Bacterial Diversity from Benthic Mats of Antarctic Lakes As a Source of New Bioactive Metabolites

Marine Genomics 2 (2009) 33–41

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Marine Genomics

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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 acetate were previously described and/or antifungal activities and showing a chemical novelty worthy (Gaspari et al., 2005). In the case of LPE with methanol, microbial broths of further investigation (Biondi et al., 2008). This was one of the few (10 mL) were extracted with an equal volume of methanol and were studies focused on the intensive characterization of Antarctic microbial centrifuged at 2000 g for 10 min. Cell-free supernatants were isolates by testing their bioactivity with the validated protocols of an concentrated to dryness under a N2 flow in a Turbo-Vap unit and industrial high throughput screening (Ashbolt, 1990; Biondi et al., dissolved in 1 mL 25% DMSO in water to be used directly in the assay. For 2008; Bull et al., 2000; Nichols et al., 1999). In this work we extended LPE by methyl–ethyl–ketone, microbial broths (10 mL) were extracted this antimicrobial screening to 723 heterotrophic bacteria isolated with 1.6 volumes of methyl–ethyl–ketone and centrifuged at 2000 g for from the benthic mats collected in ten lakes from Larsemann Hills, 10 min. The organic phase was concentrated to dryness under a N2 flow Vestfold Hills and McMurdo Dry Valleys, with the aim of discovering in a Turbo-Vap unit and dissolved in 1 ml 25% DMSO in water to be used new anti-infective producers among these Antarctic microorganisms. directly in the assay.

2. Materials and methods 2.4. Evaluation of antimicrobial activity

2.1. Microorganisms The following human pathogens from American Type Culture Collection (ATCC) and the Merck Culture Collection (MB, MY) were Bacteria were isolated on marine agar 2216 (Difco) and on R2A used as target organisms: Staphylococcus aureus ATCC19636, MB5393 agar (Oxoid) and classified using a chemotaxonomic and phylogenetic and EP167 MSSA (Novick, 1990); Enterococcus faecium MB4147 and study by the Laboratory of Microbiology of the University of Ghent MB5571; Escherichia coli MB4926 and MB5746 (envA/tolC); Pseudo- (Van Trappen et al., 2002). All the strains had been characterized by monas aeruginosa PAO1 (Holloway et al., 1979); Candida albicans whole-cell fatty acid analysis and were grouped in 41 fatty acid MY1055; Aspergillus fumigatus ATCC13073; Cryptococcus neoformans clusters, including 31 strains with unique profiles. Forty representa- ATCC90112 and MY2062. tive strains were further characterized by 16S rDNA sequence analysis. Procedures used for the antimicrobial screening by microtiter Strains showing pairwise sequence similarities of less than 97% to assay in liquid have been previously described (Biondi et al., 2008; their nearest validly named neighbours were considered belonging to Gaspari et al., 2005). Media were cation-adjusted Mueller Hinton yet unnamed new taxa or to species for which no sequences are yet broth (CAMHB, Difco) for S. aureus and E. faecium; sodium phosphate available (Stackebrandt and Goebel, 1994). Strains were deposited in buffered Terrific broth (TB, Difco) or LB Miller's broth for E. coli,and the BCCM/LMG Bacteria Collection, Laboratory of Microbiology of the LB Miller's broth for Pseudomonas aeruginosa although the primary University of Ghent (http://bccm.belspo.be). seed was grown overnight in cation-adjusted Mueller Hinton broth (CAMHB, Difco); antibiotic medium N°3 supplemented with 2% 2.2. Cultivation conditions for bacteria glucose (AM3, Difco) for C. albicans, A. fumigatus and C. neoformans. 1×104 to 5 × 105 CFU/mL of each strain were inoculated into 90 μL Bacteria were plated on Marine agar or R2A agar at 4°, 15°, 20°, 22° of appropriate culture broth added to 10 μLoftheextract(5μLin or 28 °C. Two different procedures were used for bacterial cultivation the case of P. aeruginosa) to be screened dissolved in 20% DMSO. in liquid media. Incubation time was 18–24 h, except for C. neoformans and J.L. Rojas et al. / Marine Genomics 2 (2009) 33–41 35

Fig. 1. Three distinct regions of the Antarctic continent sampled during the MICROMAT project. Benthic microbial mat samples were collected during the Antarctic summers 1997–1998 and 1998–1999.

A. fumigatus (48 h). Incubation was carried out under aerobic Elution followed a linear gradient of 17 min from 11% to 89% conditions at 35 °C or 37 °C, except for A. fumigatus (30 °C). Optical of acetonitrile with 0.5% formic acid and isocratic at 89% acetonitrile density at 620 nm was measured to detect pathogen growth in- 0.5% formic acid for the last 10 min. Full diode array UV scans from 200 hibition. One point test was used to select the “active” strains, i.e., to 900 nm were collected in 4 nm steps at 0.25 s/scan. The eluent those inhibiting more than 80% of the pathogen growth in com- from the column was collected in microtiter plates at 1 min/fraction parison with the control (set as 100%) when only DMSO/H2O (32 fractions) and used in assays. was added to the pathogen inoculum. The broth micro-dilution me- An aliquot of each fraction was evaporated to dryness and re- thod was used to confirm positive broths and to assay their potency suspended in 10% DMSO. LC–MS analysis was performed on an (Biondi et al., 2008; Gaspari et al., 2005). In the agar diffusion assay, Agilent (Santa Clara, CA) 1100 single Quadrupole LC–MS, using a the inoculum and assay plates for bacteria, yeast and filamentous Zorbax SB-C8 column (2.1×30 mm), maintained at 40 °C and with fungi were prepared as previously described (Suay et al., 2000). a flow rate of 300 μL/min. Solvent A consisted of 10% acetronitrile and After overnight incubation at 28 °C (fungi and yeasts) or 37 °C 90% water with 1.3 mM trifluoroacetic acid and ammonium formate, (bacteria), growth inhibition zones were recorded. Standard anti- while solvent B was 90% acetronitrile and 10% water with 1.3 mM biotic solutions (Sigma) were used as internal controls in the agar trifluoroacetic acid and ammonium formate. The gradient started at plates (e.g. amphotericin B, tunicamycin, penicillin G, oxytetracy- 10% B and went to 100% B in 6 min, kept at 100% B for 2 min and cline, hygromycin B and gentamycin). returned to 10% B for 2 min to initialize the system. Full diode array UV scans from 100 to 900 nm were collected in 4 nm steps at 0.25 s/scan. 2.5. Liquid chromatography and mass spectrometry of active fractions Ionization of the eluting solvent was obtained using the standard Agilent 1100 ESI source adjusted to a drying gas flow of 11 L/min Extracts were fractionated by HPLC on an Agilent (Santa Clara, CA) at 325 °C and a nebulizer pressure of 40 psi. The capillary voltage was 1100 single Quadrupole LC–MS using a Zorbax RX-C8 column 5 μm set to 3500 V. Mass spectra were collected as full scans from 150 m/z (4.6×250 mm) maintained at 40 °C and with a flow rate of 450 μL/min. to 1000 m/z, and 1000 m/z to 2000 m/z, with one scan every 0.77 s, 36 J.L. Rojas et al. / Marine Genomics 2 (2009) 33–41

Table 1 Diversity, distribution and antimicrobial activities of bacterial taxa isolated in different Antarctic areas.

Phylogenetic position FAA No. of Lake of origind Active No. of strains producing antimicrobial activities on: a b (nearest phylogenetic neighbours) cluster isolates strains S. aureus E. faecium E. coli C. albicans C. neoformans A. fumigatus testedc Bacteroidetes 136 26 Flavobacterium frigidarium (98.0%) 1 1 GR 0 0 0 0 0 0 0 Flavobacterium aquatile, F. tegetincola (95.7%) 2 5 HI 0 0 0 0 0 0 0 Saligentibacter salegens (92.9%) 4 3 FR 0 0 0 0 0 0 0 Flavobacterium gillisiae (98.4%), 5 69 FR (21), HO (2) PE (7), 17 13 2 12 0 0 1 F. hydatis (97.3%), F. frigidarium (97.5%) GR(17), WA (12), AC (1), DR (3), HI (1) RE (5) Flavobacterium gillisiae (98.4%) 6 5 PE 0 0 0 0 0 0 0 Flavobacterium hydatis (96.9%) 7 4 FR (2) WA (2) 2 2 0 1 0 0 0 Flavobacterium gillisiae (94.6%) 9 2 HO (1), FR (1) 1 1 1 0 0 0 0 Flavobacterium ﬂevense (95.3%) 10 25 AC (12), PE (4), WA (3), 43 1 10 0 0 GR (2), DR (1) RE (3) Flavobacterium tegetincola (98.2%) 11 4 AC (1), WA (2) RE (1) 0 0 0 0 0 0 0 Flavobacterium tegetincola (95.7%) 12 1 FR 0 0 0 0 0 0 0 Gelidibacter algens (99.8%) 13 4 FR (1) 00 0 00 0 0 PE (1), AC (2) Hymenobacter actinosclerus (97.3%) 14 6 FR (4) 2 2 1 0 0 0 1 PE (1) RE (1) Cyclobacterium marinum 92.5%) 15 7 FR (1) 0 0 0 0 0 0 0 AC (2) RE (4)

Gram + (high % GC) 85 10 Microbacterium keratanolyticum (96.0%) 16 9 DR (1), GR (1) RE (7) 1 1 0 0 0 0 0 Micrococcus luteus (99.7%) 17 5 DR (1), AC (2) RE (2) 1 1 0 0 0 0 0 Arthrobacter agilis (99.6%) 18 64 FR (54), HO (6) 53 3 3 0 0 DR (2), HI (1) RE (1) Clavibacter michiganensis (96.1%) 19 7 FR (3) 0 0 0 0 0 0 0 AC (1) RE (3)

Gram + (low % GC) 12 3 Bacillus oleronius (93.2%) 20 12 FR (11) OR (1) 3 1 1 3 0 0 0

α-Proteobacteria 89 12 AC (3) Porphyrobacter neustonensis (97.7%) 33 5 RE (2) 0 0 0 0 0 0 0 AC (1) Porphyrobacter neustonensis (97.6%) 34 3 RE (2) 0 0 0 0 0 0 0 Devosia riboﬂavina (97.2) 35 5 RE 0 0 0 0 0 0 0 AC (1) Sphingomonas natatoria (94.5%) 36 8 RE (7) 0 0 0 0 0 0 0 Brevundimonas subvibrioides (99.1%) 40 16 FR (2) DR (3), OR (1), 33 0 20 0 0 PE (1) RE (9) Mesorhizobium loti (96.9%), 41 52 FR (10) AC (15), OR 99 2 50 0 0 Sulﬁtobacter pontiacus (93.5%) (15), PE (4), WA (1), HI (2) RE (5)

β-Proteobacteria 60 20 Pseudomonas saccharophila (97.2%) 24 2 DR 2 2 0 2 0 0 0 Janthinobacterium lividum (99.7%) 28 13 FR (5), HO (8) 6 6 1 0 1 5 0 FR (1) AC (1) Hydrogenophaga palleronii 29 3 RE (1) 1 1 0 0 0 0 0 (98.4%), Aquaspirillum delicatum (97.8%) Hydrogenophaga palleronii 30 42 FR (10), HO (3) 11 11 1 3 1 1 1 (97.7%), Janthinobacterium DR (8), AC (12), GR (1), lividum (99.6%) PE (2) RE (6)

γ-Proteobacteria 254 49 Shewanella baltica (98.7%) 22 74 AC (18), PE (20), 19 17 0 7 0 2 0 WA (11), HI (20) RE (5) Pseudomonas anguilliseptica, 23 74 FR (11) AC (13), PE (13), 10 5 1 8 0 0 0 P. migulae (95.8%) WA (22), GR (1), HI (5) RE (9) Alteromonas macleodii (93.9%) 25 6 PE (4), WA (1), HI (1) 0 0 0 0 0 0 0 Pseudomonas orientalis (99.5%) 26 37 FR (1), HO (10) DR (3), 13 6 7 7 0 0 3 GR (1), HI (5) RE (17) Pseudomonas syringae (98.6%) 31 2 GR 0 0 0 0 0 0 0 J.L. Rojas et al. / Marine Genomics 2 (2009) 33–41 37

Table 1 (continued)

Psychrobacter glacincola (97.5%) 38 43 FR (15), HO (1) AC (3), 52 3 40 0 0 OR (1), PE (5), HI (3) RE (15) Marinobacter hydrocarbonoclasticus 39 18 AC (7), OR (10), PE (1) 2 2 0 0 0 0 0 (95.6%)

Unknown taxonomy 39 5 Not determined 3 1 RE 0 0 0 0 0 0 0 Not determined 8 1 FR 1 1 0 0 0 0 0 Not determined 21 2 HO (1) 0 0 0 0 0 0 0 AC (1) Not determined 27 2 GR 0 0 0 0 0 0 Not determined 32 2 AC 0 0 0 0 0 0 0 Not determined 37 3 AC 0 0 0 0 0 0 0 Not determined NC 28 FR (8) OR (2), PE (3), GR (1), 43 0 20 0 0 AC (8), HI (1) RE (5) Total 675 122 95 24 60 2 8 6

a The nearest phylogenetic neighbour was identified as described in Van Trappen et al. (2002); (%) indicates the 16S rDNA sequence similarities based on pairwise alignments. b The 41 fatty acid clusters were delineated in Van Trappen et al. (2002). 28 of the tested strains (NC means not clustered) formed single branches. c Data of cluster affiliation are reported on 675 bacterial strains out of the 723 screened in this paper. The fatty acid profiles of the remaining 48 strains not listed in this table were not clearly interpretable. d Numbers of strains isolated from each lake are indicated in parenthesis. The abbreviations FR, HO, RE, AC, DR, GR, HI, PE, WA and OR stand for lakes Fryxell, Hoare, Reid, Ace, Druzby, Grace, Highway, Pendant, Watts and Organic. in both positive and negative modes. Database matching was per- temperatures (4–10–20–22–28 °C), optimal temperatures were in the formed with a proprietary application where the DAD, retention time, range of 20 to 28 °C, suggesting that all these isolates were POS and NEG mass spectra of the active samples were compared to psychrotolerant rather than psychrophilic. the UV–LC–MS data of known metabolites stored in a proprietary database where metabolite standard data were obtained using the 3.2. Antimicrobial activities exact same LC–MS conditions as the samples under analysis. A total number of 6348 extract samples were prepared from the 3. Results fermentation of the 675 bacteria in two or three media and different incubation periods. Four sample preparation methods–three liquid 3.1. Taxonomic affiliation and cultivation of the Antarctic bacteria extractions with solvents of different polarity and a solid phase extraction with a polystyrenic resin–were used to improve the The 723 bacteria cultivated and tested in the screening were isolated recovery of secondary metabolites with different molecular weights from 17 samples of benthic mats collected from 10 lakes located in three and lipo/hydrophilic properties/polarities (Biondi et al., 2008). All the distinct Antarctic regions, i.e Larsemann Hills, Vestfold Hills and samples were tested for growth inhibition against a panel of human McMurdo Dry Valleys (Fig. 1). As reported elsewhere (Van Trappen pathogen microorganisms using high throughput screening et al., 2002), most of these bacteria (647) had been characterized and approaches (HTS) either in solid or liquid assay formats. The clustered according to their fatty acid composition into 41 FAA clusters frequency of microbiological activities against each test pathogen belonging to four major bacterial branches, Bacteroidetes (FAA clusters among the heterotrophic bacteria (Table 1) was considered to be an 1 to 15), the high and low percentage G+C Gram-positives (FAA clusters indicator of their ability to produce anti-infective molecules of 16 to 20) and the alpha, beta and gamma subclasses of the Proteobacteria potential therapeutic interest. One hundred twenty two bacteria out (FAA clusters 21 to 41) (Table 1). The remaining strains represented of 675 (18%) were active against at least one of the panel pathogens, single isolates (28 strains) or presented fatty acid profiles that were and more than 80% of these active strains only produced antibacterial not easily interpretable (48 strains) (data not shown). On the basis of activities. The frequencies of antibacterial activity against the Gram- the 16 S rDNA sequence analysis of FAA cluster representative strains, positives S. aureus and E. faecium were 14% and 3.5%, respectively, many of them appeared to be yet unnamed new taxa or taxa that have whereas 8% of the isolated strains inhibited the growth of the Gram- not yet been sequenced, or were closely related to taxa isolated from negative E. coli. The few strains that showed some inhibition of the cold, aquatic environments (Van Trappen et al., 2002). tested fungal strains were active against C. neoformans, (8 strains), Due to the diversity and the number of the isolated bacteria, two A. fumigatus (6 strains) and C. albicans (2 strains). The active strains different cultivation procedures were evaluated. Medium composition were distributed among 21 FAA clusters out of the 41 above de- and incubation temperature was optimized by testing different scribed and in several lineages in the α-, β- and γ-subclasses of the nutrients in vegetative and fermentative media and by varying growth Proteobacteria, the Bacteroidetes branch, and the high and low per- and production temperatures (data not shown). Major changes in the centage G+C Gram-positives. Almost 64% of the active bacteria (79 fermentation conditions were the introduction of less concentrated isolates) were members of the six major fatty acid clusters including media and the addition of marine salts, which are common adaptations Flavobacterium spp. (FAA cluster 5), α-Proteobacteria (FAA cluster of production media composition to the cultivation of marine microbes 41), β-Proteobacteria (FAA cluster 30) and γ-Proteobacteria (FAA (Sponga et al.,1999). About half of the bacteria exhibited better growth clusters, 22, 23, and 26), all of them containing isolates widely levels in marine-derived media whereas the rest of the strains distributed in the different sampling areas (see Table 1). Most of these performed well in classical rich media. Anyhow all the strains clusters contained strains from different lakes and often from different produced enough biomass in all the media tested to guarantee a regions, suggesting their ubiquitous presence in Antarctic lakes. In good extract preparation. When growth was monitored at different contrast, 39 active cultures were associated to 15 minor clusters, 38 J.L. Rojas et al. / Marine Genomics 2 (2009) 33–41

Table 2 Origin, taxonomical afﬁliation and antibacterial activities of selected bacteria isolates (hits).

Area Lake Strain code⁎ FAA cluster Phylogenetic branch Nearest phylogenetic neighbour Antibacterial activities in Antibacterial activities in liquid agar diffusion assays (mm) inhibition assays (%) on:†† on:† S. aureus E. faecium E. coli S. aureus P. aeruginosa E. coli Dry Valleys Fryxell R-7513 18 Gram + (high % GC) Arthrobacter agilis (99.6%) 10 11 0 100 0 0 Dry Valleys Hoare R-7687 28 β-Proteobacteria Janthinobacterium lividum (99.7%) 7 8 6 94 0 100 Dry Valleys Hoare R-7941 18 Gram + (high % GC) Arthrobacter agilis (99.6%) 7 8 0 99 0 0 Vestfold Hills Pendant R-8990 22 γ-Proteobacteria Shewanella baltica (98.7%) 7 0 7 0 0 0 Vestfold Hills Ace R-8895 Not determined Not determined 7 8 7 100 34 100 Larsemann Hills Reid R-12565 26 γ-Proteobacteria Pseudomonas orientalis (99.5%) 7 6 7 40 0 0 Larsemann Hills Reid R-12533 26 γ-Proteobacteria Pseudomonas orientalis (99.5%) 6 8 6 100 23 100 Larsemann Hills Reid R-12535 26 γ-Proteobacteria Pseudomonas orientalis (99.5%) 5 0 8 100 54 65 Larsemann Hills Reid R-12597 38 γ-Proteobacteria Psychrobacter glacincola (97.5%) 5 0 6 0 0 0 Larsemann Hills Reid R-12583 NC Not determined Not determined 9 5 9 0 0 0

⁎As preserved in the research collection of the Laboratory of Microbiology, University of Ghent, Belgium. †Antibacterial activities were indicated as the diameter in mm of the inhibition zone obtained in agar diffusion assays. ††Antibacterial activities were indicated as the percentage of inhibition obtained in liquid growth inhibition assays.

including four strains forming single branches and two belonging to 3.3. Chemical diversity the non-characterized group of strains. Apparently, there was not a clear-cut correlation between the antimicrobial activity pattern and Further studies were performed with a subset of these strains that the taxonomical distribution of the bacterial strains. exhibited some relatively potent antibacterial activities against

Fig. 2. LC–MS of the active fraction isolated from Arthrobacter fragilis R-7513. MW was confirmed upon LC–MS analysis of active fraction from large volume extract fractionation using a semi-preparative column. The same MW was confirmed for the major component isolated in active fractions from strain R-7941. J.L. Rojas et al. / Marine Genomics 2 (2009) 33–41 39 bacterial human pathogens (Table 2). Their spectrum of antibacterial volumes failed to reproduce in liquid assays the activities observed activity was further explored against a wider panel of test strains in the initial screening. using both liquid and agar diffusion assays. None of the ten selected strains showed antifungal activity in the production conditions tested 4. Discussion (data not shown). Six strains were studied more in detail for their antibacterial activity. Strains R-7513 and R-7941, isolated in the lakes 4.1. Microbial diversity of bacterial strains screened Fryxell and Hoare (the McMurdo Dry Valleys), corresponded to coccoid high % GC Gram-positives and turned out to have similar fatty Antarctic heterotrophic and photosynthetic bacteria have been acid composition and a high 16 S rDNA sequence similarity (99.6%) to extensively isolated by using enrichment methods under oligotrophic the actinobacterium Arthrobacter agilis. Both strains were cultivated in and psychotropic conditions, from freshwater lakes, saline and hyper- larger volumes in the production medium CRY and methanol extracts saline lakes and ponds, soil, sandstone and sea ice (Bowman et al., were fractionated to ensure partial purification of the active 1997; De la Torre et al., 2003; Franzmann et al., 1990; Lo Giudice et al., compounds. The activity against S. aureus was recovered respectively 2007; Miteva et al., 2004; Sullivan and Palmisano, 1984; Taton et al., in 2 fractions at min 20–21. In both cases LC–MS analysis of the active 2006b; Tindall, 2004; Tindall et al., 2000; Wery et al., 2003). fractions showed similar profiles, with a principal component (high- MICROMAT studies have shown that microbial diversity living in the est UV peak) of 1514 Da, and other minor components with MW 1516, benthic mats from Antarctic lakes is dominated by cyanobacteria, 1532 and 1580, suggesting that they produced related compounds being most of them novel and/or endemic species (Taton et al., 2003, (Fig. 2). High-resolution mass spectrometry of purified extracts 2006b). Many novel phylotypes, including two novel genera and 11 confirmed an exact mass of MW 1514 for the compound without novel species have been described among these heterotrophic bac- any match in natural products dictionaries. The exact mass suggested teria (Van Trappen et al., 2002, 2003, 2004a,b,c,d, 2005). These new compounds with different possible molecular formulas containing phylotypes can be added to others described from similar benthic multiple sulfur atoms. In all cases the presence of sulfurs and the microbial assemblages (McCammon and Bowman, 2000; Reddy et al., molecular weight range suggested that they could be members of the 2000, 2002, 2003; Sheridan et al., 2003; Shivaji et al., 2005). In con- cyclic thiazolyl peptide class of antibiotics, being related to com- trast, few but highly specialized microorganisms dominated among pounds such as siomycin, cyclothiazomycin or thiopeptin, with similar eukaryotes, often belonging to endemic Antarctic taxa (Gottlich et al., antibacterial spectrum against Gram-positive bacteria, including 2001; de Hoog et al., 2005). Following our recent effort to obtain from methicillin resistant bacteria (Z. Guan, personal communication). this biota a unique microbial collection to be exploited for pharma- The strain R-7687 belongs to the class of β-Proteobacteria and was ceutical indications, this study was aimed to a first evaluation of the isolated from Lake Hoare in the McMurdo Dry Valleys. In addition to its potential of heterotrophic Antarctic bacteria as a novel source of activity on Gram-positive pathogens, this strain differs from the previous antibiotics. On the basis of 16 S rDNA sequence analysis of FAA cluster ones by its additional activity against E. coli.ThisactivityagainstE. coli representative strains, most of them were associated with taxa that observed in the crude methanol extracts from the strain cultivated in had not been sequenced yet or are yet unnamed new taxa, related to two production media was lost in the course of the fractionation, only the genera Alteromonas, Bacillus, Clavibacter, Cyclobacterium, Flavo- recovering the activity against S. aureus. This activity eluted in four bacterium, Marinobacter, Mesorhizobium, Microbacterium, Pseudomo- fractions that showed similar MS spectrum with a principal component nas, Saligentibacter, Sphingomonas and Sulfitobacter. Their nearest (1206 MW) without UV spectrum that did not match any known phylogenetic neighbours often belonged to taxa isolated from cold, compound in the natural products library (data not shown). aquatic environments, such as Shewanella baltica, Psychrobacter The strain R-8895 from Ace Lake in the Vestfold Hills, not glacincola, Sulfitobacter pontiacus, Flavobacterium frigidarium, Flavo- characterized taxonomically, showed activity both against Gram- bacterium gillisiae, Saligentibacter salegens, Gelidibacter algens (Van positive and Gram-negative pathogens. After fractionation of the Trappen et al., 2002). methanol extract, both activities were recovered in one fraction eluting at 26 min. MS analysis of this active fraction did not show the presence 4.2. Cultivation approaches of any component that could be associated to this dual activity. The strains R-12565, R-12533 and R-12535 were γ-Proteobacteria An essential requirement was the development of cultivation and related to Pseudomonas orientalis, and were isolated from Lake Reid in screening techniques, to allow the identification of strains producing the Larsemann Hills. They were also particularly active against Gram- interesting bioactivities, and ensure the supply of sufficient material positive and Gram-negative pathogens in the primary hits. The for the biological and chemical characterization of the active meta- original activities produced against S. aureus and E. coli in agar bolites. A similar screening approach was applied in parallel to diffusion assays were only recovered in liquid inhibition assays in two Antarctic cyanobacteria (Biondi et al., 2008) and filamentous fungi of the strains (R-12533 and R-12535). The activities against S. aureus and yeasts isolated from the same mats (M. Brunati, data in and E. coli produced by R-12535 co-eluted in fractions 23 to 25 publication). In contrast to the poor growth of cyanobacteria isolated whereas in the case of strain R-12533, activities were lost after from the same sources, the heterotrophic bacteria included in this fractionation of the crude extract. The mass spectra analysis of the study did not show any specific nutrient requirements to grow, and chromatographic peaks corresponding to the active fractions from produced enough biomass to support the following extraction and strain R-12535 revealed the presence of two groups of molecules with screening protocols. Whereas some of them may prefer oligotrophic major components (1595 MW and 1673 MW respectively) that co- and saline enriched conditions, their oligotrophic way of life, i.e. elute in fraction 24 but that can be resolved in fractions 23 and 25 inability to propagate at elevated nutrient conditions and tempera- respectively (data not shown). When these fractions were tested ture, has been shown to be a transient characteristic, as reported for against a wider panel of Gram-negative strains, an activity against bacteria isolated from polar seas (Mergaert et al., 2001). In fact, in our P. aeruginosa was also confirmed in fraction 25. None of the com- laboratory conditions heterotrophic bacteria grew better at 20 °C than pounds detected in these fractions could be associated to any known at lower temperatures, suggesting that they are psychrotolerant and antibacterial compound when compared to proprietary databases or not psychrophilic (Biondi et al., 2008; Van Trappen et al., 2002). This in the Dictionary of Natural Products. phenomenon was previously observed with Antarctic marine bacteria The remaining four isolates selected as primary hits belonged that exhibited optimal growth levels at temperatures higher than to taxa isolated from cold, aquatic environments such as Shewanella those of their natural environment (Delille and Perret, 1989; Delille, baltica and Psychrobacter glacincola, but when cultivated in larger 1995; O'Brien et al., 2004). 40 J.L. Rojas et al. / Marine Genomics 2 (2009) 33–41

To increase the chance of finding novel bioactive chemical entities, similar molecular weight and tentatively associated to the class of the these heterotrophic bacteria were cultivated in different media in thiazolyl peptides produced by two strains of Arthrobacter.Thegenus order to trigger a variety of metabolic pathways (high throughput Arthobacter belongs to the order Actinomycetales, an industrially relevant cultivation). Moreover their biomass or culture medium was extracted group of high G+C gram positive bacteria known for bioconversions and by a plethora of procedures to favour the recovery of active meta- for secondary bioactive metabolite production. In particular, Arthrobac- bolites with different molecular weights and lipo/hydrophilic proper- ter is considered an ubiquitous genus recurrently isolated in Arctic and ties/polarities (Biondi et al., 2008; Gaspari et al., 2005). Antarctic regions (Miteva et al., 2004; Wery et al., 2003). Although some reports have already shown that antimicrobial activities are also 4.3. Antimicrobial activities produced from strains obtained from these cold environments and that they may play an antagonistic role in the maintenance of local As general trend these bacteria exhibited a relatively high rate of microbial communities (O'Brien et al., 2004; Lo Giudice et al., 2007), antimicrobial activities (18% of the strains tested) within the activity there is limited information about the type of antimicrobials produced ranges observed with other bacterial populations tested in industrial by these bacteria and only some molecules (i.e. peptide antibiotic screenings (Bills et al., 2009). Nevertheless it has to be mentioned that complex 02-8) have been described some far with wide antibacterial they were relatively weak as deduced from the size of the inhibition spectrum activity (Dictionary of Natural Products, 2003). This is the first zones or the percentages of inhibition obtained in liquid assays, when report on a preliminary characterization of bioactive metabolites from compared to those obtained from other bacterial groups such as Arthrobacter strains isolated from the Antarctic region. In the case of Actinomycetes, one of the most prolific bacteria producers the bioactive extracts obtained respectively from the β-andγ-Proteo- of antibiotics (Strohl, 1997; Berdy, 2005). The occurrence of activities bacteria R-7687 and R-12535, none of the preliminary MS spectra could could not be exclusively associated with any specific phylogenetic be associated with any known compound, suggesting a chemical novelty group, and it was found distributed across all major bacterial groups. associated to the activity of these Antarctic bacteria. Further studies will Nevertheless the β-Proteobacteria presented the highest activity rates be required to explore more in depth the chemical diversity of the active (33%), whereas the lowest hit rates were observed in the group of compounds identified in this study. high % G+C Gram-positive bacteria (11%). With regard to the activities produced, the majority of the active Acknowledgments strains inhibited bacterial growth, and very few antifungal activities were observed. The frequency of bioactivities against Gram-positive This work was supported by the European Community (MICRO- pathogens (14% and 3.5% versus S. aureus and E. faecium) was lower MAT Project BIO4-98-0040, http://www.nerc-bas.ac.uk/public/ than the one detected in the Antarctic cyanobacteria screening (29% mlsd/micromat). Thanks for the samplings are due to Kathy Welsh against S. aureus)(Biondi et al., 2008). In addition less than 1% of the (USA–McMurdo Dry Valleys Long Term Ecosystem Research Program heterotrophic bacteria produced some activity against the tested project), Dominic Hodgson and Pip Noon (British Antarctic Survey, fungal pathogens in contrast to what was obtained with cyanobacteria UK), Johanna Laybourn-Parry, Gareth Murtagh, Paul Dyer, Ingmar (4% and 20% of the screened cyanobacteria inhibited the growth of C. Janse, Tracey Henshaw, Wendy Quayle (University of Nottingham, UK neoformans and A. fumigatus, respectively). Consistently, extracts from with logistic support from the Australian Antarctic Division). We the heterotrophic bacteria were rarely found cytotoxic to mammalian thank all the colleagues of MICROMAT consortium, and a special cells (data not shown) (Biondi et al., 2008; Marinelli et al., 2004). acknowledgment is due to the coordinator Annick Wilmotte, Interestingly a relatively high number of bioactivities against the gram University of Liège, for her support and collaboration. We also thank negative E. coli was observed among the heterotrophic bacteria (8% of C. Rivas and P. Gómez for technical assistance. the tested strains). None of the Antarctic cyanobacteria recently screened showed any activity against Gram negatives (Biondi et al., References 2008). Although it is always difficult to evaluate and compare screening data with those generated by other authors due to the Ashbolt, N.J., 1990. Antarctic biotechnology—what is the potential? Aust. J. 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