Michael Goodfellow, School of Biology, Newcastle University, Newcastle-

2. Major Themes Importance of selective isolation. Extent of actinobacterial diversity. New approaches to selective isolation of specific taxa Selective isolation of actinomycetes from desert soils. Prospects for the future.

3. WHY ISOLATE ACTINOBACTERIA? Recognition and identification of animal (eg. actinomadurae, mycobacteria) and plant pathogens (eg. clavibacteria, curtobacteria). Recycling of waste materials and turnover of organic matter (eg. arthrobacters, streptomycetes). Mutualistic nitrogen fixation (eg. frankiae, micromonosporae and certain angiospermous plants). Production of bioactive compounds (eg. salinisporae and streptomycetes). Vaccine production (eg. BCG from Mycobacterium bovis). Agents of bioremediation (eg. gordoniae, rhodococci) and biocontrol of fungal pathogens (eg. streptomycetes). Ripening of cheese (eg. brevibacteria, microbacteria).

4. RELATIVE IMPORTANCE OF ACTINOBACTERIA “If one had to answer the question” – Which group of microorganisms has been the most helpful in medicine and agriculture? – The loud and clear answer would be “the actinomycetes”. Arne Demain (1988).

8. THE KNOWN EXTENT OF ACTINOBACTERIAL DIVERSITY IS THE TIP OF THE ICEBERG Lines of evidence Many actinobacterial-related phylogenetic lines of descent can be detected in community DNA isolated from environmental samples. There is clear evidence from culture independent approaches of the existence of deep rooted actinobacterial lineages. New actinobacterial genera continue to be isolated and characterised. A wealth of undiscovered actinobacterial diversity exists in neglected habitats. Implications New selective isolation strategies are needed to representatively sample actinobacterial communities in natural habitats. Selective isolation, characterisation and screening are pivotal in search and discovery programmes, especially since a strong correlation has been observed between taxonomic and chemical diversity (e.g. Salinispora and Streptomyces species).

10. NUMBER OF SPECIES IN SELECTED ACTINOMYCETE GENERA Genus Bergey’s Manual 1974 1989 2011 INDUSTRIALLY IMPORTANT Actinocorallia - - 6 Actinoplanes 6 11 30 Amycolatopsis - - 26 Gordonia - - 32 Micromonospora 12 8 30 Rhodococcus - 12 22 Saccharomonospora - 3 8 Streptomyces 463 418 553 MEDICALLY IMPORTANT Actinomadura - 16 35 Actinomyces 5 10 37 Corynebacterium 7 16 104 Mycobacterium 29 54 126 Nocardia 6 8 72

12. Stach, J.E.M., Maldonado, L.A., Ward, A.C., Goodfellow, M. & Bull, A. T. (2003). New primers for the class Actinobacteria: application to marine and terrestrial environments. Environmental Microbiology 5, 828-841. Primers were designed and evaluated for the class Actinobacteria. In silico testing showed that the primers had a perfect match with 82% of actinobacterial genera. The primers amplified an ˜ 640 bp stretch of 16S rRNA genes of representatives of 26 out of 27 actinomycete genera tested, the exception was Rubrobacter radiotolerans DSM 5868T. An Actinobacteria Amplification Resource was generated to provide a visual guide to aid in the amplification of 16S rDNA.

13. APPLICATION OF PRIMERS TO DNA EXTRACTED FROM MARINE AND TERRESTRIAL SAMPLES One hundred clones were generated from two marine and two terrestrial environmental samples. Dereplication using single-strand conformation polymorphism screening reduced the numbers of clones to eighty-five. Phylogenetic analysis of dereplicated clones. 16S rDNA similarity and DNA:DNA pairing correlations showed that almost all of the dereplicated actinobacterial clones represented new species or novel genera. Clones were assigned to the subclass Acidimicrobidae (31), the suborder Corynebacterineae (3) and to the families Actinosynnemataceae, Micrococcaceae, Micromonosporaceae, Nocardioidaceae and Pseudonocardiaceae. Results reinforce the view that current culture based techniques underestimate actinomycete diversity in the environment.

14. Stach, J.E.M., Maldonado, L.A., Masson, D.G., Ward, A.C., Goodfellow, M. & Bull, A. T. (2003). Statistical approaches for estimating actinomycete diversity in marine sediments. Applied and Environmental Microbiology 69, 6189-6200. A deeper knowledge of the diversity, distribution and ecology of actinobacterial in natural habitats is needed to inform bioprospecting and diversity management strategies Actinomycete diversity in a deep sea sediment (3,814 m) was investigated by constructing actinobacterial specific 16S rDNA clone libraries from sediment sections taken below the seafloor. Clones were assigned to operational taxonomic groups with >99% 16S rDNA similarity. Diversity statistics were used to determine how the level of dominance, species richness and genetic diversity varied with depth.

15. Statistical approaches for estimating actinomycete diversity in marine sediments Principal Results Species richness decreased with sediment depth with a concomitant decrease in genetic diversity Each of the sediment core sections contained unique phylogenetic lineages Clones grouped into Operational Taxonomic Units are likely to be derived from distinct species corresponding to separate ecological lineages Implications of diversity estimates for bioprospecting Presence or absence of species in environmental samples – from molecular census data – will inform the design of cultivation strategies Comparison of the cultural subset to the molecular data provides a measure of how many of the target species are available for screening Coverage curves and richness estimators provide a means to assess how much further sampling is required. Phylogenetic diversity estimates will aid in the identification of species that are cosmopolitan or endemic

17. CLASSICAL APPROACHES TO SELECTIVE ISOLATION OF ACTINOMYCETES Collection of Environmental Samples Pretreatment of Samples Extraction of Propagules Inoculation onto Non-Selective and Selective Media Incubation and Colony Selection Identification of Colonies

23.  SOME NEW STRATEGIES FOR THE SELECTIVE ISOLATION OF NOVEL ACTINOBACTERIA Ecological approaches: Sampling from neglected habitats. Dilution to extinction. Mining of phenotypic databases: Supplementation of basal media with taxon specific selective agents. Media formulation designed to inhibit the growth of ubiquitous actinobacteria. Nutritional and tolerance methods: Addition of complex carbon sources. Design of media for the isolation of acidophilic, alkaliphilic and halophilic organisms. Others Use of different gelling agents. Gel encapsulation. Selection based on the use of actinophage. Sucrose-gradient centrifugation.

24. Salt, M., Hugenholz, P. & Janssen, P.H. (2002). Cultivation of globally distributed soil bacteria from phylogenetic lineages previously only detected in cultivation – independent surveys. Environmental Microbiology 4, 654-666. Used a polymeric growth substrate (xylan) and unusually long incubation times to isolate bacteria from soil samples. Seventy-one isolates were identified by comparative 16S rRNA gene sequence analysis. Isolates included globally distributed but previously uncultured phylogenetically novel soil bacteria. Novel members of the class Actinobacteria were considered to form a new order of the subclass Rubrobacteridae, a new family of the suborder Frankineae and a new family of the suborder Streptosporangineae.

26. DISPERSION AND DIFFERENTIAL CENTRIFUGATION TECHNIQUE Soil Shake in ionic detergent and chelating ion exchange resin Low speed centrifugation Shake in Tris buffer Low speed centrifugation Ultrasonication Low speed centrifugation Shake in water Low speed centrifugation Residue

27. Nocardia Trevisan 1889VP Aerobic, Gram-positive actinomycetes. Acid to partially acid- alcohol fast organisms. Substrate mycelium fragments into coccoid to rod-shaped, nonmotile elements. Aerial hyphae nearly always formed. Wall chemotype IV, A1? peptidoglycan, N-glycolated muramic acid. Straight chain saturated and unsaturated fatty acids with tuberculostearic acid. Mycolic acids: 46 to 60 carbons, mycolic acid esters with 12 to 18 carbon atoms. Predominant menaquinone is MK8 (H4, cycl). Major polar lipids are DPG, PE, PI and PIM’s. DNA base composition is within the range 64 to 72 mol % GC.

29.  SELECTIVE ISOLATION OF NOCARDIAE Basal medium: Diagnostic Sensitivity Agar. Antifungal antibiotics: Actidione and nystatin (each at 50 µg ml-1). Antibacterial antibiotics: (a) Demethylchlortetracycline (5 µg ml-1). (b) Methacycline (10 µg ml-1). (c) Chlortetracycline (45 µg ml-1) + Demethylchlortetracycline (5 µg ml-1). (d) Chlortetracycline (45 µg ml-1) + Methacycline (10 µg ml-1). Incubate isolation plates at 25° C for 14 days.

32. COMPUTER- ASSISTED FORMULATION OF SELECTIVE ISOLATION MEDIA Selective agents Target organisms References Aminobutyric acid and rhamnose Streptomyces violaceusniger Williams & Vickers (1984) Dextran and L-histidine Streptomyces violaceoruber Duangmal et al. (2005) and pencillin clade Nalidixic acid and novobiocin Amycolatopsis species Tan et al. (2006) Raffinose and histidine Streptomyces aureus and Manfio et al. (2003) Sreptomyces sanglieri Oleandomycin and sodium chloride Streptomyces atroolivaceus Williams & Vickers (1988) 2-Aminoethanol and raffinose Streptacidiphilus spp. Rodriguez et al. (unpublished) (medium buffered at pH4.5)

33. PROFILE OF THE GENUS AMYCOLATOPSIS LECHEVALIER ET AL. 1986 Aerobic, Gram positive, non-acid fast actinomycetes. Substrate mycelium fragments into squarish elements. Aerial hyphae may be sterile or differentiate into squarish to oval spore-like structures. Wall chemotype IV, A1g peptidoglycan, N-acetylated muramic acid. Complex fatty acid pattern: straight chain, monounsaturated, iso-, anteiso- and branched chain components. Predominant menaquinones – MK9 (H2, H4, H6). Major polar lipids – DPG (variable), PE, PG, PI and PIM’s. DNA base composition is between 66-69%.

36. Selective Isolation and Characterisation of  Novel Amycolatopsis Strains Choice of selective agents. Formulation of selective media. Characterization of representative isolates. Proof of novelty. Polyphasic study of members of putative new species. Publication of new species in the International Journal of Systematic and Evolutionary Microbiology.

37. Selective Isolation Media Based on Information Taken from a Numerical Taxonomic Database

45. One route to discovering novel actinobacteria is to investigate environments that have been un- and under-explored: Acidic and alkaline ecosystems. Anaerobic habitats. Desert biomes. Freshwater habitats. High and low temperature environments. Littoral sediments. Low nutrient sites. Marine habitats. Mutualistic associations. Saline habitats. Polluted soils.

46. Map of Chile Showing Location of the Atacama Desert.

47. Comparison of the Core Arid Region of the Atacama (above) Desert Which Bears Similarity to the Landscape in Mars (below).

48. Atacama Desert Sampling Locations

49. Physico-Chemical Properties of the Soils

50. Media used for the Selective Isolation of Actinobacteria

51. Total Viable Bacterial Counts (cfu g-1 soil) and Percentage of Putative Actinobacteria.

52. Environmental Sampling

53. 16S rRNA Amycolatopsis Neighbour-Joining Tree

56. 3D Relationships of 16S rRNA Genes of Streptomycetes

57. SELECTIVE ISOLATION OF ACTINOBACTERIA: WHERE ARE WE NOW? Good news Procedures are available for the isolation of: Actinoplanes, Amycolatopsis, Dactylosporangium, Dietzia, Gordonia, Micromonospora, Nocardia, Rhodococcus, Saccharomonospora, Saccharopolyspora and Streptomyces strains. Reliable procedures are needed for the isolation of members of most actinomycete genera: Actinomadura, Actinopolyspora, Kitasatospora, Pseudonocardia, Streptosporangium, Thermomonospora and Verrucosispora.

58. PROSPECTS FOR THE FUTURE Some new approaches: Plating onto low nutrient media – seawater nutrient media; acidic nutrient media. Isolation from unusual habitats (e.g. insects and plant tissues). Enrichment culture. In situ procedures. Cultivation using electron acceptors and substrate gradients. Zinder (2002) “The golden era of culturable microbiology lies ahead”

59.  The Creativity of Nature is Incomparable and Remains Underexploited

60. VISION FOR THE TWENTY-FIRST CENTURY “If I could do it all again, I would be a microbial ecologist. Ten billion bacteria live in a grain of ordinary soil. They represent thousands of species, almost none of which are known to science. Into that world I would go with the aid of a modern microscope and molecular analysis. I would cut my way through clonal forests sprawled across grains of sand, and track predators and prey in order to discover new life ways and alien food webs” Edward O. Wilson (1994) Naturalist

61. Number of microbes on Earth 5 x 1030 Number of stars in the Universe 7 x 1021 Number of microbes in all humans 6 x 1023 Number of microbial cells in one human gut 1014 Number of cells in one human 1013 Number of bacteriophage on Earth 1031 “The remarkable number of microbes urgently calls for a transition from random, anecdotal and small-scale surveys towards a systematic and comprehensive exploration of our planet”. “To explore and seek to understand how the Earth breaths, grows, evolves, renews and sustains life – all essentially the work of the microbial world – is the great adventure now beckoning to us”. Nikos C. Krypides Nature Biotechnology, July 2009