Environmental genomics of solar saltern crystallizers. The very beginnings

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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)

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Environmental genomics (Metagenomics, Ecogenomics)

• Application of large scale sequencing and
  annotation to environmental DNA
• Culture independent, 16S rRNA independent study
  of genetic diversity

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SOLAR SALTERNS IN SANTA POLA, ALICANTE
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The microbiota of the crystallizers (gt30
salinity)
1 ?m
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Phase contrast
Electron microscopy
SPhT
DAPI
FISH
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Salinibacter ruber
Crystallizer
ARCH915 EHB338 Haloquadratum
HRCU1502
Crystallizer
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CONSTRUCTION OF AN ENVIRONMENTAL GENOMIC LIBRARY
SAMPLE EXTRACTION
SAMPLE FRACTIONATION (2 ?m filters)
DNA EXTRACTION
GENOMIC LIBRARY CONTRUCTION
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CONSTRUCTION OF THE GENOMIC LIBRARY
Pcc1fos Vector Map
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BLAST hits of end sequences Halobacterium sp.
NRC-1 167 Haloferax 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 93 Total 306
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Comparison to genomes of Haloquadratum walsbyi
(Martinsried)and Salinibacter ruber (TIGR)

• Both at gap-closing annotation stage

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BLAST hits of ends assigned to Haloquadratum
(very high similarity, most gt95 nuc identity
) Haloarchaea (mostly Halobacterium sp.
NRC-1) 72 (including gvpF and Cruxrhodopsin) Arch
aea 2 Assorted bacteria 18 No hits
25 Total 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
1 Assorted bacteria
11 No hits
20 Total 85 out of 306
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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
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Haloquadratum walsbyi 16S rRNA
AF477930 22 pond, May 1999
56
AF477945 31 pond, May 1999
35
X84084 31 pond, 1995
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Haloquadratum walsbyi 31 pond, September 2001
Fosmid 2B
35 pond, November 2003
Fosmid 7B
58
AF477940 31 pond, May 1999
90
AF477927 31 pond, May 1999
AF477935 31 pond, May 1999
AF477925 22 pond, May 1999
AF477934 22 pond, May 1999
Haloferax volcanii
0.01
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Overall 2B-7B and H. walsbyi similarity 98.9
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, 16sRNA 27. 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
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H. Walsbyi contig382
Identity ranges between 70 and 100 aa identity
Fosmid 10E2
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Bacteriorhodopsin Containing Fosmid 10H09
Top BLAST hits
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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

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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

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Bacteriorhodopsin Containing Fosmid 10E02
Top Blast Hits