16S phylogeny of the
Streptomycetaceae
Alan Ward
16S phylogeny of the Streptomycetaceae
Outline
• Motivation
• Streptomyces taxonomy
• 16S
• Whole genome sequencing
• A bigger 16S phylogeny
• Where is the diversity
• The type strains
• Uncultured diversity
• Where are:
• The whole genomes
• Kitasatospora and Streptacidiphilus
• Marine actinomycetes
• Thermophiles
• The Streptomyces violaceusniger clade
• Who makes nigericin
16S phylogeny of the Streptomycetaceae
Why?
Hypothesis
those claiming the same antibiotic in different species
“are mistaken in species identification or the nature of
the antibiotic” Krassilnikov5
best producers of natural product antibiotics are members
of the actinomycetes,
and
according to
et al.
To address
this
forHugenholz
Streptomyces,
For example,
a survey
ofhypothesis,
the literature
linked
the “species”
(1998), approximately 30% or more of these have been
names need
Streptomyces
antibioticus,
griseus
and S. of
betime
able
addressS.This
the
relationship
discovered
atto
one
ortoanother.
has been the result
hygroscopicus, withstrains
the production
of 13, 32, and 46 different
one another
of some fifty years worth
of to
intense
antibiotic screening
structural types of natural products, respectively. Examples of
programs conducted by pharmaceutical industry natural
compounds produced by strains characterized as S.
products scientists Strohl, 2004
hygroscopicus include rapamycin, spectinomycin, bialophos,
hygromycin, and a variety of polyethers. Strohl 2004
16S phylogeny of the Streptomycetaceae
16S phylogeny of the Streptomycetaceae
Stackebrandt and Goebel (1994) Int J Syst
Bacteriol 44, 846–849 - prokaryotes
Stach et al. (2003) App Environ Microbiol 69,
6189-6200 - actinobacteria
16S phylogeny of the Streptomycetaceae
more Whole genome projects
•
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•
•
•
•
•
•
•
•
•
•
•
Streptomyces sp. H668
Streptomyces sp. 371
Streptomyces sp. HGB0020
Streptomyces sp. HPH0547
Streptomyces sp. 118.1MFTsu1.1
Streptomyces sp. 142MFCol3.1
Streptomyces sp. 142MFCol3.1
Streptomyces sp. 351MFTsu5.1
Streptomyces sp. AP10 JX101691
Streptomyces cinnamoneus
Streptomyces thermoviolaceus
Streptomyces sp. M17
Streptomyces sp. M43
Streptomyces sp. CNH099
Streptomyces sp. CNH189
Streptomyces sp. CNP082
Streptomyces sp. CNS654
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
Streptomyces sp. CNS615 •
Streptomyces sp. CNS335 •
Streptomyces sp. CNT371 •
Streptomyces sp. CNR698 •
Streptomyces sp. CNB091 •
Streptomyces sp. CNQ766 •
Streptomyces sp. CNS335 •
Streptomyces sp. CNS615 •
Streptomyces sp. CNT302 •
Streptomyces sp. CNT372 •
Streptomyces sp. CNY228
Streptomyces sp. CNY243
Streptomyces sp. PVA 94-07
Streptomyces sp. GBA 94-10
Streptomyces sp. CNS606
Streptomyces sp. CNT318
Streptomyces sp. TAA204
Streptomyces sp. CNQ329
Streptomyces sp. CNT360
Streptomyces sp. CNQ865
Streptomyces sp. TAA486
Streptomyces sp. CNH287
Streptomyces sp. TAA040
Streptomyces sp. CNQ-525
Streptomyces sp. GXT6
Streptomyces sp. PAMC26508
…
16S phylogeny of the Streptomycetaceae
Whole genome phylogeny
Mohammad Tauqeer Alam, Maria Elena Merlo, Eriko Takano, Rainer Breitling (2010) Genome-based
phylogenetic analysis of Streptomyces and its relatives. Molecular Phylogenetics and Evolution 54 763–772
Streptomyces acidiscabies 84-104
Streptomyces bingchenggensis BCW-1
Streptomyces cattleya NRRL 8057
Streptomyces chartreusis NRRL 12338
Streptomyces chartreusis NRRL 3882
Streptomyces clavuligerus ATCC 27064
Streptomyces coelicolor A3(2)
Streptomyces flavogriseus IAF45CD, ATCC 33331
Streptomyces griseoaurantiacus M045
Streptomyces griseus griseus NBRC 13350
Streptomyces griseus XyelbKG-1 1
Streptomyces hygroscopicus jinggangensis 5008
Streptomyces ipomoeae 91-03
Streptomyces lysosuperificus ATCC 31396
Streptomyces pristinaespiralis ATCC 25486
Streptomyces roseosporus NRRL 11379
Streptomyces scabiei 87.22
Streptomyces sp. S4
Streptomyces sp. SA3_actF
Streptomyces sp. SA3_actG
Streptomyces sp. SirexAA-E
Streptomyces sp. TOR3209
Streptomyces sp. Tu6071
Streptomyces sp. W007
Streptomyces sp. Wigar10
Streptomyces sviceus ATCC 29083
Streptomyces tsukubaensis NRRL 18488
Streptomyces turgidiscabies Car8
Streptomyces venezuelae ATCC 10712
Streptomyces violaceusniger Tu 4113
Streptomyces viridosporus T7A, ATCC 39115
16S phylogeny of the Streptomycetaceae
Tree generation
• Downloaded 7,610 pre-aligned streptomycete 16S from RDP in Genbank
FASTA format (in 2010) – remove 17 ambiguous sequences
• Edit labels to exclude invalid characters and consist of a unique numeric
code, the species name, the strain name, the culture collection number
and the Genbank sequence code
• Generate the pairwise alignment distance matrix
• Calculate the RDP alignment distance matrix and compare the pairwise
and multiple alignment distances
• Repeat
– Manually re-align and calculate the distance matrix
– Calculate a tree (NJ-Jukes & Cantor initially then Maximum Likelihood)
– Sort the sequences to tree order
• Until the total distance in the distance matrix stops decreasing
16S phylogeny of the Streptomycetaceae
Multiple alignment
16S phylogeny of the Streptomycetaceae
16S phylogeny of the Streptomycetaceae
Whole genome projects
16S phylogeny of the Streptomycetaceae
Higher order clusters?
Kitasatospora
Streptacidiphilus
16S phylogeny of the Streptomycetaceae
16S phylogeny of the
Streptomycetaceae
16S phylogeny of the Streptomycetaceae
Streptomyces violaceusniger clade
16S phylogeny of the Streptomycetaceae
Streptomyces violaceusniger clade
16S phylogeny of the Streptomycetaceae
HPLC analysis of Streptomyces violaceusniger clade
DAD1
DAD1
DAD1
DAD1
DAD1
DAD1
DAD1
DAD1
A
A,
B,
C,
D,
E,
F,
G,
H,
Sig=210,10 Ref =of f
Sig=230,10 Ref =of f
Sig=260,20 Ref =of f
Sig=280,20 Ref =of f
Sig=310,20 Ref =of f
Sig=360,40 Ref =of f
Sig=435,40 Ref =of f
Sig=500,40 Ref =of f
(Z:\P08\019-2001.D)
(Z:\P08\019-2001.D)
(Z:\P08\019-2001.D)
(Z:\P08\019-2001.D)
(Z:\P08\019-2001.D)
(Z:\P08\019-2001.D)
(Z:\P08\019-2001.D)
(Z:\P08\019-2001.D)
Ela
100
No. of producing s trains
mAU
1200
1000
Aza
800
He
x
600
400
Hexaene
90
Pentaene
80
Elaiophylin
70
Geldanamycin
60
Azalomycin F
50
Unknown 1
40
Unknown 2
30
Unknown 3
20
Unknown 4
10
200
NL19
0
2
DAD1
DAD1
DAD1
DAD1
DAD1
DAD1
DAD1
DAD1
B
Unknown 5
0
A,
B,
C,
D,
E,
F,
G,
H,
4
Sig=210,10 Ref =of f
Sig=230,10 Ref =of f
Sig=260,20 Ref =of f
Sig=280,20 Ref =of f
Sig=310,20 Ref =of f
Sig=360,40 Ref =of f
Sig=435,40 Ref =of f
Sig=500,40 Ref =of f
6
8
10
12
14
(Z:\P16\019-1901.D)
(Z:\P16\019-1901.D)
(Z:\P16\019-1901.D)
(Z:\P16\019-1901.D)
(Z:\P16\019-1901.D)
(Z:\P16\019-1901.D)
(Z:\P16\019-1901.D)
(Z:\P16\019-1901.D)
16
min
SGG
OM
Production Media
M4
Unknown 6
Unknown 7
Number of strains producing each secondary
metabolite in different production media.
mAU
500
Pen
HPLC-DAD chromatograms showing major
metabolites produced by members of the S.
violaceusniger clade A, Streptomyces sp. DSM 4137
and B, WS-P18C9. Aza = azalomycin F, Ela =
elaiophylin, Gel = geldanamycin, Hex = hexaene
polyene and Pen = pentaene polyene.
400
300
Gel
200
100
0
2.5
5
7.5
10
12.5
15
min
16S phylogeny of the Streptomycetaceae
Streptomyces violaceusniger clade
Elaiophylin
Lankacidin
16S phylogeny of the Streptomycetaceae
Blastp analysis of AntiSMASH for elaiophylin gene cluster
16S phylogeny of the Streptomycetaceae
AntiSMASH Streptomyces himastatinicus
16S phylogeny of the Streptomycetaceae
16S phylogeny of the Streptomycetaceae
Conclusions
Motivation
Streptomyces taxonomy
16S
Whole genome sequencing
A bigger 16S phylogeny
•Where is the diversity
The type strains
Uncultured diversity
•Where are:
The whole genomes
Kitasatospora and Streptacidiphilus
Marine actinomycetes
Thermophiles
The Streptomyces violaceusniger clade
Who makes nigericin
16S phylogeny of the Streptomycetaceae
Acknowledgements
Stephen Haydock – for introducing us to Streptomyces DSM 4137
Porntipa Aiemsum-ang and Yashawant Kumar – PhD students at Newcastle
Peter Fiedler
Ern Lacy
The software developers
Genbank/EMBL
databases/blast/PubMed/links
RDP 16S data
SeaView
ClustalX Muscle MAFFT
PhyML FastTree ESPRIT
Dendroscope
antiSMASH
The sequencers for
whole genomes and gene clusters
The chemists for
molecules and structures
Culture collections/microbiologists for
microorganisms