Environmental genomics of solar saltern crystallizers. The very beginnings Francisco...
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Transcript of Environmental genomics of solar saltern crystallizers. The very beginnings Francisco...
Environmental genomics of solar saltern crystallizers.
The very beginnings
Francisco Rodríguez-Valera
Arantxa López-López and Boris A Legault
División de Microbiología y Grupo de Genómica Evolutiva, Universidad Miguel Hernández, San Juan de Alicante (Spain)
Thane R. Papke ; Adrian K. Sharma and W.Ford DoolittleBiochemistry and Molecular Biology, Dalhousie University,
Halifax, Nova Scotia (Canada)
Environmental genomics (Metagenomics, Ecogenomics)
• Application of large scale sequencing and annotation to environmental DNA
• Culture independent, 16S rRNA independent study of genetic diversity
SAMPLE EXTRACTION
SAMPLE FRACTIONATION (2 m filters)
DNA EXTRACTION
GENOMIC LIBRARY CONTRUCTION
CONSTRUCTION OF AN ENVIRONMENTAL GENOMIC LIBRARY
Pcc1fos Vector Map
CONSTRUCTION OF THE GENOMIC LIBRARY
Around 3000 clones storedin microtiter plates at -80ºCin glycerol (2000 after hurricane)
BLAST hits of end sequences
Halobacterium sp. NRC-1 167Haloferax volcanii 3 (phosphate kinase, aldehyde dehydrogenase, thermosome subunit 3)Haloferax mediterranei 2 (gvpF, nitrate reductase)Haloarcula vallismortis 1 (Cruxrhodopsin)Methanosarcina barkeri 2 (Restric. endonuclease, transposase)Methanothermobacter thermoautotrophicus 1 (valyl-tRNA synthase)Cyanobacteria (Nostoc etc.) 4 (glycosyl transferase, ABC transporter, lactoyl glutatione lyase,
nitrous oxidase)Assorted bacteria 33 (mostly transport proteins)No hit 93Total 306
Comparison to genomes of Haloquadratum walsbyi
(Martinsried)and Salinibacter ruber
(TIGR)Both at gap-closing annotation stage
BLAST hits of ends assigned to Haloquadratum (very high similarity, most >95% nuc identity )
Haloarchaea (mostly Halobacterium sp. NRC-1) 72 (including gvpF and Cruxrhodopsin)Archaea 2Assorted bacteria 18 No hits 25Total 117 out of 306
BLAST hits of ends assigned to Salinibacter (similarity low nuc 50-60% identity e.g. Yersinia/Escherichia)
Haloarchaea (mostly Halobacterium sp. NRC-1) 53!!!Archaea 1Assorted bacteria 11No hits 20Total 85 out of 306
Salinibacter ruber 16S rRNA
Strain E11
Strain C12
Strain C5
Strain C4r
Strain C9
Strain A7
Strain C16
Strain C25A
Strian E
Strain E3
Strain PR1
Strain C14
Strain A1
POLA-18
EHB-3
Strain M1
POLA-13
Salinibacter ruber DSMZ 13855
Strain M8
EHB-4
EHB-2
EHB-5
Rhodothermus marinus
0.02
Haloquadratum walsbyi 16S rRNA
AF477945 31% pond, May 1999
X84084 31% pond, 1995
AF477940 31% pond, May 1999
AF477927 31% pond, May 1999
AF477935 31% pond, May 1999
AF477925 22% pond, May 1999 AF477934 22% pond, May 1999 Haloferax volcanii
90
56
58
35
46
0.01
Haloquadratum walsbyi 31% pond, September 2001
AF477930 22% pond, May 1999
Fosmid 2BFosmid 7B 35% pond, November 2003
1. Kef-type K+ transp. system NAD-bind.component [Rubrobacter xylanophilus]2. Haloquadratum specific [Haloquadratum]3. Haloquadratum specific [Haloquadratum]4. Vng6193 [Halobacterium]5. Haloquadratum specific [Haloquadratum]6. Vng2555c (with ferodoxin domain) [Halobacterium]7. 6-phosphogluconate dehydrogenase YqeC [Halobacterium] 8. Aminopeptidase homolog PepB3 [Halobacterium] 9. Signal-transducing histidine kinase homolog KinA2 [Halobacterium] 10. Haloquadratum specific [Haloquadratum]11. Vng0738h [Halobacterium] 12. 30S ribosomal protein S6E [Halobacterium] 13. Heat shock protease protein Hsp5 [Halobacterium] 14. Vng2515h [Halobacterium] 15. Vng2519h [Halobacterium] 16. Stress related protein [Halobacterium] 17. Vng2521h [Halobacterium] 18. Vng2522c [Halobacterium] 19. Vng2523h [Halobacterium] 20. Vng2524h [Halobacterium] 21. Dihydroorotase PyrC [Halobacterium] 22. Isochorismatase EntB [Halobacterium] 23. Vng2539h [Halobacterium]
24. Nicotinic acid phosphoribosyltransferase [Halobacterium] 25. Vng2543c [Halobacterium] XX. rnpB ribonuclease P RNA 26. Haloquadratum specific [Haloquadratum]XX. tRNA-Cys, 5sRNA, 23sRNA, tRNA-Ala, 16sRNA27. Vng2508c (non-histone chromosomal protein) [Halobacterium] 28. Haloquadratum specific [Haloquadratum]29. Dihydroorotate dehydrogenase PyrD [Halobacterium]30. Valyl-tRNA synthetase ValS [Methanothermobacter
thermautotrophicus]31. Phenylalanyl-tRNA synthetase subunit beta PheY
[Halobacterium] 32. Phenylalanyl-tRNA synthetase subunit alpha PheS
[Halobacterium] 33. Tryptophanyl-tRNA synthetase TrpS1 [Halobacterium] 34. tRNA intron endonuclease EndA [Halobacterium] 35. pre-mRNA splicing helicase Brr2 [Halobacterium] 36. Vng2211h (DNA topoisomerase?) [Halobacterium] 37. Phosphoserine phosphatase SerB [Halobacterium] 38. Phosphoglycerate dehydrogenase SerA1 [Halobacterium] 39. Threonine synthase ThrC1 [Halobacterium] 40. Transcriptional reg. with DNA-binding domain [Methanopyrus
kandleri]41. Vng2306h [Halobacterium] 42. Haloquadratum specific [Haloquadratum]43. Vng2497h [Halobacterium]44. Haloquadratum specific [Haloquadratum]
Overall 2B-7B and H. walsbyi similarity 98.9%
1. Cell Division Cycle Protein48-1 (Thermococcus)2. CHP VGN1468 (Halobacterium)3. Imidazoleglycerol-phosphate dehydratease; HisB (Halobacterium)4. CHP VGN2296c (Halobacterium)5. Neutral Proteinase; Npt (Halobacterium)6. CHP VGN2298a (Halobacterium)7. Helicase; Rad3b (Halobacterium)8. Ornithine Carbamoyltransferase (Rubrobacter)9. Succinyl-diaminopimelate desuccinylase (Deinococcus)10. Acetylornithine deacetylase; argF (Pyrococcus)11. Acetylornithine aminotransferase (Thermus)12. N-Acetylglutamate kinase (Deinococcus)13. N-Acetyl-gamma-glutamyl-phophate reductase (Thermus)14. DNA Polymerase D (Haloferax)15. CHP VGN2376 (Halobacterium)16. Aspartokinase II alpha subunit (Halobacterium)17. Cytochrome p450 (Halobacterium)
18. CHP VNG2440h (Halobacterium)19. CHP VNG2386c (Halobacterium)20. Membrane Spanning Protein (Bacillus)21. Excision Nuclease chain B; UvrB (Halobacterium)22. CHP VGN2392h (Halobacterium)23. Signal Sequence Peptide; Sec11 (Halobacterium)24. Cell Division Control Protein 6; CDC6 (Halobacterium)25. GTP-Binding Protein (Halobacterium)26. CHP VNG2408c (Halobacterium)27. CHP VNG2406c (Halobacterium)28. CHP2403h (Halobacterium)29. Rad55 (Halobacterium)30. 24-Sterol C-Methyltransferase; scm (Halobacterium)31. Tiosulphate sulfurtransferase; tssA (Halobacterium)32. Thiosulphate sulfurtransferase; tssB (Halobacterium)33. Uncharacterized membrane protein (Nostoc)
Fosmid 10E2
H. Walsbyi contig382
Identity ranges between 70 and 100% aa identity
10 11 12 14
13
15 16
17
18
19 20 21 22 23
24
25 26 271 2 3
4 5 6
7 8 9
1. Phosphoenoylpyruvate carboxylase (Xanthomonas)2. CHP VGN1451c (Halobacterium)3. Orotatephosphoribosyl transferase (Haloferax)4. CHP VGN1205c (Halobacterium)5. Cytochrome p450 (Halobacterium)6. Oxidoreductase (Halobacterium)7. CHP MTH1897 (Methanobacterium)8. Antifreeze Glycopeptide (Fish)9. CHP VGN0725h (Halobacterium)10. CHP (Streptococcus)11. Archaerhodopsin (Halorubrum)12. CHP VGN1468h (Halobacterium)13. Cell division protein; CDC-48 (Halobacterium)14. Xylanase/chitin deacetylase (Burkhodaria)
15. Amidotransferase (Mesorhizobium)16. Methyltransferase (Streptomyces)17. Enol-CoA-isomerase (Actinomycete)18. CHP VGN0447h (Halobacterium)19. Membrane protease (Rubrobacter)20. CHP (Clostridium)21. Succinoglycan biosynthesis transport (Halobacterium)22. Universal stress protein (Halobacterium)23. Transposable element (Drosophila)24. Phosphoglycerate dehydrogenase (Halobacterium)25. Phosphoenol pyruvate carboxylase (Rhodopseudomonas)26. Kinase (Halobacterium)27. Glyceraldehyde-3-phosphate dehydrogenase (Halobacterium)
Halobacterial Bacterial Eukaryotic Archaeal
Top BLAST hits
Bacteriorhodopsin Containing Fosmid 10H09
Conclussions
• Close relatives of the Haloquadratum isolate sequenced are predominant
• Distant relatives of the strain of Salinibacter sequenced are also common (EHB2?), they show as many hits to haloarchaea as Haloquadratum!!!
• About 1/3 of the genes are not present in either genome, they could come from different organisms or strains with different gene pools
Acknowledgments
• Funding from EU project GEMINI (FRV) and Genome Canada (WFD)
• Access to H. walsbyi genome , D. Oesterhelt and F. Pfeiffer, MPI Martinsried
• Access to S. ruber genome, Karen Nelson and Emmanuel Mongodin, TIGR, Rockville
1 3 4 17
18 19 20 21 22 33
23
24
30 32
3129
2825 272616
15
14
1312119872
5 6
10
Bacteriorhodopsin Containing Fosmid 10E02
Halobacterial Bacterial Archaeal
1. Cell Division Cycle Protein48-1 (Thermococcus)2. CHP VGN1468 (Halobacterium)3. Imidazoleglycerol-phosphate dehydratease; HisB (Halobacterium)4. CHP VGN2296c (Halobacterium)5. Neutral Proteinase; Npt (Halobacterium)6. CHP VGN2298a (Halobacterium)7. Helicase; Rad3b (Halobacterium)8. Ornithine Carbamoyltransferase (Rubrobacter)9. Succinyl-diaminopimelate desuccinylase (Deinococcus)10. Acetylornithine deacetylase; argF (Pyrococcus)11. Acetylornithine aminotransferase (Thermus)12. N-Acetylglutamate kinase (Deinococcus)13. N-Acetyl-gamma-glutamyl-phophate reductase (Thermus)14. DNA Polymerase D (Haloferax)15. CHP VGN2376 (Halobacterium)16. Aspartokinase II alpha subunit (Halobacterium)17. Cytochrome p450 (Halobacterium)
18. CHP VNG2440h (Halobacterium)19. CHP VNG2386c (Halobacterium)20. Membrane Spanning Protein (Bacillus)21. Excision Nuclease chain B; UvrB (Halobacterium)22. CHP VGN2392h (Halobacterium)23. Signal Sequence Peptide; Sec11 (Halobacterium)24. Cell Division Control Protein 6; CDC6 (Halobacterium)25. GTP-Binding Protein (Halobacterium)26. CHP VNG2408c (Halobacterium)27. CHP VNG2406c (Halobacterium)28. CHP2403h (Halobacterium)29. Rad55 (Halobacterium)30. 24-Sterol C-Methyltransferase; scm (Halobacterium)31. Tiosulphate sulfurtransferase; tssA (Halobacterium)32. Thiosulphate sulfurtransferase; tssB (Halobacterium)33. Uncharacterized membrane protein (Nostoc)
Top Blast Hits