Isolation and Characterization of a New Streptomyces Strain LG10 from an Unexploited Algerian Saharan Atlas
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ADVANCED RESEARCH IN LIFE SCIENCES 5, 2021, 36-42 www.degruyter.com/view/j/arls DOI:10.2478/arls-2021-0027 Research Article Isolation and Characterization of a New Streptomyces strain LG10 from an Unexploited Algerian Saharan Atlas Saïd Belghit1,2, Omrane Toumatia2,3, Mahfoud Bakli4 , Boubekeur Badji2 , Abdelghani Zitouni2 , Florence Mathieu5 , Laura Smuleac6 , Noureddine Bouras1,2* 1Département de Biologie, Faculté des Sciences de la Nature et de la Vie et Sciences de la Terre, Université de Ghardaia, Ghardaïa, Algeria 2Laboratoire de Biologie des Systèmes Microbiens (LBSM), Ecole Normale Supérieure de Kouba, Alger, Algeria 3Agro Pastoralism Research Center (APRC) Djelfa, Algeria 4Département des Sciences de la Nature et de la Vie, Faculté des Sciences et Technologie, Université Belhadj Bouchaib d’Ain Temouchent, Ain Temouchent, Algeria 5Laboratoire de Génie Chimique, Université de Toulouse, CNRS, Toulouse, France 6Banat’s University of Agriculture Science and Veterinary Medicine “King Michael I of Romania”, Faculty of Agriculture,119 Calea Aradului, 300645, Timisoara, Romania Received March, 2021; Revised May, 2021; Accepted June, 2021 Abstract An actinobacterial strain named LG10 was isolated from a Saharan Atlas soil (Laghouat, Algeria). The aerial hyphae were yellowish-white on all culture media with rectiflexibiles spore chains, suggested that this bacterium attached to Streptomyces. Furthermore, LG10 contained chemical characteristics that were diagnostic for the genus Streptomyces, such as the presence of LL-diaminopimelic acid isomer (LL-DAP) and glycine amino acid. The hydrolysates of whole-cell included non-characteristic sugars. Comparative analysis of the 16S rDNA sequence displayed a similarity level of 100% with Streptomyces puniceus NRRL ISP-5058T. The antimicrobial activity of the LG10 strain was better in the culture medium MB5. Streptomyces strains are good sources of bioactive compounds with multiple biological activities. Keywords: isolation, soil, taxonomy, Streptomyces, Saharan Atlas. Introduction were producers of interesting biological activities Actinobacteria are particularly interesting for the [5, 6]. In this work, we report the taxonomy of a production of bioactive substances such as Streptomyces strain labelled LG10 originated from antibiotics. Indeed, it has been estimated that a Saharan Atlas sample. more than 45% of antimicrobial products are secreted by actinobacteria [1]. Majority of these Material and Methods compounds were produced by Streptomyces [2]. Isolation of the actinobacterial strain Previous studies have indicated the richness of In our study, the aim is to isolate and identify actinobacteria in Algerian Sahara [3, 4], and it has actinobacteria that have antimicrobial bioactivities. been reported that many isolates from these soils Therefore, we have used six soil samples collected from different sites of Algeria. Based on morphological characteristics, 175 isolates of * Corresponding author: Noureddine Bouras, Email: actinobacteria including 123 Streptomyces were [email protected] purified and studied for their antimicrobial © 2021 S. Belghit et al., published by De Gruyter Open. activities. After screening of all these strains, 28 of This work was licensed under the Creative Commons Attribution-NonCommercial- NoDerivs 3.0 License them have showed an interesting antagonistic 36 DOI: 10.2478/arls-2021-0027 ARLS, 5, 2021, 36-42 microbial activity. Among these isolates, the without casein peptone) and MB5 (Bennett actinobacterium LG10 was isolated using chitin- without meat and yeast extracts). The antagonistic vitamins agar medium [7], from a sample collected activities were highlighted by the double layer from Laghouat (33°48′N, 2°52′E) in the north edge technique [19]. Actinobacterium LG10 was of the Algerian Sahara (Saharan Atlas). Penicillin seeded inside a circle diameter of 10 mm in the (25 µg/mL) and cycloheximide (50 µg/mL) were middle of the Petri dish containing one of the agar supplemented to culture medium to avoid media mentioned above. After 8 to 14 days of bacterial and fungal development, respectively. incubation at 28 °C, the cultures (3 replicates for The Petri dishes were incubated at 28°C for 21 each medium) were flooded with 3 mL of semi- days. solid ISP2 medium (10 g/L of agar) previously inoculated with the target microorganism (Candida Taxonomic study albicans IPA200 or Bacillus subtilis ATCC 6633). Phenotypical, chemical and physiological Petri dishes re-incubated at 28 °C and the characterization diameter of the inhibition zones was determined in Cultural and morphological characteristics were mm (including the diameter of the seeded isolate) explored using the method of Shirling and Gottlieb after 1 day for Bacillus subtilis and 2 days for [8], on various ISP (International Streptomyces Candida albicans. Project) media, nutrient agar and Bennett medium [9]. Results and Discussion For the chemical analysis, the isolate was cultivated on ISP-2 broth medium at 28°C for 96 Phenotypic traits, chemical and physiological hours. Determination of the diaminopimelic acid characteristics isomerism was realized depending to the methods The cultural characteristics are shown in (Table of Becker et al. [10], The constitution of whole-cell 1). A good growth was exhibited by the strain sugars were identified as mentioned by LG10 on ISP2, ISP3, ISP4, Bennett and less Lechevalier and Lechevalier [11] and growth on nutrient agar after incubation period of phospholipids were defined corresponding to the 14 days at 28 °C. The aerial hyphae were method Minnikin et al. [12]. Fifty-eight (58) tests yellowish-white on almost all culture media, with were evaluated as reported by Locci [13]. straight and flexuous spore chains containing between 10 and 50 spores. Substrate hyphae DNA extraction, PCR amplification and were not exhibited fragmentation and it was sequence study colored with beige or brown to yellowish brown DNA extraction of pure isolate LG10 was done according to the culture media. The cell-wall of following to the method of Liu et al. [14]. The LG10 strain was determined of type IC which amplification of 16S rDNA was performed as consisted of no characteristic sugars composition reported by Belghit et al. [15]. The PCR product (ribose, glucose and galactose), and possessed was purified and then sequenced by the Company LL-DAP isomer and glycine [20]. The phospholipid of MilleGen (Toulouse, France). The 16 rDNA profile is of type PII containing phosphatidyl sequence obtained was matched with those ethanol amine [21]. The physiological available in EzBioCloud database [16]. A characteristics of the LG10 strain are cited in phylogenetic tree was constructed using MEGA (Table 2). It was able to degrade the carbohydrate version 6.0 package [17] as described by Lu et al. compounds used except: adonitol, lactose, [18]. arabinose, mannose, ribose, erythritol, melibiose, myo-inositol, raffinose, sucrose and sorbitol. It Impact of some culture media on antimicrobial was able to use all amino-acids tested as unique activity sources of nitrogen except for serine; all organic In order to determine the best culture medium for compounds used except for guanine and all antimicrobial activities production, ten culture sodium salts except for benzoate and succinate. It media were examined. Two standard media: ISP2 was able to grow at pH 5 and pH 9, at 45 °C and medium (MA0) composed of malt extract, yeast at 10% of NaCl, but not at 15% of NaCl, at 60 °C extract, agar; and Bennett medium (MB0) and in the presence of phenol (0.001%), crystal composed of glucose, casein peptone, meat violet (0.05%) or sodium azide (0.001%). The extract, yeast extract and agar. Eight derived strain was insensitive to all antibiotics utilized media as following: MA1 (ISP2 without glucose), (except vancomycin, penicillin and kanamycin). It MA2 (ISP2 without malt extract), MA3 (ISP2 not produced the melanoid pigments on ISP6 and without yeast extract), MB1 (Bennett without meat ISP7 media. Due to its phenotypic traits, extract), MB2 (Bennett without yeast extract), physiological and chemical features, the strain MB3 (Bennett without glucose), MB4 (Bennett LG10 exhibited characteristics that are typical 37 DOI: 10.2478/arls-2021-0027 ARLS, 5, 2021, 36-42 representatives of the genus Streptomyces. and 55 subspecies [22]. Currently, this genus composed of 968 species Table 1. Cultural characteristics of the strain LG10 Medium Growtha Macromorphology Micromorphology AM SM SP ISP2 +++ yellowish white beige None RF Type, 10 to 50 spores per chain ISP3 +++ Yellowish white Brown Trace RF Type, 10 to 50 brownish spores per chain ISP4 +++ Yellowish white Yellowish Trace RF Type, 10 to 50 brown brownish spores per chain Bennett +++ Yellowish white Yellowish None RF Type, 10 to 50 brown spores per chain Nutrient agar ++ White Light None RF Type, 10 to 50 yellow spores per chain a +++: abundant, ++: moderate, AM: Areal Mycelium, SM: Substrate Mycelium, SP: Soluble Pigments, RF: Rectus Flexibilis. Table 2. Physiological features of the LG10 strain Test Result Test Result Degradation of: Histidine + Starch + Phenylalanine + Esculine + Proline + Gelatine + Serine − Xanthine + Decarboxylation of sodium salts Adenine + Pyruvate + Tyrosine + Citrate + Guanine − Acetate + Utilization of glucides (1% w/v) Tartrate + Rhamnose + Oxalate − Mannitol + Succinate − Xylose + Benzoate − Trehalose + Growth in the presence of: (µg/mL) Galactose + Streptomycin (10) + Fructose + Rifampicin (5) + Glucose + Erythromycin (10)