III Workshop Comparative Microbial Genomics & Taxonomy

Forest Rohwer, SDSU (USA)Tom Coenye, Ghent University (Belgium)

III Workshop Comparative Microbial Genomics & Taxonomy

Forest Rohwer, SDSU (USA)Tom Coenye, Ghent University (Belgium)

IV Workshop Comparative Microbial Genomics and Taxonomy

IV Workshop Comparative Microbial Genomics and Taxonomy

Attendees & Background

General info

• Morning Coffee-break – first floor

• Labs 5 and 6

• Certificates on Friday

• Program

Time Monday Tuesday Wednesday Thursday Friday

08.30-09.30

Opening - Fabiano Rob Rob Tom Presentation

09.30-10.30

David Ussery Rob Rob Tom preparation

10.30-11.00

Coffee break Coffee break Coffee break Coffee break Coffee break

11.00-12.30

Nei Pereira Jr. Tom Tom Rob Presentations

12.30-13.30

Lunch Lunch Lunch Lunch Lunch

13.30-14.30

Rob David Ussery David Ussery David Ussery Pesentations

14.30-15.30

Tom Computer exercise 2 Computer exercise 3 Computer exercise 4 Discussion

15.30-16.00

Coffee break Coffee break Coffee break Coffee break Coffee break

16.00-17.30

Computer exercise 1Computer exercise 2 cont.

Computer exercise 3 cont.

Computer exercise 4 cont.

17.30-19.00

Dinner Dinner Dinner Dinner

19.00-21.00

Student project Student project Student project Student project

Program

Faculty board

• Forest Rohwer, SDSU (USA)

Rob Edwards, SDSU (USA)

• Tom Coenye, Ghent University (Belgium)

• Dave Ussery, CBS-DTU (Denmark)

• Karin Lagesen, CBS-DTU (Denmark-Norway)

• Nei Pereira Jr, UFRJ (BR)

• Fabiano Thompson, UFRJ (BR)

Why CMGT?

• Taxonomy– Underpins of Biology– Biodiversity

• Comparative Genomics– Whole genome sequences– Metagenomics

Bacterial taxonomy

• Historical phases?

• Numerical Taxonomy – 1950s-1960s. – Sneath & Sokal 1962. Numerical Taxonomy.– Phenotypic data (100 to 200 caracteres) and

construction of dendrograms with the help of computers.

– More objective taxonomic schemes.

• Polyphasic Taxonomy

• Genomic Taxonomy

Bacterial taxonomy

90 % of the species described in the 7th edition of The Bergey’s manual (1957) were reclassified (1974) or removed from the list of valid names(Skerman et al., 1980)

1974

Some (positive) consequences of the numerical taxonomySome (positive) consequences of the numerical taxonomy

Some (negative) consequences of the numerical taxonomySome (negative) consequences of the numerical taxonomy

Some (negative) consequences of the numerical taxonomySome (negative) consequences of the numerical taxonomy

1970. New paradigmaRita R. Cowell

DNA-DNA hybridization

integration of information from the

molecular to the ecological level

Polyphasic TaxonomyPolyphasic Taxonomy

Wayne et al. (1987). Report of the Ad Hoc Committee on Reconciliation of Approaches to Bacterial Systematics. IJSB 37:453-464.

HDD becomes the “Gold Standard”Species definition: Strains of the same species show at least 70 % hibridizations between their genomes.

% S

imil

ari

ty s

eq

. 16S

% Similarity HDD

80604020 100

94

96

98

100

> 70 % simil. HDD > 97 % 16S similarity

< 97 % simil. 16S

New species

16S and DDH in polyphasic taxonomy16S and DDH in polyphasic taxonomy

Taxonomic Taxonomic resolutionresolution

Family

Species

Strain

Genus

Supra-Family

Tools

RFLP

LFRFA

RIBOTYPIN

G

AFLP, AP-P

CR, DAF, R

APD, ARDRA

PHAGE AND B

ACTERIOCIN

TYPING

SEROLOGY

ZYMOGRAMS

SDS - PAGE

DNA-DNA H

YBRIDIZATIO

NS

% G+C

tDNA-P

CR

POLYAMINES, Q

UINONES

FAME

CELL WALL S

TRUCTURE

PHENOTYPIC A

NALYSIS

DNA-rRNA H

YBRIDIZATIO

NS

rRNA SEQUENCIN

G

DNA PROBES

DNA SEQUENCIN

G

AFLPAFLP - Principle - PrincipleAFLPAFLP - Principle - Principle

(De Vos et al., 1994; Janssen et al., 1995)

Band pattern normalization

Electroforeses

Similarity matrix calculation and dendrogram building

100806040200

R-1586

A1A2

A3

A4

A5

A7-V. wodanis

A8

A9A10-L. pelagiaA11-V. logei

A12-V. cincinnatiensis

A13-V. nigr./V. orie.

A15-V. fisc./P. ilio.A16

A17-V. scophthalmi

A20-V. metschnikovii

A26A27-V. fluvialisA28A29-V. furnissii

A30

A31A32

A34A35-V. aestuarianus

A6-V. medi./V. shiloi

A21-V. gazo./V. salm.A22-S. costicola A23

A24-V. vulnificusA25-V. proteolyticus

A33

A36-V.harv./V. trac.

A37

A38-L. anguillarum

A39-V. ordaliiA69

A68

A67-V. halioticoli

A64A65

A66

A63-V. ichthyoenteri

V. aerogenes

A61

A60

A62-V. alginolyticus

A58-V. pena./V. rumo./V. tape.

A57-V. diazotrophicus

A59-V. tubiashii

A55

A54-V. myti./P. leio.

A56-P. angu./P. dams.

A52A51

A53

A48-V. natriegensA49-V. diabolicus

A50-V. splendidus

A45A46A47

A43-V. pectenicidaA42-V. parahaemolyticus

A44-V. nereis

A41-V. mimicusA40-V. cholerae

100806040200

A14-V. campbellii

R-3681

A18-V. navarrensisA19-P. phosphoreum

V. hollisae

Ward Dendrogram (Dice), 506 strains.

Genomic diversity

R-1586

A1A2

A3

A4

A5

A7-V. wodanis

A8

A9A10-L. pelagiaA11-V. logei

A12-V. cincinnatiensis

A13-V. nigr./V. orie.

A15-V. fisc./P. ilio.A16

A17-V. scophthalmi

A20-V. metschnikovii

A26A27-V. fluvialisA28A29-V. furnissii

A30

A31A32

A34A35-V. aestuarianus

A6-V. medi./V. shiloi

A21-V. gazo./V. salm.A22-S. costicolaA23

A24-V. vulnificusA25-V. proteolyticus

A33

A36-V.harv./V. trac.

A37

A38-L. anguillarum

A39-V. ordaliiA69

A68

A67-V. halioticoli

A64A65

A66

A63-V. ichthyoenteri

V. aerogenes

A61

A60

A62-V. alginolyticus

A58-V. pena./V. rumo./V. tape.

A57-V. diazotrophicus

A59-V. tubiashii

A55

A54-V. myti./P. leio.

A56-P. angu./P. dams.

A52A51

A53

A48-V. natriegensA49-V. diabolicus

A50-V. splendidus

A45A46A47

A43-V. pectenicidaA42-V. parahaemolyticus

A44-V. nereis

A41-V. mimicusA40-V. cholerae

A14-V. campbellii

R-3681

A18-V. navarrensisA19-P. phosphoreum

V. hollisae

Related to V. tubiashii

Related toV. harveyi/V. campbellii

Related to V. splendidus

Related toV. halioticoli

Related toG. hollisae

Unknown clusters

Related toV. pelagius

Related to V. proteolyticus

Genomic diversity

Ward Dendrogram (Dice), 506 strains.

Ubiquitous

Infections in corals

And in other marine

organisms

Why to care about species descriptions?

M 25°C 28°C

Phylogenetic position of V. coralliilyticusPhylogenetic position of V. coralliilyticus

Species descriptionSpecies description

Abrolhos Bank

Nursery, goods and services, bioproducts

Extinction of the coral Mussismilia

local + global impactsFrancini-Filho, Moura, Leão, Thompson

Year

Abrolhos: Endemic corals form chapeirões (“cogumelos”)

Aprox. 70 % is Mussismilia braziliensis & M. hispida.

Mussismilia spp.

R-265 R-319

Corumbau AMP-3 Mh 27D Corumbau AMP-3 Mh 27A P.dilatata saudvel+antib D7 R-329 R-322 R-309 R-300 R-666 R-292 R-304 R-318 R-326 P.dilatata saudvel+antib B1b P.dilatata saudvel+antib B1a

R-294 Corumbau AMP-3 Mh 27C R-288 R-303 R-313 R-325 R-635 P.dilatata saudvel D4 Corumbau AMP-3 Mh 27B Corumbau AMP-3 Mh 27E Corumbau Roi-Roi Mb 40B doente Corumbau Roi-Roi Mb 40E doente

R-296 R-302 R-299

R-301 R-1 R-228 R-232 R-234 R-235 R-262 R-263 R-283 R-284 R-290 R-293 R-306 R-310 R-312 R-314 R-315 R-316 R-317 R-321 R-323 R-324

R-308 LMG4409T Vibrio alginolyticus

R-320 R-295 R-298 R-331

P.dilatata doente A1 R-241

LMG2850T Vibrio parahaemolyticus LMG21460T Vibrio rotiferianus P.dilatata saudvel D1 P.dilatata saudvel D2

P.dilatata doente A2 R-330 LMG4044T Vibrio harveyi R-328 R-327 R-307 R-305 R-257 R-246 R-242 R-230

R-311 R-644 P.dilatata saudvel+antib D5

R-603 P.dilatata saudvel+antib D6

R-612 LMG11216T Vibrio campbelii

Corumbau AMP-2 Mh 35B R-233 P.dilatata doente C

Corumbau AMP-1 Mh 50A P.dilatata doente+antib A3

P.dilatata saudvel+antib D2 R-260

LMG20370 Vibrio harveyi R-227 R-239

R-280 P.dilatata saudvel+antib D4

R-248 P.dilatata doente+antib A2

Corumbau AMP-3 Mh 28A2 R-254

LMG21557T Vibrio fortis P.dilatata doente+antib A1

Corumbau AMP-1 Mb 45A R-252

LMG10936T Vibrio tubiashii P.dilatata saudvel+antib D3

P.dilatata doente+antib A4 LMG20984T Vibrio coralliilyticus

P. dilatata saudve+antib D1 P.dilatata doente+antib C2(2) P.dilatata doente+antib C1 P.dilatata doente+antib C3 P.dilatata doente+antib C2

LMG20536T Vibrio neptunius Corumbau Roi-Roi Mb 42B Corumbau Roi-Roi Mh 42B Corumbau AMP-1 Mb 45D Corumbau AMP-1 Mb 45B Corumbau AMP-1 Mb 45D(2)

R-240 LMG19703T Vibrio shilonii

LMG11258T Vibrio mediterranei ATCC7744 Vibrio fischeri

R-231

0.05

Vibrios of Corumbau, Porto Seguro and São Sebastião

A single genome may play diferent roles, mutualistic or pathogenic, modulated by environmental conditions.

NJ pyrH

V. coralliil.V. coralliil.

Maiden et al. (1998) MLST

MLEE

100 % reproducible

DB in WWW

Pop. genetics(clonal x panmitic)

Genomic taxonomy: MLSA

John Maynard Smith (1920-2004)

Maynard Smith et al. (2000) see the Neisseria species as clusters, partially differente, but sharing some identity with others via HGT and thus conclude that "there are no such entities as species in these pathogenic bacteria." Maynard Smith et al. (2000) also recomended a study of the genetic and phenotypic variation of a heterogenous taxon as Nesseria to be mandatory to phylosofers who believe in natural entities (species), for all cladistics who believe in the universal validity of phylogenetic classification, and for all´pheneticists, whatever they believe! In the end we are forced to adopt a pragmatic strategy, and see the Neisseria as a pool of clusters mutually compartilable.

Is this perception applicable to other bacterial groups?

Países

Australia

Bélgica

Brasil

China

Equador

Japão - Sawabe

México - Gomez-Gil

Nova Zelândia

Filipinas

Espanha

Tailândia

EUA

Fonte

Peixes

Moluscos

Corais

Aprox. 70 anos

N = 120 strains

Taxonomy of Vibrio harveyi

“Diagnostic” phenotypic features (%)

V. harveyi (n=9) V. campbellii (n=33) V. rotiferianus (n=5)

 -Cyclodextrin 100 15·2 100

cis-Aconitic acid 77·8 24·2 (6·1) 0

Citric acid 88·9 3·0 0

Glucose 6-phosphate 100 69·7 100

Hydroxy L-proline 55·6 3·0 0

N-Acetyl-D-galactosamine 88·9 12·1 (3·0) 0

Quinic acid 0 24·2 (3·0) 0

Sucrose 88·9 15·2 100

Taxonomy of Vibrio harveyi

Molecular identification of Vibrio harveyi-related isolates associated with diseased aquatic organisms

DDH between V. harveyi & V. campbellii

1 2 3 4 5 6 7 8 9

1. LMG 4044T

2. CAIM 372 64·03. CAIM 128 60·9

96·44. CAIM 333 57·

594·6

90·25. CAIM 415 64·

999·0

90·4

92·96. CAIM 113 64·

690·1

86·3

83·4

87·57. LMG 11216T 69·

079·2

71·3

74·0

81·7

81·88. R-14899 59·

294·2

91·9

89·7

93·0

86·0

78·09. LMG 20369 59·

387·7

79·3

81·1

90·3

89·9

76·1

88·910. LMG 16835 64·

987·0

80·4

80·7

91·4

82·7

77·6

80·4

85·6

Taxonomy of Vibrio harveyi

82

3O

DD

Z2

0.02

(A) topA (B) pyrH

CA

IM1

28

3

0.005

(C) ftsZ

0.01

(D) mreB

>98.4%

>99.3%

100%

>99.0%

>97.0%

>99.0%

>96%

>99.5%

>99.0%

LMG78

90wbz7

ODDZ14

AS93

SDZ1

2 R8

30LM

G196

43AS

71O

DDZ6

CAIM

1173

CAIM

973

1847

CAIM

1 07 5

CAIM

1511

CAIM

1510

DZ6

S92

CAIM

79SF

2003

R149

47

SZD16

SDZ14

CAIM70

0

ODDZ2ODDZ8CAIM1

Wt44AS59CAIM2LMG19714CAIM1266CAIM1761R264CAIM1508CAIM1159Wt11SDZ20ODEZ12S35

R825DZ5

R259

R831CAIM1333

CAIM107

TK1091BAZ5

DZ1TEZ1

TDZ13R826BZ62

AS1310436

ODDZ10

LMG

4044TCAIM

1766 S30

AS11

S20

SDZ1

8 R8

27LM

G209

77

0802

TEZ2

DZ3CAIM16

14DZ2

R828

R829

LMG

2036

9

CAIM198 CA

IM1 2

8 3LM

G11

216T

R376

R300 CA

IM15

00

R149

02

CAIM155

CAIM10

74

CAIM3

CAIM149

CAIM105LMG11256CAIM372CAIM9CAIM392

CAIM109CAIM249

CAIM1558

CAIM115

R14899CAIM

150CAIM

401CAIM

757R633CAIM134

CAIM4LMG16835

LMG

20370R14913

LMG

21460T

1975

1979

1976197807721000

1977

0.005

>99.5%

>99.0%

>99.7%

Blue: V. harveyi; Red: V. campbellii; Green: V. rotiferianus

R82

5S

30

1847

R82

8C

AIM

1508

CAIM401CAIM134

1976

0.005

(G) gapAAS

131

CAI

M17

61

SDZ18

SF2003

CAIM

107C

AIM1266

R828

LMG

7890

LMG

2146

0TC

AIM

1159

CAI

M11

73C

AIM

4

CAIM149

DZ2

CAIM1510CAIM1511TEZ2

OD

DZ10

SDZ16

0.01

(E) recA

OD

DZ8

OD

DZ6

CAIM973SDZ18

R82

5C

AIM

1511

SDZ2

0R

829

wbz7WT11

1979

1978

R376

0.01

(F) gyrB

98.0%

>96.0%

>94.0%

>99.0%

>98.5%

>97.0%>99.4%

100%

Blue: V. harveyi; Red: V. campbellii; Green: V. rotiferianus

MLSA: sampling of the bacterial genome Markers for identification and evolutionary inferences

ftsZ

topAgapA

pyrH

rpoAmreB

recA

V. parahaemolyticus3.3Mb

gyrB

Genomic taxonomy: whole genome of vibrios

D. UsseryD. Ussery

0

25

50

75

100

chole

rae-

chole

rae

algino

lyticu

s-alg

inolyt

icus

fisch

eri-f

ische

rim

imicu

s-m

imicu

spa

raha

emoly

ticus

-par

ahae

moly

ticus

vulni

ficus

-vuln

ificus

harv

eyi-h

arve

yich

olera

e-m

imicu

sca

mpb

ellii-h

arve

yialg

inolyt

icus-

para

haem

olytic

usalg

inolyt

icus-

harv

eyi

fisch

eri-s

almon

icida

algino

lyticu

s-vu

lnific

usha

rvey

i-vuln

ificus

chole

rae-

vulni

ficus

mim

icus-

vulni

ficus

para

haem

olytic

us-m

imicu

salg

inolyt

icus-

mim

icus

chole

rae-

para

haem

olytic

usalg

inolyt

icus-

chole

rae

harv

eyi-m

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san

gust

um-p

rofu

ndum

chole

rae-

harv

eyi

algino

lyticu

s-sa

lmon

icida

para

haem

olytic

us-s

almon

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algino

lyticu

s-fis

cher

ipa

raha

emoly

ticus

-fisc

heri

chole

rae-

fisch

eri

chole

rae-

salm

onici

dam

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s-sa

lmon

icida

vulni

ficus

-fisc

heri

vulni

ficus

-salm

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daha

rvey

i-fisc

heri

angu

stum

-fisc

heri

prof

undu

m-fi

sche

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m-s

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s-an

gust

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mch

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e-an

gust

umha

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mm

imicu

s-an

gust

umpa

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-ang

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mpa

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-pro

fund

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tum

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m

Species

Interspecies

Intergenera

Species

Interspecies

Red = AAI; yelloow = Karlin´s signature dissimilarity; Green= Usserys proteome. The taxonomic resolution of AAI is down to the intergenera level, whereas Karlin´s has a resolution at interspecies level.

Microbial Biodiversity & Taxonomy Group

Observation 1

Observation 2

observation 3

Bacterial Taxonomy is modeled through the observed features and measures of microrganisms

Polyphasic Taxonomy: applying the rules of the pragmatism

Recurrent clusters

IN

TER

AC

TIO

NIN

TER

AC

TIO

N R

ELIA

BILIT

YR

ELIA

BILIT

Y

LEVEL 1: DNA, rRNALEVEL 1: DNA, rRNA

Level 2: proteinsLevel 2: proteins

Level 3: chemotaxonomyLevel 3: chemotaxonomy

Nível 4: fenotipagemNível 4: fenotipagem

Sequencing, hibridization, tiping, %G+C

SDS-PAGE, serology, MLEE

FAME, PyMS, Poliamines

BIOLOG™, API, antibiograms, S & T tolerance

Taxonomic information

P

Gene repertoires in the tree of life.

Pangenomes

Estrategy MLST

Candidate genes?

Aprox. 200 genes (Lerat et al., 2003) in Proteobacteria.

Criteria: ubiquitous, single copy, resistant to HGT, informative, correlate with whole genome similarity (Zeigler, 2003).

recN (r² = .965, P <.001)

recN and thdF (r² = .986, P <.001)

recN, thdF and rpoA(r² = .989, P <.001)

Zeigler (2003) IJSEM 53:1893-1900

Gen

om

e S

imil

ari

ty

Similaridade AFLP

20 40 60 80 100

Sim

ilari

dade

HD

D

2040

6080

100

Polinomial regression of DDH X AFLP similarity (N=234).

CONCLUSION: AFLP is an alternative for the taxonomy of bacteria (for ex. Agrobacterium, Aeromonas, Bacillus, Burkholderia, Vibrios, Xanthomonas)

Genomic taxonomy: whole genomes

a) abcZ , b) adk , c) aroE , d) fumC , e) gdh , f) pdhC e g) pgm.