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Successful Drug Discovery Informed by
Actinobacterial Systematics
Michael Goodfellow, School of Biology,
Newcastle University, Newcastle upon Tyne
Newcastle
University
Abyssomicins
Structures of Abyssomicins Biological activityHPLC-DAD analysis of culture filtrateextract Antibacterial activity (MIC):
Growth inhibition of Gram-positive bacteria
Staphylococcus aureus (MRSA) 4 µg/ml
Staphylococcus aureus (iVRSA) 13 µg/ml
Mode of action:
inhibitior of pABA biosynthesis
covalent binding to Cys263 of PabB
(4-amino-4-deoxychorismate synthase) by
Michael addition D
C
B
CH3H3C
O
OO
HO
CH3
O7
O
CH3H3C
O
OO
HO
CH3
O7
O
CH3H3C
O
OO
HO
CH3
OO
N
HO
CH3H3C
OO
HO
CH3
OO
HO
CH3H3C
O
OO
HO
CH3
OO
NHO
CH3H3C
O
OO
HO
CH3
OO
G H
atrop-C
min2 4 6 8 10 12 14 16
mAU
0
100
200
300
400
500
C
AB
DAD1 G, Sig=435,40 DAD1 F, Sig=360,40
DAD1 C, Sig=260,20
DAD1 A, Sig=210,10
DAD1 D, Sig=280,20 DAD1 , Sig=310,20
DAD1 H, Sig=500,40
DAD1 B, Sig=230,10
Pro
xim
icin
A
nm300 400 500
mAU
0
50
100
150
200
250
DAD1, 7.446 (300 mAU,Dn1) of 002-0101.D
Rt 7.5 min
atrop-C
nm300 400 500
mAU
0
25
50
75
100
125
150
175
DAD1, 7.382 (196 mAU,Up2) of 002-0101.D
CRt 7.4 min
Verrucosispora
maris AB-18-032
Sea of Japan
-289 m
V. maris AB-18-032T
S. leeuwenhoekii C34T
Atacama Desert
Actinobacteria from Atacama Desert
In This Talk I will Consider:
• Actinobacteria 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.
3
Need for a New Generation of Antibiotics
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).
Drug Target Sample extracts
Dereplication Filters
H I T
Compound Evaluation &
Characterization
New
Biological
Material
Novel
Screens
L E A D
Twin Track Approach
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 Streptomyces
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 clustersHopwood et al., 2002. Nature 417:141-147.
Specialised Metabolites produced by Streptomyces coelicolor A3(2)
(cultivated in R2 for 5 days)
Actinorhodin
∆act ∆red
Prodiginine
Actinorhodin
Prodiginine
1955 / Actinorhodin
1980 / Undecylprodigiosin
1978 / Calcium Dependent Antibiotic1976 / Methylenomycin(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
8
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).
Integrated use of genotypic and phenotypic data:
Chemotaxonomy
Genomics
Molecular Systematics
Numerical Taxonomy
Outcomes:
Sound infrastructure for pure and applied microbiology and for designing and implementing
scientific policies.
Polyphasic Taxonomy
1960-2014
9
Polyphasic Taxonomy =Sound Classification
Stable Nomenclature
Reliable Identification
Phylum Actinobacteria
5 Classes
19 Orders
50 Families
220 Genera
3,000 Species
Hierarchic Classification of Actinobacteria based on Phylogeny
Goodfellow et al. 2012. Bergey’s Manual of Systematic Bacteriology, 2nd Edition, Volume 5, Springer, New York.
Bergey’s Manual Trust is
a nonprofit organisation
that is supported by
royalty income.
The next edition of
Bergey’s Manual will be
an electronic one that will
be frequently updated.
Our Strategy
Extreme/Neglected habitats
Selective isolation
Selection of isolates
Dereplication technologies
Novel actinobacteria
Commercial success
Screening
Dereplication of active compounds
New natural products
Culture-dependent bioprospecting strategy
Current bottleneck
• Recent approaches to drug discovery (eg.
combinatorial chemistry and fragment based
drug design) have yet to make a significant
impact.
• Need to screen novel actinobacteria to avoid
costly rediscovery of known bioactive
compounds.
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.
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).
Low concentrations of organic matter, high salinity, the
presence of inorganic oxidants and high UV radiation
conditions.
The underlining premise is that these extreme conditions
will have given rise to a unique actinobacterial diversity and
consequential chemical diversity.
ALMALomas Byas
Yungay
Salar de
Atacama
Sampling Sites
Salar de Atacama
Hyper-arid area
YungayExtreme hyper-arid area
ALMA
High altitudes, Very high UV radiation
Lomas Bayas
Most extreme hyper arid area
Selective Isolation of Actinobacteria
16
Media Selective agents Target organism (s) References
Glucose-yeast extract
agar
Rifampicin (20µg ml-1) Actinomadura spp. Athalye et al. (1981)
Gause’s No.1 agar Nalidixic acid (10µg ml-1) Rare or uncommon
actinomycetes
Zakharova et al. (2003)
Humic acid-vitamin agar Humic acid (1g/L) Streptosporangiaceae
spp.
Hayakawa & Nonomura
(1984)
Oligotrophic agar Low carbon and nitrogen
sources
Rare actinomycetes Busarakam (2013)
Raffinose-histidine agar Common Streptomyces spp. do
not grow well in the presence
of these nutrients
Rare or uncommon
streptomycetes
Vickers et al. (1984)
Starch-casein agar High carbon to nitrogen ratio Streptomyces spp. Küster and Williams (1964)
SM1and SM2* agars Neomycin (4 µg ml-1), D(+)
sorbitol (1%, w/v)*
Amycolatopsis spp. Tan et al. (2006)
Cycloheximide and nystatin (each at 25 µg ml-1) used to control fungi.
Selective Isolation
Range of selective isolation media
Sprinkle plates Dilution plates
Isolation of Streptomyces Strains
Isolation of Amycolatopsis Strains
SM 1 agar
Oligotrophic agar
Starch-casein vitamin agar
Humic-acid-vitamin-agar
Isolation of Modestobacter StrainsArginine-vitamin agar
Starch-casein-vitamin agar
Humic-acid-vitamin agar
Oligotrophic agar
R2A 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
Soil
Media
Gause’s No.1 Humic acid agar
Oligotrophicagar
Starch-casein agar
SM1 agar
Salar de Atacama
1.1x104 1.3x104 0.3x102 0.1x102 0.3x102
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, genus 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 group
code
Aerial spore
mass pigment
Substrate
mycelial colour
Colour of
diffusible
pigment
Salar de Atacama Yungay
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),
G14-3 (257e), G14-4
(258e), G14-5 (259e) and
G14-6 (260e)
KB1d, KB2d, KB8d,
KB13d,
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
Aerial spore mass colour Substrate mycelial colour Diffusible pigment Melanin production Strains
Yellow white Dark yellow brown - - H9, H39
Yellow white Pale orange yellow - - H45
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.
Multi-membered colour-group
Single-membered colour-group
Isolate H9
ALMA subsurface soil sample 4 at 4000 m above sea level.
Isolate H45
ALMA subsurface soil sample 6 at 5000 m above sea level.
•113 representative strains isolated from the ALMA sites were assigned to 9 multi- and 57
single-membered colour-groups.
Oatmeal agar PYEIA Strains
Aerial spore mass colour Substrate mycelial colour Diffusible pigment Melanin
production
Dark gray Gray orange Pale green yellow - LB55, LB60
White Dark purple gray & Black Blackish purple - LB63
LB55 isolated from Lomas Bayas LB63 isolated from Lomas Bayas
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.
Multi-membered colour-group
Single-membered colour-group
•52 representative strains isolated from Lomas Bayas soil were assigned to 9 multi- and 26
single-membered colour-groups.
Agar Plug Assays
Isolate C38
Starch-casein isolation plate
Zones of Inhibition (mm in diameter) produced by Representative Novel Streptomyces Strains isolated from ALMA and Salar de Atacama Soils
Isolates Bacillus
subtilis
Escherichia
coli
Pseudomonas
fluorescens
Staphylococcus
aureus
Saccharomyces
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
synthesis
Cell wall
synthesis
DNA
synthesis
Fatty acid
synthesis
RNA
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).
Actinomadura rifamycini IFO 14183T(U49003)
Isolate G4-15 (Cell wall)
Isolate BA8 (Cell wall)
Isolate B45 (Cell wall)
Isolate B68 (Cell wall)
Actinomadura apis IM17-1T(AB557596)
Actinomadura cremea JCM 3308T(AF134067)
Actinomadura yumaensis JCM 3369T (AF163122)
Actinomadura livida JCM 3387T(AF163116)
Actinomadura sputi IMMIB L-889T (FM957483)
Actinomadura vinacea JCM 3325T(AF134070)
Actinomadura rugatobispora IFO14382T(U49010)85
53
ME
100
*
*
99
0.002
*
99
100ME
ME
ME50
Novel Actinomadura isolates from the hyper-arid
Salar de Atacama soil
30
N. candida HMC10T(DQ285421)
N.rubra DSM 43768T(AF277200)
N. turkmeniaca DSM 43926T(AF277201)
N. antimicrobica YIM 61105T(FJ157184)
Isolate BA6 (Cell wall & Cell envelope)
Isolate B37 (Cell wall & Cell envelope)
N. salmonea DSM 43678T(X97892)
N. endophytica YIM 65601T(GU367158)
N. maritima FXJ7.203T(GU002054)
60
69
ME
64
ME
*
0.002
45
*
*
75
72
31
Novel Nonomuraea isolates from the hyper-arid
Salar de Atacama soil
S. flavidoviresns subclade
S. michigannensis subclade
S. thioluteus subclade
S. albus subclade
S. macrosporus subclade
KNN89 (KB208 edit)
S. nanhaiensis subclade
S. radiopplugnans subclade
S. somaliensis DSM 40738T (AJ007403)
S. griseosporeus NBRC 13458T (AJ781333)
S. glomeratus LMG 19903T (AJ781754)
S. chiangmaiensis suclade
S. lannensis TA 4-8T (AB562508)
S. griseocarnatus subclade
KNN2-4 (G11-4 edit)
Streptomyces new subclade
S. speibonae PK-BlueT (AF452714)
S. parvulus subclade
S. albogriseolus subclade
S. lusitanus subclade
S. atrovirens NRRL B-16357T DQ26672
S. glaucus subclade
S. aurantiogriseus subclade
S. djkartensis NBRC 15409T AB184657
S. carpinensis NBRC 12414T AB184574
S. viridiviolaceus subclade
S. caecalis subclade
KNN42 (AT17 edit)
KNN35-1 (G34-1 edit2)
KNN35-2 (G34-2 edit2)
S. braxilliensis subclade
KNN64-5 (G8-5 edit)
KNN38-1 (G7-1 edit)
C59
KNN26 (G79 edit)
KNN13 (AT5 edit)
S. fimbriatus NBRC 15411T (AB184659
99
95100
83
5193
96
68
98
99
94
95
88
97
83
96
52
90
94
58
89
83
50
89
0.002
Novel Streptomyces isolated from the hyper-arid Salar de Atacama soil
C38
C34
C79
C58
Isolate C38
S. lannensis TA4-8T
S. chiangmaiensis TA4-1T
S. chromofuscus NBRC 12851T
S. speibonae PK-BlueT
S. griseoincarnatus LMG 19316T
S. erythrogriseus LMG 19406T
S. albogriseolus NRRL B-1305T
S. viridodiastaticus NBRC 13103T
S. longispororuber NBRC 13488T
S. thermocarboxydus DSM 44293T
S. lusitanus NBRC 13484T
S. coeruleorubidus NBRC 12844T
S. coerulescens ISP 5146T
S. spinoverrucosus NBRC 14228T
S. lomondensis NBRC 15426T
S. parvulus NBRC 13193T
S. hyderabadensis OU-40T
S. leeuwenhoekii C34T
S. somaliensis DSM 40738T
S. griseosporeus NBRC 13458T
S. glomeratus LMG 19903T
S. mexicanus CH-M-1035T
S. albus subsp. albus NRRL B-2365T
100
100
100
74
86
63
0.005
Bar 1 µ
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).
Isolate KNN41
Isolate KMM11-1
Isolate KNN11-5
Isolate KNN9-3
Isolate KNN6-6
Isolate KNN6-9
Isolate C38 (atacamycins)
Isolate C58 (novel cyclic lasso peptides)
Isolate C79 (four new specialised metabolites)
Isolate KNN10-5
Isolate KNN25
Isolate KNN10-4
Isolate KNN48-1
Isolate KNN24-1
Isolate KNN2-6
Isolate C34 (chaxamycins)
61
60
0.0002
Streptomyces sp. C38
Streptomyces sp. C59
Streptomyces sp. C79 Streptomyces leeuwenhoekii 16S rRNA gene clade
S. leeuwenhoekii
Putative new species
SM 1 agar
Oligotrophic agar
Starch-casein vitamin agar
Humic-acid-vitamin-agar
Members of Amycolatopsis methanolica 16S rRNA gene clade isolated from hyper-arid
Salar de Atacama soil
Selective mediaKNN50-11KNN50-16KNN50-9KNN61-1
A. thermalba SF45T (HQ668525)KNN50-17KNN50-13KNN50-10KNN50-4KNN50-2KNN50-1KNN49-12KNN49-11KNN49-6A. granulosa GY307T (AF466101)
A. viridis GY115T (AF466095)
A. thermoflava N1165T (AF052390)
A. methanolica 239T (AQUL01000001)
A. eurytherma NT 202T (AJ000285)
A. tucumanensis ABOT (DQ886938)
A. thermophila GY088T (AY129774)
A. endophytica KLBMP 1221T (HM153799)
A ruanii NGM112T HQ668524
KNN50-18
KNN50-14
KNN50-6
KNN49-3
KNN49-5
KNN49-26
KNN50-7
KNN50-8
KNN49-10
KNN49-1
KNN50-5
KNN50-13
KNN50-15
GY024
GY142
A. orientalis subsp. orientalis IMSNU 20058T (AJ400711)
Actinokineospora riparia NRRL B-16432T (AF 114802)
100
100
87
66
100
99
53
99
0.005
*
*
*
**
***
**
*
Novel Blastococcus, Geodermatophilus and Modestobacter isolates from the extreme hyper arid Yungay soil
Arginine-vitamin agar Humic-acid-vitamin agar Oligotrophic agar R2A agar
ISP2ISP1 ISP3 ISP4
ISP5 ISP6 ISP7
Starch casein agar
Selective media
Modestobacter colour-group 46, 12 isolates
M. versicolor CP153-2T (AJ871304)
M. marinus 42H12-1T (EU181225)
M. roseus KLBMP 1279T ( JQ819258)
Modestobacter colour-group 45, 4 isolates
M. multiseptatus AA-826T (Y18646)
B. jejuensis KST3-10T (DQ200983)
B. endophyticus YIM 68236T (GQ494034)
B. aggregates ATCC 25902T (L40614)
Isolate KNN47
B. saxobsidens BC448T (AJ316571)
G. terrae PB261T (JN033773)
G. solani PB34T (JN033772)
G. taihuensis 3-wff-81T (JX294478)
G. normandii CF5/3T (HE654546)
G. telluris CF9/1/1T (HE815469)
G. saharensis CF5/5T (HE654551)
G. arenarius CF5/4T (HE654547)
G. nigrescens YIM 75980T (JN656711)
G. ruber CPCC 201356T (EU438905)
G. obscurus DSM 43160T (CP001867)
G. siccatus CF6/1T (HE654548)
Isolate KNN44-3
Isolate KNN44-1
Isolate KNN44-4
99
100
92
87
94
79
100
92
51
98
95
99
77
54
86
76
87
8499
58
7879
0.005
**
***
*
*
*
ML
*
**
**
*
* **
**
Novel Micromonospora isolates from the extreme hyper-arid Lomas Bayas soil
http://www0.nih.go.jp/saj/DigitalAtlas2/
Isolate LB-1 GZ-4
Isolate LB-1 IS-10
Micromonospora saelicesensis Lupac 09T(AJ783993)
Isolate LB-1 R2-3
Isolate LB-1 SC-4Micromonospora chokoriensis 2-19/6T(AB241454)
Micromonospora lupini lupac 14NT(AJ783996)
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
Micromonospora chalcea DSM 43026T(X92594)64
99
*
86
91
91
58
95
81 95
58
0.002
*
**
***
*
**
Actinobacterial Genera Isolated from the Sampling Sites
Salar de Atacama(Hyper-arid soil)
ALMA(Hyper-arid soil)
Yungay
(Extreme hyper-arid soil)
Lomas Bayas
(Most extreme hyper-arid soil)
*Actinomadura+ Amycolatopsis
*Kribbella
*Lechevalieria
*Nocardiopsis
*Nonomuraea
*Saccharothrix+Streptomyces
*Actinomadura+Amycolatopsis
*Cryptosporangium
*Nocardia
*Pseudonocardia+Streptomyces
*Blastococcus
*Couchioplanes
*Geodermatophilus
*Kribbella
*Modestobacter
*Pseudonocardia+Streptomyces
*Actinocorralia
*Micromonospora+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.
Rapid assignment of isolates to colour-groups.
Selection of isolates showing bioactivity in plug assays.
Choice of creative isolates that form distinct 16S rRNA gene
lineages.
Growth of selected isolates on production media.
Preparation of extracts for chemical screening.
Many
Fewer
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.
MR289_KNN64_5_ISP2 #248 RT: 8.23 AV: 1 NL: 2.07E5 microAU
200 220 240 260 280 300 320 340 360 380 400
wavelength (nm)
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
Re
lative
Ab
so
rba
nce
226.00
279.00
336.00
394.00
MR289_KNN64_5_ISP2 #288 RT: 9.57 AV: 1 NL: 1.64E5 microAU
200 220 240 260 280 300 320 340 360 380 400
wavelength (nm)
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
Re
lative
Ab
so
rba
nce
228.00
284.00
339.00
393.00
MR289_KNN64_5_ISP2 #310 RT: 10.30 AV: 1 NL: 1.36E5 microAU
200 220 240 260 280 300 320 340 360 380 400 420
wavelength (nm)
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
Re
lative
Ab
so
rba
nce
227.00
354.00
265.00
394.00
MR289_KNN64_5_ISP2 #364 RT: 12.10 AV: 1 NL: 1.23E5 microAU
200 220 240 260 280 300 320 340 360 380 400
wavelength (nm)
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
Re
lative
Ab
so
rba
nce
227.00
336.00
352.00
RT: 0.00 - 30.00
0 5 10 15 20 25 30
Time (min)
0
10000
20000
30000
40000
50000
60000
70000
80000
90000
100000
110000
120000
130000
140000
150000
160000
170000
180000
190000
uAU
6.67
0.50
7.07
9.576.23
5.47 10.30
4.53 12.102.97
26.0012.302.07 25.7323.9022.00
14.00 17.53
26.43 29.77
NL:1.92E5
Total Scan PDA MR289_KNN64_5_ISP2
UV trace
UV trace
UV traceUV trace
LCMS analysis of metabolite extract from
Streptomyces isolate KNN64-5 after shaking in ISP2
broth for 14 days at 28oC.
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.
MS trace
LCMS analysis of metabolite extract from Amycolatopsis ruanii KNN50-8 after
shaking in medium 19 broth for 14 days at 28oC.
Strain KNN50-8 produces major and minor specialised novel metabolites. The
strain was isolated from the hyper-arid Salar de Atacama.
Major new metabolite
UV spectrum of the major metabolite
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
Primary Screening of ALMA and Lomas Bayas Isolates against
Panel of Pathogens
Percentage of the 132 actinbacteria that showed antimicrobial activity
against pathogens
• 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.
• 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.
LCMS analysis of metabolite extract from Lechevalieria isolate H45 after shaking
in medium 410 broth for 14 days at 28oC
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 45and 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.
Phylogenetic Analyses
B
A
46J O N G S I K C H U N L A B . S E O U L N A T I O N A L U N I V .
Released Genomes in National Center for Biotechnology Information (NCBI)
(1995-2014)15,842 genomes (as of January 2014)
0
1000
2000
3000
4000
5000
6000
7000
8000
NGS
>7,000 genomes
In 2013
GENOMICS
47J O N G S I K C H U N L A B . S E O U L N A T I O N A L U N I V .
Genomics for Systematics:
The Present Status
Species with valid
names
Species with 16S rRNA
gene sequence (type
strain)
Species with genome
sequence
Species with genome
sequence (type strain)
Bacteria 11,275 10,887 2,951 2,085
Archaea 429 423 179 165
Total
(Prokaryotes)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.
Modestobacter Isolate ASC16 Streptomyces leeuwenhoekii C34T
Preliminary Analysis of Whole Genome Sequence Data
Habitat: isolated from the extreme hyper-arid Yungay soil
Genomic size: ~ 5.57 Mb
G+C content: 74.1%
Protein coding genes: 5,445
Secondary metabolite profile : few predicted biosynthetic gene
clusters
Stress genes: resistance to UV radiation and desiccation
Spore forming genes: present
Habitat: isolated from hyper-arid Salar de Atacama soil
Genomic size: ~ 8.66 Mb
G+C content: 72.1%
Protein coding genes: ~7,800
Secondary metabolic profile: 31 predicted biosynthetic gene
clusters including one for chaxamycins
Stress genes: present
Spore forming genes: present
Culture Independent Studies based on Pyrosequencing
Family Genera
Actinoplanaceae Couchioplanes
Cellulomonadaceae Actinotalea, Cellulomonas, Oerskovia
Cryptosporangiaceae Cryptosporangium
Dietziaceae Dietzia
Geodermatophilaceae Blastococcus, Geodermatophilus, Modestobacter
Glycomycetaceae Glycomyces
Microbacteriaceae Agrococcus, Agromyces, Microbacterium, Rathayibacter
Micrococcaceae Micrococcus
Micromonosporaceae Actinoplanes, Catellatospora, Microbiospora, Micromonospora, Pilimelia,
Virgisporangium
Nocardiaceae Nocardia, Rhodococcus
Nocardioidaceae Aeromicrobium, Friedmanniella, Kribbella, Nocardioides, Pimelobacter
Nocardiopsaceae Nocardiopsis
Pseudonocardiaceae Actinomycetospora, Amycolatopsis, Lechevalieria, Lentzea, Pseudonocardia,
Saccharothrix
Streptomycetaceae Streptomyces
Streptosporangiaceae Acrocarpospora, Nonomuraea
Thermomonosporaceae Actinoallomurus, Actinocorallia, Actinomadura
* Genera isolated classified using culture dependent approaches are marked in bold
Primary analysis suggests that there are at least 97 different genera in the soil samples, as exemplified below:
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
