Systematic and Applied Microbiology 34 (2011) 606–616 Contents lists available at ScienceDirect Systematic and Applied Microbiology j ourna l ho mepage: www.elsevier.de/syapm Detection, selective isolation and characterisation of Dactylosporangium strains from diverse environmental samples Byung-Yong Kim, Jenileima Devi Kshetrimayum, Michael Goodfellow ∗ School of Biology, Newcastle University, Newcastle upon Tyne NE1 7RU, UK a r t i c l e i n f o Article history: Received 14 February 2011 Keywords: Actinomycetes Diversity Selective isolation Dactylosporangium Rare-actinomycetes a b s t r a c t A culture-independent, nested PCR procedure based on genus-specific oligonucleotide primers detected the presence of members of the genus Dactylosporangium in 14 out of 21 diverse environmental sam- ples. Clones generated from the 14 positive environmental samples formed distinct phyletic lines in the dactylosporangial 16S rRNA gene tree. Presumptive dactylosporangiae were isolated from 7 of these samples using a medium designed to be selective for members of the genus Dactylosporangium, namely Streptomyces Isolation Medium supplemented with gentamicin and antifungal antibiotics. One hundred and two out of 219 representative presumptive dactylosporangiae were considered as authentic mem- bers of the genus Dactylosporangium as they gave PCR amplification products using the genus-specific primers and had chemical features typical of dactylosporangiae. Representative of the Dactylosporangium isolates formed distinctive phyletic lines in the dactylosporangial 16S rRNA gene tree, contained the non-ribosomal peptide and type-I polyketide synthase genes and inhibited the growth of Bacillus subtilis, Kocuria rhizophila and Staphylococcus aureus strains. It is evident from these results that the genus Dacty- losporangium is underspeciated, widely distributed in natural habitats and is a potentially rich source of novel secondary metabolites. © 2011 Elsevier GmbH. All rights reserved. Introduction There is an urgent need to find new drugs, especially antibiotics, to control the spread of antibiotic resistant pathogens [16,52,60] and to treat life-threatening diseases such as cancer [50]. Amongst prokaryotes, filamentous actinomycetes, notably streptomycetes, remain a unique source of new antibiotics [4,21,48] and this is likely to continue as full genome sequences of model actinomycete strains contain over 20 natural product biosynthetic gene clusters for the production of known or predicted secondary metabolites, as exemplified by studies on Amycolatopsis mediterranei U32 [74], Saccharopolyspora erythraea NRRL 23338 [51], Salinispora tropica CNB 440 [68], Streptomyces avermitilis [32] and “Streptomyces coeli- color” A3(2) [7]. In contrast, few, if any, such gene clusters have been detected in whole genomes of other prokaryotes [21]. It is becoming increasingly difficult to discover new antibiotics from common actinomycetes as screening such organisms tends to lead to the costly rediscover of known secondary metabolites [11,40]. This problem can be addressed by using standard proce- dures for the selective isolation of novel actinomycetes from poorly studied habitats [8,30,49], by applying novel procedures for the ∗ Corresponding author. Tel.: +44 191 222 7706; fax: +44 191 222 5228. E-mail addresses: m.goodfellow@ncl.ac.uk, m.goodfellow@newcastle.ac.uk, jan.fife@newcastle.ac.uk (M. Goodfellow). selective isolation and characterisation of uncommon and rare acti- nomycetes [18,64,65] and by detecting the presence of novel, rare and target actinomycetes in environmental samples using culture- independent methods prior to the application of selective isolation procedures [43,61]. These strategies have lead to the isolation of novel actinomycetes with the capacity to produce diverse new bioactive compounds [15,21,34,35]. Effective taxon-specific strategies are required for the selec- tive isolation of neglected filamentous actinomycetes, not least for the isolation of members of the genus Dactylosporangium which encompasses strains known to produce anti-allergic drugs [66], antibiotics, such as dactimicin [58] and tiacumicins [67], and a plant growth inhibitor [38]. The genus Dactylosporangium encom- passes aerobic, filamentous actinomycetes which form motile spores in spore vesicles borne on short sporangiophores on sub- strate hyphae and have hydroxy and/or meso-diaminopimelic acids in whole-organism hydrolysates, but lack mycolic acids [37,70]. Dactylosporangiae have been isolated from a range of terres- trial habitats [23,24] but have rarely featured in pharmacological screening programmes as they grow slowly and are difficult to iso- late in large numbers. The present study was designed to establish the extent of dacty- losporangial taxonomic diversity in diverse environmental samples using a culture-independent procedure prior to developing a reli- able strategy for the selective isolation and characterisation of dactylosporangiae known to be present in environmental samples. 0723-2020/$ – see front matter © 2011 Elsevier GmbH. All rights reserved. doi:10.1016/j.syapm.2011.03.008