little rotters: adventures with plant-pathogenic bacteria

Little RottersAdventures with Plant-Pathogenic Bacteria

Leighton Pritchard1,2,3

1Information and Computational Sciences,2Centre for Human and Animal Pathogens in the Environment,3Dundee Effector Consortium,The James Hutton Institute, Invergowrie, Dundee, Scotland, DD2 5DA

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These slides will be made available athttp://www.slideshare.net/leightonp

http://www.slideshare.net/leightonp

Table of Contents

1 IntroductionHey, Hey We’re The James Hutton Institute!The Tangled Taxonomy of Soft-Rot EnterobacteriaThe Insidious Dickeya Menace

2 GenomicsThe £250k Genome25 Dickeya Genomes

3 Classification and DiagnosticsDiagnosis: Plant Murder IANI Are You OK? Are You OK ANI?Diagnosis: Plant Murder II

4 Systems BiologyPlant Pathology’s Next Top Model?

5 Synthetic BiologyFrom Food (Waste) To Fuel

6 Acknowledgements

The James Hutton Institute


Formed in 2011 by merger

Scottish Crop Research Institute (Dundee)

Macaulay Land Use Research Institute (Aberdeen)

Comprises Biomathematics and Statistics Scotland (BioSS)

Hosts University of Dundee Division of Plant Sciences

http://www.hutton.ac.uk/

http://www.hutton.ac.uk/


Mission

To deliver the highest quality integrated and innovative sciencethat contributes knowledge, products and services to meet themultiple demands on land and natural resources.

Food security

Societal and agricultural impactExtensive burden/cost (P. infestans ≈e1bn in Europe)Emerging pathogens (imports, climate change)

Environmental sustainability

Pesticide minimisation and withdrawalDurable resistance via breeding (and/or GM)

Plant-Pathogen Interactions (potato)

Phytophthorainfestans/oomycetes & fungi

Myzus persicae/aphids

Erwinia, Dickeya,Pectobacterium spp./Bacteria

PCN/nematodes

Table of Contents






6 Acknowledgements

Soft-Rot Enterobacteria (SRE) a

aToth et al. (2006) Annu. Rev. Phytopath. doi:10.1146/annurev.phyto.44.070505.143444

Erwinia, Dickeya and Pectobacterium spp.

Plant Cell Wall Degrading Enzymes (PCWDEs)

http://dx.doi.org/10.1146/annurev.phyto.44.070505.143444

Tangled Taxonomy of SRE a b

aPritchard et al. (2016) Anal. Methods doi:10.1039/c5ay02550h

bWilliams et al. (2010) J. Bact. doi:10.1128/JB.01480-09

Gammaproteobacteria, Enterobacteria taxonomy difficult toresolve, in general

Historical classification mostly polyphasic/phenotypic

Soft rot enterobacteria (SRE) all originally Erwinia spp.

SRE now three distinct genera (Dickeya, Pectobacterium,Erwinia)

Pectobacterium used to be E. chrysanthemi

Dickeya used to be P. chrysanthemi

Old names hold over in the literature, collections, etc.

http://dx.doi.org/10.1039/c5ay02550h

http://dx.doi.org/10.1128/JB.01480-09

Tangled Taxonomy of SREa

aCzajkowski et al. (2015) Ann. Appl. Biol. doi:10.1111/aab.12166

http://dx.doi.org/10.1111/aab.12166

Table of Contents






6 Acknowledgements

Dickeya spp. moving across Europeab

aToth et al. (2011) Plant Path. doi:10.1111/j.1365-3059.2011.02427.x

bParkinson et al. (2015) Eur. J. Plant Path. doi:10.1007/s10658-014-0523-5

D. dianthicola is established across Europe

D. solani is an emerging, encroaching threat

http://dx.doi.org/10.1111/j.1365-3059.2011.02427.x

http://dx.doi.org/10.1007/s10658-014-0523-5

Legislationa


European and Mediterranean Plant Protection Organisation(EPPO)

Member states should regulate D. dianthicola and E. amylovora asquarantine pests (A2 list)

Seed Potatoes (Scotland) Amendment Regulations (2010)

Zero tolerance policy for all Dickeya spp. on potatoes in Scotlandto ensure production of ‘clean’ (disease-free) seed potatoproduction for export

: EUPHRESCO consortium: control and epidemiology


Scottish Government Controlsa

ahttp://www.gov.scot/Topics/farmingrural/Agriculture/plant/18273/PotatoHealthControls/

PotatoQuarantineDiseases/Dickeya

No positive tests for D. solani since 2010

http://www.gov.scot/Topics/farmingrural/Agriculture/plant/18273/PotatoHealthControls/PotatoQuarantineDiseases/Dickeya

http://www.gov.scot/Topics/farmingrural/Agriculture/plant/18273/PotatoHealthControls/PotatoQuarantineDiseases/Dickeya

Table of Contents






6 Acknowledgements

2003: E. carotovora SCRI1043 a

aBell et al. (2004) Proc. Natl. Acad. Sci. USA doi:10.1073/pnas.0402424101

£250k collaboration between SCRI, University ofCambridge, WT Sanger Institute

Single isolate: E. carotovora subsp. atroseptica SCRI1043

The first sequenced enterobacterial plant pathogen (32authors!)

All repeats and gaps bridged and sequenced directly

Result: a single, complete, high-quality 5Mbp circularchromosome at 10.2X coverage: 106,500 reads

http://dx.doi.org/10.1073/pnas.0402424101

2003: E. carotovora subsp. atroseptica

Compared against all 142 available bacterial genomes

GenomeDiagram/SciArt a b c

aPritchard et al. (2006) Bioinformatics doi:10.1093/bioinformatics/btk021

bShemilt (2009) in“Digital Visual Culture: Theory and Practice” ISBN 978-1-84150-248-9

cShemilt (2010) in “Art Practice in a Digital Culture”, ISBN 978-0-7546-7623-2

Influence

Free open-source comparativegenomics visualisation library

Impact

Artwork (prints, audio-visualinstallation) exhibited in UK andinternationally

http://dx.doi.org/10.1093/bioinformatics/btk021

Functional adaptation in Pbaa

aToth et al. (2006) Ann. Rev. Phytopath. doi:10.1146/annurev.phyto.44.070505.143444


Table of Contents






6 Acknowledgements

2013: Dickeya spp. a b

aPritchard et al. (2013) Genome Ann. 1 (4) doi:10.1128/genomeA.00087-12

bPritchard et al. (2013) Genome Ann. 1 (6) doi:10.1128/genomeA.00978-13

Sequenced and annotated 25 new isolates of Dickeya

25 Dickeya isolates, at least six species

Multiple sequencing methods: 454, Illumina (SE, PE)

Minor publications (6, 8 authors)

Results: 12-237 fragments containing 4.2-5.1Mbp, at 6-84Xcoverage, 170k-4m reads

Automated annotation Initially RAST with manualcorrection

http://dx.doi.org/10.1128/genomeA.00087-12

http://dx.doi.org/10.1128/genomeA.00978-13

2013: Dickeya spp.

Within-genus comparisons: large-scale synteny and rearrangement

Within-species comparisons: e.g. indels, HGT

Pangenome

Species Weak prune Strong prune

Core 2201 2201D. chrysanthemi 32 36D. dadantii 11 14D. dianthicola 102 127D. paradisiaca 404 441D. solani 120 157D. zeae 33 40

Accessory: RBBH only with all other membersof same speciesWeak: All RBBH < 80% ID, 40% coverage

Strong: Trim complete graph by Mahalanobis

distance until minimal cliques found

2013: Dickeya spp. a

avan der Wolf et al. (2014) Int. J. Syst. Evol. Micr. 64:768-774 doi:10.1099/ijs.0.052944-0

Within-genus comparisons: whole genome-based speciesdelineation

http://dx.doi.org/10.1099/ijs.0.052944-0

Table of Contents






6 Acknowledgements

Introduction of D. dianthicola a

aParkinson et al. (2015) Eur. J. Plant Pathol. doi:10.1007/s10658-014-0523-5

VNTR identified from genomes used to trace D. dianthicolaintroduction through historical isolates of ornamentals; potato

http://dx.doi.org/10.1007/s10658-014-0523-5

VNTR Minimum Spanning Tree a

aParkinson et al. (2015) Eur. J. Plant Pathol. doi:10.1007/s10658-014-0523-5

Central nodes associated with: older, ornamental accessions.Distal nodes associated with: recent, potato accessions.

http://dx.doi.org/10.1007/s10658-014-0523-5

Ddi introduction through ornamentals

Earliest (1957) strains already show VNTR diversity

No recorded Dickeya potato infection until 1975

Greater VNTR diversity on ornamentals than potato

Ornamental to potato cross-infection supported by threeVNTR profiles in both host types

Ornamental plants likely provided a route for introduction ofthe broad host range pathogen Dickeya to EU potato crops.

There is strict prohibition and regulation of potato import intothe EU; there is not equivalent regulation of ornamentalimports

Legislation by taxonomy a


European and Mediterranean Plant Protection Organisation(EPPO)

Member states should regulate D. dianthicola and E. amylovora asquarantine pests (A2 list)

Why legislate by taxonomy?

“Easy” to incorporate into legislation: binary (yes/no)classification

Assumption: taxonomy can be determined precisely

Assumption: taxonomy is a proxy for disease risk

Those assumptions do not necessarily hold


Problems a b c


bToth et al. (2006) Ann. Rev. Phyto. doi:10.1146/annurev.phyto.44.070505.143444

cDeans et al. (2015) PLoS Biol. doi:10.1371/journal.pbio.1002033

Is existing bacterial taxonomy/nomenclature correct?

Is a species concept even relevant for bacteria?

Taxonomy is ‘vertical’, but pathogenicity may be ‘laterally’transferred (plasmid-borne, etc.)

Mapping from taxonomy to phenotype is not one-to-one

Testing for disease phenotypes not straightforward

Relationship between gene complement and disease not fullyunderstood



http://dx.doi.org/10.1371/journal.pbio.1002033

Brute Force Primer Design

1 Design large numbers of primers to (draft) genomes from thetarget groups

2 Test cross-hybridisation of primer sets in silico against targetand off-target groups

3 Screen primers against broader set of off-target sequences

4 Classify primer sets according to in silico specificity

5 Evaluate specificity against unseen panel of target/off-targetorganisms

qPCR Primer Design: 1

1 If needed, convert drafts to (pseudo)chromosomes andidentify CDS

2 Define target and related off-target groups3 Define classes within target groups

targets

o�-targets

classi�cation

V

IV

III

II

I

genomes


1 Bulk predict primer sets on all chromosomes (Primer3)2 Design only thermodynamically plausible primers3 Over 1000 primer sets per chromosome

targets

o�-targets

classi�cation

V

IV

III

II

I

genomes


1 Predict cross-amplification in silico (primersearch)2 Classify primers by cross-amplification profile3 Additional screen against off-target database (BLAST)

targets

o�-targets

classi�cation

V

IV

III

II

I

genomes

IIIIIIIVV


1 Select diagnostic primer sets2 Evaluate in vitro against panel of previously “unseen” isolates

of known class3 Report performance metrics

targets

o�-targets

classi�cation

V

IV

III

II

I

IIIIIIIVV

primer sets validation gels

III IV V +ve -ve

III IV V +ve -ve

III IV V +ve -ve

III IV V +ve -ve

II

V

I

III

Classification is a problem! a

aPritchard et al. (2013) Plant Path. doi:10.1111/j.1365-3059.2012.02678.x

qPCR design gave no diagnostic primers for several species.

Misassigned species in GenBank made ‘training’ impossible.

1

1ML tree, recA

http://dx.doi.org/10.1111/j.1365-3059.2012.02678.x

Consequences of misclassification a b


bVarghese et al. (2015) Nucl. Acids Res. doi:10.1093/nar/gkv657

Failed classification has real-world consequences:

False positives (type I errors):clean samples rejected: economic costfarms quarantined/close: economic/societal cost

False negatives (type II errors):(irreversible) introduction of infectious materialpotential for novel host jumps and spread

“Gold-standard”, correctly classified training and test sets essentialto estimate classifier error rates.

MiSI: 18% of NCBI genomes: bacterial species misclassified


http://dx.doi.org/10.1093/nar/gkv657

Table of Contents






6 Acknowledgements

DNA-DNA hybridisation a b

aMorello-Mora and Amann (2001) FEMS Micro. Rev. doi:10.1016/S0168-6445(00)00040-1

bChan et al (2012) BMC Microbiol. doi:10.1186/1471-2180-12-302

“Gold Standard” forprokaryotic taxonomy,since 1960s. “70%identity ≈ same species.”

Denature DNA from twoorganisms.

Allow to anneal.Reassociation ≈ similarity,measured as ∆T ofdenaturation curves.

Proxy for sequence similarity - replace with genome analysis?

http://dx.doi.org/10.1016/S0168-6445(00)00040-1

http://dx.doi.org/10.1186/1471-2180-12-302

Average Nucleotide Identity (ANIm) a

aRichter and Rossello-Mora (2009) Proc. Natl. Acad. Sci. USA doi:10.1073/pnas.0906412106

1. Align genomes(MUMmer)2. ANIm: Mean% identity of allmatches

DDH:ANImlinear

70%ID ≈95%ANIm

http://dx.doi.org/10.1073/pnas.0906412106

ANIm

Average identity of all ‘homologous’ regions

Approximates a limiting case of MLST/MLSA/multigenecomparisons

Straightforward principle

Classification not dependent on dataset composition (unliketree methods)

D. solani ANIm a

avan der Wolf et al. (2014) Int. J. Syst. Evol. Micr. doi:10.1099/ijs.0.052944-0

Defining D. solani as a new species, with ANIm:

http://dx.doi.org/10.1099/ijs.0.052944-0

34 Dickeya spp. ANIm a


Ninespecies-levelgroups (twonovel)

Three speciesmisidentifiedin GenBank

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55 Pectobacterium spp. ANIm a


Tenspecies-levelgroups (fournovel)

P. carotovorum

split: several

species

P. wasabiae

split: two

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P. atrosepticum SCRI1043P. atrosepticum NCPPB 3404P. atrosepticum JG1008P. atrosepticum 21AP. atrosepticum CFBP 6276P. atrosepticum NCPPB 549P. atrosepticum ICMP 1526P. carotovorum PC1P. carotovorum UGC32P. betavasculorum NCPPB 2793P. betavasculorum NCPPB 2795P. carotovorum M022P. wasabiae CFBP 3304P. wasabiae NCPPB 3701P. wasabiae NCPPB3702P. wasabiae CFIA1002P. wasabiae WPP163P. wasabiae RNS08.42.1AP. sp. SCC3193 SCC3193P. carotovorum BC D6P. carotovorum YC D49P. carotovorum BC S2P. carotovorum YC D29P. carotovorum YC D65P. carotovorum CFIA1001P. carotovorum PCC21P. carotovorum YC D46P. carotovorum YC T31P. carotovorum YC D62P. carotovorum YC T3P. carotovorum CFIA1009P. carotovorum YC D52P. carotovorum YC D21P. carotovorum YC D64P. carotovorum YC D60P. carotovorum CFIA1033P. carotovorum PBR1692P. carotovorum LMG 21371P. carotovorum BD255P. carotovorum ICMP 19477P. carotovorum LMG 21372P. carotovorum KKH3P. carotovorum NCPPB3841P. carotovorum NCPPB 3839P. carotovorum BC S7P. carotovorum YC T1P. carotovorum NCPPB 3395P. carotovorum YC D57P. carotovorum BC T2P. carotovorum ICMP 5702P. carotovorum NCPPB 312P. carotovorum YC D16P. carotovorum YC T39P. carotovorum WPP14P. carotovorum BC T5

0.00

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Criticisms of ANIma


95% threshold ‘arbitrary’

Taxonomic classification, not phylogenetic reconstruction

No functional (or gene-based) interpretation: still needpangenome classification and analysis

Only considers ‘homologous’ regions:

Define ‘homologous’misled by HGT/LGT?misled by distant relationships/low extent of homology?

Coverage plots help interpretation: exclude HGT/LGT lowhomology bias.


55 Pectobacterium spp. ANIma


All isolatesalign over>50% ofwholegenome

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YC

D57

P. c

arot

ovor

um W

PP

14P

. car

otov

orum

BC

T5

P. c

arot

ovor

um C

FIA

1033

P. c

arot

ovor

um P

C1

P. c

arot

ovor

um L

MG

213

71P

. car

otov

orum

BD

255

P. c

arot

ovor

um P

BR

1692

P. c

arot

ovor

um IC

MP

194

77P

. car

otov

orum

LM

G 2

1372

P. w

asab

iae

CF

BP

330

4P

. was

abia

e N

CP

PB

370

1P

. was

abia

e N

CP

PB

3702

P. s

p. S

CC

3193

SC

C31

93P

. was

abia

e W

PP

163

P. w

asab

iae

RN

S08

.42.

1AP

. was

abia

e C

FIA

1002

P. a

tros

eptic

um C

FB

P 6

276

P. a

tros

eptic

um N

CP

PB

340

4P

. atr

osep

ticum

NC

PP

B 5

49P

. atr

osep

ticum

ICM

P 1

526

P. a

tros

eptic

um S

CR

I104

3P

. atr

osep

ticum

JG

100

8P

. atr

osep

ticum

21A

P. c

arot

ovor

um N

CP

PB

3841

P. c

arot

ovor

um N

CP

PB

383

9P

. car

otov

orum

M02

2P

. bet

avas

culo

rum

NC

PP

B 2

793

P. b

etav

ascu

loru

m N

CP

PB

279

5

P. carotovorum M022P. carotovorum YC T1P. carotovorum KKH3P. carotovorum UGC32P. carotovorum CFIA1033P. carotovorum NCPPB3841P. carotovorum NCPPB 3839P. carotovorum BC S7P. carotovorum ICMP 19477P. carotovorum BD255P. carotovorum LMG 21372P. carotovorum PBR1692P. carotovorum LMG 21371P. carotovorum PC1P. carotovorum ICMP 5702P. carotovorum NCPPB 312P. carotovorum YC D57P. carotovorum BC T2P. carotovorum YC D16P. carotovorum WPP14P. carotovorum BC T5P. carotovorum YC D62P. carotovorum YC T31P. carotovorum YC D52P. carotovorum YC T3P. carotovorum YC D64P. carotovorum YC D21P. carotovorum YC D60P. carotovorum YC T39P. carotovorum CFIA1001P. carotovorum YC D46P. carotovorum YC D49P. carotovorum BC D6P. carotovorum YC D65P. carotovorum BC S2P. carotovorum YC D29P. carotovorum PCC21P. carotovorum CFIA1009P. atrosepticum ICMP 1526P. atrosepticum CFBP 6276P. atrosepticum SCRI1043P. atrosepticum NCPPB 3404P. atrosepticum NCPPB 549P. atrosepticum JG1008P. atrosepticum 21AP. wasabiae WPP163P. sp. SCC3193 SCC3193P. wasabiae RNS08.42.1AP. wasabiae CFIA1002P. wasabiae CFBP 3304P. wasabiae NCPPB 3701P. wasabiae NCPPB3702P. carotovorum NCPPB 3395P. betavasculorum NCPPB 2793P. betavasculorum NCPPB 2795

0.00

0.25

0.50

0.75

1.00

AN

Im_a

lignm

ent_

cove

rage


34 Dickeya spp. ANIma


Most isolatesalign over>50% ofwholegenome

Community:two outlierspecies arequestionableassignments

Dic

keya_d

adanti

i_Ech

703_u

id59363

Dic

keya_p

ara

dis

iaca

_NC

PPB

_2511

Dic

keya_a

quati

ca_D

W_0

440

Dic

keya_a

quati

ca_C

SL_

RW

240

Dic

keya_s

ola

ni_

MK

10

Dic

keya_s

ola

ni_

AM

YI0

1D

icke

ya_s

ola

ni_

AM

WE01

Dic

keya_s

ola

ni_

MK

16

Dic

keya_s

ola

ni_

GB

BC

2040

Dic

keya_s

ola

ni_

IPO

2222

Dic

keya_d

ianth

icola

_NC

PPB

_453

Dic

keya_d

ianth

icola

_GB

BC

2039

Dic

keya_d

ianth

icola

_IPO

980

Dic

keya_d

ianth

icola

_NC

PPB

_3534

Dic

keya_d

adanti

i_N

CPPB

_2976

Dic

keya_d

adanti

i_3937_u

id52537

Dic

keya_s

pp_N

CPPB

_3274

Dic

keya_s

pp_M

K7

Dic

keya_d

adanti

i_N

CPPB

_3537

Dic

keya_d

adanti

i_N

CPPB

_898

Dic

keya_z

eae_A

PM

V01

Dic

keya_z

eae_A

JVN

01

Dic

keya_z

eae_A

PW

M01

Dic

keya_d

adanti

i_Ech

586_u

id42519

Dic

keya_z

eae_C

SL_

RW

192

Dic

keya_z

eae_N

CPPB

_3532

Dic

keya_z

eae_N

CPPB

_2538

Dic

keya_z

eae_N

CPPB

_3531

Dic

keya_z

eae_M

K19

Dic

keya_s

pp_N

CPPB

_569

Dic

keya_c

hry

santh

am

i_N

CPPB

_402

Dic

keya_c

hry

santh

am

i_N

CPPB

_516

Dic

keya_z

eae_E

ch1591_u

id59297

Dic

keya_c

hry

santh

am

i_N

CPPB

_3533

Dickeya_dadantii_Ech703_uid59363Dickeya_paradisiaca_NCPPB_2511Dickeya_aquatica_DW_0440Dickeya_aquatica_CSL_RW240Dickeya_dianthicola_GBBC2039Dickeya_dianthicola_NCPPB_3534Dickeya_dianthicola_NCPPB_453Dickeya_dianthicola_IPO980Dickeya_solani_GBBC2040Dickeya_solani_IPO2222Dickeya_solani_MK10Dickeya_solani_MK16Dickeya_solani_AMYI01Dickeya_solani_AMWE01Dickeya_dadantii_NCPPB_3537Dickeya_dadantii_NCPPB_898Dickeya_spp_NCPPB_3274Dickeya_spp_MK7Dickeya_dadantii_NCPPB_2976Dickeya_dadantii_3937_uid52537Dickeya_zeae_APMV01Dickeya_zeae_AJVN01Dickeya_dadantii_Ech586_uid42519Dickeya_zeae_APWM01Dickeya_zeae_MK19Dickeya_zeae_NCPPB_3532Dickeya_zeae_NCPPB_2538Dickeya_zeae_NCPPB_3531Dickeya_zeae_CSL_RW192Dickeya_spp_NCPPB_569Dickeya_zeae_Ech1591_uid59297Dickeya_chrysanthami_NCPPB_3533Dickeya_chrysanthami_NCPPB_402Dickeya_chrysanthami_NCPPB_516

0.00

0.25

0.50

0.75

1.00

AN

Im_a

lignm

ent_

covera

ge


34 Dickeya spp. ANIma


Combineidentity andcoverage in asingle(Hadamard)measure.

Non

e

None

GC

F_0

0036

5305

.1_D

DI4

53_v

1.0_

geno

mic

GC

F_0

0097

5305

.1_D

dRN

S04

.9V

1.0_

geno

mic

GC

F_0

0043

0955

.1_D

DIIP

O98

0_1.

0_ge

nom

ic

GC

F_0

0036

5405

.1_D

DI3

534_

1.0_

geno

mic

GC

F_0

0036

5405

.2_D

DI3

534_

1.0_

geno

mic

GC

F_0

0040

6085

.1_D

XX

RW

240_

1.0_

geno

mic

GC

F_0

0036

5365

.1_D

DIG

BB

C20

39_1

.0_g

enom

ic

GC

F_0

0140

1705

.1_D

sl_1

.0_g

enom

ic

GC

F_0

0083

1935

.1_A

SM

8319

3v1_

geno

mic

GC

F_0

0036

5285

.1_D

SO

MK

10_1

.0_g

enom

ic

GC

F_0

0164

4705

.1_A

SM

1644

70v1

_gen

omic

GC

F_0

0047

4655

.1_D

icke

ya1.

0_ge

nom

ic

GC

F_0

0051

1285

.1_S

SP

AC

E_g

enom

ic

GC

F_0

0141

7915

.1_D

sl_1

.0_g

enom

ic

GC

F_0

0036

5345

.1_D

SO

MK

16_1

.0_g

enom

ic

GC

F_0

0140

1695

.1_D

sl_1

.0_g

enom

ic

GC

F_0

0150

6125

.1_A

SM

1506

12v1

_gen

omic

GC

F_0

0040

0565

.1_D

SO

GB

BC

2040

_1.0

_gen

omic

GC

F_0

0040

0795

.1_D

SO

IPO

2222

_1.0

_gen

omic

GC

F_0

0040

6265

.1_D

DA

3537

_1.0

_gen

omic

GC

F_0

0040

6145

.1_D

DA

898_

1.0_

geno

mic

GC

F_0

0014

7055

.1_A

SM

1470

5v1_

geno

mic

GC

F_0

0040

6185

.1_D

DA

2976

_1.0

_gen

omic

GC

F_0

0078

4735

.1_A

SM

7847

3v1_

geno

mic

GC

F_0

0040

6205

.1_D

XX

3274

_1.0

_gen

omic

GC

F_0

0118

7965

.1_A

SM

1187

96v1

_gen

omic

GC

F_0

0040

6305

.1_D

XX

MK

7_1.

0_ge

nom

ic

GC

F_0

0118

7975

.1_A

SM

1187

97v1

_gen

omic

GC

F_0

0077

4065

.1_A

SM

7740

6v1_

geno

mic

GC

F_0

0080

3195

.1_A

SM

8031

9v1_

geno

mic

GC

F_0

0040

4105

.1_D

iZe_

1.0_

geno

mic

GC

F_0

0026

4075

.1_A

SM

2640

7v1_

geno

mic

GC

F_0

0081

6045

.1_A

SM

8160

4v1_

geno

mic

GC

F_0

0040

6045

.1_D

ZE

RW

192_

1.0_

geno

mic

GC

F_0

0040

6225

.1_D

ZE

3531

_1.0

_gen

omic

GC

F_0

0002

5065

.1_A

SM

2506

v1_g

enom

ic

GC

F_0

0040

0525

.1_D

ZE

3532

_1.0

_gen

omic

GC

F_0

0040

6165

.1_D

ZE

2538

_1.0

_gen

omic

GC

F_0

0040

6325

.1_D

ZE

MK

19_1

.0_g

enom

ic

GC

F_0

0038

2585

.1_D

iZ_M

S1_

geno

mic

GC

F_0

0040

6125

.1_D

XY

569_

1.0_

geno

mic

GC

F_0

0040

6105

.1_D

CH

402_

1.0_

geno

mic

GC

F_0

0040

6245

.1_D

CH

3533

_1.0

_gen

omic

GC

F_0

0002

3565

.1_A

SM

2356

v1_g

enom

ic

GC

F_0

0078

4725

.1_A

SM

7847

2v1_

geno

mic

GC

F_0

0040

6065

.1_D

CH

516_

1.0_

geno

mic

GC

F_0

0040

0505

.1_D

PA

2511

_1.0

_gen

omic

GC

F_0

0002

3545

.1_A

SM

2354

v1_g

enom

ic

GC

A_0

0040

6085

.2_A

SM

4060

8v2_

geno

mic

GC

F_0

0040

6285

.1_D

XX

DW

0440

_1.0

_gen

omic

GCF_000023545.1_ASM2354v1_genomic

GCF_000400505.1_DPA2511_1.0_genomic

GCF_000406285.1_DXXDW0440_1.0_genomic

GCA_000406085.2_ASM40608v2_genomic


GCF_000406245.1_DCH3533_1.0_genomic


GCF_000406065.1_DCH516_1.0_genomic

GCF_000406105.1_DCH402_1.0_genomic

GCF_000406125.1_DXY569_1.0_genomic

GCF_000406165.1_DZE2538_1.0_genomic

GCF_000400525.1_DZE3532_1.0_genomic

GCF_000406325.1_DZEMK19_1.0_genomic

GCF_000382585.1_DiZ_MS1_genomic


GCF_000406225.1_DZE3531_1.0_genomic

GCF_000406045.1_DZERW192_1.0_genomic



GCF_000404105.1_DiZe_1.0_genomic

GCF_000430955.1_DDIIPO980_1.0_genomic

GCF_000365305.1_DDI453_v1.0_genomic

GCF_000975305.1_DdRNS04.9V1.0_genomic

GCF_000365405.2_DDI3534_1.0_genomic

GCF_000365405.1_DDI3534_1.0_genomic

GCF_000365365.1_DDIGBBC2039_1.0_genomic

GCF_000406085.1_DXXRW240_1.0_genomic


GCF_000406185.1_DDA2976_1.0_genomic


GCF_000406145.1_DDA898_1.0_genomic

GCF_000406265.1_DDA3537_1.0_genomic




GCF_000406305.1_DXXMK7_1.0_genomic


GCF_000406205.1_DXX3274_1.0_genomic


GCF_001401695.1_Dsl_1.0_genomic

GCF_000365345.1_DSOMK16_1.0_genomic


GCF_000400795.1_DSOIPO2222_1.0_genomic

GCF_000400565.1_DSOGBBC2040_1.0_genomic

GCF_000365285.1_DSOMK10_1.0_genomic

GCF_000511285.1_SSPACE_genomic

GCF_000474655.1_Dickeya1.0_genomic




0.27 0.27 0.26 0.26 0.26 0.26 0.26 0.26 0.29 0.26 0.26 0.26 0.26 0.26 0.26 0.26 0.26 0.26 0.26 0.25 0.25 0.26 0.26 0.24 0.26 0.26 0.27 0.27 0.26 0.25 0.21 0.21 0.21 0.19 0.19 0.19 0.2 0.2 0.19 0.2 0.21 0.23 0.22 0.21 0.22 0.22 1 1 0.16 0.16

0.27 0.26 0.26 0.26 0.26 0.26 0.26 0.25 0.26 0.25 0.26 0.26 0.26 0.26 0.26 0.26 0.26 0.26 0.26 0.25 0.24 0.26 0.26 0.24 0.26 0.26 0.27 0.27 0.25 0.25 0.2 0.21 0.21 0.19 0.19 0.19 0.2 0.2 0.19 0.2 0.21 0.23 0.21 0.21 0.22 0.22 1 1 0.15 0.15

0.29 0.29 0.3 0.3 0.3 0.29 0.29 0.28 0.29 0.28 0.28 0.29 0.28 0.29 0.29 0.29 0.28 0.28 0.28 0.28 0.28 0.28 0.3 0.3 0.3 0.3 0.29 0.3 0.3 0.29 0.29 0.28 0.28 0.29 0.29 0.29 0.28 0.28 0.28 0.27 0.3 0.29 0.29 0.29 0.29 0.3 0.15 0.15 0.98 1

0.3 0.29 0.3 0.3 0.3 0.29 0.29 0.28 0.27 0.28 0.28 0.29 0.28 0.29 0.29 0.29 0.28 0.28 0.29 0.28 0.28 0.29 0.3 0.3 0.3 0.3 0.3 0.3 0.3 0.29 0.29 0.29 0.29 0.3 0.29 0.29 0.28 0.28 0.29 0.28 0.31 0.29 0.3 0.29 0.3 0.3 0.15 0.15 1 0.99

0.65 0.66 0.64 0.63 0.63 0.63 0.62 0.61 0.6 0.62 0.63 0.63 0.63 0.63 0.63 0.63 0.62 0.62 0.62 0.61 0.62 0.66 0.65 0.64 0.61 0.62 0.62 0.63 0.61 0.61 0.6 0.61 0.62 0.62 0.62 0.61 0.64 0.64 0.61 0.63 0.61 0.83 1 1 0.9 0.9 0.22 0.22 0.32 0.32

0.65 0.65 0.64 0.63 0.63 0.63 0.62 0.61 0.6 0.62 0.62 0.63 0.62 0.63 0.63 0.63 0.62 0.62 0.63 0.61 0.61 0.65 0.65 0.63 0.6 0.62 0.62 0.63 0.61 0.61 0.61 0.61 0.61 0.62 0.62 0.61 0.63 0.64 0.61 0.63 0.61 0.83 1 1 0.9 0.9 0.22 0.22 0.32 0.32

0.66 0.65 0.63 0.63 0.63 0.64 0.62 0.62 0.6 0.62 0.63 0.63 0.63 0.63 0.63 0.63 0.63 0.63 0.63 0.61 0.61 0.64 0.64 0.63 0.61 0.62 0.62 0.62 0.61 0.6 0.61 0.61 0.61 0.61 0.61 0.61 0.63 0.63 0.61 0.62 0.61 0.84 0.91 0.89 1 0.9 0.22 0.22 0.32 0.32

0.63 0.64 0.61 0.62 0.61 0.62 0.61 0.59 0.59 0.59 0.61 0.61 0.61 0.61 0.62 0.62 0.61 0.61 0.61 0.61 0.6 0.63 0.64 0.63 0.59 0.61 0.6 0.61 0.6 0.59 0.6 0.6 0.6 0.6 0.61 0.6 0.63 0.62 0.6 0.6 0.61 0.82 0.88 0.87 0.87 1 0.22 0.22 0.32 0.32

0.66 0.66 0.64 0.64 0.64 0.65 0.63 0.61 0.59 0.61 0.62 0.62 0.62 0.62 0.63 0.62 0.62 0.62 0.62 0.6 0.59 0.64 0.63 0.62 0.6 0.6 0.6 0.61 0.6 0.59 0.6 0.61 0.61 0.61 0.61 0.61 0.62 0.62 0.6 0.6 0.61 1 0.82 0.81 0.83 0.83 0.23 0.23 0.31 0.31

0.55 0.54 0.53 0.55 0.55 0.54 0.53 0.52 0.55 0.52 0.53 0.54 0.53 0.54 0.54 0.54 0.53 0.53 0.54 0.52 0.51 0.53 0.55 0.55 0.53 0.55 0.54 0.55 0.54 0.52 0.56 0.55 0.55 0.54 0.57 0.53 0.55 0.55 0.55 0.54 1 0.54 0.54 0.54 0.54 0.56 0.19 0.19 0.29 0.3

0.56 0.56 0.54 0.55 0.55 0.54 0.54 0.56 0.58 0.56 0.57 0.57 0.57 0.57 0.57 0.57 0.57 0.57 0.57 0.55 0.56 0.57 0.56 0.59 0.54 0.57 0.56 0.57 0.56 0.55 0.8 0.8 0.8 0.82 0.85 0.84 0.93 1 0.91 0.89 0.59 0.6 0.61 0.61 0.6 0.62 0.2 0.2 0.29 0.29

0.56 0.55 0.54 0.55 0.55 0.54 0.53 0.55 0.58 0.56 0.57 0.57 0.56 0.57 0.57 0.57 0.57 0.57 0.57 0.56 0.54 0.57 0.57 0.59 0.54 0.57 0.56 0.57 0.56 0.55 0.81 0.8 0.8 0.82 0.85 0.84 1 0.93 0.9 0.88 0.6 0.6 0.61 0.6 0.6 0.62 0.19 0.19 0.3 0.3

0.56 0.55 0.55 0.56 0.56 0.54 0.54 0.55 0.54 0.56 0.56 0.57 0.56 0.57 0.57 0.57 0.56 0.57 0.57 0.55 0.54 0.57 0.57 0.58 0.55 0.57 0.56 0.57 0.56 0.55 0.82 0.81 0.81 0.84 0.87 0.84 0.93 0.93 1 0.89 0.61 0.6 0.6 0.59 0.6 0.6 0.19 0.19 0.31 0.31

0.57 0.56 0.55 0.55 0.55 0.55 0.54 0.56 0.54 0.56 0.57 0.57 0.57 0.57 0.58 0.57 0.57 0.57 0.57 0.56 0.55 0.57 0.58 0.59 0.56 0.57 0.56 0.56 0.56 0.55 0.81 0.82 0.82 0.83 0.85 0.84 0.92 0.93 0.9 1 0.6 0.61 0.63 0.62 0.62 0.62 0.2 0.2 0.3 0.3

0.58 0.58 0.56 0.57 0.57 0.56 0.55 0.57 0.6 0.57 0.58 0.58 0.58 0.58 0.58 0.58 0.58 0.58 0.58 0.57 0.55 0.59 0.58 0.59 0.57 0.58 0.57 0.57 0.57 0.56 0.82 0.81 0.83 0.84 0.87 1 0.88 0.89 0.86 0.85 0.6 0.62 0.62 0.61 0.61 0.63 0.2 0.2 0.32 0.32

0.57 0.56 0.55 0.56 0.56 0.55 0.54 0.55 0.58 0.55 0.56 0.56 0.56 0.56 0.56 0.57 0.56 0.56 0.56 0.55 0.55 0.56 0.56 0.58 0.55 0.57 0.57 0.57 0.56 0.56 0.85 0.85 0.85 0.88 1 0.83 0.86 0.87 0.86 0.83 0.63 0.6 0.6 0.6 0.6 0.61 0.19 0.19 0.31 0.31

0.58 0.57 0.56 0.56 0.56 0.56 0.55 0.54 0.53 0.55 0.56 0.56 0.55 0.56 0.56 0.56 0.55 0.55 0.56 0.55 0.53 0.56 0.56 0.57 0.55 0.57 0.57 0.57 0.56 0.55 0.84 0.85 0.86 1 0.89 0.82 0.85 0.85 0.84 0.83 0.6 0.61 0.61 0.61 0.6 0.61 0.19 0.19 0.32 0.32

0.56 0.56 0.54 0.54 0.54 0.54 0.52 0.53 0.52 0.53 0.54 0.54 0.54 0.54 0.54 0.54 0.54 0.54 0.54 0.51 0.51 0.54 0.54 0.55 0.53 0.53 0.53 0.53 0.53 0.52 0.96 1 1 0.83 0.83 0.78 0.8 0.8 0.79 0.78 0.59 0.59 0.58 0.58 0.58 0.59 0.2 0.2 0.3 0.3

0.56 0.56 0.54 0.54 0.55 0.54 0.53 0.54 0.52 0.54 0.54 0.55 0.54 0.55 0.55 0.55 0.54 0.54 0.55 0.52 0.51 0.54 0.55 0.56 0.54 0.54 0.53 0.54 0.53 0.53 0.95 1 1 0.83 0.84 0.77 0.81 0.8 0.8 0.79 0.6 0.59 0.59 0.58 0.59 0.59 0.2 0.2 0.3 0.3

0.56 0.56 0.54 0.56 0.56 0.54 0.53 0.54 0.53 0.54 0.55 0.55 0.55 0.55 0.55 0.55 0.55 0.55 0.55 0.53 0.52 0.55 0.55 0.56 0.53 0.55 0.54 0.55 0.54 0.53 1 0.96 0.98 0.83 0.86 0.79 0.82 0.82 0.81 0.79 0.62 0.59 0.59 0.58 0.59 0.61 0.21 0.21 0.3 0.31

0.99 0.98 1 0.95 0.95 0.97 0.95 0.72 0.71 0.73 0.73 0.74 0.73 0.74 0.74 0.74 0.74 0.74 0.74 0.7 0.69 0.73 0.73 0.72 0.7 0.71 0.71 0.72 0.71 0.69 0.56 0.57 0.57 0.58 0.57 0.56 0.58 0.57 0.57 0.56 0.61 0.66 0.66 0.65 0.64 0.64 0.27 0.27 0.33 0.33

1 1 0.95 0.95 0.95 0.95 0.94 0.72 0.71 0.73 0.74 0.74 0.74 0.75 0.74 0.74 0.74 0.74 0.74 0.7 0.69 0.73 0.73 0.72 0.71 0.71 0.71 0.72 0.71 0.69 0.57 0.57 0.58 0.58 0.57 0.56 0.57 0.58 0.56 0.56 0.61 0.66 0.64 0.64 0.64 0.64 0.27 0.27 0.32 0.32

1 1 0.95 0.97 0.97 0.96 0.95 0.72 0.77 0.73 0.74 0.74 0.74 0.74 0.74 0.74 0.74 0.74 0.74 0.7 0.69 0.73 0.73 0.72 0.71 0.72 0.71 0.72 0.71 0.7 0.57 0.58 0.58 0.58 0.57 0.56 0.57 0.58 0.56 0.56 0.61 0.66 0.65 0.64 0.64 0.65 0.27 0.27 0.32 0.32

0.99 1 0.95 1 1 0.96 0.95 0.72 0.72 0.73 0.74 0.75 0.74 0.75 0.75 0.75 0.74 0.75 0.75 0.72 0.7 0.74 0.74 0.73 0.72 0.72 0.72 0.73 0.72 0.7 0.58 0.58 0.58 0.58 0.59 0.58 0.58 0.58 0.59 0.57 0.63 0.66 0.65 0.64 0.65 0.64 0.27 0.27 0.33 0.34

0.99 1 0.95 1 1 0.96 0.95 0.72 0.72 0.73 0.74 0.75 0.74 0.75 0.75 0.75 0.74 0.75 0.75 0.72 0.7 0.74 0.74 0.73 0.72 0.72 0.72 0.73 0.72 0.7 0.59 0.58 0.58 0.58 0.59 0.58 0.58 0.58 0.58 0.57 0.63 0.66 0.65 0.64 0.65 0.65 0.27 0.27 0.33 0.34

0.96 0.97 0.95 0.93 0.94 1 1 0.7 0.74 0.71 0.73 0.72 0.72 0.72 0.72 0.72 0.72 0.72 0.72 0.7 0.69 0.72 0.72 0.71 0.7 0.7 0.71 0.71 0.7 0.68 0.55 0.55 0.55 0.56 0.56 0.55 0.56 0.57 0.55 0.54 0.6 0.64 0.62 0.61 0.62 0.63 0.27 0.26 0.32 0.32

0.9 0.89 0.88 0.87 0.87 1 0.94 0.66 0.69 0.67 0.68 0.68 0.68 0.68 0.68 0.68 0.68 0.68 0.68 0.64 0.63 0.67 0.67 0.67 0.65 0.66 0.66 0.67 0.66 0.64 0.51 0.52 0.52 0.52 0.52 0.52 0.52 0.52 0.51 0.51 0.56 0.6 0.59 0.58 0.59 0.59 0.25 0.25 0.29 0.29

0.67 0.67 0.65 0.65 0.65 0.67 0.64 0.67 0.71 0.68 0.69 0.69 0.69 0.69 0.69 0.69 0.69 0.69 0.69 0.67 0.65 0.68 0.7 1 0.7 0.73 0.72 0.73 0.72 0.71 0.53 0.53 0.53 0.53 0.54 0.53 0.56 0.56 0.54 0.54 0.57 0.57 0.58 0.58 0.58 0.59 0.23 0.23 0.3 0.3

0.75 0.74 0.72 0.73 0.74 0.74 0.73 0.79 0.82 0.79 0.81 0.81 0.81 0.81 0.81 0.82 0.81 0.81 0.81 0.82 0.84 0.85 1 0.77 0.77 0.78 0.76 0.77 0.78 0.77 0.57 0.58 0.58 0.58 0.59 0.58 0.6 0.59 0.59 0.59 0.63 0.64 0.66 0.65 0.65 0.66 0.27 0.27 0.33 0.33

0.76 0.76 0.74 0.75 0.75 0.75 0.73 0.81 0.81 0.81 0.84 0.84 0.83 0.83 0.83 0.83 0.83 0.83 0.83 0.9 0.89 1 0.87 0.77 0.77 0.79 0.78 0.79 0.79 0.78 0.59 0.58 0.58 0.59 0.6 0.61 0.62 0.61 0.6 0.59 0.62 0.67 0.67 0.67 0.66 0.67 0.27 0.27 0.32 0.32

0.73 0.72 0.71 0.71 0.71 0.71 0.71 0.78 0.81 0.78 0.83 0.79 0.79 0.8 0.8 0.8 0.79 0.79 0.8 0.9 1 0.89 0.86 0.74 0.76 0.76 0.79 0.76 0.76 0.74 0.55 0.55 0.55 0.56 0.59 0.56 0.59 0.6 0.57 0.57 0.6 0.62 0.64 0.63 0.63 0.64 0.26 0.26 0.31 0.32

0.72 0.72 0.7 0.71 0.71 0.7 0.7 0.78 0.81 0.78 0.8 0.8 0.8 0.8 0.8 0.8 0.79 0.8 0.8 1 0.88 0.87 0.82 0.74 0.74 0.76 0.75 0.76 0.75 0.74 0.55 0.55 0.54 0.56 0.57 0.57 0.59 0.58 0.57 0.56 0.59 0.61 0.62 0.61 0.61 0.64 0.25 0.26 0.31 0.31

0.76 0.75 0.73 0.74 0.74 0.74 0.72 0.8 0.83 0.8 0.82 0.82 0.82 0.82 0.83 0.83 0.82 0.82 0.82 0.78 0.77 0.8 0.81 0.83 0.87 0.92 0.91 0.92 0.97 1 0.59 0.59 0.59 0.6 0.62 0.6 0.62 0.62 0.6 0.6 0.63 0.64 0.65 0.64 0.64 0.65 0.27 0.27 0.33 0.34

0.75 0.75 0.72 0.73 0.73 0.74 0.72 0.79 0.78 0.79 0.81 0.81 0.81 0.81 0.81 0.81 0.8 0.81 0.81 0.77 0.76 0.79 0.8 0.83 0.86 0.91 0.9 0.91 1 0.94 0.57 0.58 0.58 0.59 0.6 0.59 0.61 0.61 0.6 0.59 0.63 0.64 0.64 0.63 0.63 0.64 0.27 0.27 0.34 0.34

0.75 0.75 0.73 0.73 0.73 0.74 0.72 0.79 0.82 0.79 0.81 0.81 0.81 0.81 0.81 0.81 0.81 0.81 0.81 0.77 0.75 0.79 0.78 0.82 0.84 0.95 1 1 0.9 0.88 0.58 0.57 0.57 0.59 0.61 0.58 0.61 0.61 0.59 0.58 0.63 0.63 0.65 0.64 0.64 0.65 0.28 0.28 0.33 0.34

0.75 0.74 0.73 0.73 0.73 0.74 0.73 0.79 0.82 0.79 0.81 0.81 0.8 0.81 0.81 0.81 0.8 0.81 0.81 0.77 0.79 0.78 0.78 0.82 0.85 0.96 1 1 0.9 0.88 0.58 0.57 0.57 0.59 0.6 0.58 0.61 0.61 0.59 0.58 0.63 0.63 0.65 0.64 0.64 0.65 0.28 0.28 0.33 0.33

0.75 0.75 0.72 0.72 0.73 0.74 0.72 0.79 0.82 0.79 0.8 0.81 0.8 0.81 0.81 0.81 0.8 0.8 0.8 0.77 0.75 0.78 0.79 0.82 0.85 1 0.95 0.95 0.9 0.88 0.58 0.57 0.57 0.59 0.6 0.59 0.61 0.61 0.59 0.59 0.63 0.63 0.64 0.63 0.63 0.64 0.28 0.27 0.33 0.34

0.77 0.76 0.74 0.75 0.75 0.76 0.75 0.8 0.84 0.81 0.83 0.83 0.82 0.83 0.83 0.83 0.82 0.82 0.83 0.79 0.79 0.8 0.81 0.82 1 0.89 0.88 0.88 0.89 0.87 0.58 0.6 0.6 0.59 0.61 0.6 0.61 0.61 0.6 0.6 0.64 0.65 0.65 0.64 0.66 0.66 0.29 0.28 0.35 0.35

0.77 0.76 0.75 0.75 0.75 0.76 0.74 0.93 1 1 1 1 0.99 1 1 1 1 0.99 1 0.81 0.79 0.82 0.82 0.78 0.79 0.81 0.8 0.81 0.8 0.79 0.58 0.58 0.58 0.58 0.59 0.59 0.61 0.61 0.59 0.59 0.62 0.65 0.65 0.64 0.65 0.65 0.27 0.27 0.32 0.32

0.77 0.76 0.75 0.75 0.75 0.76 0.73 0.93 1 0.99 1 1 0.99 1 1 1 1 0.99 1 0.81 0.79 0.82 0.82 0.78 0.79 0.81 0.8 0.81 0.8 0.79 0.58 0.58 0.58 0.58 0.6 0.59 0.61 0.61 0.59 0.59 0.62 0.65 0.65 0.64 0.65 0.65 0.27 0.27 0.32 0.32

0.77 0.77 0.75 0.75 0.75 0.76 0.73 0.93 1 1 1 1 1 1 1 1 0.99 1 1 0.81 0.79 0.82 0.82 0.78 0.79 0.81 0.8 0.81 0.8 0.79 0.58 0.58 0.58 0.58 0.59 0.59 0.61 0.61 0.59 0.59 0.62 0.65 0.65 0.64 0.65 0.65 0.27 0.27 0.32 0.32

0.78 0.77 0.75 0.75 0.75 0.76 0.73 0.94 0.98 0.99 1 1 0.99 1 1 1 0.99 1 1 0.81 0.79 0.83 0.82 0.77 0.79 0.8 0.8 0.81 0.8 0.79 0.58 0.58 0.58 0.58 0.59 0.59 0.61 0.61 0.59 0.59 0.62 0.65 0.65 0.64 0.65 0.64 0.27 0.27 0.32 0.32

0.77 0.76 0.74 0.75 0.75 0.75 0.73 0.93 0.97 0.99 0.99 0.99 0.99 1 1 1 0.99 1 1 0.81 0.78 0.82 0.82 0.77 0.79 0.8 0.8 0.81 0.8 0.78 0.58 0.58 0.58 0.58 0.59 0.59 0.61 0.61 0.59 0.59 0.62 0.64 0.64 0.63 0.64 0.65 0.27 0.27 0.32 0.32

0.77 0.76 0.74 0.74 0.75 0.75 0.73 0.93 0.96 0.98 0.98 0.99 0.98 0.99 0.99 0.99 0.98 1 1 0.8 0.78 0.82 0.82 0.77 0.78 0.8 0.8 0.8 0.8 0.78 0.57 0.58 0.58 0.57 0.59 0.58 0.61 0.61 0.59 0.58 0.62 0.64 0.64 0.63 0.64 0.64 0.27 0.27 0.32 0.32

0.77 0.76 0.74 0.74 0.74 0.75 0.73 0.93 0.98 1 1 1 1 1 1 1 1 1 1 0.8 0.78 0.81 0.81 0.77 0.78 0.79 0.79 0.8 0.79 0.78 0.57 0.58 0.58 0.57 0.59 0.58 0.6 0.6 0.59 0.58 0.61 0.64 0.64 0.63 0.64 0.63 0.27 0.27 0.31 0.32

0.77 0.77 0.75 0.75 0.75 0.76 0.73 0.94 1 1 1 1 1 1 1 1 0.99 1 1 0.82 0.79 0.83 0.82 0.78 0.79 0.81 0.8 0.81 0.8 0.79 0.58 0.58 0.59 0.58 0.6 0.59 0.61 0.61 0.59 0.59 0.62 0.65 0.65 0.64 0.65 0.65 0.27 0.27 0.32 0.32

0.78 0.77 0.75 0.75 0.75 0.76 0.74 0.94 1 1 1 1 1 1 1 1 1 1 1 0.82 0.79 0.83 0.82 0.78 0.79 0.81 0.8 0.81 0.8 0.79 0.58 0.58 0.59 0.58 0.6 0.59 0.61 0.61 0.59 0.59 0.62 0.65 0.65 0.64 0.65 0.65 0.27 0.27 0.32 0.32

0.77 0.77 0.75 0.75 0.75 0.76 0.75 0.94 1 1 1 1 1 1 1 1 1 1 1 0.82 0.82 0.84 0.82 0.78 0.79 0.81 0.8 0.82 0.8 0.79 0.58 0.58 0.59 0.58 0.6 0.59 0.61 0.61 0.59 0.59 0.62 0.65 0.65 0.64 0.65 0.65 0.27 0.27 0.32 0.32

0.77 0.76 0.74 0.74 0.74 0.75 0.73 1 0.93 0.94 0.95 0.95 0.95 0.96 0.96 0.96 0.95 0.95 0.96 0.81 0.78 0.82 0.82 0.77 0.78 0.8 0.8 0.8 0.8 0.78 0.58 0.58 0.58 0.58 0.59 0.59 0.61 0.61 0.59 0.59 0.62 0.65 0.64 0.63 0.65 0.64 0.27 0.27 0.32 0.32

0.78 0.83 0.75 0.76 0.76 0.8 0.78 0.95 1 1 1.1 1.1 1.1 1 1 1 1 1 1 0.86 0.83 0.84 0.87 0.83 0.83 0.85 0.85 0.86 0.81 0.83 0.58 0.58 0.58 0.58 0.63 0.63 0.65 0.65 0.59 0.58 0.67 0.64 0.64 0.64 0.64 0.65 0.28 0.31 0.32 0.35

0.0

0.2

0.4

0.6

0.8

1.0

AN

Im_h

adam

ard


pyani software a

ahttp://widdowquinn.github.io/pyani

Python ANImodule andscript

Activedevelopment

Paralleliseson clusters

Preprintcoming soon(simulatedgenomes; lotsof bacterialtaxonomy!)

http://widdowquinn.github.io/pyani

Table of Contents






6 Acknowledgements

qPCR Primer Design

With ‘correct’ classifications, we can design accurate diagnostics

targets

o�-targets

classi�cation

V

IV

III

II

I

genomes

IIIIIIIVV

Dickeya primer evaluation a

aPritchard et al. (2013) Plant Path. doi:10.1111/j.1365-3059.2012.02678.x

Primers designed to 29 sequenced Dickeya isolatesEvaluated against panel of 70 unseen isolates100% sensitivity; 0-4% FDR

http://dx.doi.org/10.1111/j.1365-3059.2012.02678.x

E. coli diagnostic primers a b

ahttps://github.com/ehec-outbreak-crowdsourced/BGI-data-analysis/wiki

bKwan et al. (2011) http://precedings.nature.com/documents/6663/version/1

Summer 2011, E. coli EHEC O104:H4 outbreak, Germany

3950 affected, 53 deaths; rapid production of sequence data,crowd-sourcing of analyses

https://github.com/ehec-outbreak-crowdsourced/BGI-data-analysis/wiki

http://precedings.nature.com/documents/6663/version/1

E. coli primer evaluation a

aPritchard et al. (2012) PLoS One doi:10.1371/journal.pone.0034498

Primers designed to nine crowdsourced draft outbreak E. coliO104:H4 assemblies

21 clinical outbreak, 32 HUSEC/EPEC isolates

Combined primers specific at sub-serotype level100% sensitivity, 9-22% FDR for individual primers; 100% specificity and sensitivity for paired primers

http://dx.doi.org/10.1371/journal.pone.0034498

find differential primers a

ahttp://widdowquinn.github.io/find differential primers/

Software freely available at GitHub

Parallises across cluster

http://widdowquinn.github.io/find_differential_primers/

Table of Contents






6 Acknowledgements

Comparative metabolism a b

aBiopython KGML/KEGG visualisation module

bhttps://github.com/widdowquinn/Notebooks-Bioinformatics

Metabolic potential is a clue to community function & host range

https://github.com/widdowquinn/notebooks/blob/master/Biopython_KGML_intro.ipynb

https://github.com/widdowquinn/Notebooks-Bioinformatics

Differential Metabolic Capacity

Annotated metabolic capacitypredicts host range

Growth curves confirm predicted

carbon use

Transposon mutant grids for 17

sequenced Dickeya

Dynamic Metabolic Modelling

Flux Balance Analysis modelsfor sequenced Dickeya

whole-organism metabolic fluxprediction of KO effectssubstrate usage/productionflux optimisation (SynBio)

Potential Influence

Systems-level understanding of environmental interactions ofplant pathogens

Funding

RESAS 5-year programme

Table of Contents






6 Acknowledgements

Food or Fuel? a

aMohr & Rahman et al. (2013) Energy Policy doi:10.1016/j.enpol.2013.08.033

Biofuels: ”Riches to Rags”1st generation: fuel from food crops2nd generation: fuel from cellulosic crops, e.g. miscanthus,willowStealing food, or stealing land/water?

http://dx.doi.org/10.1016/j.enpol.2013.08.033

Food and Fuel? a

aMohr & Rahman et al. (2013) Energy Policy doi:10.1016/j.enpol.2013.08.033

Waste material = carbon-neutral feedstock

Agricultural waste as feedstock?

2nd generation fuel from food crops?

Maize stover, straw, sugarcane bagasse, etc.

http://dx.doi.org/10.1016/j.enpol.2013.08.033

Processing Waste a b c

aBeall & Ingram (1993) J. Indust. Micro. 11:151-155

bZhou et al. (1999) Appl. Environ. Microbiol. 65:2439-2445

cEdwards et al. (2011) Appl. Environ. Microbiol. doi:10.1128/AEM.05700-11

Soft rot pathogens

(PCWDEs): hydrolases and lyases

Engineer pathogens for ethanol production? a

Express PCWDEs in ethanologenic E. coli? b,c

PCWDE libraries for synthetic biology?

SynBio automated platforms for engineered microbial pathways(e.g. Cellulect, UoEdinburgh)understanding enzyme structure-function relationships

http://link.springer.com/article/10.1007/BF01583716

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC91359/

http://dx.doi.org/10.1128/AEM.05700-11

CAZy a

aLombard et al. (2014) Nucl. Acids Res. doi:10.1093/nar/gkt1178

CAZy: Carbohydrate-Active Enzymes database (http://www.cazy.org/)

5 Dickeya, 14 Erwinia, 9 Pectobacterium genomes

63 Dickeya, 66 Erwinia, 74 Pectobacterium families

Survey/mine natural diversity of CAZymes

http://dx.doi.org/10.1093/nar/gkt1178

http://www.cazy.org/

Pathogen Diversity a

aPritchard et al. (2016) Anal. Methods doi:10.1039/C5AY02550H

48 Dickeya, 38 Erwinia, 57 Pectobacterium genomesSurvey/mine natural diversity of PCWDEs: ‘evolvability’

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Pectobacterium_atrosepticum_SCRI1043_uid57957Pectobacterium_atrosepticum_NCPPB8549Pectobacterium_atrosepticum_NCPPB3404Pectobacterium_atrosepticum_21APectobacterium_atrosepticum_JG10-08Pectobacterium_carotovorum_PC1_uid59295Pectobacterium_carotovorum_subsp_carotovorum_NCPPB312Pectobacterium_carotovorum_subsp_oderiferum_NCPPB3841Pectobacterium_carotovorum_subsp_oderiferum_NCPPB3839Pectobacterium_carotovorum_subsp_carotovorum_NCPPB3395Pectobacterium_carotovorum_PCC21_uid174335Pectobacterium_carotovorum_subsp_brasiliensis_B5Pectobacterium_carotovorum_subsp_brasiliensis_B4Pectobacterium_betavasculorum_NCPPB2293Pectobacterium_betavasculorum_NCPPB2795Pectobacterium_wasabiae_NCPPB3702Pectobacterium_wasabiae_NCPPB3701Pectobacterium_wasabiae_WPP163_uid41297Pectobacterium_SCC3193_uid193707Dickeya_solani_AMYI01Dickeya_solani_AMWE01Dickeya_solani_GBBC2040Dickeya_solani_IPO2222Dickeya_solani_MK16Dickeya_solani_MK10Dickeya_dianthicola_NCPPB_3534Dickeya_dianthicola_GBBC2039Dickeya_dianthicola_NCPPB_453Dickeya_dianthicola_IPO980Dickeya_spp_NCPPB_3274Dickeya_spp_MK7Dickeya_dadantii_NCPPB_2976Dickeya_dadantii_NCPPB_898Dickeya_dadantii_NCPPB_3537Dickeya_dadantii_3937_uid52537Pantoea_ananatis_AJ13355_uid162073Pantoea_ananatis_LMG_20103_uid46807Pantoea_ananatis_PA13_uid162181Pantoea_ananatis_uid86861Erwinia_amylovora_CFBP1430_uid46839Erwinia_amylovora_ATCC_49946_uid46943Erwinia_Ejp617_uid159955Erwinia_pyrifoliae_Ep1_96_uid40659Erwinia_pyrifoliae_DSM_12163_uid159693Dickeya_dadantii_Ech703_uid59363Dickeya_paradisiaca_NCPPB_2511Dickeya_aquatica_DW_0440Dickeya_aquatica_CSL_RW240Erwinia_tasmaniensis_Et1_99_uid59029Pantoea_At_9b_uid55845Pantoea_vagans_C9_1_uid49871Erwinia_billingiae_Eb661_uid50547Dickeya_zeae_APMV01Dickeya_zeae_AJVN01Dickeya_zeae_CSL_RW192Dickeya_zeae_NCPPB_3531Dickeya_dadantii_Ech586_uid42519Dickeya_zeae_APWM01Dickeya_zeae_NCPPB_2538Dickeya_zeae_MK19Dickeya_zeae_NCPPB_3532Dickeya_spp_NCPPB_569Dickeya_chrysanthami_NCPPB_402Dickeya_chrysanthami_NCPPB_516Dickeya_zeae_Ech1591_uid59297Dickeya_chrysanthami_NCPPB_3533

0.00

0.25

0.50

0.75

1.00

AN

Im_p

erc

enta

ge_i

denti

ty

http://dx.doi.org/10.1039/C5AY02550H

Protein Structures a b

aChapon et al. (2001) J. Mol. Biol. doi:10.1006/jmbi.2001.4787

bLarson et al. (2003) Biochem. doi:10.1021/bi034144c

Several landmark enzyme structures from Dickeya

First GH5 xylanase structure (1NOF)Cel5 (1EGZ)

Obtain novel structures for Dickeya CAZymes

http://dx.doi.org/10.1006/jmbi.2001.4787

http://dx.doi.org/10.1021/bi034144c

Generate Diversity

Gene shuffling/directed evolutionSaturating site-directed mutagenesis

Functional space theoretically available to enzyme family

structure/sequence

Functional space explored

in nature

Gene Shuffling a

aCrameri et al. (1998) Nature doi:10.1038/34663

Generate novel diversity and select for substrate specificity

http://dx.doi.org/10.1038/34663

Positional epistasis a

aMcLaughlin et al. (2012) Nature doi:10.1038/nature11500

Context-dependence of mutation and function: epistasis“hotspots”/pathways for control of substrate specificity

Saturated single substitutions, with substrate assay screens

http://dx.doi.org/10.1038/nature11500

Protein Sectors a b

aPritchard & Dufton (2000) J. Theor. Biol. doi:10.1006/jtbi.1999.1043

bHalabi et al. (2009) Cell doi:10.1016/j.cell.2009.07.038

Sequence diversity and protein structure enables:

Correlated mutation/conservation analysisDecomposition of structure into ”sectors”

Sectors: subdomain structural/functional organisation ofproteins

http://dx.doi.org/10.1006/jtbi.1999.1043

http://dx.doi.org/10.1016/j.cell.2009.07.038

Anticipated Outputs

Libraries of carbohydrate-processing enzymes for SynBio

Survey of natural PCWDE diversityNovel specificity variants from gene shufflingSaturated site-specific mutagenesis libraries for screeningagainst novel substratesIP?

Structure-function insight

New PCWDE enzyme structuresSector and epistasis maps for PCWDEsReverse-engineering of carbohydrate-processing enzymes

Empirically-improved enzymes

Gene-shuffling targeted to novel specificityForward-engineering of carbohydrate-processing enzymesIP?

Acknowledgements

Dickeya/Erwinia/PectobacteriumSteve Baeyen (ILVO)Emma Campbell (JHI)Sean Chapman (JHI)John Elphinstone (Fera)Tracey Gloster (St Andrews)Rachel Glover (Fera)Sonia Humphris (JHI)Martine Maes (ILVO)Katrin Mackenzie (BioSS)Neil Parkinson (Fera)Minna Pirhonen (Helsinki)Gerry Saddler (SASA)Elaine Shemilt (Duncan ofJordanstone)Ian Simpson (Edinburgh)Ian Toth (JHI)Jan van der Wolf (Wageningen)Johan van Vaerenbergh (ILVO)Frank Wright (BioSS)Eirini Xemantilotou (StAndrews/JHI)NematodesPeter Cock (JHI)John Jones (JHI/St Andrews)Robbie Rae (John Moores)Peter Thorpe (JHI)

PhytophthoraMiles Armstrong (Dundee)Anna Avrova (JHI)Jim Beynon (Warwick)Paul Birch (Dundee)David Cooke (JHI)Kath Denby (York)Eleanor Gilroy (JHI)Sarah Green (Forest Research)Edgar Huitema (Dundee)Rory McLean (Dundee)Hazel McLellan (Dundee)Sophien Kamoun (TSL)Gail Preston (Oxford)Paul Sharp (Edinburgh)Jens Steinbrenner (Warwick)Pieter van West (Aberdeen)Steve Whisson (JHI)Computational and SystemsBiologyDavid Broadhurst (EdithCowan)Peter Cock (JHI)Mark Dufton (Strathclyde)Roy Goodacre (Manchester)Douglas Kell (Manchester)David Martin (Dundee)Iain Milne (JHI)Pedro Mendes (Manchester)

E. coli/other bacteriaFlorence Abram (Galway)Martina Bielaszewska (Muenster)Fiona Brennan (Galway)Ken Forbes (Aberdeen)Nicola Holden (JHI)Ashleigh Holmes (JHI)Paul Hoskisson (Strathclyde)Helge Karch (Muenster)Norval Strachan (Aberdeen)David Studholme (Exeter)Nick Waters (Galway)Metagenomics/CommunitiesNatalie Ferry (Salford)Thomas Freitag (JHI)Ryan Joynson (Liverpool)John Mitchell (St Andrews)Les Noble (Aberdeen)Jim Prosser (Aberdeen)V Anne Smith (St Andrews)PotatoMicha Bayer (JHI)Glenn Bryan (JHI)Graham Etherington (TSL)Ingo Hein (JHI)Florian Jupe (JHI)Jonathan Jones (TSL)Dan Maclean (TSL). . .and many others. . .

Licence: CC-BY-SA

By: Leighton Pritchard

This presentation is licensed under the Creative CommonsAttribution ShareAlike licensehttps://creativecommons.org/licenses/by-sa/4.0/

https://creativecommons.org/licenses/by-sa/4.0/