1. GENOMIC VARIABILITY WITHIN A POPULATION OF Alteromonasmacleodii FROM A SINGLE DEEPMEDITERRANEAN SAMPLEFrancisco Rodriguez-Valera

2. IntroduccinWelch et al, 2002Ussery, D 3. Human The great divide: constreined sex (a.k.a. meiosis) 70% Drosophila70% genes incommon 4. All this diversity only withdifferent alleles of the samegenes 5. Metagenome B +viriomeGlobal GenomeThe shape of prokaryotic biodiversity Metagenome A+ viriome Pan-genome species ASpecies A corePan-genome species BSpecies B coreSpecies C coreMetagenome C +viriome 6. Periodic-Selection (Atwood et al, 1951) The unit of selection: The clone? Giovannoni Nature 430: 515 (2004) 7. Periodic selection clonal sweep 8. How to reconcile PS with large pan-genomes? Study the genes found in theaccessory pool 9. Solar SalternEvapora.Carbonate Gypsum HaliteRatioDomain DomainDomainNoIntermediate (Vi/Vo) 36-70 g/l70-140 g/l 220-290 g/l>290 g/llife 140-220 g/l Diatoms Haloarchaea Cyanobacteria Haloquadratum Green Algae Sulphur Phototrophic Bacteria Artenia Salina walsbyiDunaliella up to 70-95% of the population Along many years: unique low GC content 10. H. walsbyi is a unique specieNum. Mbp over 95% id (in 70% length) = 398.8A 1009590%id858075 24 6810 12 14 16 182022 24 2628 30Haloquadratum walsbyi HSBQ001 chr. (3132494 bp)B CHouse-keeping genes Metagenome AtpB SecY Tef2 RadA Num. Seqs.Num. Seqs.%id398.8Mp%id 11. Environmental pan-genome of H. walsbyiGI 1GI 2GI 3 GI 4(promer)(BLASTN)H. Walsbyi (3132494 bp) 12. e-fosmids of H. walsbyiVariability of GI 1 Cell surface glycoprotein 100 90 80 70 605040 30 %sim. Cell surface adhesin Putative S-layer protein Dentin-sialophosphoprotrein 23-cyclic-nucleotide 2-phosphodiesterase IS element240631279189 Ig-like domain-containing proteinH.w. C23 Protease ABC-type cobalamin/iron(III)eHw-1Hmu2H.w. 239281312087 HBSQ001eHw-4eHw-7 eHw-559 eHw-5 eHw-12eHw-6 eHw-9 13. METAGENOMIC ISLANDS GOS marine metagenomes GENOMIC ISLANDSO-chain LPS Giant protein TransportersEXTRACELLULAR COMPONENTS IN GENOMIC ISLANDSPiliExo-polysaccharideOther Prochlorococcus marinus MED4Prochlorococcus marinus MIT9301 Pelagibacter ubique HTCC1062100%% sim.Pelagibacter ubique HTCC1062(1308759 bp) 50% Burkholderia sp. 383 chr.1Shewanella sp. MR-4 PiliO-chain LPS Giant Exo- Pili Transportersprotein polysaccharide related Pelagibacter ubique HTCC1002contigs100% Aeromonas hydrophila subs. hydrophila ATCC 7966 Synechococcus sp. WH812% sim. 50%Transmembrane/ O-chain LPSPili Respiratory system GiantOuter membrane TransportersFe transporter proteinproteinsPili Solar salter metagenomes Pelagibacter ubique HTCC7211100% Salinibacter ruber DSM 13855Haloquadratum walsbyi DSM 16790 100 100% sim. 9595Y Axis TitleY Axis Title 9090 858550% 8080O-chain LPSPili Zn transporter, PhosphonatePiliO-chain75Exo-polysaccharide75 Cell surfaceCell surfaceand Phosphate metabolism LPS 0500000 1000000 1500000 2000000 2500000 3000000 3500000 glycoproteins 0 5000001000000 15000002000000glycoproteins25000003000000 X Axis Title X Axis Title 14. Low recruiting genes (MGIs and islets) Coding for exposed features: O-chain of LPS,glycoproteins etc, pili, flagella Transporters Sensors and regulators (behaviour) Sub-niche specialization e.g. microaerophilicgrowth 15. Alteromonas macleodii Proteobacterium isolated by Baumannfrom surface waters near Hawaii in the60s Common isolate Heterotrophic aerobe, wide range ofsubstrates as C,E source, 2-3 m long,typical copiotroph r strategist Mostly found in the particulate fraction 16. 160 isolates on MA characterized by 16SrRNA sequencing , 28% A. macleodii2%Alteromonas macleodii DE strain genome4% 2% 2%Alteromonas sp. SequencedSample from6%Bacillus sp.1000 m deep5%28%Sulfitobacter sp.South Adriatic10%Rhodobacteraceae28%5% bacterium SCSWE048%Oceanobacillusiheyensis3 other strains17/05/2003; 22:14 R/V UraniaBottom ca. 1100 m sequenced now200 L retrieved by rossete(single cast) from 1000 m AD1000 Metagenomicfosmid library ca. 38,000clones (1.75 Gbp or 600prokaryotic genomes)Sampling site 17. Variation of A. macleodii in a single sample of 200 L If we consider that the cell density atthe location is 105 cells per ml andaccept the figure of 5% belonging toA. macleodii, in 200 liters we willhave about 5x108 cells of A.macleodii (about the population thatyou have in a common 100 mllaboratory culture) 18. End sequencing of ca 8000 fosmids 15968 individual reads 424 (2.6%) could be affiliated to A.macleodii DE (>98% nuc identity over300 nucs), average 99.04%PurificacinLpez-Garca 19. Alteromonas macleodii DE1x106 2x1063x106 4x10657,2 GC%44,933,40,16 GC%0 skew-0,16Microaerobic MetalFlagellins LPS O-ChainPeripheralextracellular Phage Nitrate respirationrespirationresistance Hg resistance Giant proteinLPS O-chainpolysaccharide CRISPR Hydrogenases(pEPS)eFosmids 6 MbNot AD1synthenic AD2rRNAstRNAs100%95%AD 180%100%English95%Channel67380%100%Balearic95%Sea 4580%100% Black95% Sea 1180% 20. 4216 Genes annotated in 182 fosmids of A. macleodii sequenced. Similarity#ORFs 100 1032 (24.47%) 98-1001837 (43.57%)BlastN 90-98 405 (9.60%) 80-90 132 (3.13%) 70-80 25 (0.59%) >cytoplasmic Acetyl CoA Diguanalate TransglycosilasecyclaseSTLSmr DomainABC Transporter carboxylasecontaining p. RTX ToxinDNA pol Cell cycle p.Kef-type K+ FGAMThrombospondin type 3 subunit MesJ transport system Synthetase repeat:OmpA/MotB Synonymous151 181 38 69 110 155 31 32 1216 28 1015 757531 33 Non-synymous 28 19 6 3 16 23 906 44103151862 Mer B,A,ReAmacleodiiAD1000-7-B08A. macleodii DE98% 98%60%78%83% 83%1.483.12199% 1.518.371A. macleodii DE HlyDSterol 3.203.823HKLuxRUmuC desaturaseMer B,A,RFliDFliCFlgL FlgK FlgJ FlgL FlgG FlgE FlgCFlgA FlgN Transposase FlaG FlgH FlgF FlgD FlgBFlgMCheR/VGI 9 32. Different exposed receptors, flagelins and theirglycosilation BUT ALSObehaviour at the level of flagellum synthesis i.e. thelineage represented by the sequenced strain and thatrepresented by fosmid eAmacleodii AD1000-39-B06could have different responses to environmental signalsthat either induce or block synthesis of the flagellum i.e.produce flagella under different conditions. 33. Constant-DiversityNature Reviews Microbiology ,November 2009 34. San Francisco Bay salt pond POM Alteromonas macleodii and CoCO2 + H2O 300 major organic compoundsProblem: Solution:only the transporters will to have manyoccupy the 3 Mpb of oneindividual cellDNA? lineages sharing the feast(3132494 pb)Problem: what about clonal sweeps? Solution:phages One lineage will be more efficient in consuming the limiting nutrient 35. Red Queen andConstant-Diversity 36. What does natural selection act upon genes,individuals, groups of individuals (such as a kin group), or the species as a whole? The unit of selection in the prokaryotic worldmight be a complex consortium that includes severalclonal cellular lineages sharing a core genome butdiffering in an adaptive gene complements thatallows the population a much more efficientexploitation of available resources. These pan-clonalpopulations are maintained equalized by phagepredation in an analogous way as the immunesystem in a mammal maintains in check tumors. Ifthis is so, phage populations should be consideredas belonging to the same selection unit. 37. Alicante (Spain),EvolutionaryUniversity Miguel HernndezGenomics GroupAna Beln Rohit GhaiPost-doc. Post-doc. Inma Garca Pre-doc.Carol Mizuno Aitor Gonzaga Pre-doc. Pre-doc.Sandra Rocamora Pre-doc. 38. Thank you very much! Alicante (Spain), University Miguel HernndezAitor GonzagaAna-BelnMartn-CuadradoRohit Ghai 39. Evapora.Carbonate Gypsum HaliteRatioDomain Intermediate Domain Domain NoBACTERIOME36-70 g/llife*Santa pola saltern : (Vi/Vo)70-140 g/l 140-220 g/l 220-290 g/l>290 g/lDNA Bulk DNA0.22 micronssp37, sp33, sp32, Time-series 5 microns extraction pyrosequencing1041 Mpb(400pb) *Torrevieja saltern: 145 MpbDiatomsParticles / small ArchaeaHaloarchaeaeukaryotes(& Bacteria)* Santa pola saltern:Cyanobacteria Metagenomic Precipitation sp37 clones Mg & KGreen AlgaeSulphur Phototrophic Bacteriafosmids libraryArtenia Salina(35 Kb)*Torrevieja saltern: 5952 clonesDunaliellaGI 1GI 2GI 3 GI 4 100 Haloquadratum walsbyi chromosome% id90 (3132494 bp)Nu. SNP80246 8 10 12 1416 18 202224262830Lengt (bp) 100 90 80 7060504030%sim. eHw-1 H. walsbyi DSM 16790 Cell surface glycoprotein Cell surface adhesin00 Putative S-layer protein Dentin-sialophosphoprotrein 1010 23-cyclic-nucleotide diesterase 2020 IS element C23 Ig-like domain 2406312791890 10000 20000 30000 010000 20000 30000 40000 50000 60000 70000 ProteaseeHw-4H. walsbyi C23 ABC-type cobalamin/iron(III)eHw-1 00 1010 HBSQ001 Hmu22020239281 3120870 10000 2000030000 4000001000020000 30000eHw-4eHw-5eHw-559 00 1010eHw-7 20200 10000 20000 30000 010000 2000030000eHw-559eHw-9 eHw-6 00eHw-510 10 20 200 10000 2000030000010000 2000030000eHw-12 eHw-7eHw-120 0eHw-6 1010 2020 eHw-90 010000 2000030000 1000020000 30000 40. OBJETIVEHOW MANY DIFFERENTHaloquadratum walsbyiclones exists? Core-genomehowdifferent Pan-genomeIn situ, from thedo they they are? of H. walsbyi metagenomevary alongtime? are equallyAccesory-genome distributed ? 41. What we have?: SequencesTwo genomes Haloquadratum walsbyi strains sequenced(in theory)HBSQ001 C234 SAGs of H.+ (2000)Sanger(2011) 454 pyro.walsbyi 3132494 bp3148034 bpMetagenomic collections of Santa Pola saltern :DNA Bulk DNA pyrosequencing (FLX, 400pb) 5 m0.22 m extraction sp37 (2008) sp33 (2010) sp32 (2007)prokariots time-series (2005)1041 Mpb+Torrevieja (Alicante) Saltern (2010) 145 MbpFosmids libraries. 33 Sequenced fosmidsAnalysis of 2947fosmid endsDNAMetagenomic fosmids library (35 Kb)5 m 0.22 m extractionsp (2002) 2000 clonessp37 (2008) 5952 clones23 fosmids (2006) (Sanger)prokariots Torrevieja saltern: 2880 clones10 fosmids GI1 (2009) FLX