APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Sept. 2011, p. 6710–6714 Vol. 77, No. 18 0099-2240/11/$12.00 doi:10.1128/AEM.00326-11 Copyright © 2011, American Society for Microbiology. All Rights Reserved. Isolation, Taxonomy, and Antagonistic Properties of Halophilic Actinomycetes in Saharan Soils of Algeria  Atika Meklat, 1,2 Nasserdine Sabaou, 1 Abdelghani Zitouni, 1 Florence Mathieu, 2 and Ahmed Lebrihi 2 * Laboratoire de Recherche sur les Produits Bioactifs et la Valorisation de la Biomasse, Ecole Normale Supe ´rieure de Kouba, B.P. 92, 16050 Kouba, Alger, Algeria, 1 and Laboratoire de Ge ´nie Chimique UMR 5503 (CNRS/INPT/UPS), INP/ENSAT de Toulouse, Universite ´ de Toulouse, 1 Avenue de l’Agrobiopo ˆle, B.P. 32607, 31326 Castanet-Tolosan Cedex, France 2 Received 14 February 2011/Accepted 7 July 2011 The diversity of a population of 52 halophilic actinomycetes was evaluated by a polyphasic approach, which showed the presence of members of the Actinopolyspora, Nocardiopsis, Saccharomonospora, Streptomonospora, and Saccharopolyspora genera. One strain was considered to be a new member of the last genus, and several other strains seemed to be new species. Furthermore, 50% of strains were active against a broad range of indicators and contained genes encoding polyketide synthetases and nonribosomal peptide synthetases. Actinomycetes are economically and biotechnologically priceless prokaryotes. They have provided many important bioactive compounds (27). Several studies of the ecology of actinomycetes have shown that these microorganisms are wide- spread in nature and may occur in extreme environments. Thus, groups of acidophilic and alkaliphilic, psychrophilic and thermophilic, halophilic and haloalkaliphilic, and xerophilic actinomycetes have been described (1, 12, 25). Several genera and species of novel halophilic actinomycetes have been men- tioned in the literature (1, 9, 14, 29, 30). Despite these findings, little is known about the diversity of actinomycetes in saline environments. A broad range of biologically active molecules are synthesized by polyketide synthetase (PKS) and nonribo- somal peptide synthetase (NRPS) pathways (2, 18). The explo- ration of new soils and habitats has been recommended to screen for rare microorganisms able to produce several anti- biotics (21). The Algerian Sahara is one such extreme environ- ment. The aims of the present work were to study the biodi- versity of halophilic actinomycetes in Saharan soils and to highlight their potential to produce bioactive substances. Isolation and phenotypical identification of actinomycetes. Eighteen nonrhizospheric soil samples were collected from various Saharan regions in the south of Algeria: Adrar, Bechar, Djelfa, El Golea, El Oued, Ghardaia, Laghouat, Ouargla, and Tolga. Most of them were saline, with an electrical conductivity (1/5 soil/water ratio [wt/vol] at 25°C) between 2 and 55 mS/cm. Actinomycete strains were isolated by dilution at 30°C for 2 to 6 weeks on two selective media: complex medium agar (7) and humic acid-vitamin agar (11), both supplemented with 20% NaCl and 50 mg/liter of actidione. The morphological charac- teristics of actinomycetes were studied on International Strep- tomyces Project (ISP) medium 2, ISP medium 4, and complex medium agar (7, 26) containing 20% NaCl. Sixty-two physio- logical tests were used to characterize actinomycete strains. Degradation of different organic substrates and resistance to some chemical and physical agents were determined as de- scribed by Locci (17). The results of the physiological study were analyzed by numerical taxonomy. The degree of similarity between strains was calculated by simple matching (SM), and clustering was performed by the unweighted-pair group method using average linkages (UPGMA) in the SPSS package (v.16.0.1). For the chemical study of cell constituents, analyses of diaminopimelic acid and whole-cell sugars were carried out using the methods of Becker et al. (3) and Lechevalier and Lechevalier (13). Phospholipids were analyzed according to the procedure developed by Minnikin et al. (20). Variable levels of cultivable halophilic actinomycetes were observed, ranging from 10 2 to 10 3 CFU per g (dry weight) of soil, which represented only 0.001 to 0.1% of the total actino- mycetes in these soils (23, 24). In total, 52 actinomycetes were collected from 18 analyzed soil samples and were detected in both of the media used. The preliminary investigation based on morphological and chemical analysis of the 52 isolates of ac- tinomycetes allowed some of these strains to be tentatively classified into several genera. Sixteen strains belonged to No- cardiopsis (19), six strains to Saccharomonospora (22), and three strains to Streptomonospora (8). Twenty-seven strains could not be accurately identified at the genus level at this stage. The physiological classification based on the similarity SM (S SM ) coefficient and on UPGMA clustering gave 4 cluster groups, designated I to IV, and one single strain at the 75% similarity (S) level (Fig. 1). Two major (14 and 18 strains) and six minor (2 to 6 strains) subclusters were circumscribed at the 81% S level (Fig. 1). Clusters II, III, and IV contained strains of Streptomonospora, Nocardiopsis, and Saccharomonospora, respectively. All strains of cluster I and the single strain H31 were not identified at the genus level during the preliminary classification. The numerical taxonomy used in this study en- abled rational discrimination between strains but did not per- mit identification. Phylogenetic studies of actinomycetes. Genomic DNA of 30 strains, belonging to different clusters established by numerical taxonomy, was extracted for 16S rRNA gene analysis according * Corresponding author. Mailing address: Laboratoire de Ge ´nie Chimique UMR 5503 (CNRS/INPT/UPS), INP/ENSAT de Toulouse, Universite ´ de Toulouse, 1 Avenue de l’Agrobiopo ˆle, B.P. 32607, 31326, Castanet-Tolosan Cedex, France. Phone: (335) 34 32 39 00. Fax: (335) 34 32 39 01. E-mail: lebrihi@ensat.fr.  Published ahead of print on 15 July 2011. 6710 on June 23, 2017 by guest http://aem.asm.org/ Downloaded from