Novel Bioactive Metabolites from a Marine Derived Bacterium Nocardia Sp. ALAA 2000 Mervat M
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J. Antibiot. 61(6): 379–386, 2008 THE JOURNAL OF ORIGINAL ARTICLE ANTIBIOTICS Novel Bioactive Metabolites from a Marine Derived Bacterium Nocardia sp. ALAA 2000 Mervat M. A. El-Gendy, Usama W. Hawas, Marcel Jaspars Received: November 4, 2007 / Accepted: June 10, 2008 © Japan Antibiotics Research Association Abstract Extracts of the Egyptian marine actinomycete, represent an enormous resource for the discovery of Nocardia sp. ALAA 2000, were found to be highly chemotherapeutic agents. Given the diversity of marine bioactive. It was isolated from the marine red alga organisms and habitats, marine natural products encompass Laurenica spectabilis collected off the Ras-Gharib coast of a wide variety of chemical classes such as terpenes, the Red Sea, Egypt. According to detailed identification polyketides, acetogenins, peptides and alkaloids of varying studies, the strain was classified as a member of the genus structures, representing biosynthetic schemes of stunning Nocardia. The cultivation and chemical analysis of this variety. Over the past 30 to 40 years, marine organisms species yielded four structurally related compounds have been the focus of a worldwide effort for the discovery namely, chrysophanol 8-methyl ether (1), asphodelin; 4,7Ј- of novel natural products [1]. Marine microorganisms bichrysophanol (2) and justicidin B (3), in addition to (actinobacteria) are sources of novel compounds with often a novel bioactive compound ayamycin; 1,1-dichloro-4- unique structures and potential therapeutic applications. ethyl-5-(4-nitro-phenyl)-hexan-2-one (4) which is unique The Actinomycetes are widely distributed in natural and in contain both chlorination and a rarely observed nitro manmade environments and are also well known as a rich group. The compounds were isolated by a series of source of antibiotics and bioactive molecules, which are of chromatographic steps and their structures of 1ϳ3 secured considerable importance in chemotherapy [2, 3]. by detailed spectroscopic analysis of the MS and NMR data Nocardia species, which was reported as an antibiotic whereas that of 4 was elucidated by single crystal X-ray producer, is one of the most promising sources of bioactive diffraction studies. These compounds displayed different compounds because among sixteen Actinomycete strains potent antimicrobial activity against both Gram-positive that were tested for their potentiality to produce bioactive and Gram-negative bacteria as well as fungi with MIC compounds that inhibit the growth of the pathogen Vibrio ranging from 0.1 to 10 mg/ml. damsela, Nocardia brasiliensis showed the largest inhibition zone and highest activity [4]. Keywords Nocardia sp. ALAA 2000, ayamycin, Nocardia brasiliensis has revealed such new bioactive justicidin B, NMR, X-ray, antimicrobial metabolites as a new antifungal and antibacterial compound, indole alkaloid named brasilidine A and new cytotoxic antibiotics named brasiliquinones (A, B and Introduction C29). A new Transvalencin A, a thiazolidine zinc complex antibiotic was isolated from Nocardia transvalensis IFM The oceans cover more than 70% of the earth’s surface and 10065 while, the remaining strains produced a chelating M. M. A El-Gendy: Chemistry of Natural and Microbial M. Jaspars (Corresponding author), U. W. Hawas: Department Products Department, National Research Centre, Dokki, Cairo, Chemistry, University of Aberdeen, AB 24 3UE Aberdeen, Egypt Scotland, UK, E-mail: [email protected] 380 compound N,N-ethylenediaminedisuccinic acid (EDDS) strain was found to produce chrysophanol 8-methyl ether and nocaracins [5, 6]. (1) [7], asphodelin; 4,7Ј-bichrysophanol (2) [8] identified We have recently started a program aimed at as two known anthraquinone metabolites; justicidin B (3) investigating the bioactive secondary metabolites of the as arylnaphthalene lignanolide [9], reported here to be Egyptian marine derived microbial strains as a part of our isolated for the first time from microorganisms; and a novel continued efforts to exploit marine Actinomycetes natural bioactive water soluble compound, ayamycin, 1,1-dichloro- products for drug discovery and development. We have 4-ethyl-5-(4-nitro-phenyl)-hexan-2-one (4). The absolute been isolating strains of Actinomycetes from marine algae stereochemistry of a novel compound 4 has been assigned of the Red Sea, Egypt. Based on different chemical and through X-ray crystallographic analysis. The compounds biological screening of the crude extracts of these different were potentially active against Gram-positive and Gram- marine Actinomycetes, we have found one marine strain negative bacteria as well as fungi. with high bioactivity against different human, animal or The taxonomy of the producing strain ALAA 2000 as plant pathogens. It was found to belonging to the genus well as the extraction, purification, structure elucidation Nocardia, Nocardia sp. ALAA 2000, and it was selected and biological activity of four antimicrobial molecules for the further investigations. (1ϳ4) from ALAA 2000 strain are described. According to physical and chemical analysis data, the Fig. 1 A; Structures of compounds 1ϳ4. B; Selected HMBC correlations for compound 1. C; Selected HMBC correlations for compound 2 (i) at Dϭ50 ms and (ii) at Dϭ200 ms. D; elected HMBC correlations for compound 3. 381 Taxonomy of the Producing Strain Materials and Methods Cell morphology of Nocardia sp. ALAA 2000 was observed under a Zeiss light microscope at ϫ1,000, with Collection of Samples cells grown on inorganic salts-starch agar (ISP medium 4) Samples of the marine red alga Laurenica spectabilis from after incubating the strain at 35°C for 3 days. The strain a depth of 1ϳ10 m were collected at Ras-Gharib coast of stained Gram-positive and examined for a balanced set of Red Sea, Egypt. The algal samples were placed inside phenotypic properties, using procedures described in sterile polyethylene bags underwater and transferred to the previous study [12ϳ19]. Color determinations were carried lab aseptically in ice boxes. The samples of algae were out according to ISCC-NBS color charts [20]. immediately processed at the Chemistry of Natural and Chemotaxonomically: The presence of the isomeric form microbial products Department of the National Research of diaminopimelic acid, and the composition of cell-wall Center, Egypt. sugars was analyzed by the previous method [21]. Polar lipids extracted, examined by two dimensional thin layer Isolation of Marine Actinobacteria chromatography, the frist dimension with chloroform/ Algae associated actinobacteria were isolated by following methanol/water (65 : 24 : 4) and in the second dimension the method outlined by Ananda et al. [1]. Initially, the algal with chloroform/methanol/acetic acid/water (80 : 12 : 15 : samples were washed with jets of filtered and autoclaved 4). Polar lipids were identified using published procedures seawater until they were visibly free of debris. [22]. Menaquinones were extracted, purified and identified Subsequently the algal surface was sterilized by a rapid [23]. Mycolic acids were extracted and analyzed by one- wash of 70% EtOH and immediately immersed in dimensional TLC and developed in petroleum autoclaved and filtered seawater and then aspirated. One benzene/acetone (95 : 5, v/v) as described previously [24] gram of algal tissue was removed with a sterile scalpel and and fatty acids analysisas described before [25]. the tissue was immediately transferred to 99 ml sponge Molecular characteristics, in order to characterize strain dissociation medium (2.7% NaCl, 0.008% KCl, 0.01% Merv21695 by molecular methods, GϩC content of the Na2SO4, pH 8). The samples were soaked for 20 minutes DNA and 16S rDNA sequencing were carried out. The and then the tissue and diluents were macerated and the GϩC contents were determined using the thermal homogenate was plated on starch nitrate agar medium of denaturation (Tm) method in standard saline citrate (SSC) the following composition (g/liter): Starch 20; KNO3 1; by the method of Mandel & Marmur [26]. K2HPO4 0.5; MgSO4·7H2O 0.5; NaCl 0.5; FeSO4·7H2O Chromosomal DNA was isolated from the test strain 0.01; agar 15 [10]. The isolation medium was prepared by using a procedure of Chun and Goodfellow [27], slightly using 100% seawater and contained 25 and 75 mg/ml of modified from that of Pitcher et al. [28]. filter-sterilized cycloheximide and nystatin, respectively, 16S rRNA gene amplification and sequencing from the as antifungal agent [11], using a serial dilution plate test strain were carried out using a procedure of Chun et al. technique. The plates were incubated at room temperature [29], and Kim et al. [30]. for 14 days. Actinomycetes were recognized by their tough dry colonies, branched vegetative mycelium, aerial Biological Assays mycelium when present and spore formation, and only The antimicrobial spectrum and activity of Nocardia sp. colonies with these characteristics were continued from the ALAA 2000 secondary metabolites were determined by the third day onwards up to the fourteenth day and included in agar plate diffusion assay [31]. Ten ml of the filtrate broth this study. was then applied to sterile paper disc (Whatman) of 6 mm diameter, placed on the surface of indicator test plate and Organisms and Maintenance incubated at the temperature that permitted optimal growth Biomass of the ALAA 2000 strain was prepared in shake of the test organisms. For determination of antibacterial and flasks of tryptic soy broth at 35°C for 7 days. At maximum antifungal activities, indicator bacteria were grown growth, the broth cultures were checked for purity, overnight in LB medium, yeasts were grown in YPG harvested by centrifugation, washed twice in TE buffer (pH medium (10 g/liter yeast extract, 10 g/liter peptone, 7) and kept at 5°C. The strain was maintained on tryptic 100 g/liter glucose) for 24 hours, and fungi were grown in soy agar plates and as spore suspensions (20%, v/v, potato dextrose agar (PDA) for 3ϳ4 days. The MIC of the glycerol). pure compounds was expressed as mg/ml by the dilution method. 382 Fig. 2 Typical time course of bioactive secondary metabolites produced by Nocardia sp.