Atika Meklat

Noureddine Bouras
email: bouras@yahoo.fr

Salim Mokrane

Abdelghani Zitouni

Nadjette Djemouai

Hans-Peter Klenk

Nasserdine Sabaou

Florence Mathieu

Isolation, Classification and Antagonistic Properties of Alkalitolerant Actinobacteria from Algerian Saharan Soils

Keywords: Alkalitolerant actinobacteria, Saharan soils, diversity, antimicrobial activity

The Sahara, one of the most extreme environments on Earth, constitutes an unexplored source of alkalitolerant actinobacteria. ln this worl<, we studied the diversity of alkalitolerant actinobacteria in various soils collected from different regions of the Algerian Sahara. A total of 29 alkalitolerant actinobacterial strains were isolated by using a complex agar medium. The diversity of these acti nobacteria was evaluated using a polyphasic approach, which included morphological, chemotaxo nomic, physiological (numerical taxonomy) and 16S rRNA gene analyses. The isolates which were assigned to the genus Nocardiopsis, shared relatively low 16S rRNA gene sequences similarities compared to closely related species suggesting that they belonged to putatively new species. Ali of the strains were tested for antibiotic activity against a broad range of microorganisms and screened for genes encoding polyketide synthases and non-ribosomal peptide synthetases and found to have the potential to produce secondary metabolites. Consequently, the study supports the view that extrerne environments contain many nove! actinobacteria, which represent an unex plored source for the discovery of biologically active compounds.

Introduction

Actinobacteria are a group of microorganisms that are wide spread in nature and are able to occupy diverse habitats such as soils ( [START_REF] Li | Studies on antibacterial activ ity and diversity of cultivable actinobacteria isolated from mangrove soil in Futian and Maoweihai of China[END_REF], plants tissues [START_REF] Chankharnhaengdecha | Endophytic actinomycetes: a nove! source of potential acyl homoserine lactone degrading enzymes[END_REF], lichens [START_REF] Yamamura | Actinomycetospora irio motensis sp. nov., a novel actinomycete isolated from a lichen sam pie[END_REF] and extreme aquatic and terrestrial ecosystems [START_REF] Dhakal | Marine rare acti nobacteria: isolation, characterization, and strategies for harnessing bioactive compounds[END_REF][START_REF] Goodfellow | Rare taxa and dark microbial matter: nove! bioactive actinobacteria abound in Atacama Desert soils[END_REF][START_REF] Idris | Actinobacterial rare biospheres and dark matter revealed in habitats of the Chilean Atacama desert[END_REF]. Extremophilic actino bacteria can be divided into acidophilic [START_REF] Golinska | Streptacidiphilus toruniensis sp. nov., isolated from a pine forest soil[END_REF], alkaliphilic (Sel ya nin et al 2005; [START_REF] Thumar | Isolation and partial purification of an antirnicrobial agent from halotolerant alkaliphilic Streptomyces aburaviensis strain Kut 8[END_REF], haloalka liphilic [START_REF] Gohel | Molecular phylogeny and diversity of the sait tolerant alkaliphilic actinobacteria inhabiting coastal Gujarat, lndia[END_REF], halophilic (Al- [START_REF] Tai | Nocardiopsis halophila sp. nov., a new halo philic actinomycete isolated from soil[END_REF][START_REF] Chun | Nocardiopsis kunsanensis sp. nov., a moderately halophilic actinomy cete isolated from a saltern[END_REF][START_REF] Hamedi | Systematic and bio technological aspects of halophilic and halotolerant actinomycetes[END_REF]Meldat et al. 2011Meldat et al. , 2013)), psychrophilic (Miinnisto et al. 2000), thermophilic [START_REF] Zhou | Thermoactinospora rubra gen. nov., sp. nov., a thermophilic actinomycete isolated from Tengchong, Yunnan prov ince, south west China[END_REF]) and xerophilic [START_REF] Montero Calasanz | Geodermatophilus arenarius sp. nov., a xerophilic actinomycete isolated from Saharan desert sand in Chad[END_REF] groups.

Many alkaliphilic and alkalitolerant actinobacteria have long been known to live in various habitats induding neutral as well as alkaline environments [START_REF] Jiang | Actinomycete diversity in unusual habitats[END_REF]. These actinobacteria are capable of growing in alkaline lakes [START_REF] Yang | Nocardiopsis valliformis sp. nov., an alkaliphilic actinomycete isolated from alkali lake soil in China[END_REF], alkaline soils ( [START_REF] Li | Kocuria aegyptia sp. nov., a nove) actinobacte rium isolated from a saline, alkaline desert soil in Egypt[END_REF][START_REF] Zhang | Nesterenkonia a/ka liphila sp. nov., an alkaliphilic, halotolerant actinobacteria isolated from the western Pacifie Ocean[END_REF]; sait alkaline lakes [START_REF] Li | Streptomyces sodiiphilus sp. nov., a novel alkaliphilic actinomycete[END_REF][START_REF] Mwirichia | Isolation and characterisation of bacteria from the haloalkaline Lake Elmenteita, Kenya[END_REF]Zhang et al 2013b) and soda lakes [START_REF] Duckworth | Dietzia natronolimnaios sp. nov., a new member of the genus Dietzia isolated from an East African soda Jake[END_REF][START_REF] Grant | Distribution and diversity of soda Jake alkaliphiles[END_REF][START_REF] Groth | Bogoriella caseilytica gen. nov., sp. nov., a new alkaliphilic actinomycete from a soda Jake in Africa[END_REF][START_REF] Jones | Microbial diversity of soda lakes[END_REF][START_REF] Sorokin | Nitriliruptor a/kaliphilus gen. nov., sp. nov., a deep lineage haloalka liphilic actinobacterium from soda lakes capable of growth on ali phatic nitriles, and proposai of Nitriliruptoraceae fam. nov. and Nitriliruptorales ord. nov[END_REF]. This group of actinobacteria can be divided into three major categories, namely alkaliphilic, mod erately alkaliphilic and alkalitolerant actinobacteria that grow optimally at pH 10-11 and pH 7-10, but show poor growth at pH 7.0; and pH 6-11, respectively [START_REF] Jiang | Actinomycete diversity in unusual habitats[END_REF].

Several novel genera and species of alkaliphilic and alkalito leratnt actinobacteria have been described such as Bogoriella caseilytica [START_REF] Groth | Bogoriella caseilytica gen. nov., sp. nov., a new alkaliphilic actinomycete from a soda Jake in Africa[END_REF], Kocuria aegyptia [START_REF] Li | Kocuria aegyptia sp. nov., a nove) actinobacte rium isolated from a saline, alkaline desert soil in Egypt[END_REF], Nesterenkonia alba [START_REF] Luo | Nesterenkonia alba sp. nov., an alkaliphilic actinobacte rium isolated from the black liquor treatment system of a cotton pulp mill[END_REF], Nesterenkonia alkaliphila (Zh ang et al. 2015), Nitriliruptor alkaliphilus [START_REF] Sorokin | Nitriliruptor a/kaliphilus gen. nov., sp. nov., a deep lineage haloalka liphilic actinobacterium from soda lakes capable of growth on ali phatic nitriles, and proposai of Nitriliruptoraceae fam. nov. and Nitriliruptorales ord. nov[END_REF], Nocardiopsis algeriensis [START_REF] Bouras | Nocardiopsis algeriensis sp. nov., an alkalitolerant actinomycete isolated from Saharan soil[END_REF], Nocardiopsis alkaliphila [START_REF] Hozzein | Nocardiopsis alkaliphila sp. nov., a nove! alkaliphilic actinomycete isolated from desert soil in Egypt[END_REF]), Nocardiopsis metallicus [START_REF] Schippers | Nocardiopsis metallicus sp. nov., a metal leaching actinomycete isolated from an alkaline slag dump[END_REF], Nocardiopsis valliformis [START_REF] Yang | Nocardiopsis valliformis sp. nov., an alkaliphilic actinomycete isolated from alkali lake soil in China[END_REF], Streptomyces aburaviensis [START_REF] Thumar | Isolation and partial purification of an antirnicrobial agent from halotolerant alkaliphilic Streptomyces aburaviensis strain Kut 8[END_REF], Streptomyces fukangensis (Zhang et al. 2013b) and Streptomyces sodiiphilus [START_REF] Li | Streptomyces sodiiphilus sp. nov., a novel alkaliphilic actinomycete[END_REF].

Ail of these represent species that share 16S rRNA gene sequence similarities with their current closest phylogenetic neighbors below the 97%, 98.2% and 98.65 thresholds rec ommended by [START_REF] Wayne | Report of the ad hoc committee on rec onciliation of approaches to bacterial systematics[END_REF], [START_REF] Kolthoff | When should a DDH experiment be mandatory in microbial taxonomy?[END_REF] and [START_REF] Kim | Towards a taxonomie coher ence between average nucleotide identity and 16S rRNA gene sequence similarity for species demarcation of prokaryotes[END_REF], respectively supporting thresh olds used to detect prospective new speci es.

Alkaliphiles and alkalitolerant actinobacteria are an interest ing group that produce valuable natural compounds, such as antibi otics [START_REF] Ali | NW A52 A, a macrolide anti biotic produced by the alkalophile Nocardiopsis dassonvillei W A52[END_REF][START_REF] Dieter | Pyrocoll, an antibiotic, antiparasitic and antitumor compound produced by a nove! alkaliphilic Streptomyces $train[END_REF][START_REF] Ding | Griseusins F and G, spiro naphthoquinones from a tin mine tailings derived alkalophilic Nocardiopsis species[END_REF][START_REF] Helaly | Warkmycin, a nove! angucycline antibioti c pro duced by Streptomyces sp. Acta 2930*[END_REF][START_REF] Nair | Faeriefungin: a new broad spectrum antibiotic from Streptomyces griseus var. auto trophicus[END_REF][START_REF] Sanghvi | Isolation and partial pu rifi cation of erythromycin from alkaliphilic Streptomyces werraensis isolated from Rajkot, India[END_REF][START_REF] Solanki | Bioactive compounds from marine actinomycetes[END_REF][START_REF] Thumar | Isolation and partial purification of an antirnicrobial agent from halotolerant alkaliphilic Streptomyces aburaviensis strain Kut 8[END_REF][START_REF] Wang | New pyran 2 ones from alkalophilic actinomycete, Nocardiopsis alkali phila sp. Nov. YIM 80379[END_REF], anticancer, antitumor and immunosuppressive agents [START_REF] Dieter | Pyrocoll, an antibiotic, antiparasitic and antitumor compound produced by a nove! alkaliphilic Streptomyces $train[END_REF][START_REF] Li | Griseusin D, a new pyranonaphthoquinone derivative from a alka philic Nocardiopsis sp[END_REF], antioxidants [START_REF] Quadri | Detection of melanin producing thermo alkaliphilic Streptomyces from limestone quarries of the Deccan traps[END_REF], antiparasitics [START_REF] Dieter | Pyrocoll, an antibiotic, antiparasitic and antitumor compound produced by a nove! alkaliphilic Streptomyces $train[END_REF], cytotoxic compounds [START_REF] Tsujibo | Isolation of cytotoxic substance, kalafungin from an alkalophilic actinomycete, Nocardiopsis dassonvillei subsp. prasina[END_REF], enzymes [START_REF] Ara | Proteolytic activity of alkaliphilic, sait tolerant actinomycetes from various regions in Saudi Arabia[END_REF][START_REF] Jani | Optimization of extracellular protease production from alkali thermo tolerant actinomycetes: Saccharomonospora viridis SJ 21[END_REF][START_REF] Mehta | Production of alkaline protease from an alkaliphilic actinomycete[END_REF][START_REF] Ningthoujam | Screening, identification of best producers and optimization of extracellular proteases from moderately halophilic alkalithermotoler ant indigenous actinomycetes[END_REF][START_REF] Sorokin | Nitriliruptor a/kaliphilus gen. nov., sp. nov., a deep lineage haloalka liphilic actinobacterium from soda lakes capable of growth on ali phatic nitriles, and proposai of Nitriliruptoraceae fam. nov. and Nitriliruptorales ord. nov[END_REF][START_REF] Thumar | Organic solvent tolerance of an alkaline protease from sait tolerant alkaliphilic Streptomyces c/avuligerus strain Mit 1[END_REF], herbicides [START_REF] Souagui | Optimization of antifungal production by an alkaliphilic and haloto lerant actinomycete, Streptomyces sp. SY BSS, using response surface methodology[END_REF], insecticides [START_REF] Nair | Faeriefungin: a new broad spectrum antibiotic from Streptomyces griseus var. auto trophicus[END_REF]) and nanoparticles [START_REF] Ahmad | Intracellular synthesis of gold nanoparticles by a nove) alkalotolerant actinomycete, Rhodococcus species[END_REF].

A wide range of bioactive secondary metabolites are bio synthesized by the non-ribosomal peptide synthetase (NRPS) and polyketide synthase (PKS) pathways. Polyketides are structurally diverse secondary metabolites that have demon strated efficacy in a wide range of therapeutic applications (Ayuso- [START_REF] Sacido | New PCR primers for the screen ing of NRPS and PKS I systems in actinomycetes: detection and dis tribution of these biosynthetic gene sequences in major taxonomie groups[END_REF][START_REF] Fischbach | Assembly line enzymology for polyke tide and nonribosomal peptide antibiotics: logic, machinery, and mechanisms[END_REF][START_REF] Gomes | Biotechnology of polyketides: new breath of life for the nove! antibiotic genetic path ways discovery through metagenomics[END_REF][START_REF] Ketela | An efficient approach for screening minimal PKS genes from Streptomyces[END_REF]. In turn, NRPS gene clusters have been found to synthesize a diverse selection of biologically active non-ribosomal peptides, in particular antibiotics [START_REF] Cane | Harnessing the biosynthetic code: combinations, permutations, and mutations[END_REF]Grünewald and Marahiel 2013). The presence of NRPS and PKS genes has been documented in many studies. From a population of 52 halophilic desert actinobacteria, half of the strains were bio active and harbored genes encoding for NRPS and PKS compounds [START_REF] Meklat | Isolation, taxonomy, and antagonistic properties of halophilic actinomycetes in Saharan soils of Algeria[END_REF]. In another study, the PKS and NRPS genes detected in 53 actinobacteria isolated from Qinghai-Tibet Plateau indicated diversified potential bio active products [START_REF] Ding | Culturable actinomycetes from desert ecosystem in northeast of Qinghai Tibet Plateau[END_REF]. In contrast, little is known about the products of NPRS or PKS genes in alkali philic or alkalitolerant actinobacteria; thus, the screening for these genes would allow for the generation of new drugs.

One of the main strategies in the search for new sources of bioactive compounds is the isolation of non-streptomy cete actinobacteria; also known as rare actinobacteria which are often difficult to isolate and cultivate; from underex plored and uncommon habitats [START_REF] Baltz | Gifted microbes for genome mining and natural prod uct discovery[END_REF][START_REF] Bundale | Rare actinobacteria: a potential source of bioactive polyketides and pepti des[END_REF][START_REF] Goodfellow | Rare taxa and dark microbial matter: nove! bioactive actinobacteria abound in Atacama Desert soils[END_REF]. In this context, the Sahara is a good example of the extreme habitats that is unfavorable for life, apart from microbial life that has become adapted to the harsh conditions. lt is clear from studies on Saharan soils that they are a rich source of members of rare genera that have been shown to produce many antibiotics [START_REF] Bouras | Precursor directed biosynthesis of new dithiolopyrrolone ana logs by Saccharothrix algeriensis NRRL B 24137[END_REF][START_REF] Meklat | Isolation, taxonomy, and antagonistic properties of halophilic actinomycetes in Saharan soils of Algeria[END_REF][START_REF] Meklat | A nove) strain of Actinopolyspora morti vallis with antibacterial activity isolated from a Saharan soil[END_REF][START_REF] Sabaou | Les sols des oasis du Sahara algérien, source d'actinomycètes rares producteurs d'antibio tiques[END_REF][START_REF] Zitouni | Nocardiopsis and Saccha rothrix genera in Saharan soils in Algeria: isolation, biological activities and partial character ization of antibiotics[END_REF].

Nocardio psis species are widespread in extreme habitats such as the Atacama [START_REF] Goodfellow | Rare taxa and dark microbial matter: nove! bioactive actinobacteria abound in Atacama Desert soils[END_REF][START_REF] Idris | Actinobacterial rare biospheres and dark matter revealed in habitats of the Chilean Atacama desert[END_REF], the Qinghai-Tibet [START_REF] Ding | Culturable actinomycetes from desert ecosystem in northeast of Qinghai Tibet Plateau[END_REF] and Sahara [START_REF] Bouras | Nocardiopsis algeriensis sp. nov., an alkalitolerant actinomycete isolated from Saharan soil[END_REF][START_REF] Hozzein | Nocardiopsis alkaliphila sp. nov., a nove! alkaliphilic actinomycete isolated from desert soil in Egypt[END_REF][START_REF] Li | Kocuria aegyptia sp. nov., a nove) actinobacte rium isolated from a saline, alkaline desert soil in Egypt[END_REF][START_REF] Meklat | Isolation, taxonomy, and antagonistic properties of halophilic actinomycetes in Saharan soils of Algeria[END_REF][START_REF] Montero Calasanz | Geodermatophilus arenarius sp. nov., a xerophilic actinomycete isolated from Saharan desert sand in Chad[END_REF][START_REF] Zitouni | Nocardiopsis and Saccha rothrix genera in Saharan soils in Algeria: isolation, biological activities and partial character ization of antibiotics[END_REF] deserts. They are known to produce a variety of bioactive compounds such as tumor inducers, anticancer substances and immunomodulators and novel extracellular enzymes such as amylases, inulinases, chitinases, proteases, xylanases, glucanases and cellulases [START_REF] Bennur | Nocardiopsis species as potential sources of diverse and nove! extracellular enzymes[END_REF].

The aim of the present study is to investigate the biodiver sity of alkalitolerant actinobacteria isolated from Algerian Saharan soils, to determine the relationship of these alkalito lerant actinobacteria by using a pol yp hasic approach and to evaluate their potential to produce bioactive substances.

Materials and methods

Soi/ samples

Seventeen non-rhizospheric soil samples were collected from various Saharan regions in the South of Algeria. They were taken from depths of up to 20 cm, after removing approxi mately 3 cm of the soil surface. The samples were placed in sterile polyethylene bags that were closed tightly and stored at 4 °C until analysis. The geographic locations and the physico chemical properties of the soils are given in Table 1. Soil pH was determined by using a Jenway pH meter (soil: distilled water = 1: 2.5). For determination of soil organic carbon (in %), the Walld ey and Black wet oxidation method (1934) was used Soil total nitrogen to organic carbon (in %) was determined by the Kjeldahl method [START_REF] Bremner | Nitrogen total[END_REF]. Total CaC0 3 content was determined using a weak acid dissolution followed by measurement of the pH of the extractant After incubation at 30 °C for 1-2 weeks, the suspected colonies were picked up and purified on CM agar (pH 10.0) and incubated at 30 °C. The purified strains were stored on the same medium at 4 °C and also at -20 °C as 20% (v/v) glycerol suspensions.

Morphological characteristics

Twenty nine actinobacterial strains were grown on two International Sptreptomyces Project (ISP) culture media [START_REF] Shirling | Methods for characterization of Streptomyces species[END_REF], namely yeast extract-malt extract-dextrose agar (ISP2) and inorganic salts starch agar (ISP4), as well as CM agar. The cultures were grown for 3weeks at 30 °C and pH 10.0.

Ali strains were observed by light microscopy (Motic; Bl Series). A 15 days culture of strain B33 that was grown on ISP2 medium was fixed in glutaraldehyde (2.5%) for 1 h and then post-fixed in osmium tetroxide vapor (2%). After using ethanol and tert-butanol, the dehydrated sample was sput ter-coated with gold-palladium and then observed under a scanning electron microscope (model S-450; Hitachi).

Physiological characteristics

Ali actinobacterial strains were characterized by using sixty three physiological tests. Nocardiopsis algeriensis [START_REF] Bouras | Nocardiopsis algeriensis sp. nov., an alkalitolerant actinomycete isolated from Saharan soil[END_REF] was examined under the same conditions for the same characters. Ali tests were made at pH 7.5 (except those of pH resistance). Utilization of twenty two carbohydrates and decarboxylation of nine organic acids were determined according to the methods of [START_REF] Gordon | Nocardia coeliaca, Nocardia autotrophica, and the nocardin strains[END_REF]. Degradation of adenine, gelatin, guanine, hypoxanthine, milk casein, starch, testosterone, Tween 80, L-tyrosine and xanthine were studied as described by [START_REF] Goodfellow | Numerical taxonomy of some nocardioform bac teria[END_REF] and [START_REF] Marchal | Les milieux de culture pour l'isolement et l'identifi cation biochimique des bactéries[END_REF]. Lysozyme sensitivity was eval uated by the method of [START_REF] Gordon | Resistance to rifampicin and lysozyme of strains of some species of Mycobacterium and Nocardia as a taxo nomic tool[END_REF]. Production of melanoid pigments was tested on peptone yeast extract iron agar (ISP6) and tyrosine agar (ISP7) (Shirling and Gottlieb 1%6). Growth at two different tem peratures (30 and 45 °C) and three NaCl concentrations (0, 7 and 10% w/v), and in the presence of chloramphenicol (25 µg/mL), erythromycin (10 µg/mL), kanamycin (5 µg/mL), MNaOH.

Numerical taxonomy

The results of the physiological study were analyzed by numerical taxonomy. The data were coded in a binary sys tem (1/0) and examined with the SPSS package (v.16.0.1), using the simple matching (S s M) coefficient (Sokal and Michener 1958), which includes both positive and negative similarities. Clustering was achieved using the unweighted pair group method with arithmetic averages (UPGMA) algo rithm [START_REF] Sneath | Numerical Taxonomy: The Principles and Practice of Numerical Classification[END_REF].

Chemical an alysis of ce// constituents

For the chemical analysis, the biomass of each strain was harvested by centrifugation at 3.500 rpm of cultures growing on CM broth (pH 7.5) at 30 °C for 6days on a rotary shaker (250 rpm). The isomeric form of diaminopimelic acid and the presence (or not) of glycine in the cell-wall were ascer tained as described by [START_REF] Becker | Rapid differentiation between Nocardia and Streptomyces by paper chroma tography of whole cell hydrolysates[END_REF]. The composition of whole-cell sugars was determined as described by [START_REF] Lechevalier | Chemical composition as a cri terion in the classifi cation of aerobic actinomycetes[END_REF]. Phospholipids were ana lyzed using the procedure of [START_REF] Minnikin | Polar lipid composition in the classifi cation of Nocardia and related bac teria[END_REF]. The experiment was done firstly by streaking a straight line of the actinobacterium inoculum across the surface of the medium in 90 mm diameter plates and incubating at 30 °C for 10 days. After the growth of the strains, target microorganisms were seeded in streaks crossing the actino bacterium culture. The antimicrobial activity was recorded by measuring the length of inhibition between target micro organisms and the actinobacterial colony margins.

Phylogenetic studi es

Genomic

Detection of PKS-1, PKS-11, enediyne PKS and NRPS sequences

The series of primers listed in Table 2 were

Results

Isolation of actinobaderial strains and distribution in soils

On the basis of characteristic colonial morphology, mainly the ability to form aerial and substrate mycelia, microorgan isms putatively identifi ed as actinobacteria were selected.

Except for the neutral pH of the soil sample Lal of Laghouat, all of the samples had a basic pH ranging from 8.0 to 9.3. In addition, very low amounts of carbon and nitrogen were observed in the analyzed soil samples, and some of them such as Gh l (Ghardaïa), Ad3 (Adrar) Ou3 (Ouargla) Bel, Bc4 and Bc6 (Béchar) and DJl (Djelfa) were saline soils (Table 1).

In the adopted cultural conditions, moderate levels of cultivable actinobacteria were observed ranging from 1.0 to 63.0 10 2 CFU per g (dry weight) of soil. Actinobacteria were found in all the soil samples. In total, 29 actinobacteria were isolated from 17 anal yze d soil samples (Table 3). There was no correlation between the distribution of actinobacteria and pH as the highest number (63.0 10 2 ) was found at neutral pH (the soil sample Lal of Laghouat).

Morphological and chemotaxonomic studies

The preliminary investigation based on morphological and chemical analysis (diarninopimelic acid isomers, sugars and phospholipids) of the 29 actinobacteria allowed these strains to be tentatively classified at the genus level. Ali strains formed a branched substrate myceliurn that fragmented into coccoid and rod-shaped elements. Aerial h yp hae were zig zag shaped at the beginning of sporulation and fragmented at maturity into branched and straight to fl exuous spore chains with rod-shaped spores (Figure 1, Supplementary Figure Sl). These strains contained meso-diarninopimelic acid in the cell wall, phosphatidylcholine as the diagnostic phospholipid in the cell membrane, and ribose and glucose (no diagnostic sugars) in the whole-cells. All of these prop erties are consistent with the isolates being assigned to the genus Nocardiopsis [START_REF] Meyer | Genus Nocardiopsis[END_REF].

1 (B33) 2 (B12, B14) 1 (B30) 1 (B20) 1 (B5) 1 (86) 3 (B9, B10, B11) 1 (B28) 4 (B15, B16, B17, B 18) 1 (B25) 1 (84) 3 (B1, B2, B3) 3 (B22, B23, B24) 1 (B29) 2 (B26, B27) 2 (B7, B8) 1 (B19)

Physiological and numerical study

The physiological classification based on the S s M coefficient and UPGMA clustering method was examined in detail as it gave the most compact aggregate groups. The 30 strains (29 isolated strains and Nocardiopsis algeriensis DSM 45462 T ) were assigned to 7 cluster-groups, designated I to VII, and three single strains at the 91 % similarity (S) level (Figure 2). This dendrogram showed that strain B33 was grouped in cluster II with N. algeriensis DSM 45462 T .

All the isolated strains shared the following characteris tics: they grew within the pH range of 7-11, and in the presence of 0-7% of NaCl. Furthermore, most strains (except Bl, B3, B13, B24, B25 and B26) were able to grow at pH 12. However, only strains B3, B9, B13, B15, B20 and B28 were able to grow at pH 6. All of the strains were resist ant to penicillin (25 µg/mL), degraded adenine, casein, gel atin, glucose, tyrosine and Tween 80, were sensitive to kanamycin (5 µg/mL) but did not use adonitol, benzoate, butyrate, erythritol, inositol, melezitose, testosterone and starch as a sole carbon sources.

Molecular taxonomy

From a total of 29 actinobacteria, 19 strains belonging to different physiological groups (clusters) were subjected to molecular analysis. The phylogenetic tree (Figure 3

Antimicrobial activity and detection of biosynthetic gene sequences

Antimicrobial activity of ail stains was evaluated on CM agar (at pH 7.5) using the streak method against various microorganisms (Table 4). The results showed that 96.5% of the strains were active against at least one of the tested tar get microorganisms. The most frequent activity was Thermobifida halotolerans YIM 90462 T was used as an outgroup. Bar, 0.005 nt substitutions per site. 4). In total, 15 strains (51.7%) yielded at least one type of these biosynthetie sequences. NRPS and PKS-11 were the most frequent genes; th ey were detected in 10 and 9 strains, respectively (34.5 and 31 %). However, PKSE and PKS-I genes were detected only in 2 strains (6.9%).

Discussion

A small number of alkalitolerant actinobacteria were isolated from 17 different Saharan soils; similar results were obtained by [START_REF] Meklat | Isolation, taxonomy, and antagonistic properties of halophilic actinomycetes in Saharan soils of Algeria[END_REF] for Saharan halophilie actinobacteria. Nevertheless, previous studies have reported the abundance of non-extremophilie actinobacteria (10 6 to 10 7 CFU per g of soil) in Saharan soils [START_REF] Sabaou | Distribution quantitative et qualitative des actinomycètes dans les horizons de sol de surface et profonds d'une palmeraie algérienne[END_REF][START_REF] Sabaou | Les sols des oasis du Sahara algérien, source d'actinomycètes rares producteurs d'antibio tiques[END_REF].

The assignment of the isolates to genera was based on morphological and chemical characteristics while the numer ical taxonomie data permitted their separation but not their identification. Nineteen isolates representing the numerieally defined clusters were the subject of 16S rRNA gene sequenc ing studies. Only one strain (B33) was distinguished from the type strain of its nearest validly named species [START_REF] Kim | Towards a taxonomie coher ence between average nucleotide identity and 16S rRNA gene sequence similarity for species demarcation of prokaryotes[END_REF]) whereas the remaining 18 strains showed simi larities above the 98.65% hence these isolates need to be the subject of more detailed molecular systematie studies based on DNA-DNA reassociation or whole-genome sequenci ng to determine their taxonomie status.

The phylogenetie study revealed that strain B33 was distinct from other strains and the validly published Nocardiopsis spe cies. This strain exhibited a similarity value of 98.4% with N. algeriensis DSM 45462 T and N. lucentensis, a value below the 98.65% threshold [START_REF] Kim | Towards a taxonomie coher ence between average nucleotide identity and 16S rRNA gene sequence similarity for species demarcation of prokaryotes[END_REF]) hence this isolate can be considered to be a presumptive new Nocaridiopsis species. The results of 16S rRNA gene sequence similarity of the remaini ng 18 sequenced strains ra ng ed from 99.1% to 99.9% (supplemen tary Table Sl

Disclosure statement

No potential conflict of interest was reported by the author(s).

Sequence

  µg/mL) and streptomycin (l0µg/mL) were determined on nutrient agar. The following buffers were used to test the pH range of growth on nutrient broth: pH 6.0, 7.0 and 8.0, 0.1 M KH 2 PO 4 /0.l M NaOH; pH 9.0 and 10.0, 0.1 M NaHCOy'0. l M Na 2 CO 3 ; pH 11.0, 0.05 M Na 2 HPOJ0.l M NaOH; and pH 12.0, 0.2 M KCV0.2

  DNA of 19 selected strains, belonging to different dusters established by numerical taxonomy, was extracted for 16S rRNA gene analysis according to the method of Liu et al. (2000). 16S rRNA genes were PCR-amplified in 50 µL of reaction mixture, using 25-50 ng of genomic DNA, 0.5 µM of the forward FC27 and reverse RC1492 primers (Table 2), lX PCR buffer, 10 µM deoxynudeoside triphos phate mixture and 0.4 U Taq DNA polymerase. The amplifi cation was performed according to the following conditions: initial denaturation of template DNA at 98 °C for 4 min fol lowed by 30 cycles of denaturation at 94 °C for 1 min, pri mer annealing at 52 °C for 1 min and extension at 72 °C for 2 min. At the end of cycling, the reaction mixture was held at 72 °C for l0min to achieve the final elongation and then cooled to 4 °C. The PCR products were analyzed by agarose gel electrophoresis, and then submitted to the Beck.man Coulter Genomics Company (United Kingdom) for purifica tion and sequencing. The sequences obtained were compared with sequences present in the EzTaxon-e server (http:/ /eztaxon-e.ezbiocloud net/; Kim et al. 2012). Phylogenetic analysis was conducted using MEGA version 5 (Tamura et al. 2011). The 16S rRNA gene sequences of the 19 strains were aligned against neigh boring nucleotide sequences using the CLUSTAL W (with default parameters) (Thompson et al. 1994). A phylogenetic tree was constructed by using the neighbor-joining method (Saitou and Nei 1987) with the Jukes and Cantor (1969) model. Bootstrap analysis (Felsenstein 1985) was performed to evaluate the reliability of the tree topology. Antimicrobial assay on solid medium The 29 strains of actinobacteria were tested against four bac teria (Badllus subtilis ATCC 6633, Staphylococcus aureus CIP 7625, Klebsiella pn eumoniae CIP 8291 and Micrococcus luteus ATCC 9314), four fungi (Umbelopsis ramanniana NRRL 1829, Penicillium expansum B831P, P. glabrum and Aspergillus carbonarius M333) and one yeast (Saccharomyces cerevisiae ATCC 4226) to evaluate their antimicrobial activ ities after cultivation on ISP 2 agar.

  used to detect the genes encoding polyketide synthases I and II (PKS-I and PKS-11), enediyne polyketide synthase (PKSE) and non-ribo somal peptide synthetase (NRPS) genes for all the isolated strains. The 50-µL PCR reaction mixture contained 20-40 ng of DNA template, 0.4 µM of deoxynucleoside triphosphate mixture, 2 µM of each primer, lOX reaction buffer contain ing MgCh, 2.5 U of Taq DNA polymerase and 5% dimethyl sulfoxide (DMSO). The PCR thermal cycle program included an initial denaturation at 98 °C for 4 min, followed by 30 cycles, with a denaturation step at 94 °C for 1 min, an annealing step of 1 min, at 57.5 °C with KlF/M6R (Ayuso Sacido and Genilloud 2005), at 58 °C with KSa/KSP (Metsa Ketela et al. 1999), at 62 °C with EdyA/EdyE (Liu et al. 2003) and at 57 °C with A3F/ A7R (Ayuso-Sacido and Genilloud 2005), followed by an extension step during 1 min at 72 °C. Final extension was performed at 72 °C for 10 min. Ali of the amplification products were examined by agar ose gel electrophoresis (0.8%), and bands of 1200-1400 pb, 600 pb, 1400 pb and 700-800 pb were classified as products of PKS-I, PKS-11, PKSE and NRPS genes, respectively.

Figure 1 .

 1 Figure 1. Scanning electron micrograph of strain 833 T grown on ISP 2 medium for 15 days at 30 °C. Bar 5 µm.

Figure 2 .

 2 Figure 2. Dendrogram derived from UPGMA cluster analysis of 63 phenotypic characters showing the relationship between 29 strains of alkalitolerant actino bac te ria isolated from Sahara n soils.

Figure 3 .

 3 Figure 3. Phylogenetic tree derived from nearly complete 16S rRNA gene sequences showing relationships between the isolates of alkaliphilic actinobacteria and their phylogenetic neighbors. The tree was constructed using the neighbor joining method (Saitou and Nei 1987). Asterisks indicate branches of the tree that were also found using maximum parsimony (Fitch, 1977) tree making algorithm. Bootstrap values (� 50 %) based on 1000 resamplings are shown at branch nodes.Thermobifida halotolerans YIM 90462 T was used as an outgroup. Bar, 0.005 nt substitutions per site.

  Many antibioties produced by actinobacteria have been synthesized through a pathway involving NRPS and/or PKS, hence the 29 strains were screened by PCR for the presence of PKS-I, PKS-11, PKSE and NRPS sequences (Table

  ) and hence are below or approximately the same as the similarities between the closely related species of validly published Nocardiopsis species and are related to known spe cies as in the case of isolates B2, B9, B15 and B22. However in these cases, additional studies are needed to confi rm these deductions. In this context it is interesting to note that very high 16S rRNA gene sequence similarities have been found between the type strains of validly published Nocardiopsis spe cies, as exemplified by Nocardiopsis valliformis and N. exhalans (99.9%) (Ya ng et al 2008), N. sinuspersici and N. arvandica (99.9%) (Hamedi et al. 2011), N. halop hila and N. baichengen sis (99.9%) (Li et al. 2006), N. litoralis and N. kunsanensis (99.6%) (Chen et al. 2009), N. metallicus and N. exhalans (99.4%) (Schippers et al 2002), N. metallicus and N. prasina (99.3%) (Schippers et al. 2002) and N. synnemataformans and N. dassonvillei (99.3%) (Yassin et al. 1997). Most of the tested isolates showed antibacterial and anti fu ng al activities which are in line with earlier findings (Bennur et al 2016; Ibrahim et al. 2018; Zitouni et al 2005). Strains B2, B9 and B20 showed relatively stro ng inhibition against the test mieroorganisms and may well be the source of novel antibiotics. To date, Nocardiopsis strains have been shown to be the source of new secondary metabolites, as shown by the production of 3-trehalosamine (Evtushenko et al. 2000), griseusin D (Li et al. 2007), macrolide W A52-A (Ali et al. 2009), thiopeptide (E ng elhardt et al. 2010), nocapyr one derivatives (Schneemann et al. 2010), diketopiperazine derivatives (Zhang et al. 2013a), 4-oxo-1,4-dihydroquinoline-3carboxamide and N-acetyl -anthranilic acid (Tian et al. 2014), angucyclinones (Hadj Rabia-Boukhalfa et al 2017) and others (Ibrahim et al. 2018). The screening of NRPS, PKS-I, PKS-11 and PKSE genes revealed a low presence in Nocardiopsis strains. NRPS sequences are extensively distributed amo ng actinobacterial taxa, whereas PKS-I genes are concentrated in fewer genera (Ayuso-Sacido and Genilloud 2005; Meldat et al. 2011; Metsa-Ketela et al. 1999). Dedication This paper is dedieated to the late Dr. Nasserdine Sabaou (1956-2019) for his genuine love, dedication, and service to science. He was a superb researcher and professor of Mierobiology at the École Normale Supérieure de Kouba, Alger (Algeria) and the Ex-head of the Laboratoire de Biologie des Systèmes Microbiens (LBSM). He published many papers on Saharan actinobacteria and their metabolites.

Table 1 . Geographical locations of sampling sites and physicochemical properties of soils. Soil characteristics Region Latitude Longitude Sample pH Carbon(%) Nitrogen (%) CaC0 3 (%) EC" Texture b El Oued 33 °19'N 6 °52'E E01 8.1 0.62 0.04 16.8 0.3 s Ghardaïa 32 °19'N 3 °40'E

 1

				Gh1	8.3	0.45	0.04	12.8	4.9	Sl
	Adrar	27 °51'N	ü°19'W	Gh2 Gh6 Ad3	8.0 8.2 8.6	0.66 0.19 0.80	0.08 0.01 0.04	10.0 3.6 4.0	1.9 0.2 6.5	Sl s Sl
				Ad4	8.5	0.15	0.06	3.2	0.4	Sl
				Ad5	8.8	0.81	0.05	6.0	0.2	Sl
	Ouargla Béchar	32°0'N 31 °34'N	5 °16'E 2°16'W	Ou3 Ou4 Bel Bc2 Bc3	8.8 8.7 9.3 8.9 8.5	0.80 0.15 0.15 0.45 0.15	0.04 0.06 0.06 0.04 0.07	6.0 3.2 5.6 6.4 10.0	33.9 3.9 20.4 0.2 0.4	Sl s lS s s
				Bc4	8.4	0.40	0.02	6.4	10.2

lS Bc5 8.2 0.15 0.07 12.0 3.0 Sl Bc6 8.9 0.32 0.07 8.4 13.3 Sil La ghouat 33 °49'N 2 °55'E la1 7.0 3.38 0.18 8.8 1.4 Sil Djelfa 34 °43'N 3 °13'E Dj1 8.9 0.88 0.04 7.2 8.4 l •Ec: electrical conductivity (1/5 at 25 °0 in ms/cm. bîexture acc ording to United States Department of Agriculture (USDA): l: loam

; lS: loamy sand; S: sandy; Sil: silt loam; Sl: sandy loam.

Table 2 . PCR primers used to amplify 16S rRNA, PKSs and NRPS genes.

 2

	Primer name
	FQ7
	RC1492
	KlF
	M6R
	KScx
	KSP
	EdyA
	EdyE
	A3F
	A7R

Table 3 . Distribution of alkalitolerant actinobacterial strains in Saharan soils.

 3

		Number of
		actinobacterial	Number of
		colonies (10 2 CFU/g of	strains isolated
	Soil sample	soil dry weight)	(isolate code)
	EOl	1.0
	Ghl	8.3
	Gh2	47.0
	Gh6	1.0
	Ad3	2.0
	Ad4	2.0
	Ad5	4.5
	Ou3	13.0
	Ou4	4.0
	Bel	1.0
	Bc2	1.0
	Bc3	3.0
	Bc4	2.7
	Bc5	1.0
	Bc6	54.2
	lal	63.0
	Djl	1.0

Table 4 . Antimicrobia activities and PKSs/NRPS genes of a ka ito erant actinobacteria iso ated from Saharan soi s.

 4

	Activity againsr'

7 6