Successful Drug Discovery Informed by Actinobacterial Systematics

Verrucosispora HPLC-DAD analysis of culture filtrate Structures of Abyssomicins Biological activity T DAD1, 7.382 (196 mAU,Up2) of 002-0101.D V. maris AB-18-032 mAU CH3 CH3 T extract H3C H3C Antibacterial activity (MIC): S. leeuwenhoekii C34 maris AB-18-032 175 mAU DAD1 A, Sig=210,10 150 C DAD1 B, Sig=230,10 O O DAD1 C, Sig=260,20 125 7 7 500 Rt 7.4 min DAD1 D, Sig=280,20 O O O O Growth inhibition of Gram-positive DAD1 , Sig=310,20 100 Abyssomicins DAD1 F, Sig=360,40 C 75 DAD1 G, Sig=435,40 Staphylococcus aureus (MRSA) 4 µg/ml DAD1 H, Sig=500,40 50 400 O O 25 O O Staphylococcus aureus (iVRSA) 13 µg/ml 0 CH CH3 300 400 500 nm 3 DAD1, 7.446 (300 mAU,Dn1) of 002-0101.D 300 mAU Mode of action: C HO atrop-C HO 250 atrop-C CH3 CH3 CH3 CH3 200 H C H C H C  inhibitior of pABA biosynthesis

200 Rt 7.5 min H3C 3 3 3 Proximicin A Proximicin 150 HO O HO O O O O O O O O O A 100 O  covalent binding to Cys263 of PabB 100 N 50 O O HO O O Sea of Japan B O O N O O (4-amino-4-deoxychorismate synthase) by 0 CH CH3 CH3 CH3 3 300 400 500 nm HO HO HO HO Michael addition -289 m 0 B D G H 2 4 6 8 10 12 14 16 min

Newcastle Michael Goodfellow, School of Biology, University Newcastle University, Newcastle upon Tyne Atacama Desert In This Talk I will Consider:

• as a key group in the search for new therapeutic drugs.

• Taxonomic approaches to the selection of actinobacteria from extreme habitats.

• Atacama desert soil as a source of creative actinobacteria.

• Selection of novel actinobacteria for chemical screening.

• Conclusions and some new directions of travel.

Actinobacteria from Atacama Desert Need for a New Generation of

2008-2012

New antibacterial agents approved by the Food and Drug Administration (FDA) in the United States between 1983-2009. Adapted from Bassetti et al. (2011). 3 Twin Track Approach

Novel New Drug Target Screens Sample extracts Biological Material Dereplication Filters

H I T

Compound Evaluation & Characterization

L E A D Goodfellow & Fiedler (2010). Antonie van Leeuwenhoek 98:119-142. Actinobacteria

What are they? • A phylogenetically defined, metabolically active group of slow-growing, gram-positive bacteria which have DNA rich in guanine plus cytosine (> 55% mol. GC), unique 16S and 23S rRNA signatures and conserved indels in some proteins.

Why prioritize them for drug discovery? • Unravelled capacity to synthesize natural products with a wide spectrum of bioactivity. • Account for almost half of all microbial bioactive compounds; nearly 80% produced by strains. • Culture-independent studies show that <1% of actinobacterial taxa in natural habitats have been cultivated hence scope for the discovery of new chemical entities from cultivable novel taxa is enormous. • Diverse populations of novel actinobacteria present in extreme habitats are able to produce new specialised metabolites. Whole Genome Sequence of Streptomyces coelicolor Strain A3 (2)

Contains 27 natural product biosynthetic gene clusters Hopwood et al., 2002. Nature 417:141-147. Specialised Metabolites produced by Streptomyces coelicolor A3(2)

Actinorhodin Actinorhodin 1955 / Actinorhodin

1980 / Undecylprodigiosin

∆act ∆red

Prodiginine (cultivated in R2 for 5 days) Prodiginine

1976 / Methylenomycin 1978 / Calcium Dependent (SCP1 encoded) We do a poor job in interpreting a cell’s potential from its genomic sequence.

However, we are getting better. Prokaryotic Systematics is not a Luxury

Prokaryotic systematics is a core discipline practiced by few. However, the implementation of taxonomic concepts and practices underpins taxonomic approaches to drug discovery: • Well delineated natural classification. • Provision of a stable, universally accepted nomenclature. • Establishing cultivable actinobacterial diversity in natural habitats. • Improved procedures for selective isolation of novel and neglected taxa. • Rational choice of strains for bioprospecting and biotechnology. • Inferring the course of prokaryotic evolution. • Legislation (e.g. Hazard groups, patents of living biological material, Convention on Biological Diversity).

8 Polyphasic 1960-2014

Integrated use of genotypic and phenotypic data:

Chemotaxonomy Sound Classification Genomics Polyphasic Taxonomy = Stable Nomenclature Molecular Systematics Reliable Identification Numerical Taxonomy

Outcomes: Sound infrastructure for pure and applied microbiology and for designing and implementing scientific policies.

9 Hierarchic Classification of Actinobacteria based on Phylogeny

Phylum Actinobacteria

5 Classes

19 Orders Bergey’s Manual Trust is The next edition of a nonprofit organisation Bergey’s Manual will be that is supported by 50 Families an electronic one that will royalty income. be frequently updated.

220 Genera

3,000 Species

Goodfellow et al. 2012. Bergey’s Manual of Systematic Bacteriology, 2nd Edition, Volume 5, Springer, New York. Our Strategy

Extreme/Neglected habitats

Selective isolation

Selection of isolates

Dereplication technologies

Novel actinobacteria • Recent approaches to drug discovery (eg. combinatorial chemistry and fragment based drug design) have yet to make a significant Screening impact.

Dereplication of active compounds • Need to screen novel actinobacteria to avoid costly rediscovery of known bioactive New natural products compounds. Current bottleneck Commercial success

Culture-dependent bioprospecting strategy Why Focus on Actinobacterial Diversity from Extreme Habitats?

• Isolation of actinobacteria from extreme biomes rests on the premise that harsh environmental conditions give rise to unique taxa (species, genera) with novel chemistry.

• Evidence of a coupling between taxonomic and chemical diversity means that taxonomic diversity can be used as a surrogate for chemical diversity.

• Screening common actinobacteria from well studied habitats leads to the costly rediscovery of known bioactive compounds

Decrease in the number of new drugs coming onto the market

http://smellslikescience.com/a-need-for-new-antibiotics/ Actinobacteria for Low Throughput Screens

Objectives: Focus on rare and previously unknown taxa, including novel species of Streptomyces.

Key taxonomic methods designed to meet these aims:

• Choice of selective isolation procedures.

• Selection of cultivation media.

• Recognition of novel taxa by comparative sequencing of conserved genes / proteins, notably 16S rRNA genes.

Screening strategies:

• Cell based targets- plug assays using panel of pathogenic strains.

• Reporter Bacillus subtilis strains to detect cellular targets.

• Choice of production media.

• Chemical analysis of extracts for novel specialised metabolites.

Discovery dependent on dereplication at all stages of the procedure. Atacama Desert

The Atacama Desert located in northern Chile is the oldest and driest desert on the planet. ALMA Lomas Byas Most Atacama Desert soils and regoliths are either hyper- arid (ratio of mean annual rainfall to mean annual evaporation is < 0.05) , or extreme hyper-arid (corresponding ratio < 0.002).

Yungay Low concentrations of organic matter, high salinity, the

presence of inorganic oxidants and high UV radiation Salar de conditions. Atacama

The underlining premise is that these extreme conditions will have given rise to a unique actinobacterial diversity and consequential chemical diversity. Sampling Sites

Salar de Atacama Yungay Hyper-arid area Extreme hyper-arid area

ALMA Lomas Bayas High altitudes, Very high UV radiation Most extreme hyper arid area Selective Isolation of Actinobacteria

Media Selective agents Target organism (s) References

Glucose-yeast extract Rifampicin (20µg ml-1) spp. Athalye et al. (1981) agar Gause’s No.1 agar Nalidixic acid (10µg ml-1) Rare or uncommon Zakharova et al. (2003) actinomycetes Humic acid-vitamin agar Humic acid (1g/L) Streptosporangiaceae Hayakawa & Nonomura spp. (1984)

Oligotrophic agar Low carbon and nitrogen Rare actinomycetes Busarakam (2013) sources

Raffinose-histidine agar Common Streptomyces spp. do Rare or uncommon Vickers et al. (1984) not grow well in the presence streptomycetes of these nutrients Starch-casein agar High carbon to nitrogen ratio Streptomyces spp. Küster and Williams (1964)

SM1and SM2* agars Neomycin (4 µg ml-1), D(+) spp. Tan et al. (2006) sorbitol (1%, w/v)*

Cycloheximide and nystatin (each at 25 µg ml-1) used to control fungi. 16 Selective Isolation Range of selective isolation media

Sprinkle plates Dilution plates Isolation of Streptomyces Strains Isolation of Amycolatopsis Strains

Oligotrophic agar

Humic-acid-vitamin-agar SM 1 agar

Starch-casein vitamin agar Isolation of Modestobacter Strains Arginine-vitamin agar Humic-acid-vitamin agar

R2A agar

Oligotrophic agar Starch-casein-vitamin agar Actinobacteria (cfu/g dry weight soil) growing on Selective Media inoculated with Suspensions of Atacama Desert Soils and Incubated at 28oC for 3 Weeks

Media Soil Gause’s No.1 Humic acid Oligotrophic Starch-casein SM1 agar agar agar agar

Salar de 1.1x104 1.3x104 0.3x102 0.1x102 0.3x102 Atacama

Yungay 3x102 5x103 3.3x102 2.3x102 4.7x102

Actinobacteria were not isolated on glucose-yeast extract or raffinose-histidine agars. Dereplication

Term used for differentiating phenotypically similar isolates (and metabolites) to select representatives to facilitate efficient screening and to minimize costs and time in maintaining large culture collections.

• Choice of methods: Chemical: fatty acid analyses, pyrolysis and MALDI-TOF mass spectrometry. Molecular: rep-PCR, specific primers, multilocus sequence analysis and 16S rRNA and whole-genome sequencing. Morphology: use of predictive colonial features (colour-grouping).

• Outcome: Significant increase in hits. Colour-grouping

Production of melanin pigments

Antony-Babu et al. (2010). Antonie van Leeuwenhoek 97, 231-239. Colour-grouping of Streptomyces isolates from Salar de Atacama and Yungay Soils

Growth on oatmeal agar Strain numbers and source of isolates

Colour of Salar de Atacama Yungay Colour group Aerial spore Substrate diffusible code mass pigment mycelial colour pigment Code Code KNN11 yellowish white yellowish white none G18-1 (63a), G18-2 (74a), - G18-3 (149c), G18-4 (155b), G52 (62a) nd G91(a) KNN23 yellowish white deep orange yellow gray yellow none ASC2b, KB111b

KNN48 yellowish white light orange yellow light orange yellow G14-1 (57c), G14-2 (65a), KB1d, KB2d, KB8d, G14-3 (257e), G14-4 KB13d, (258e), G14-5 (259e) and G14-6 (260e)  None of the isolates produced melanin pigments on peptone-yeast extract-iron agar.

 174 out of 224 representatives of the genus Streptomyces isolated from the hyper-arid Salar de Atacama soil (71%) were assigned to 36 multi- and 11 single-membered colour- groups (82.3% of isolates).

 58 out of 105 representatives of the genus Streptomyces isolated from Yungay soil (55.2%) were assigned to 8 multi- and 5 single-membered colour-groups (22.8% of isolates).

 The remaining isolates was assigned to colour-groups containing isolates from both locations. Colour-grouping of Isolates from ALMA soils

Assignment of isolates from ALMA soil samples to colour-groups based on growth on oatmeal and peptone-yeast extract-iron agar plates incubated at 28oC for 14 days.

Aerial spore mass colour Substrate mycelial colour Diffusible pigment Melanin production Strains

Yellow white Dark yellow brown - - H9, H39 Multi-membered colour-group Yellow white Pale orange yellow - - H45

Single-membered colour-group

•113 representative strains isolated from the ALMA sites were assigned to 9 multi- and 57 single-membered colour-groups.

Isolate H9 Isolate H45 ALMA subsurface soil sample 4 at 4000 m above sea level. ALMA subsurface soil sample 6 at 5000 m above sea level. Colour-grouping of Isolates from Lomas Bayas soil

Assignment of isolates from the Lomas Bayas soil sample to colour-groups based on growth on oatmeal and peptone-yeast extract-iron agar plates incubated at 28oC for 14 days.

Oatmeal agar PYEIA Strains Aerial spore mass colour Substrate mycelial colour Diffusible pigment Melanin production Dark gray Gray orange Pale green yellow - LB55, LB60 Multi-membered colour-group White Dark purple gray & Black Blackish purple - LB63

Single-membered colour-group

•52 representative strains isolated from Lomas Bayas soil were assigned to 9 multi- and 26 single-membered colour-groups.

LB55 isolated from Lomas Bayas LB63 isolated from Lomas Bayas Agar Plug Assays

Starch-casein isolation plate

Isolate C38 Zones of Inhibition (mm in diameter) produced by Representative Novel Streptomyces Strains isolated from ALMA and Salar de Atacama Soils

Isolates Bacillus Escherichia Pseudomonas Staphylococcus Saccharomyces subtilis coli fluorescens aureus cereviseae ALMA H9 16 12 18 - 18 H45 12 14 15 - 22 H71 13 11 19 12 32 Salar de Atacama

KNN26 18 17 12 24 22 KNN35-1 24 20 20 28 25 KNN90 22 16 22 25 24

 113 representatives isolated from ALMA soils were screened; 6 showed activity against all 5 pathogens and 77 (68%) activity against at least one of the pathogenic strains.

 121 representatives of the Salar de Atacama colour-groups were screened and 72 (60%) showed activity against at least one of the pathogenic strains. Zones of Inhibition (mm in diameter) produced by Representative Novel Streptomyces strains isolated from Hyper-Arid Salar de Atacama Soil against Bacillus subtilis Reporter Strains

Isolates Cell envelope DNA Fatty acid RNA synthesis synthesis synthesis synthesis synthesis G9-5 13 10 15 14 17

G10-1 9 8 10 10 10

G10-5 18 12 15 16 15

G16-1 7 2 10 8 8

G36-1 13 11 15 14 12

G64 10 9 19 12 10

This approach is based on cells that carry -galactosidase genes bound to promotors that specifically respond to certain types of antibiotics (Fischer et al. 2004, Genome Research 14:90-98). Novel Actinomadura isolates from the hyper-arid Salar de Atacama soil

Actinomadura rifamycini IFO 14183T(U49003) 0.002 * Isolate G4-15 (Cell wall) 100 MEIsolate BA8 (Cell wall)

* 99 Isolate B45 (Cell wall)

99 Isolate B68 (Cell wall)

Actinomadura apis IM17-1T(AB557596)

Actinomadura cremea JCM 3308T(AF134067)

ME Actinomadura yumaensis JCM 3369T (AF163122)

50 Actinomadura livida JCM 3387T(AF163116) ME 100 Actinomadura sputi IMMIB L-889T (FM957483) ME 53 Actinomadura vinacea JCM 3325T(AF134070) * Actinomadura rugatobispora IFO14382T(U49010) 85

30 Novel Nonomuraea isolates from the hyper-arid Salar de Atacama soil

0.002 N. candida HMC10T(DQ285421) * 45 N.rubra DSM 43768T(AF277200) ME 72 N. turkmeniaca DSM 43926T(AF277201) * 60 N. antimicrobica YIM 61105T(FJ157184)

69Isolate BA6 (Cell wall & Cell envelope)

ME Isolate B37 (Cell wall & Cell envelope) 75 T * N. salmonea DSM 43678 (X97892) 64 N. endophytica YIM 65601T(GU367158)

N. maritima FXJ7.203T(GU002054)

31 Novel Streptomyces isolated from the hyper-arid Salar de Atacama soil

S. flavidoviresns subclade 100 95 S. michigannensis subclade Isolate C38 88 99 S. thioluteus subclade 94 97 S. albus subclade 95 S. macrosporus subclade 68 KNN89 (KB208 edit) 98 83 C34 96 S. nanhaiensis subclade 93 S. radiopplugnans subclade 51 Bar 1 µ S. somaliensis DSM 40738T (AJ007403) S. griseosporeus NBRC 13458T (AJ781333) T S. glomeratus LMG 19903T (AJ781754) S. lannensis TA4-8 74 T S. chiangmaiensis suclade S. chiangmaiensis TA4-1 T S. lannensis TA 4-8T (AB562508) S. chromofuscus NBRC 12851 S. griseocarnatus subclade T 83 S. speibonae PK-Blue T 0.002 KNN2-4 (G11-4 edit) S. griseoincarnatus LMG 19316 Streptomyces new subclade 100S. erythrogriseus LMG 19406T 89 C38 63 T S. speibonae PK-BlueT (AF452714) S. albogriseolus NRRL B-1305 T S. parvulus subclade 100 S. viridodiastaticus NBRC 13103 S. albogriseolus subclade S. longispororuber NBRC 13488T S. lusitanus subclade 0.005 S. thermocarboxydus DSM 44293T 50 S. atrovirens NRRL B-16357T DQ26672 86 S. lusitanus NBRC 13484T S. glaucus subclade T 89 S. coeruleorubidus NBRC 12844 S. aurantiogriseus subclade 100 S. coerulescens ISP 5146T 58 S. djkartensis NBRC 15409T AB184657 S. spinoverrucosus NBRC 14228T S. carpinensis NBRC 12414T AB184574 S. lomondensis NBRC 15426T C58 S. viridiviolaceus subclade S. parvulus NBRC 13193T S. caecalis subclade S. hyderabadensis OU-40T KNN42 (AT17 edit) S. leeuwenhoekii C34T KNN35-1 (G34-1 edit2) 94 S. somaliensis DSM 40738T 90 KNN35-2 (G34-2 edit2) T S. braxilliensis subclade S. griseosporeus NBRC 13458 52 S. glomeratus LMG 19903T 99 KNN64-5 (G8-5 edit) T KNN38-1 (G7-1 edit) S. mexicanus CH-M-1035 T C59 S. albus subsp. albus NRRL B-2365 96 KNN26 (G79 edit) 83 KNN13 (AT5 edit) C79 S. fimbriatus NBRC 15411T (AB184659 Phylogenetic tree inferred from concatenated partial sequences of the house-keeping genes atpD, gyrB, recA, rpoB and trpB in MEGA 5.2 (Tamura et al., 2011) using the maximum-likelihood method based on the General Time Reversible model (Nei & Kumar, 2000). Streptomyces sp. C38

Streptomyces sp. C79 Streptomyces leeuwenhoekii 16S rRNA gene clade

Isolate KNN41 0.0002 61Isolate KMM11-1 Isolate KNN11-5 Putative new species Isolate KNN9-3 Isolate KNN6-6 Isolate KNN6-9

60Isolate C38 (atacamycins) Isolate C58 (novel cyclic lasso peptides) Isolate C79 (four new specialised metabolites) Isolate KNN10-5 Isolate KNN25 S. leeuwenhoekii Isolate KNN10-4 Isolate KNN48-1 Isolate KNN24-1 Streptomyces sp. C59 Isolate KNN2-6 Isolate C34 (chaxamycins) Members of Amycolatopsis methanolica 16S rRNA gene clade isolated from hyper-arid Salar de Atacama soil KNN50-11 66*KNN50-16 Selective media KNN50-9 KNN61-1 Starch-casein vitamin agar A. thermalba SF45T (HQ668525) KNN50-17 KNN50-13 99* KNN50-10

0.005 KNN50-4 KNN50-2 KNN50-1 *53 KNN49-12 KNN49-11 KNN49-6 SM 1 agar A. granulosa GY307T (AF466101) * 87* A. viridis GY115T (AF466095) A. thermoflava N1165T (AF052390) A. methanolica 239T (AQUL01000001) * * A. eurytherma NT 202T (AJ000285) 100* A. tucumanensis ABOT (DQ886938) A. thermophila GY088T (AY129774) A. endophytica KLBMP 1221T (HM153799) A ruanii NGM112T HQ668524 KNN50-18 Oligotrophic agar KNN50-14 KNN50-6 KNN49-3 * KNN49-5 100 KNN49-26 *99 KNN50-7 KNN50-8 KNN49-10 KNN49-1 KNN50-5 Humic-acid-vitamin-agar KNN50-13 KNN50-15 GY024 100* GY142 A. orientalis subsp. orientalis IMSNU 20058T (AJ400711) riparia NRRL B-16432T (AF 114802) Novel Blastococcus, Geodermatophilus and Modestobacter isolates from the extreme hyper arid Yungay soil

Selective media

Arginine-vitamin agar Humic-acid-vitamin agar Oligotrophic agar R2A agar Starch casein agar

79 Modestobacter colour-group 46, 12 isolates *78 M. versicolor CP153-2T (AJ871304) T *58 M. marinus 42H12-1 (EU181225) 76* M. roseus KLBMP 1279T ( JQ819258) *84 0.005 *99 Modestobacter colour-group 45, 4 isolates 87 * M. multiseptatus AA-826T (Y18646) ML B. jejuensis KST3-10T (DQ200983) 86 B. endophyticus YIM 68236T (GQ494034) B. aggregates ATCC 25902T (L40614) 54 Isolate KNN47 *99 B. saxobsidens BC448T (AJ316571) G. terrae PB261T (JN033773) 100* G. solani PB34T (JN033772) 99* G. taihuensis 3-wff-81T (JX294478) * G. normandii CF5/3T (HE654546) 92 G. telluris CF9/1/1T (HE815469) 94* * G. saharensis CF5/5T (HE654551) 98 G. arenarius CF5/4T (HE654547) * T * 87 G. nigrescens YIM 75980 (JN656711) 77 G. ruber CPCC 201356T (EU438905) ISP1 ISP2 ISP3 ISP4 G. obscurus DSM 43160T (CP001867) *95 92* G. siccatus CF6/1T (HE654548) 51* Isolate KNN44-3 * 100 Isolate KNN44-1 ISP5 ISP6 ISP7 79* Isolate KNN44-4 Novel Micromonospora isolates from the extreme hyper-arid Lomas Bayas soil

Isolate LB-1 GZ-4 * * 91 Isolate LB-1 IS-10 58 0.002 T 95* Micromonospora saelicesensis Lupac 09 (AJ783993) * Isolate LB-1 R2-3 58 * * Isolate LB-1 SC-4 * 81 95 Micromonospora chokoriensis 2-19/6T(AB241454) * 91 Micromonospora lupini lupac 14NT(AJ783996) 86 Micromonospora mirobrigensis WA201T(AJ626950) Micromonospora purpureochromogenes DSM 43821T(X92611) Micromonospora aurantiaca ATCC 27029T(CP002162) Micromonospora marina JSM1-1T(AB196712) Micromonospora humi P0402T(GU459068) Micromonospora tulbaghiae TVU1T(EU196562) * Isolate LB-1 SC-7 99* 64 Micromonospora chalcea DSM 43026T(X92594)

http://www0.nih.go.jp/saj/DigitalAtlas2/ Actinobacterial Genera Isolated from the Sampling Sites

Salar de Atacama ALMA Yungay Lomas Bayas (Hyper-arid soil) (Hyper-arid soil) (Extreme hyper-arid (Most extreme soil) hyper-arid soil) *Actinomadura *Actinomadura *Blastococcus *Actinocorralia + Amycolatopsis +Amycolatopsis *Couchioplanes *Micromonospora *Kribbella * *Geodermatophilus +Streptomyces *Lechevalieria * *Kribbella *Nocardiopsis * *Modestobacter *Nonomuraea +Streptomyces *Pseudonocardia *Saccharothrix +Streptomyces +Streptomyces

*Novel members of rare and +established genera.

Members of 17 genera isolated to date. Dereplication of Isolates and Preparation of Extracts for Chemical Screening

 Representatives of colony types taken from isolation plates. Many

 Rapid assignment of isolates to colour-groups.

 Selection of isolates showing bioactivity in plug assays. Fewer  Choice of creative isolates that form distinct 16S rRNA gene lineages.

 Growth of selected isolates on production media.

 Preparation of extracts for chemical screening. Few Media for Submerged Culture of Selected Novel Isolates  Medium 19: enhances production of novel drug leads from Streptomyces species.

 Medium 410: promotes optimal growth and production of drug candidates from filamentous actinobacterial genera.

 Starch-casein broth: supports growth of most streptomycetes.

 Yeast extract-malt extract broth (ISP medium 2): promotes production of novel drug leads from filamentous actinobacteria.

Choice of production media for the expression of specialised metabolites is heavily dependent on assignment of isolates to the correct taxa. RT: 0.00 - 30.00 6.67 NL: 190000 1.92E5 LCMS analysis ofTotal Scanmetabolite extract from 180000 PDA MR289_KN N64_5_ISP 170000 Streptomyces isolate KNN2 64-5 after shaking in ISP2 160000 150000 broth for 14 days at 28oC. 140000

130000

0.50 MR289_KNN64_5_ISP2 #364 RT: 12.10 AV: 1 NL: 1.23E5 microAU 227.00 120000 100

95 336.00 110000 90 85 352.00 80 UV trace 100000 75

70

uAU 90000 MS trace 65 60 80000 55 50

45

70000 RelativeAbsorbance 40

35 60000 30 25

20 50000 7.07 15 10 40000 9.57 5 6.23 0 200 220 240 260 280 300 320 340 360 380 400 5.47 wavelength (nm) 30000 10.30 4.53 12.10 20000 2.97 12.30 26.00 2.07 22.00 23.90 25.73 10000 14.00 17.53 26.43 29.77 0 0 5 10 15 20 25 30 Time (min)

MR289_KNN64_5_ISP2 #288 RT: 9.57 AV: 1 NL: 1.64E5 microAU MR289_KNN64_5_ISP2 #310 RT: 10.30 AV: 1 NL: 1.36E5 microAU 227.00 MR289_KNN64_5_ISP2 #248 RT: 8.23 AV: 1 NL: 2.07E5 microAU 228.00 100 100 226.00 100 95 95 95 90 90 90 85 85 85 80 354.00 80 80 75 75 75 70 70 284.00 70 65 65 65 60 UV trace 60 UV trace UV trace 60 55 55 55 50 50 339.00 50 45

45 RelativeAbsorbance 45 40

279.00 RelativeAbsorbance

RelativeAbsorbance 40 40 35 35 35 30 30 30 25 25 25 20 265.00 394.00 20 20 15 336.00

15 15 10 393.00 10 10 5

5 5 0 394.00 200 220 240 260 280 300 320 340 360 380 400 420 0 0 wavelength (nm) 200 220 240 260 280 300 320 340 360 380 400 200 220 240 260 280 300 320 340 360 380 400 wavelength (nm) wavelength (nm)

Strain KNN64-5 produces 5-6 derivatives of novel aromatic or polyene compounds. The strain was isolated from the Salar de Atacama soil and forms a distinct branch in the S. fimbriatus 16S rRNA gene clade. LCMS analysis of metabolite extract from Amycolatopsis ruanii KNN50-8 after shaking in medium 19 broth for 14 days at 28oC.

Major new metabolite

UV spectrum of the major metabolite

Strain KNN50-8 produces major and minor specialised novel metabolites. The strain was isolated from the hyper-arid Salar de Atacama. Primary Screening of ALMA and Lomas Bayas Isolates against Panel of Pathogens

Percentage of the 132 actinbacteria that showed antimicrobial activity against pathogens

Pathogen B. subtilis E. coli P. fluorescens S. aureus S. cerevisiae ALMA 52.2 11.5 21.2 30 60

Lomas Bayas 18.8 0 4.7 23.4 15.6

• The 16 isolates that showed activity against the Escherichia coli strain were selected for further study. LCMS analysis of metabolite extract from Streptomyces isolate H9 after shaking in starch-casein broth for 14 days at 28oC

• The strain was isolated from ALMA site 4 (4000 m surface soil) on Gause’s No.1 broth after 14 days at 28oC

• Streptomyces isolate H9 produces novel specialised metabolites belonging to diverse chemical scaffolds. LCMS analysis of metabolite extract from Lechevalieria isolate H45 after shaking in medium 410 broth for 14 days at 28oC

• The strain was isolated from ALMA site 6 (5000 m subsurface soil) on Guase’s No.1 agar after 14 days at 28oC

• Lechevalieria isolate H45 produces specialised metabolites belonging to diverse novel chemical scaffolds. Phylogenetic Analyses

Neighbour–joining tree based on almost complete 16S rRNA gene sequences showing relationships between (A) Streptomyces isolate H9 and closely related Streptomyces type strains and (B) Lechevalieria isolate 45 and the type strains of its nearest neighbours. Asterisks (*) indicate branches of the tree that were recovered with the maximum-likelihood and maximum-parsimony tree-making algorithms.

A

B GENOMICS

Released Genomes in National Center for Biotechnology Information (NCBI)

8000

7000 >7,000 genomes In 2013 6000

5000

4000

3000 NGS 2000

1000

0

(1995-2014) 15,842 genomes (as of January 2014)

JONGSIK CHUN LAB . SEOUL NATIONAL UNIV . 46 Genomics for Systematics: The Present Status

Species with valid Species with 16S rRNA Species with genome Species with genome names gene sequence (type sequence sequence (type strain) strain)

Bacteria 11,275 10,887 2,951 2,085

Archaea 429 423 179 165

Total 11,704 11,310 3,130 2,250 ()

Percent 100% 96.6% 26.7% 19.2%

We need a strategy for describing all of the prokaryotes (~ 1 million species), not just the small fraction that have been cultivated.

JONGSIK CHUN LAB . SEOUL NATIONAL UNIV . 47 Preliminary Analysis of Whole Genome Sequence Data Modestobacter Isolate ASC16 Streptomyces leeuwenhoekii C34T

Habitat: isolated from the extreme hyper-arid Yungay soil Habitat: isolated from hyper-arid Salar de Atacama soil

Genomic size: ~ 5.57 Mb Genomic size: ~ 8.66 Mb

G+C content: 74.1% G+C content: 72.1%

Protein coding genes: 5,445 Protein coding genes: ~7,800

Secondary metabolite profile : few predicted biosynthetic gene Secondary metabolic profile: 31 predicted biosynthetic gene clusters clusters including one for chaxamycins

Stress genes: resistance to UV radiation and desiccation Stress genes: present

Spore forming genes: present Spore forming genes: present Culture Independent Studies based on Pyrosequencing

Primary analysis suggests that there are at least 97 different genera in the soil samples, as exemplified below:

Family Genera Actinoplanaceae Couchioplanes Cellulomonadaceae Actinotalea, Cellulomonas, Oerskovia Cryptosporangium Dietziaceae Dietzia Geodermatophilaceae Blastococcus, Geodermatophilus, Modestobacter Glycomycetaceae Glycomyces Microbacteriaceae Agrococcus, Agromyces, Microbacterium, Rathayibacter Micrococcaceae Micrococcus Micromonosporaceae Actinoplanes, Catellatospora, Microbiospora, Micromonospora, Pilimelia, Virgisporangium Nocardia, Nocardioidaceae Aeromicrobium, Friedmanniella, Kribbella, Nocardioides, Pimelobacter Nocardiopsaceae Nocardiopsis , Amycolatopsis, Lechevalieria, , Pseudonocardia, Saccharothrix Streptomycetaceae Streptomyces Streptosporangiaceae Acrocarpospora, Nonomuraea Actinoallomurus, Actinocorallia, Actinomadura

* Genera isolated classified using culture dependent approaches are marked in bold Key Conclusions

• Atacama Desert soils contain novel filamentous actinobacteria with the ability to synthesise new specialised metabolites.

• Natural product hits enhanced by dereplication of isolates and extracts.

• Distribution of actinobacterial taxa provide insights into their taxonomic roles.

• Further support for the taxonomic approach to drug discovery. HENCE NEED FOR HIGHLY TRAINED MICROBIAL SYSTEMATISTS. Some New Directions of Travel • Community DNA extracted from different habitats to determine extent of actinobacterial diversity in Atacama Desert soils.

• Innovative and rapid selective isolation methods needed to isolate and recognise rare and neglected taxa present in environmental samples.

• Improved methods required for the expression of natural product gene clusters.

• Structural analyses of interesting bioactive compounds.

• Polyphasic taxonomy of novel creative isolates.

• Establish the extent of coupling between taxonomic and chemical diversity.

• Focus on creative novel strains for systems and synthetic biology.

• Search for adaptive genes and drivers of actinobacterial speciation in Atacama Desert soils. Acknowledgements

 Juan Asenjo and Barbara Andrews, University of Chile, Santiago, Chile.

 Luis Cáceres, University of Antofagasta, Chile.

Jungsik Chun, Seoul National University, Korea.

Genevieve Girard and Gilles van Wezel, Leiden University, The Netherlands.

 David Labeda, USDA-ARS, Peoria, USA.

 Marcel Jaspars, Aberdeen University, Scotland.

 Barny Whitman, University of Georgia, USA.

 Ros Brown, Kanungnid Busarakam and Hamidah Idris, Newcastle University, England.

And for support from the Leverhulme Trust and The Royal Society. Thank You