Marine Genomics 2 (2009) 33–41 Contents lists available at ScienceDirect Marine Genomics journal homepage: www.elsevier.com/locate/margen Bacterial diversity from benthic mats of Antarctic lakes as a source of new bioactive metabolites Jose Luis Rojas a, Jesús Martín a,1, José Rubén Tormo a,1, Francisca Vicente a,1,MaraBrunatib,2, Ismaela Ciciliato b,3, Daniele Losi b,4, Stefanie Van Trappen c, Joris Mergaert c, Jean Swings c, Flavia Marinelli d, Olga Genilloud a,⁎,1 a CIBE, Merck Research Laboratories, Merck Sharp and Dohme de España S.A., Josefa Valcárcel 38, E-28027 Madrid, Spain b Vicuron Pharmaceuticals (formerly Biosearch Italia S.p.A), Via R. Lepetit 34, 21040 Gerenzano, Varese, Italy c Laboratorium voor Microbiologie, Universiteit Gent, K. L. Ledeganckstraat 35, B-9000 Gent, Belgium d DBSM, University of Insubria, J.H. Dunant 3, 21100 Varese, Italy article info abstract Article history: During the MICROMAT project, the bacterial diversity of microbial mats growing in the benthic environment Received 23 September 2008 of Antarctic lakes was accessed for the discovery of novel antibiotics. In all, 723 Antarctic heterotrophic Received in revised form 22 January 2009 bacteria belonging to novel and/or endemic taxa in the α-, β- and γ-subclasses of the Proteobacteria, the Accepted 2 March 2009 Bacteroidetes branch, and of the high and low percentage G+C Gram-positives, were isolated, cultivated in different media and at different temperatures, and then screened for the production of antimicrobial Keywords: activities. A total of 6348 extracts were prepared by solid phase extraction of the culture broths or by biomass Antarctic lakes Microbial mats solvent extraction. 122 bacteria showed antibacterial activity against the Gram-positives Staphylococcus Bacteria aureus and to a lower extent Enterococcus faecium, and versus the Gram-negative Escherichia coli.Fewof High throughput screening these strains showed also some antifungal activity against Cryptococcus neoformans, Aspergillus fumigatus Antibiotics and to a lower extent Candida albicans.LC–MS fractionation of extracts from a subset of strains (hits) that exhibited relatively potent antibacterial activities evidenced a chemical novelty that was further investigated. Two strains of Arthrobacter agilis produced potent antibacterial compounds with activity against Gram- positives and possibly related to novel cyclic thiazolyl peptides. To our knowledge, this is the first report of new antibiotics produced by bacteria from benthic microbial mats from Antarctic lakes. With no doubts these microbial assemblages represent an extremely rich source for the isolation of new strains producing novel bioactive metabolites with the potential to be developed as antibiotic compounds. © 2009 Elsevier B.V. All rights reserved. 1. Introduction approaches continue to enrich the variety of bioactive metabolites, the yield of novel useful drugs is decreasing and new sources of natural Natural products have been proven to be the richest source of novel products need to be investigated (Lam, 2007; Newman and Cragg, 2007). bioactive compounds. Historically, most bioactive products of microbial Antarctica, the coldest and windiest continent, is a remote, hostile origin derived from few taxonomic groups and terrestrial habitats (Berdy, and uninhabited area that together with its surrounding marine sites, 2005; Lam, 2007). In these decades, microbial natural products research offers a timely opportunity to investigate a still unexplored microbial stimulated the development of integrated approaches combining specific biodiversity (Brambilla et al., 2001; Marinelli et al., 2004; Taton et al., isolation methods and the access to geographically diverse sources and to 2003, 2006a,b; Tindall, 2004; Tindall et al., 2000; Van Trappen et al., different ecological niches. More recently other initiatives targeting the 2002). The infrequent combination of selection pressures has lead to exploitation of the metabolic potential of environmental gene libraries the evolution of novel biochemical adaptations and the possibility of without undergoing the step of culturing microbes (Lefevre et al., 2008; indigenous species (Ellis-Evans and Walton, 1990; Vincent, 2000). The MacNeil et al., 2001; Wang et al., 2000). Although all these new production of antibiotics and toxins may confer a competitive survival advantage in this environment as the accumulation of pigments offers ⁎ Corresponding author. a protection against strong UV radiation. The benthic mats from E-mail address: [email protected] (O. Genilloud). Antarctic lakes have accumulated for thousands of years and are 1 Present address: Fundación Centro de Excelencia MEDINA, Parque Tecnológico virtually undisturbed due to the particular climatic conditions and the Ciencias de la Salud, 18100 Granada, Spain. absence of higher metazoans. In the course of the MICROMAT project, 2 Present address: Fondazione Istituto Insubrico di Ricerca per la Vita, Via R. Lepetit, focused on the characterization and potential biotechnological 34 21040 Gerenzano, Varese, Italy. “ ” 3 Present address: Via Orazio 7, Busto Arsizio 21052, Italy. exploitation of the cultivated and not-yet-cultivated diversity of 4 Present address: Via Carso 28/B, Rovellasca, 22069, Italy. bacteria and fungi living in microbial mats at the bottom of Antarctic 1874-7787/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.margen.2009.03.005 34 J.L. Rojas et al. / Marine Genomics 2 (2009) 33–41 lakes, more than 1500 strains were isolated from mats sampled in a Procedure 1: A preculture step was carried out in 50-mL Erlenmeyer dozen of lakes differing in age and physico-chemical characteristics flasks containing 20 mL of medium and flasks were incubated for 72– and located in three distinct regions of the Antarctic continent (Fig. 1). 96 h at 22 °C or 28 °C and 150 rpm. Preculture media were Marine Broth The Larsemann Hills (Lakes Reid, Manning and Sarah Tarn) are (Difco) or rich media such as S/BIS (in g/L: glucose,10; peptone, 4; yeast a series of granite and gneiss peninsulas into Prydz Bay (Eastern extract, 4; MgSO47H2O, 0.5; K2HPO4,4;KH2PO4, 2; 1000 mL distilled Antarctica) with fjords and lakes directly (currents, inlets) or in- water) (Carelli et al., 1995) and V6 (in g/L: meat extract, 5; peptone, 5; directly (sea spray) subjected to marine influences; most of them casein hydrolysate, 3; glucose, 20; NaCl, 1.5; 1000 mL distilled water) thaw for up to two months in summer and during this time are (Marinelli et al., 1995). Growth was carried out for 96–120 h in 500 subjected to considerable wind driven mixing (Taton et al., 2006a). Erlenmeyer flasks containing 50 mL of three different fermentation The Vestfold Hills (Lakes Ace, Organic, Druzby, Pendant, Watts, media: Marine Broth and two other media Mare 1 and Mare 2 previously Highway and Grace) constitute a low-lying area where hundreds of developed for the screening of marine bacteria (Sponga et al., 1999). water bodies are found in the valleys, with salinities ranging from Flasks were incubated at 22 °C or at 28 °C and 150 rpm and time courses fresh to hypersaline (ten times seawater) (Bowman et al., 2000; of pH, glucose consumption and morphology variation were registered. Taton et al., 2006a). In McMurdo Dry Valleys, South Victoria Land, After 96 h of fermentation, cultures were harvested and treated ac- lakes Fryxell and Hoare are hundreds of thousand years old; they cording to the process sample preparation below described. do not lose their ice-cover, are mostly oligotrophic and are per- Procedure 2: The preculture step was performed in seed tubes with manently stratified (Brambilla et al., 2001; De la Torre et al., 2003; 10 mL medium MBY (Narinx et al., 1997) and tubes were incubated for Taton et al., 2006a). 72 h at 20 °C and 220 rpm. Fermentations were performed in tubes The diversity of heterotrophic bacteria isolated from these benthic containing 10 mL of three different production media: CGY (in g/L: mats, was studied by fatty acid analysis (FAA) and 16S rDNA se- casitone, 5; glycerol, 5; yeast extract 1; 1000 mL distilled water), MBY, quencing (Van Trappen et al., 2002). In addition to the extremely high CRY (Obata et al., 1999). Bacterial broths were harvested at 2 phylogenetic diversity with strains belonging to the α-, β- and γ- incubation times (3 and 7 d) for liquid extraction with methanol subclasses of the Proteobacteria, the high and low percent G+C Gram- and methyl–ethyl–ketone as indicated below. Fermentations in pro- positives, and the Cytophaga–Flavobacterium–Bacteroides branch, duction media were scaled-up to 150 mL in 500 mL flasks and novel phylotypes were discovered including e.g. novel species be- incubated for 3 or 7 days at 20 °C and 220 rpm. longing to Flavobacterium, Algoriphagus, and members of novel genera such as Loktanella and Gillisia (Van Trappen et al., 2003, 2004a,b,c,d, 2.3. Extract preparation methods 2005). Novel and endemic cyanobacteria were isolated from the same mats and their classification on the basis of morphological and mo- Fermentation broths were extracted according to different sample lecular taxonomy has been previously reported (Taton et al., 2003, preparation methods. Solid phase extraction (SPE) on polystyrenic resin 2006b). We recently screened these Antarctic cyanobacteria for anti- HP-20 (Mitsubishi Chemical Co., Tokyo, Japan) by methanol and liquid microbial and anticancer activity, finding interesting antibacterial phase extractions (LPE) with ethyl