A Novel Strain of Actinopolyspora Mortivallis with Antibacterial Activity Isolated from a Saharan Soil

Ann Microbiol (2012) 62:1049–1057 DOI 10.1007/s13213-011-0346-y ORIGINAL ARTICLE A novel strain of Actinopolyspora mortivallis with antibacterial activity isolated from a Saharan soil Atika Meklat & Nasserdine Sabaou & Noureddine Bouras & Abdelghani Zitouni & Cathrin Spröer & Hans-Peter Klenk & Florence Mathieu & Ahmed Lebrihi Received: 21 March 2011 /Accepted: 29 August 2011 /Published online: 17 September 2011 # Springer-Verlag and the University of Milan 2011 Abstract A new halophilic actinomycete strain, designated hybridization confirmed that it belonged to A. mortivallis. H16, has been isolated from a hypersaline Saharan soil in This strain showed high activity against Klebsiella Ouargla province (southern Algeria) and characterized pneumoniae and optimally secreted antibiotics during the taxonomically using a polyphasic approach. The strain mid-stationary growth phase in liquid culture. The anti- grew at 18–50°C, pH 5–9, and 7–32% (w/v) NaCl. It biotics were extracted with n-butanol and separated on produced abundant aerial mycelia, which formed long silica gel plates by ethanol–ammonia–water (mobile chains of rod-shaped spores at maturity, and fragmented phase). The results of bioautography revealed the presence substrate mycelia. The strain contained chemotaxonomic of five antibiotics. The chemical revelations showed that markers that were diagnostic for the genus Actinopolyspora, these antibiotics were glycosylated polycyclic aromatic such as meso-diaminopimelic acid, arabinose, and galactose, compounds containing amine groups and hydroxamic phosphatidylcholine as a diagnostic phospholipid, and acids. The UV-visible and mass spectra of the most active predominant menaquinones MK-9(H4) and MK-10(H4). antibiotics were determined. The predominant fatty acids were anteiso- and iso-C17:0, iso-C15:0, and 9-methyl-C16:0. Phylogenetic analysis, based Keywords Halophilic actinomycete . Actinopolyspora on 16S rDNA gene sequences, confirmed that strain H16 is a mortivallis . Saharan soil . Taxonomy. Antibiotic activities . member of the genus Actinopolyspora and most closely Microbial natural products related to A. mortivallis (98.7% identity). DNA–DNA Introduction A. Meklat : N. Sabaou (*) : N. Bouras : A. Zitouni Laboratoire de Recherche sur les Produits Bioactifs et la Valorisation de la Biomasse, Ecole Normale Supérieure de Kouba, Actinomycetes are a group of microorganisms that are BP 92, 16 050 Vieux-Kouba, Algiers, Algeria widely distributed in nature. Several studies on the e-mail: [email protected] ecology of actinomycetes have revealed that these microorganisms may occur in unusual habitats, such as C. Spröer : H.-P. Klenk German Collection of Microorganisms saline aquatic and terrestrial environments (Okazaki and and Cell Cultures (DSMZ), Okami 1975). The ecological distribution of actinomy- Inhoffenstraße 7B, 38124 Braunschweig, Germany cetes from Saharan soils has been studied in Algeria and their biodiversity in those soils demonstrated (Sabaou et F. Mathieu : A. Lebrihi Laboratoire de Génie Chimique UMR 5503 (CNRS/INPT/UPS), al. 1992, 1998). ENSAT/INP de Toulouse, Université de Toulouse, Antibiotics are produced by microorganisms, and actino- 1 avenue de l’Agrobiopôle, Castanet–Tolosan Cedex, France mycetes are likely the most important group of antibiotic- producing organisms (Demain 2006). This group, in A. Lebrihi Université Moulay Ismail, particular members of the genus Streptomyces, has been Marjane 2, BP 298 Meknes, Maroc studied in great depth, which has led to the discovery of a 1050 Ann Microbiol (2012) 62:1049–1057 large number of novel antibiotics (Okami and Hotta 1988). Cultural and micro-morphological characteristics In recent years, the rate of discovery of new antibiotics in the genus Streptomyces has been declining, and the number Cultural characteristics were determined after 1, 2, and 3 of drug-resistant pathogens have increased. The isolation of weeks of incubation on ISP-2, ISP-4 (Shirling and Gottlieb yet-unknown actinomycetes producing antibiotics appears to 1966), CMA, and nutrient agar media. The colors of the be essential in order obtain alternative antipathogenic agents substrate and aerial mycelia and any soluble pigments for those antibiotics that are becoming increasingly ineffective produced were determined by comparison with chips from and to find novel commercially valuable antibiotics. Several ISCC-NBS color charts (Kelly and Judd 1976). Spores and members of the families Micromonosporaceae (Actinoplanes, mycelia were examined by light microscopy (B1 series; Micromonospora) and Pseudonocardiaceae (Amycolatopsis, Motic, Xiamen, China) and scanning electron microscopy Saccharopolyspora) are considered among the major producers (model S450; Hitachi, Tokyo, Japan) after 3 weeks of of commercially important biomolecules (Solanki et al. growth on ISP-2 medium. 2008). The selective isolation and characterization of rare taxa from unexplored habitats could represent a supplementary Chemotaxonomic characterization source of novel bioactive molecules. In this context, the saline soils of south Algeria make up a large part of the Sahara and For the chemotaxonomic analyses, strain H16 was grown in may be of interest as a source of such bioactive molecules. complex medium broth (CMB) containing 15% (w/v) NaCl A broad range of biologically active molecules are at 30°C for 10 days on a rotary shaker (250 rpm). Biomass synthesized through pathways mediated by the polyketide was harvested by centrifugation at 3,500 rpm and washed synthetases (PKS)- and nonribosomal peptide synthetases several times with distilled water. Analysis of diaminopimelic (NRPS). The genes for these enzymes have been detected in acid and whole-cell sugars was carried out using the methods many actinomycetes producing antibiotics (Ayuso-Sacido and of Becker et al. (1964) and Lechevalier and Lechevalier Genilloud 2005;Metsä-Keteläetal.1999). Recent explora- (1970). Phospholipids were analyzed according to the tions of microbial antibiotic potential have focused on the procedures developed by Minnikin et al. (1977). The cellular screening of PKS and NRPS genes (Gontang et al. 2010; fatty acid composition was studied as described by Sasser Janso and Carter 2010; Qin et al. 2009). (1990) using the microbial identification system (MIDI). Here we describe the isolation of a new Actinopolyspora strain, designated H16, from a hypersaline Saharan soil Physiological characterization sample and its identification by conventional and molecular methods, together with the production and the partial Seventy physiological tests were used to characterize characterization of the corresponding antibiotics. actinomycete strains. Production of melanoid pigments was tested on ISP-6 and ISP-7 media (Shirling and Gottlieb 1966). Degradation of adenine, gelatin, guanine, hypoxanthine, Materials and methods milk casein, starch, testosterone, Tween 80, tyrosine, and xanthine was studied as described by Goodfellow (1971)and Actinomycete strain Marchal et al. (1987). Utilization of 23 carbohydrates and decarboxylation of nine organic acids were determined During an investigation of actinomycete diversity in according to the methods of Gordon et al. (1974). Lysozyme Saharan soils, strain H16 was isolated from a hypersaline sensitivity was evaluated by the method of Gordon and Barnett soil sample (electrical conductivity 33.9 mS cm–1) collected (1977). Growth at different temperatures (18, 25, 30, and from Ouargla province (southern Algeria), by plating 1:10 45°C), pH (5, 7, and 9), and NaCl concentrations (0, 7, 10, 15, serial dilutions of the sample on humic acid–vitamins agar 20, 25, 28, and 32% w/v) and in the presence of chloram- (Hayakawa and Nonomura 1987) supplemented with 20% phenicol (25 μgmL–1), erythromycin (10 μgmL–1), (w/v) NaCl at 30°C for 25 days. The choice of this medium kanamycin (5 μgmL–1), penicillin (25 μgmL–1), and was based on the good results obtained previously in our streptomycin (10 μgmL–1) was determined on nutrient agar laboratory for the isolation and selection of actinomycetes medium. All media used for physiological tests contained (Zitouni et al. 2005). The strain was purified and 15% (w/v) NaCl (except for the NaCl concentration test). maintained on complex medium agar (CMA) described by Chun et al. (2000) (casamino acids, 7 g; yeast extract, 10 g; DNA preparation, PCR amplification, sequencing, sodium citrate, 3 g; magnesium sulfate, 10 g; potassium and phylogenetic analysis chloride, 2 g; iron sulfate, 1 mL of a solution of 4.98% w/v; agar 20 g; distilled water, 1 L; pH 7.2) supplemented with Strain H16 was grown in 100 mL of the CMB supple- 20% (w/v) NaCl at 4°C. mented with 15% (w/v) NaCl. Biomass was harvested by Ann Microbiol (2012) 62:1049–1057 1051 centrifugation (8,000 rpm for 10 min) and washed twice with and Klebsiella pneumoniae E40), two filamentous fungi double-distilled water. Chromosomal DNA was prepared (Aspergillus niger CX33N and Penicillium expansum using a DNA extraction kit (JetFlex, Germany). The 16S B831P), and one yeast (Candida albicans M1). Strains rRNA gene was enzymatically amplified using the oligonu- of S. aureus and E. coli were from the collection of the cleotide primers 10-30 F (5′-GAGTTTGATC-CTGGCTCA- Pasteur Institute in Paris (France); those of A. baumanii, 3′) and 1500R (5′-AGAAAGGAGGTGATCCAGCC-3′), as E. cloacae,andK. pneumoniae were isolated from sick described by Rainey et al. (1996). Amplification was carried patients in hospitals of Algeria. The strains of filamentous out

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