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

SOLAR SALTERNS IN SANTA POLA, ALICANTE

1 m

The microbiota of the crystallizers (>30% salinity)

SPhTSPhT

Phase contrast Electron microscopy

FISHDAPI

Crystallizer

ARCH915 EHB338 Haloquadratum HRCU1502

Crystallizer

Salinibacter ruber

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