16S phylogeny of the Streptomycetaceae Alan Ward

16S phylogeny of the Streptomycetaceae

Alan Ward

• 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 StreptomycetaceaeOutline

Why?Hypothesisthose 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 Hugenholz et al. (1998), approximately 30% or more of these have been discovered at one time or another. This has been the result of some fifty years worth of intense antibiotic screening programs conducted by pharmaceutical industry natural products scientists Strohl, 2004

For example, a survey of the literature linked the “species” names Streptomyces antibioticus, S. griseus and S. hygroscopicus, with the production of 13, 32, and 46 different structural types of natural products, respectively. Examples of compounds produced by strains characterized as S. hygroscopicus include rapamycin, spectinomycin, bialophos, hygromycin, and a variety of polyethers. Strohl 2004

To address this hypothesis, for Streptomyces, need to be able to address the relationship of

strains to one another



Stackebrandt and Goebel (1994) Int J Syst Bacteriol 44, 846–849 - prokaryotes Stach et al. (2003) App Environ Microbiol 69, 6189-6200 - actinobacteria

• 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 Streptomycetaceaemore Whole genome projects

Streptomyces acidiscabies 84-104 Streptomyces scabiei 87.22Streptomyces bingchenggensis BCW-1 Streptomyces sp. S4Streptomyces cattleya NRRL 8057 Streptomyces sp. SA3_actFStreptomyces chartreusis NRRL 12338 Streptomyces sp. SA3_actGStreptomyces chartreusis NRRL 3882 Streptomyces sp. SirexAA-EStreptomyces clavuligerus ATCC 27064 Streptomyces sp. TOR3209Streptomyces coelicolor A3(2) Streptomyces sp. Tu6071Streptomyces flavogriseus IAF45CD, ATCC 33331 Streptomyces sp. W007Streptomyces griseoaurantiacus M045 Streptomyces sp. Wigar10Streptomyces griseus griseus NBRC 13350 Streptomyces sviceus ATCC 29083Streptomyces griseus XyelbKG-1 1 Streptomyces tsukubaensis NRRL 18488Streptomyces hygroscopicus jinggangensis 5008 Streptomyces turgidiscabies Car8Streptomyces ipomoeae 91-03 Streptomyces venezuelae ATCC 10712Streptomyces lysosuperificus ATCC 31396 Streptomyces violaceusniger Tu 4113Streptomyces pristinaespiralis ATCC 25486 Streptomyces viridosporus T7A, ATCC 39115Streptomyces roseosporus NRRL 11379

16S phylogeny of the StreptomycetaceaeWhole 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

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 StreptomycetaceaeMultiple alignment


16S phylogeny of the StreptomycetaceaeWhole genome projects

16S phylogeny of the StreptomycetaceaeHigher order clusters?

Kitasatospora

Streptacidiphilus


16S phylogeny of the

Streptomycetaceae

16S phylogeny of the StreptomycetaceaeStreptomyces violaceusniger clade


16S phylogeny of the StreptomycetaceaeHPLC analysis of Streptomyces violaceusniger clade

min2 4 6 8 10 12 14 16

mAU

0

200

400

600

800

1000

1200

DAD1 A, Sig=210,10 Ref =of f (Z:\P08\019-2001.D) DAD1 B, Sig=230,10 Ref =of f (Z:\P08\019-2001.D) DAD1 C, Sig=260,20 Ref =of f (Z:\P08\019-2001.D) DAD1 D, Sig=280,20 Ref =of f (Z:\P08\019-2001.D) DAD1 E, Sig=310,20 Ref =of f (Z:\P08\019-2001.D) DAD1 F, Sig=360,40 Ref =of f (Z:\P08\019-2001.D) DAD1 G, Sig=435,40 Ref =of f (Z:\P08\019-2001.D) DAD1 H, Sig=500,40 Ref =of f (Z:\P08\019-2001.D)

Aza

Hex

Ela

A

min2.5 5 7.5 10 12.5 15

mAU

0

100

200

300

400

500

DAD1 A, Sig=210,10 Ref =of f (Z:\P16\019-1901.D) DAD1 B, Sig=230,10 Ref =of f (Z:\P16\019-1901.D) DAD1 C, Sig=260,20 Ref =of f (Z:\P16\019-1901.D) DAD1 D, Sig=280,20 Ref =of f (Z:\P16\019-1901.D) DAD1 E, Sig=310,20 Ref =of f (Z:\P16\019-1901.D) DAD1 F, Sig=360,40 Ref =of f (Z:\P16\019-1901.D) DAD1 G, Sig=435,40 Ref =of f (Z:\P16\019-1901.D) DAD1 H, Sig=500,40 Ref =of f (Z:\P16\019-1901.D)

Gel

Pen

B

0

10

20

30

40

50

60

70

80

90

100

NL19 SGG OM M4

Production Media

No

. o

f p

rod

uc

ing

str

ain

s

Hexaene

Pentaene

Elaiophylin

Geldanamycin

Azalomycin F

Unknown 1

Unknown 2

Unknown 3

Unknown 4

Unknown 5

Unknown 6

Unknown 7

Number of strains producing each secondary metabolite in different production media.

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.


Lankacidin

Elaiophylin

16S phylogeny of the StreptomycetaceaeBlastp analysis of AntiSMASH for elaiophylin gene cluster

16S phylogeny of the StreptomycetaceaeAntiSMASH Streptomyces himastatinicus


16S phylogeny of the StreptomycetaceaeConclusions

MotivationStreptomyces taxonomy16SWhole genome sequencingA bigger 16S phylogeny

• Where is the diversityThe type strainsUncultured diversity

• Where are:The whole genomesKitasatospora and StreptacidiphilusMarine actinomycetesThermophiles The Streptomyces violaceusniger clade

Who makes nigericin

16S phylogeny of the StreptomycetaceaeAcknowledgements

Stephen Haydock – for introducing us to Streptomyces DSM 4137Porntipa Aiemsum-ang and Yashawant Kumar – PhD students at NewcastlePeter FiedlerErn Lacy

The software developers The sequencers forGenbank/EMBL whole genomes and gene clusters

databases/blast/PubMed/linksRDP 16S data The chemists forSeaView molecules and structuresClustalX Muscle MAFFTPhyML FastTree ESPRIT Culture collections/microbiologists for Dendroscope microorganismsantiSMASH

Documents

16S phylogeny of the Streptomycetaceae Alan Ward