III Workshop Comparative Microbial Genomics

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III Workshop Comparative Microbial Genomics
Taxonomy Forest Rohwer, SDSU (USA) Tom Coenye,
Ghent University (Belgium)
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IV Workshop Comparative Microbial Genomics and
Taxonomy
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Attendees Background
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General info

• Morning Coffee-break first floor
• Labs 5 and 6
• Certificates on Friday
• Program

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Program
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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)

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Why CMGT?

• Taxonomy
• Underpins of Biology
• Biodiversity
• Comparative Genomics
• Whole genome sequences
• Metagenomics

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Bacterial taxonomy

• Historical phases?

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Bacterial taxonomy

• 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

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Some (positive) consequences of the numerical
taxonomy
90 of the species described in the 7th edition
of The Bergeys manual (1957) were reclassified
(1974) or removed from the list of valid
names (Skerman et al., 1980)
1974
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Some (negative) consequences of the numerical
taxonomy
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Some (negative) consequences of the numerical
taxonomy
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Polyphasic Taxonomy
1970. New paradigma Rita R. Cowell
integration of information from the molecular to
the ecological level

• DNA-DNA hybridization

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Wayne et al. (1987). Report of the Ad Hoc
Committee on Reconciliation of Approaches to
Bacterial Systematics. IJSB 37453-464. HDD
becomes the Gold Standard Species definition
Strains of the same species show at least 70
hibridizations between their genomes.
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16S and DDH in polyphasic taxonomy
gt 70 simil. HDD gt 97 16S similarity
New species
lt 97 simil. 16S
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Taxonomic resolution
Strain
Species
Genus
Family
Supra-Family
RFLP
FAME
LFRFA
GC
tDNA-PCR
SEROLOGY
SDS - PAGE
RIBOTYPING
ZYMOGRAMS
DNA PROBES
rRNA SEQUENCING
DNA SEQUENCING
PHENOTYPIC ANALYSIS
CELL WALL STRUCTURE
POLYAMINES, QUINONES
Tools
DNA-DNA HYBRIDIZATIONS
DNA-rRNA HYBRIDIZATIONS
AFLP, AP-PCR, DAF, RAPD, ARDRA
PHAGE AND BACTERIOCIN TYPING
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AFLP - Principle
(De Vos et al., 1994 Janssen et al., 1995)
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Electroforeses
Band pattern normalization
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Similarity matrix calculation and dendrogram
building
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Genomic diversity
100
80
60
40
20
0
100
80
60
40
20
0
A1
A40-V. cholerae
A41-V. mimicus
A42-V. parahaemolyticus
A2
A43-V. pectenicida
A44-V. nereis
A3
A45
A46
A4
A47
A48-V. natriegens
A49-V. diabolicus
A5
A50-V. splendidus
A6-V. medi./V. shiloi
A51
A52
A7-V. wodanis
A53
A8
A54-V. myti./P. leio.
A55
A9
A10-L. pelagia
A56-P. angu./P. dams.
A11-V. logei
A57-V. diazotrophicus
A12-V. cincinnatiensis
A58-V. pena./V. rumo./V. tape.
A13-V. nigr./V. orie.
A14-V. campbellii
A15-V. fisc./P. ilio.
A16
A59-V. tubiashii
R-1586
A17-V. scophthalmi
A18-V. navarrensis
A19-P. phosphoreum
A20-V. metschnikovii
A60
A21-V. gazo./V. salm.
A23
V. hollisae
A22-S. costicola
A61
R-3681
A24-V. vulnificus
A25-V. proteolyticus
A26
A62-V. alginolyticus
A27-V. fluvialis
A28
A29-V. furnissii
A63-V. ichthyoenteri
A30
A64
A31
A65
V. aerogenes
A32
A33
A66
A34
A35-V. aestuarianus
A36-V.harv./V. trac.
A67-V. halioticoli
A37
A68
A69
A38-L. anguillarum
A39-V. ordalii
Ward Dendrogram (Dice), 506 strains.
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Genomic diversity
A1
A40-V. cholerae
A41-V. mimicus
A42-V. parahaemolyticus
A43-V. pectenicida
A2
A44-V. nereis
A3
Related to V. tubiashii
A45
A46
A4
A47
Related to V. splendidus
A48-V. natriegens
A49-V. diabolicus
A5
A50-V. splendidus
A6-V. medi./V. shiloi
A51
A52
A7-V. wodanis
Unknown clusters
A53
Related to V. pelagius
A8
A54-V. myti./P. leio.
A55
A9
A10-L. pelagia
A56-P. angu./P. dams.
A11-V. logei
A57-V. diazotrophicus
A12-V. cincinnatiensis
A58-V. pena./V. rumo./V. tape.
A13-V. nigr./V. orie.
Related to V. proteolyticus
A14-V. campbellii
A15-V. fisc./P. ilio.
A16
A59-V. tubiashii
R-1586
A17-V. scophthalmi
A18-V. navarrensis
A19-P. phosphoreum
A20-V. metschnikovii
A60
A21-V. gazo./V. salm.
V. hollisae
A22-S. costicola
A61
A23
R-3681
A24-V. vulnificus
A25-V. proteolyticus
A26
A62-V. alginolyticus
A27-V. fluvialis
Related to V. halioticoli
A28
A29-V. furnissii
A63-V. ichthyoenteri
A30
A64
A31
Related to V. harveyi/V. campbellii
A65
V. aerogenes
A32
A66
A33
A34
A35-V. aestuarianus
A36-V.harv./V. trac.
A67-V. halioticoli
Related to G. hollisae
A37
A68
A69
A38-L. anguillarum
A39-V. ordalii
Ward Dendrogram (Dice), 506 strains.
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Why to care about species descriptions?

• Ubiquitous
• Infections in corals
• And in other marine
• organisms

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M 25C 28C
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Phylogenetic position of V. coralliilyticus
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Species description
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Abrolhos Bank
Nursery, goods and services, bioproducts
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Extinction of the coral Mussismilia
Year
Francini-Filho, Moura, Leão, Thompson
local global impacts
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Abrolhos Endemic corals form chapeirões
(cogumelos)
Aprox. 70 is Mussismilia braziliensis M.
hispida.
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Mussismilia spp.
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R-265
R-319
Vibrios of Corumbau, Porto Seguro and São
Sebastião
Corumbau AMP-3 Mh 27D
Corumbau AMP-3 Mh 27A
P.dilatata saudvelantib D7
R-329
R-322
R-309
R-300
R-666
R-292
R-304
R-318
R-326
P.dilatata saudvelantib B1b
P.dilatata saudvelantib 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
A single genome may play diferent roles,
mutualistic or pathogenic, modulated by
environmental conditions.
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 saudvelantib D5
R-603
P.dilatata saudvelantib D6
R-612
LMG11216T Vibrio campbelii
Corumbau AMP-2 Mh 35B
R-233
P.dilatata doente C
Corumbau AMP-1 Mh 50A
P.dilatata doenteantib A3
P.dilatata saudvelantib D2
R-260
LMG20370 Vibrio harveyi
R-227
R-239
R-280
P.dilatata saudvelantib D4
R-248
P.dilatata doenteantib A2
Corumbau AMP-3 Mh 28A2
R-254
LMG21557T Vibrio fortis
P.dilatata doenteantib A1
Corumbau AMP-1 Mb 45A
R-252
LMG10936T Vibrio tubiashii
P.dilatata saudvelantib D3
P.dilatata doenteantib A4
LMG20984T Vibrio coralliilyticus
V. coralliil.
P. dilatata saudveantib D1
P.dilatata doenteantib C2(2)
P.dilatata doenteantib C1
P.dilatata doenteantib C3
P.dilatata doenteantib 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)
NJ pyrH
R-240
LMG19703T Vibrio shilonii
LMG11258T Vibrio mediterranei
ATCC7744 Vibrio fischeri
R-231
0.05
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Genomic taxonomy MLSA
Maiden et al. (1998) MLST

• MLEE
• 100 reproducible
• DB in WWW
• Pop. genetics
• (clonal x panmitic)

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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 allpheneticists,
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.
John Maynard Smith (1920-2004)
Is this perception applicable to other bacterial
groups?
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Taxonomy of Vibrio harveyi
N 120 strains
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Taxonomy of Vibrio harveyi
Diagnostic phenotypic features ()
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Molecular identification of Vibrio
harveyi-related isolates associated with diseased
aquatic organisms
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Taxonomy of Vibrio harveyi
DDH between V. harveyi V. campbellii

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(B) pyrH
gt99.0
(A) topA
gt99.5
gt96
(D) mreB
gt99.3
(C) ftsZ
gt99.0
gt97.0
100
gt98.4
gt99.0
Blue V. harveyi Red V. campbellii Green V.
rotiferianus
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(G) gapA
(F) gyrB
(E) recA
gt98.5
gt96.0
gt99.0
98.0
gt94.0
gt97.0
100
gt99.4
Blue V. harveyi Red V. campbellii Green V.
rotiferianus
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MLSA sampling of the bacterial genome Markers
for identification and evolutionary inferences
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Genomic taxonomy whole genome of vibrios
D. Ussery
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Red AAI yelloow Karlins signature
dissimilarity Green Usserys proteome. The
taxonomic resolution of AAI is down to the
intergenera level, whereas Karlins has a
resolution at interspecies level.
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Microbial Biodiversity Taxonomy Group
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Polyphasic Taxonomy applying the rules of the
pragmatism
Recurrent clusters
Bacterial Taxonomy is modeled through the
observed features and measures of microrganisms
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Taxonomic information
LEVEL 1 DNA, rRNA
Sequencing, hibridization, tiping, GC
Level 2 proteins
SDS-PAGE, serology, MLEE
INTERACTION
RELIABILITY
Level 3 chemotaxonomy
FAME, PyMS, Poliamines
Nível 4 fenotipagem
BIOLOG, API, antibiograms, S T tolerance
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P
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Pangenomes
Gene repertoires in the tree of life.
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Estrategy MLST
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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).
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recN (r² .965, P lt.001) recN and thdF
(r² .986, P lt.001) recN, thdF and
rpoA (r² .989, P lt.001)
Genome Similarity
Zeigler (2003) IJSEM 531893-1900
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Polinomial regression of DDH X AFLP similarity
(N234).
CONCLUSION AFLP is an alternative for the
taxonomy of bacteria (for ex. Agrobacterium,
Aeromonas, Bacillus, Burkholderia, Vibrios,
Xanthomonas)
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Genomic taxonomy whole genomes
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a) abcZ , b) adk , c) aroE , d) fumC , e) gdh ,
f) pdhC e g) pgm.