The Journal of Antibiotics (2011) 64, 133–139 & 2011 Japan Antibiotics Research Association All rights reserved 0021-8820/11 $32.00 www.nature.com/ja ORIGINAL ARTICLE The genus Actinoallomurus and some of its metabolites Roberta Pozzi1,2, Matteo Simone2,3, Carlo Mazzetti2,3, Sonia Maffioli2, Paolo Monciardini1,4, Linda Cavaletti4, Ruggiero Bamonte4, Margherita Sosio1,2 and Stefano Donadio1,2 In the search for novel antibiotics, natural products continue to represent a valid source of bioactive molecules. During a program aimed at identifying previously unreported taxa of actinomycetes as potential source of novel compounds, we isolated hundreds of different representatives of a new group, initially designated as ‘Alpha’ and independently described as Actinoallomurus. We report on a PCR-specific method for the detection of this taxon, on appropriate growth conditions and on a pilot-screening program on 78 strains. The strains produce antibacterial or antifungal compounds at a relatively high frequency. Four strains were characterized in further detail: one produced the aromatic polyketide benanomicin B and its dexylosyl derivative; a second strain produced N-butylbenzenesulfonamide; a third strain was an efficient converter of soymeal isoflavonoids from soymeal constituents; and a fourth strain produced several coumermycin-related aminocoumarins, with coumermycin A2 as the major peak, and with some new congeners as minor components of the complex. These data suggest that Actinoallomurus strains possess several pathways for secondary metabolism and represent an attractive source in the search for novel antibiotics. The Journal of Antibiotics (2011) 64, 133–139; doi:10.1038/ja.2010.149; published online 1 December 2010 Keywords: actinomycetes; screening; secondary metabolites INTRODUCTION identification of several new taxa,7 three of which have been subseq- New antibiotics are urgently needed because of the increasing spread uently formally described.8–10 During that program we were also able of resistant pathogens and the evolution of new diseases. As microbial to isolate hundreds of different strains provisionally designated as genomes, synthetic libraries and high throughput screening have so ‘Alpha’, whose phylogenetic position suggested a new taxon within the far failed to produce a significant number of developmental candidates Actinobacteria.7 Independently, phylogenetically related strains have in the antibacterial field, it is generally acknowledged that nature will been assigned to the recently described genus Actinoallomurus.11 continue to be a significant source of novel bioactive compounds,1 We present here a characterization of ‘Alpha’ strains present in our as confirmed by a recent survey of antibiotics under clinical develop- collections, in terms of their potential to produce secondary metabo- ment.2 However, in a background of several thousands of known lites. The results from a small pilot study suggest that the genus microbial metabolites, it is necessary to introduce new strategies for Actinoallomurus has the potential to produce metabolites arising from accessing microbial strains to increase the probability of finding novel different biosynthetic pathways. compounds. Chemical screening3 and, more recently, genome mining4 are alternatives to the traditional bioassay-guided screening. In the RESULTS latter case, the probability of finding new compounds may be In the course of isolation programs, aimed at identifying actinomy- increased by the use of novel assays, of novel microorganisms or by cetes belonging to new taxa,7 we isolated from soil samples several the mere size of the library to be screened.5 In any case, dedicated actinomycete strains, with branching aerial hyphae that, as observed efforts are providing novel metabolites.6 by light microscopy on agar plates, differentiated into single spore In the past years, our attention has been drawn to previously chains along the aerial mycelium, reminiscent of some Actinomadura uncultured actinomycetes, under the assumption that strains belong- strains. Most of the strains were able to grow in the 30–37 1C ing to novel taxa, which are however phylogenetically related to temperature range at an optimal pH of 5.5. Phylogenetic analysis of prolific producers of secondary metabolites, have a high chance of selected strains indicated they belong to the recently described genus possessing diverse pathways in secondary metabolism and of affording Actinoallomurus within the Thermomonosporaceae.11 Furthermore, on novel compounds.7 A 16S-guided isolation program led to the 16S rRNA analysis, we were able to subdivide 115 strains in 32 ‘Alp’ 1KtedoGen Srl, Milano, Italy; 2NAICONS Scrl, Milano, Italy; 3ITB-CNR, Segrate, Italy and 4Vicuron Pharmaceuticals, Gerenzano, Italy Correspondence: Dr S Donadio, NAICONS Scrl, Via Fantoli 16/15, 20138 Milano, Italy. E-mail: [email protected] Dedicated to late Dr C Richard Hutchinson for his exceptional contributions to natural product biosynthesis, engineering and drug discovery. Received 3 October 2010; revised 27 October 2010; accepted 1 November 2010; published online 1 December 2010 Actinoallomurus: source of bioactive metabolites RPozziet al 134 phylotypes (Figure 1), indicating a substantial diversity within 78 different strains belonging to 26 different phylotypes. About 30% this genus. of the tested strains showed at least an antimicrobial activity in our For the rapid identification of novel isolates, we developed an conditions (Table 1). These results prompted us to further investigate Actinoallomurus-specific PCR method (see Methods). When we these strains and identify the bioactive metabolites produced. Here, we applied this method to soil samples, about 25% of soil DNA prepara- report the metabolites identified from four strains belonging to tions yielded a band of the expected size,7 suggesting that Actino- different phylotypes. The data are summarized in Table 2. allomurus strains may be quite cosmopolitan. Consistently, strains with an alpha-type morphology were isolated from many different Strain K15 soils, and the PCR method proved an effective tool for rapidly Strain K15 produced an antifungal activity and a diffusible red assigning most of them to Actinoallomurus. pigment. In all tested media, it showed good growth, and production The Thermomonosporaceae family currently comprises five genera of the red pigment and of the antifungal activity, both of which could but, to our knowledge, only strains belonging to the genus Actinoma- be efficiently recovered from the mycelium by MeOH extraction. dura have been reported to produce secondary metabolites. To HPLC fractionation of the MeOH extract, followed by LC-MS of the evaluate the ability of Actinoallomurus strains to produce bioactive active fractions, led to the identification of two peaks eluting at 14.5 compounds, we identified suitable growth conditions (see Methods). and 16.0 mins, containing the major antifungal activity (Figure 3). Examples of HPLC profiles are reported in Figure 2: different strains These peaks exhibited UV-VIS maxima at 500 nm and showed [MH+] yielded different profiles, suggesting production of different secondary of 827 and 696 m/z, respectively. Searches in the ABL database metabolites. Next, a pilot-screening program was carried out on identified the antifungal compounds benanomicin B and its dexylosyl Actinomadura alba YIM 45681T (DQ985164) Actinoallomurus purpureus NBRC 103687T (AB364588) 94 Alp2 Alp21 98 Alp11 Actinoallomurus luridus NBRC 103683T (AB364585) Alp20 Actinoallomurus spadix NBRC 14099T (AB364581) Alp3 100 Alp4 Alp33 Alp10 Alp31 Alp13 Alp24 87 Alp25 Alp34 Alp14 Alp18 Actinoallomurus coprocola NBRC 103688T (AB364579) Alp15 Actinoallomurus iriomotensis NBRC 103685T (AB364586) Alp28 Alp22 Alp27 Alp7 99 Alp8 Alp19 Alp17 Alp23 99 Alp9 Alp26 Alp30 Alp16 Actinoallomurus caesius NBRC 103678T (AB364589) Actinoallomurus amamiensis NBRC 103682T (AB364583)c 90 Actinoallomurus fulvus NBRC 103680T (AB364582) Alp32 Alp12 Alp6 99 Alp5 Actinoallomurus yoronensis NBRC 103686T (AB364584) Spirillospora rubra JCM6875T (AF163123) Actinomadura catellatispora NBRC 16341 (AF154127) Actinomadura madurae IFO 14623T (D85468) Actinomadura chokoriensis JCM 13932T (AB331730) Actinomadura latina DSM 43382T (AY035998) Actinomadura nitritigenes DSM 44137T (AY035999) Actinomadura latina DSM 43382T (AY035998) Spirillospora albida IFO 12248T (D85498) Actinomadura miaoliensis (EF116925) Actinomadura kijaniata DSM 43764T (X97890) Actinocorallia cavernae JCM13278T (AY966427) 96 100 Actinocorallia aurea IFO 14752T (AB006177) Actinocorallia herbida IFO 15485T (D85473) Thermomonospora curvata IFO 15933T (D86945) Actinomadura echinospora DSM 43163T (AJ420135) Figure 1 The ‘Alp’ phylotypes within Actinoallomurus. Phylogenetic tree of one representative from each Alp phylotype, nine Actinoallomurus species and selected representatives of other Thermomonosporaceae genera. The Journal of Antibiotics Actinoallomurus: source of bioactive metabolites RPozziet al 135 Table 2 Summary of analyzed strains Alp strain Phylotype Original bioactivity Identified metabolite(s) K15 Alp13 C. albicans Benanomicin B K216 Alp25 S. aureus N-Butylbenzenesulphonamide K241 Alp16 S. aureus Genistein and related compounds K275 Alp18 S. aureus Coumermycins Abbreviation: Alp, Alpha. de novo biosynthesis.16 Consistently, HPLC analyses of organic extracts from strain K241 grown in INA5 medium containing 5 g lÀ1 instead of 15 g lÀ1 of soybean flour showed a corresponding decrease in the Figure 2 HPLC profiles