Dtu department seminar

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Dtu department seminar

  1. 1. GENOMIC VARIABILITY WITHIN A POPULATION OF Alteromonas macleodii FROM A SINGLE DEEP MEDITERRANEAN SAMPLEFrancisco Rodriguez-Valera
  2. 2. IntroducciónWelch et al, 2002 Ussery, D
  3. 3. Human The great divide: constreined sex (a.k.a. meiosis) 70% Drosophila70% genes in common
  4. 4. All this diversity only withdifferent alleles of the samegenes
  5. 5. Metagenome B +viriome Global GenomeThe shape of prokaryotic biodiversity Metagenome A+ viriome Pan-genome species A Species A core Pan-genome species B Species B core Species C core Metagenome C +viriome
  6. 6. Periodic-Selection (Atwood et al, 1951) The unit of selection: The clone? Giovannoni Nature 430: 515 (2004)
  7. 7. Periodic selection clonal sweep
  8. 8. • How to reconcile PS with large pan- genomes?• Study the genes found in the accessory pool
  9. 9. Solar SalternEvapora. Carbonate Gypsum Halite Ratio Domain Domain Domain No Intermediate (Vi/Vo) 36-70 g/l 70-140 g/l 220-290 g/l >290 g/l life 140-220 g/l Diatoms Haloarchaea Cyanobacteria Haloquadratum Green Algae Sulphur Phototrophic Bacteria Artenia Salina walsbyi Dunaliella up to 70-95% of the population Along many years: “unique” low GC content
  10. 10. H. walsbyi is a unique specie Num. Mbp over 95% id (in 70% length) = 398.8A 100 95 90%id 85 80 75 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 Haloquadratum walsbyi HSBQ001 chr. (3132494 bp)B C House-keeping genes Metagenome AtpB SecY Tef2 RadA Num. Seqs.Num. Seqs. %id 398.8Mp %id
  11. 11. Environmental pan-genome of H. walsbyi GI 1 GI 2 GI 3 GI 4 (promer) (BLASTN) H. Walsbyi (3132494 bp)
  12. 12. e-fosmids of H. walsbyi Variability of GI 1 Cell surface glycoprotein 100 90 80 70 60 50 40 30 %sim. Cell surface adhesin Putative S-layer protein Dentin-sialophosphoprotrein 23-cyclic-nucleotide 2-phosphodiesterase IS element 240631 279189 Ig-like domain-containing protein H.w. C23 Protease ABC-type cobalamin/iron(III) eHw-1 Hmu2 H.w. 239281 312087 HBSQ001 eHw-4 eHw-7 eHw-559 eHw-5 eHw-12 eHw-6 eHw-9
  13. 13. METAGENOMIC ISLANDS GOS marine metagenomes GENOMIC ISLANDS O-chain LPS Giant protein Transporters EXTRACELLULAR COMPONENTS IN GENOMIC ISLANDS Pili Exo-polysaccharide Other Prochlorococcus marinus MED4 Prochlorococcus marinus MIT9301 Pelagibacter ubique HTCC1062100%% sim. Pelagibacter ubique HTCC1062 (1308759 bp) 50% Burkholderia sp. 383 chr.1 Shewanella sp. MR-4 Pili O-chain LPS Giant Exo- Pili Transporters protein polysaccharide related Pelagibacter ubique HTCC1002 contigs100% Aeromonas hydrophila subs. hydrophila ATCC 7966 Synechococcus sp. WH812% sim. 50% Transmembrane/ O-chain LPS Pili Respiratory system Giant Outer membrane Transporters Fe transporter protein proteins Pili Solar salter metagenomes Pelagibacter ubique HTCC7211100% Salinibacter ruber DSM 13855 Haloquadratum walsbyi DSM 16790 100 100% sim. 95 95 Y Axis Title Y Axis Title 90 90 85 8550% 80 80 O-chain LPS Pili Zn transporter, Phosphonate Pili O-chain 75 Exo-polysaccharide 75 Cell surface Cell surface and Phosphate metabolism LPS 0 500000 1000000 1500000 2000000 2500000 3000000 3500000 glycoproteins 0 500000 1000000 1500000 2000000 glycoproteins 2500000 3000000 X Axis Title X Axis Title
  14. 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. microaerophilic growth
  15. 15. Alteromonas macleodii•  Proteobacterium isolated by Baumann from surface waters near Hawaii in the ‘60s• Common isolate• Heterotrophic aerobe, wide range of substrates as C,E source, 2-3 m long, typical copiotroph r strategist• Mostly found in the particulate fraction
  16. 16. 160 isolates on MA characterized by 16S rRNA sequencing , 28% A. macleodii 2% Alteromonas macleodii DE strain genome 4% 2% 2% Alteromonas sp. SequencedSample from 6% Bacillus sp.1000 m deep 5% 28% Sulfitobacter sp.South Adriatic 10% Rhodobacteraceae 28% 5% bacterium SCSWE04 8% Oceanobacillus iheyensis 3 other strains17/05/2003; 22:14 R/V UraniaBottom ca. 1100 m sequenced now200 L retrieved by rossete(single cast) from 1000 m AD1000 Metagenomic fosmid library ca. 38,000 clones (1.75 Gbp or 600 prokaryotic genomes) Sampling site
  17. 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. 18. End sequencing of ca 8000 fosmids• 15968 individual reads• 424 (2.6%) could be affiliated to A. macleodii DE (>98% nuc identity over 300 nucs), average 99.04% Purificación López-García
  19. 19. Alteromonas macleodii DE 1x106 2x106 3x106 4x106 57,2 GC% 44,9 33,4 0,16 GC% 0 skew -0,16 Microaerobic Metal Flagellins LPS O-Chain Peripheral extracellular Phage Nitrate respiration respiration resistance Hg resistance Giant protein LPS O-chain polysaccharide CRISPR Hydrogenases (pEPS)eFosmids 6 MbNot AD1synthenic AD2rRNAstRNAs 100% 95%AD 1 80% 100%English 95%Channel673 80% 100%Balearic 95%Sea 45 80% 100% Black 95% Sea 11 80%
  20. 20. 4216 Genes annotated in 182 fosmids of A. macleodii sequenced. Similarity #ORFs 100 1032 (24.47%) 98-100 1837 (43.57%)BlastN 90-98 405 (9.60%) 80-90 132 (3.13%) 70-80 25 (0.59%) <70 781 (18.52%) 80-100 4 60-80 33 BlastP 40-60 147 Regulatory and outer layer related genes increase 20-40 149 <20 454
  21. 21. 16S 23S
  22. 22. Peripheral extracellular polysaccharide (pEPS)Fosmid eAmacleodiiAD1000-21-D04 2,600,664 99,98 2,789,943Amacleodii DE 98,04 60 85 98,65 98,45Amacleodii AD1Fosmid eAmacleodii 100 100AD1000-5-F10Fosmid eAmacleodii 99,99AD1000-42-B04Fosmid eAmacleodiiAD1000-9-F08 Phenylalanyl tRNA synthetase α/β subunit Phosphodiesterase. Msc protein Mannose ybaK/ebsC p. (MscS family) O-antigen polymerase Hydrolase guanylyltransferaseFosmid eAmacleodii Fructosamine EPS biosynthesisAD1000-21-D04 Kinase Eps P/D 99,98Amacleodii DE 69 94 85Amacleodii AD1 Ribosomal L20,35 Transcription Phenylalanyl tRNA repair Sensor histide synthetase α/β subunit kinase β-hexoaminidase EPS Cluster Polysaccharide related p. Glycosyl transferase tRNA synthetase Hypothetical p. Periplasmic Transferases p. Ribosomal proteins Integrase, IS element
  23. 23. Different exposed structures e.g. exopolysaccharide
  24. 24. Fosmids containing Ribosomal Operon II Intervening Sequence tRNA 5S Fosmid eAmacleodii 16S 23S AD1000-207-B8 99,46 98,83 100 Amacleodii DE 99,06 98,31 99,18 Amacleodii AD1 Fosmid eAmacleodii 100 100 100 ATP-dependent Methyl-accepting AD1000-231-F10 helicase HrpB chemotaxis p. Methyl-accepting RNA CheY/A/W/D Synonymous 2 chemotaxis p. Ligase Non-synonymous 0 0Fosmid eAmacleodii Chemotaxis 4 Na+/H+ Peptidyl- MembraneAD1000-207-B8 methylesterase 1 1 antiporter dipeptidase Dcp transglycolase 99,78 98,77 99,98 99,23 99,96Amacleodii DE 423.708 520.976 99,7 99,21 98,01 98 99,38 99,67Amacleodii AD1 99,9 Aconitate Glycosyltransferase hydratase LipopolysaccharideFosmid eAmacleodii heptosyltransferaseAD1000-231-F10 Synonymous 3 Non-synonymous 2 7 34 2 22 5 4 8 42 0 15 43 22 22 20 17 2 2 1 32 2 8 1 1 2 0 1 Bacterial type II secretion 0 10 1 1 0 2 system protein E:GAF GGDEF Transcriptional Cryptic haloacid Permease DMT Repressor LacI Twin-arginine translocation Branched-chain amino dehalogenase Hydrolase/ Oxidoreductase pathway signal Acid aminotransferase phosphatase Two-component system Iron-containing alcohol Phosphoglyceromutase Sensor kinase deshydrogenase Trehalose Cluster 1 2 3 4 Ribosomal proteins Trehalose Cluster Pseudoalteromonas atlantica T6c Transferase proteins tRNA tblast TblastX 46 51 56 62 67 73 78 83 89 94 Chemotaxis related proteins x% ID Pseudoalteromonas tunicata D2 rRNA 1 transcriptional regulator Transposase/Integrase 2 trehalose permease Shewanella frigidimarina NCIMB400 Hypotetical protein 3 TonB-dependent OM receptor Glycosyl transferase 4 Trehalase
  25. 25. Fosmids containing Ribosomal Operon III Sensory BOX GGDEF Bacterio-phytochrome Lactate ClusterFosmid eAmacleodii 5S 23S 16SAD1000-249-G9 4 3 2 1Fosmid eAmacleodii 68 85 98 99 98 82 91AD1000-357-C11 99 99 99 4 3 2 1 85 98 98 82 80 98Amacleodii DE 4 99 68 99 98 98 98,2 81 96 85 97Amacleodii AD1 4Fosmid eAmacleodii 99 100 99 99 98 ChrR PseudouridineAD1000-112-F00 synthase D Adenine Lipoprotein ComL Recombination GGDEF domain TRP repeat phosphoribosyltransferase Adenylate kinase RecR p. DNA polymerase III CDP-diacylglycerol--serine Caseinolyti RNA polymerase Transcriptional regulator O-phosphatidyltransferase Chaperone HtpG subunits gamma and tau c peptidase sigma 70 factor B A. macleodii ATCC 4 3 2 1 Vibrio parahemolyticus 16 4 3 2 1 Psychromonas ingrahamii 37 3 2 rRNA Lactate Cluster tRNA Integrase, IS element 1 Hyphothetical protein 2 Lactate Responsive regulator Glycosyl transferase 3 Lactate permease 4 Lactate dehydrogenase Hypothetical p.
  26. 26. Different degradative capabilities eAmacleodii AD1000-207-B8 can use trehalose while DE cannot (checkedphenotypically)
  27. 27. Blastn Blastp Sulfotransferase Poliysaccharide UDP galactopyranose Motor P-ring Acyl CoA Flagellar M-ring Flagellar biosynthesis p. switch p. FlaA precursor DegT/J reductase FlaL Sensor biosynthesis p. Chemotaxis P-loop CheW/R FliC aminotransferase kinase FliM FlgH Cluster A/Z/Y FlaM/N FlgB/C/D/E FlgJ/K/L Flagellin FlaK FlaM CheW F/G/H Maf-1 FlaR FliC FliK FliO FliA FliEAD1000-39-B06Amacleodii AD1/ 98,9% 99~100%Fosmid eAmacleodii ~72%AD1000-9-A03 96% 82% ~60% 52% ~65% 80% 96% 99~100%Amacleodii DE/ AD3/ AD4 NeuB:Antifreeze- Acylneuraminate FliC. like p. cytidylyltransferase FliD Methionyl-tRNA formyltransferase FliC Amacleodii AD1/ AD1000-39-B06 72% 99% 98% 99% Fosmid eAmacleodii FlgL AD1000-9-A03 66% 81% 85% 99% FlgK Amacleodii DE/ AD3/ AD4 FlgE FlgD FlgF FlgG FliC FlaK FlaM FlgG FlaL Sensor Flagellum Cluster Glycosyl transferase Extracellular Hypothetical p. Regulatory Transferases p. Integrase, IS element Periplasmic Phage related Cytoplasmic tRNA
  28. 28. Vibrio parahaemolyticus FlaL FlaM FlaK
  29. 29. Synonymous 241FlaK Non-synonymous 49 81% Regulatory domain DNA binding domain
  30. 30. Synonymous 128 FlaL Non-synonymous 44 85%(sensor of two-component system) FlaL Sensory box TM1 Variable region in AD1000-39-B06
  31. 31. Flagellum cluster VariabilityExtracellular>Regulation>>cytoplasmic Acetyl CoA Diguanalate Transglycosilase cyclase STL Smr Domain ABC Transporter carboxylase containing p. RTX Toxin DNA pol Cell cycle p. Kef-type K+ FGAM Thrombospondin type 3 subunit α MesJ transport system Synthetase repeat:OmpA/MotB Synonymous 151 181 38 69 110 155 31 32 12 16 28 10 15 75 75 31 33 Non-synymous 28 19 6 3 16 23 9 0 6 4 4 10 3 15 18 6 2 Mer B,A,R eAmacleodii AD1000-7-B08 A. macleodii DE 98% 98% 60% 78% 83% 83% 1.483.121 99% 1.518.371A. macleodii DE HlyD Sterol 3.203.823 HKLuxR UmuC desaturase Mer B,A,R FliD FliC FlgL FlgK FlgJ FlgL FlgG FlgE FlgC FlgA FlgN Transposase FlaG FlgH FlgF FlgD FlgB FlgM CheR/V GI 9
  32. 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. 33. Constant-Diversity Nature Reviews Microbiology , November 2009
  34. 34. San Francisco Bay salt pond POM Alteromonas macleodii and Co CO2 + H2O 300 major organic compoundsProblem: Solution:only the transporters will to have manyoccupy the 3 Mpb of oneindividual cell DNA ? 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. 35. Red Queen andConstant-Diversity
  36. 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 world might be a complex consortium that includes several clonal cellular lineages sharing a core genome but differing in an adaptive gene complements that allows the population a much more efficient exploitation of available resources. These pan-clonal populations are maintained equalized by phage predation in an analogous way as the immune system in a mammal maintains in check tumors. If this is so, phage populations should be considered as belonging to the same selection unit.
  37. 37. Alicante (Spain), EvolutionaryUniversity Miguel Hernández Genomics Group Ana Belén Rohit Ghai Post-doc. Post-doc. Inma García Pre-doc. Carol Mizuno Aitor Gonzaga Pre-doc. Pre-doc. Sandra Rocamora Pre-doc.
  38. 38. Thank you very much! Alicante (Spain), University Miguel HernándezAitor GonzagaAna-BelénMartín-CuadradoRohit Ghai
  39. 39. Evapora. Carbonate Gypsum Halite Ratio Domain Intermediate Domain Domain No BACTERIOME 36-70 g/l life *Santa pola saltern : (Vi/Vo) 70-140 g/l 140-220 g/l 220-290 g/l >290 g/l DNA Bulk DNA 0.22 microns sp37, sp33, sp32, Time-series 5 microns extraction pyrosequencing 1041 Mpb (400pb) *Torrevieja saltern: 145 Mpb Diatoms Particles / small Archaea Haloarchaea eukaryotes (& Bacteria) * Santa pola saltern: Cyanobacteria Metagenomic Precipitation sp37 clones Mg & K Green Algae Sulphur Phototrophic Bacteria fosmids library Artenia Salina (35 Kb) *Torrevieja saltern: 5952 clones Dunaliella GI 1 GI 2 GI 3 GI 4 100 Haloquadratum walsbyi chromosome % id 90 (3132494 bp) Nu. SNP 80 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 Lengt (bp) 100 90 80 70 60 50 40 30 %sim. eHw-1 H. walsbyi DSM 16790 Cell surface glycoprotein Cell surface adhesin 0 0 Putative S-layer protein Dentin-sialophosphoprotrein 10 10 23-cyclic-nucleotide diesterase 20 20 IS element C23 Ig-like domain 240631 279189 0 10000 20000 30000 0 10000 20000 30000 40000 50000 60000 70000 Protease eHw-4 H. walsbyi C23 ABC-type cobalamin/iron(III) eHw-1 0 0 10 10 HBSQ001 Hmu2 20 20 239281 312087 0 10000 20000 30000 40000 0 10000 20000 30000 eHw-4 eHw-5 eHw-559 0 0 10 10 eHw-7 20 20 0 10000 20000 30000 0 10000 20000 30000 eHw-559 eHw-9 eHw-6 0 0 eHw-5 10 10 20 20 0 10000 20000 30000 0 10000 20000 30000 eHw-12 eHw-7 eHw-12 0 0 eHw-6 10 10 20 20 eHw-9 0 0 10000 20000 30000 10000 20000 30000
  40. 40. OBJETIVE HOW MANY DIFFERENT Haloquadratum walsbyi clones exists? Core-genome how different Pan-genome “In situ”, from thedo they they are? of H. walsbyi metagenomevary alongtime? are equally Accesory-genome distributed ?
  41. 41. What we have?: SequencesTwo genomes Haloquadratum walsbyi strains sequenced (in theory) HBSQ001 C23 4 SAGs of H. + (2000) Sanger (2011) 454 pyro. walsbyi 3132494 bp 3148034 bpMetagenomic collections of Santa Pola saltern : DNA Bulk DNA pyrosequencing (FLX, 400pb) 5 m 0.22 m extraction sp37 (2008) sp33 (2010) sp32 (2007) prokariots time-series (2005) 1041 Mpb + Torrevieja (Alicante) Saltern (2010) 145 MbpFosmids libraries. 33 Sequenced fosmids Analysis of 2947 fosmid ends DNA Metagenomic fosmids library (35 Kb) 5 m 0.22 m extraction sp (2002) 2000 clones sp37 (2008) 5952 clones 23 fosmids (2006) (Sanger) prokariots Torrevieja saltern: 2880 clones 10 fosmids GI1 (2009) FLX

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