on nutrient agar medium as shown in Table 1. Moreover, the differences in the production of acids from the utilization of L-arabinose, D-cellobiose, D-fructose, D-mannitol, L-rhamnose, sucrose and xylose were also observed.

Table 1 Differential phenotypic characteristics of strain H53T compared with its closest relative halophilic species (S. lacisalsi TRM 40133T and S. qijiaojingensis YIM 91168T)
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All of the data support the designation of strain H53T as representing a novel species of the genus Saccharopolyspora, for which the name Saccharopolyspora ghardaiensis sp. nov. is proposed.

Description of S. ghardaiensis sp. nov.

S. ghardaiensis (ghar.da.i.en’sis. N.L. fem. adj. ghardaiensis, pertaining to Ghardaïa, the source of the soil from which the type strain was isolated).

Halophilic filamentous actinomycete, aerial mycelium is white color on ISP 2, nutrient agar and CM agar media. The color of the substrate mycelium is dark reddish orange on ISP 2 and nutrient agar media, and is light yellow on CM agar medium. The substrate mycelium was well developed and fragmented into non-motile cocci. The aerial mycelia form long chains of non-motile, smooth-surfaced and oval (or spherical) spores. Melanoid and other diffusible pigments are not produced in tested media. Growth occurs at 25, 28, 30, 35, 40 and 45 °C (but not at 10, 15, 20 and 47 °C), and 30 to 35 °C is the optimal temperature among the tested ones. Moreover, the growth occurs at pH 5, 6, 7 and 8 (but not at pH 4 and 9), and pH 6–7 is the optimal pH among the tested ones. Acetate, adenine, citrate, hypoxanthine, starch, testosterone and Tween 80 are degraded. Casein, gelatin, guanine and xanthine are not decomposed. Utilizes L-arabinose, D-cellobiose, erythritol, D-fructose, D-galactose, D-glucose, glycerol, maltose, adonitol, D-lactose, meso-inositol, D-mannose, D-trehalose, sucrose and D-xylose as carbon sources for growth and acid production, but not D-mannitol, D-melezitose, melibiose, D-raffinose, L-rhamnose, D-ribose, salicin and D-sorbitol. Benzoate, butyrate, oxalate, propionate, pyruvate, succinate and tartrate were not decarboxylated. L-alanine, L-proline, L-serine and tyrosine are used as a source of nitrogen. Nitrate reductase is produced. Growth occurs on nutrient agar medium in the presence of NaCl at 7, 10, 15, 20, 25, 28, 30 and 32%, but not at 0 and 35% (w/v), and 15–25% NaCl (w/v) is the optimal concentration among the tested ones. Moreover, the growth occurs in the presence of kanamycin (5 μg ml−1), erythromycin (10 μg ml−1), streptomycin (10 μg ml−1) and penicillin (25 μg ml−1), and also in the presence of 0.005% lysozyme, but not in the presence of chloramphenicol (25 μg ml−1). Chemotype IVA (meso-diaminopimelic acid, arabinose and galactose in whole-cell hydrolysates), the diagnostic phospholipid, is phosphatidylcholine. The predominant menaquinones are MK-9(H4) and MK-9(H6). The predominant cellular fatty acids are anteiso-C17:0, iso-C15:0, iso-C17:0 and cis9 iso-C17:1 The DNA G+C content of strain H53T was 72.6%. The type strain H53T (=DSM 45606T=CCUG 63370T=CECT 8304T) is isolated from a Saharan soil sample collected from Ghardaïa province (South Algeria). The GenBank accession number for the 16S rRNA gene sequence of strain H53T is KC427277.