III Workshop Comparative Microbial Genomics & Taxonomy Forest Rohwer, SDSU (USA)

1. III Workshop Comparative Microbial Genomics & Taxonomy Forest Rohwer, SDSU (USA) Tom Coenye, Ghent University (Belgium)

2. IV Workshop Comparative Microbial Genomics and Taxonomy

3. Attendees & Background

4. General info • Morning Coffee-break – first floor • Labs 5 and 6 • Certificates on Friday • Program

5. Program

6. 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)

7. Why CMGT? • Taxonomy • Underpins of Biology • Biodiversity • Comparative Genomics • Whole genome sequences • Metagenomics

8. Bacterial taxonomy Historical phases?

9. NumericalTaxonomy 1950s-1960s. Sneath & Sokal 1962. NumericalTaxonomy. Phenotypic data (100 to 200 caracteres) and constructionofdendrogramswiththe help ofcomputers. More objectivetaxonomicschemes. PolyphasicTaxonomy GenomicTaxonomy Bacterial taxonomy

11. Some (positive) consequences of the numerical 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

12. Some (negative) consequences of the numerical taxonomy

13. Some (negative) consequences of the numerical taxonomy

14. Polyphasic Taxonomy 1970. New paradigma Rita R. Cowell integration of information from the molecular to the ecological level • DNA-DNA hybridization

16. 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.

17. 100 98 % Similarity seq. 16S 96 94 20 40 60 80 100 % Similarity HDD 16S and DDH in polyphasic taxonomy > 70 % simil. HDD > 97 % 16S similarity New species < 97 % simil. 16S

18. Taxonomic resolution Strain Species Genus Family Supra-Family RFLP FAME LFRFA % G+C tDNA-PCR SEROLOGY SDS - PAGE RIBOTYPING ZYMOGRAMS DNA PROBES DNA SEQUENCING rRNA 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

19. AFLP - Principle (De Vos et al., 1994; Janssen et al., 1995)

21. Electroforeses Band pattern normalization

22. Similarity matrix calculation and dendrogram building

23. Genomic diversity 0 20 40 60 80 100 0 20 40 60 80 100 A1 A40-V. cholerae A41-V. mimicus A42-V. parahaemolyticus A43-V. pectenicida A2 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 A66 A33 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.

24. 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 Related to V. splendidus A47 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 Related to V. harveyi/V. campbellii A31 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.

25. Ubiquitous Infections in corals And in other marine organisms Why to care about species descriptions?

26. M 25°C 28°C

27. Phylogenetic position of V. coralliilyticus

30. Species description

31. Abrolhos Bank Nursery, goods and services, bioproducts

32. Extinction of the coral Mussismilia Year Francini-Filho, Moura, Leão, Thompson local + global impacts

33. Abrolhos: Endemic corals form chapeirões (“cogumelos”) Aprox. 70 % is Mussismilia braziliensis & M. hispida.

34. Mussismilia spp.

35. R-265 R-319 Vibrios of Corumbau, Porto Seguro and São Sebastião 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 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 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 V. coralliil. 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) NJ pyrH R-240 LMG19703T Vibrio shilonii LMG11258T Vibrio mediterranei ATCC7744 Vibrio fischeri R-231 0.05

36. Genomic taxonomy: MLSA Maiden et al. (1998) MLST • MLEE • 100 % reproducible • DB in WWW • Pop. genetics (clonal x panmitic)

37. 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. John Maynard Smith (1920-2004) Is this perception applicable to other bacterial groups?

38. Países Australia Bélgica Brasil China Equador Japão - Sawabe México - Gomez-Gil Nova Zelândia Filipinas Espanha Tailândia EUA Fonte Peixes Moluscos Corais Aprox. 70 anos Taxonomy of Vibrio harveyi N = 120 strains

39. 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 “Diagnostic” phenotypic features (%)

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

41. 1 2 3 4 5 6 7 8 9 1. LMG 4044T 2. CAIM 372 64·0 3. CAIM 128 60·9 96·4 4. CAIM 333 57·5 94·6 90·2 5. CAIM 415 64·9 99·0 90·4 92·9 6. CAIM 113 64·6 90·1 86·3 83·4 87·5 7. LMG 11216T 69·0 79·2 71·3 74·0 81·7 81·8 8. R-14899 59·2 94·2 91·9 89·7 93·0 86·0 78·0 9. LMG 20369 59·3 87·7 79·3 81·1 90·3 89·9 76·1 88·9 10. LMG 16835 64·9 87·0 80·4 80·7 91·4 82·7 77·6 80·4 85·6 Taxonomy of Vibrio harveyi DDH between V. harveyi & V. campbellii

42. >99.0% (B) pyrH LMG20977 LMG11216T DZ2 SDZ18 LMG4044T CAIM1766 CAIM1614 ODDZ10 R831 AS11 R827 TDZ13 AS131 S30 S20 0802 TEZ2 BZ62 0436 R826 R828 TEZ1 DZ3 R829 CAIM1500 CAIM1333 TK1091 DZ1 CAIM107 BAZ5 R259 DZ5 R825 CAIM1283 S35 ODEZ12 LMG20369 SDZ20 Wt11 CAIM1159 R376 CAIM1508 R300 R264 CAIM1761 CAIM198 CAIM1266 LMG19714 CAIM2 AS59 Wt44 R14902 CAIM1 CAIM155 ODDZ8 ODDZ2 CAIM700 SDZ14 SZD16 S92 wbz7 R14947 DZ6 AS93 SF2003 CAIM79 CAIM1074 R830 1847 AS71 ODDZ14 SDZ12 ODDZ6 CAIM973 CAIM1510 CAIM1511 LMG19643 CAIM1075 CAIM1173 CAIM3 CAIM149 LMG7890 CAIM105 LMG11256 CAIM372 CAIM9 >99.5% CAIM392 CAIM109 CAIM4 CAIM249 R633 CAIM134 CAIM1558 R14899 CAIM115 LMG16835 CAIM757 CAIM150 CAIM401 >99.0% R14913 LMG20370 1976 1977 1978 1000 0772 1979 >99.7% LMG21460T 1975 0.005 (A) topA >99.5% >96% >99.3% (D) mreB (C) ftsZ >99.0% >97.0% 100% >98.4% >99.0% Blue: V. harveyi; Red: V. campbellii; Green: V. rotiferianus

43. (F) gyrB (G) gapA (E) recA >98.5% >96.0% >99.0% 98.0% >94.0% >97.0% 100% >99.4% Blue: V. harveyi; Red: V. campbellii; Green: V. rotiferianus

46. MLSA: sampling of the bacterial genome Markers for identification and evolutionary inferences gyrB rpoA mreB ftsZ recA V. parahaemolyticus 3.3Mb pyrH gapA topA

49. Genomic taxonomy: whole genome of vibrios D. Ussery

50. Species Interspecies Intergenera Interspecies Species 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.