Origin and function of a metal resistance gene isolated from eukaryotic soil metatranscriptomes
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The document discusses a study that analyzed metal resistance genes isolated from eukaryotic soil metatranscriptomes. It found a family of cysteine-rich proteins (CRPs) that were highly expressed in metal-polluted soils. The objectives were to determine if the CRPs were metallothioneins, characterize their functional role, and identify their taxonomic origin. The study compared characteristics of the CRPs to known metallothioneins and found differences in length, cysteine content, cysteine motif occurrence and organization. Experiments were proposed to express CRP genes in E. coli and conduct in vitro metal chelation assays to help elucidate the CRPs' potential role in metal resistance.
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Origin and function of a metal resistance gene isolated from eukaryotic soil metatranscriptomes
- 1. ? ? ORIGIN(S) & FUNCTION(S) OF METAL RESISTANCE GENES ISOLATED FROM EUKARYOTIC SOIL METATRANSCRIPTOMES Supervisor : Laurence Fraissinet-Tachet Co-supervisor : Roland Marmeisse ANTOINE ZILLER
- 2. Soils contained a rich diversity of organisms SOIL BIODIVERSITY
- 3. Soils contained a rich diversity of organisms This specific biodiversity is organized in complex ecological networks SOIL BIODIVERSITY Ecological Networks
- 4. Soils contained a rich diversity of organisms This specific biodiversity is organized in complex ecological networks Eukaryotic organisms play key role in this networks SOIL BIODIVERSITY Ecological Networks Unicellular eukaryotes Fungi
- 5. Chemical pollutions are one of the main anthropic pressure (Rockström et al. 2009) CHEMICAL POLLUTIONS
- 6. Chemical pollutions are one of the main anthropic pressure (Rockström et al. 2009) Oils and metals are the main compounds found on polluted sites CHEMICAL POLLUTIONS Polluted sites pollutants occurrence 61,4% Oils 47,9% Metals
- 7. Chemical pollutions are one of the main anthropic pressure (Rockström et al. 2009) Oils and metals are the main compounds found on polluted sites Metals can be a long term stressor that change ecological networks (Ramade 1992) CHEMICAL POLLUTIONS Ecological Networks Polluted sites pollutants occurrence 61,4% Oils 47,9% Metals
- 8. Metals = atoms with a tendency to form cation species (Men+) and be bind to non-metals METALS IN CELLULAR SYSTEMS Metals Non Metals
- 9. Metals Non Metals Metals = atoms with a tendency to form cation species (Men+) and be bind to non-metals Reactive metal species (RMS) are involved in every cellular functions METALS IN CELLULAR SYSTEMS REACTIVE METAL SPECIES
- 10. Metals = atoms with a tendency to form cation species (Men+) and be bind to non-metals Reactive metal species (RMS) are involved in every cellular functions METALS IN CELLULAR SYSTEMS REACTIVE METAL SPECIES Redox chemistry Energy transfers, cell signaling Acid-base chemistry Fe, Cu, Mn, Mo, Ni Zn2+, Ni2+, Fe3+, Mn3+, Mg2+ Na+, K+, Mg2+, Ca2+, Fe2+, Mn2+, Zn2+ PROTEINS BINDED MOBILE Metals Non Metals
- 11. REACTIVE METAL SPECIES IN CELLULAR SYSTEMS Reactive metal species in excess Nucleic acids, lipids, proteins, carbohydrates Cellular system Metal binding to non- metal elements ATOMIC SCALE
- 12. REACTIVE METAL SPECIES IN CELLULAR SYSTEMS Reactive metal species in excess Nucleic acids, lipids, proteins, carbohydrates Cellular system Enzymatic catalysis MOLECULAR SCALE Reactive oxygen species production Structural metal replacement ATOMIC SCALE Metal binding to non- metal elements
- 13. REACTIVE METAL SPECIES IN CELLULAR SYSTEMS Reactive metal species in excess CELLULAR SCALE Nucleic acids, lipids, proteins, carbohydrates Cellular system Enzymatic catalysis MOLECULAR SCALE Reactive oxygen species production Structural metal replacement Redox potential and pH modifications Metabolic processes perturbations ATOMIC SCALE Metal binding to non- metal elements
- 14. REACTIVE METAL SPECIES IN CELLULAR SYSTEMS Reactive metal species in excess CELLULAR SCALE Nucleic acids, lipids, proteins, carbohydrates Cellular system Enzymatic catalysis MOLECULAR SCALE Reactive oxygen species production Structural metal replacement Redox potential and pH modifications Metabolic processes perturbations ATOMIC SCALE Gene induction PROTEINS INVOLVED IN METAL RESISTANCE MECHANISMS Metal binding to non- metal elements Metal stress reduction
- 15. METAL RESISTANCE MECHANISMS Reactive metal species Organic compounds
- 16. METAL RESISTANCE MECHANISMS Cell wall adsorption Organic compounds Chitine Reactive metal species
- 17. METAL RESISTANCE MECHANISMS Cell wall adsorption Transporters modulation Organic compounds ABC transporters, P1 ATPase Reactive metal species Chitine
- 18. METAL RESISTANCE MECHANISMS Cell wall adsorption Transporters modulation Intracellular and extracellular binding Organic compounds Metal chelators, Organic acids ABC transporters, P1 ATPase Reactive metal species Chitine
- 19. METAL RESISTANCE MECHANISMS Cell wall adsorption Transporters modulation Intracellular and extracellular binding Organic compounds Mercuric reductase Oxidation state modification ABC transporters, P1 ATPase Reactive metal species Metal chelators, Organic acids Chitine
- 20. METAL RESISTANCE MECHANISMS Cell wall adsorption Transporters modulation Intracellular and extracellular binding Organic compounds Mercuric reductase Oxidation state modification ABC transporters, P1 ATPase Mechanisms mainly described in vitro with model organisms In situ transcriptional responses ? Reactive metal species Metal chelators, Organic acids Chitine
- 21. IN SITU TRANSCRIPTIONAL RESPONSE TO METAL POLLUTIONS EUMETATOX PROJECT CEFIPRA PROJECT METAL CONTAMINATED FORMERLY POLLUTED NON POLLUTED Belgian soils Lehembre et al. 2013 TEAM : EUKARYOTIC MICROORGANISMS, ADAPTATION TO THEIR ENVIRONMENT
- 22. IN SITU TRANSCRIPTIONAL RESPONSE TO METAL POLLUTIONS EUMETATOX PROJECT CEFIPRA PROJECT METAL CONTAMINATED FORMERLY POLLUTED NON POLLUTED Belgian soils Expression in sensitive yeasts Vector sequencing from resistant strains Lehembre et al. 2013 RNA Extraction and cDNA libraries TEAM : EUKARYOTIC MICROORGANISMS, ADAPTATION TO THEIR ENVIRONMENT
- 23. TEAM : EUKARYOTIC MICROORGANISMS, ADAPTATION TO THEIR ENVIRONMENT IN SITU TRANSCRIPTIONAL RESPONSE TO METAL POLLUTIONS EUMETATOX PROJECT CEFIPRA PROJECT METAL CONTAMINATED FORMERLY POLLUTED NON POLLUTED Belgian soils RNA Extraction and cDNA libraries Expression in sensitive yeasts 2% 47-63% 35-55% Vector sequencing from resistant strains Sequences comparison with databases (BLAST) Lehembre et al. 2013 UNKOWN GENES KNOWN GENES ► not as metal resistance genes KNOWN GENES ► as metal resistance genes
- 24. TEAM : EUKARYOTIC MICROORGANISMS, ADAPTATION TO THEIR ENVIRONMENT IN SITU TRANSCRIPTIONAL RESPONSE TO METAL POLLUTIONS EUMETATOX PROJECT CEFIPRA PROJECT METAL CONTAMINATED FORMERLY POLLUTED NON POLLUTED Lehembre et al. 2013 Belgian soils Expression in sensitive yeasts 2% 47-63% 35-55% Vector sequencing from resistant strains Sequences comparison with databases (BLAST) RNA Extraction and cDNA libraries ISOLATION OF A CYSTEIN RICH PROTEINS (CRP) FAMILY UNKOWN GENES KNOWN GENES ► not as metal resistance genes KNOWN GENES ► as metal resistance genes
- 25. 1 2 5 3 7 6a 6b 8 4 46 CYSTEIN RICH PROTEINS Sub-family 1 CRP sequence9 CRP SUB-FAMILIES
- 26. 1 2 5 3 7 6a 6b 8 4 46 CYSTEIN RICH PROTEINS Sub-family 1 CRP sequence AMPLIFIED IN METAL POLLUTED AND NON POLLUTED SAMPLES 9 CRP SUB-FAMILIES
- 27. 1 2 5 3 7 6a 6b 8 4 46 CYSTEIN RICH PROTEINS METALLOTHIONEINS (MT) ? Sub-family 1 CRP sequence MDPNCSCSTGGSCTCTSSCACKNCKCTSCKKSCCSCCPVGCSKCAQGCVCKGAADKCTCCA Mammalian metallothionein 9 CRP SUB-FAMILIES AMPLIFIED IN METAL POLLUTED AND NON POLLUTED SAMPLES
- 28. METALLOTHIONEINS Small cysteine rich proteins 24-162 a.a. 15-35% Cys MT
- 29. METALLOTHIONEINS Small cysteine rich proteins Metal intracellular chelators Basal expression stress response gene induction mt promoters MRE, ARE 24-162 a.a. 15-35% Cys MT
- 30. METALLOTHIONEINS Small cysteine rich proteins Metal intracellular chelators Basal expression stress response gene induction Mainly described in pluricellular organisms (15 Families) mt promoters MRE, ARE 24-162 a.a. 15-35% Cys MT
- 31. METALLOTHIONEINS Small cysteine rich proteins Metal intracellular chelators Basal expression stress response gene induction Mainly described in pluricellular organisms (15 Families) Metal pollution bioindicators mt promoters MRE, ARE 24-162 a.a. 15-35% Cys MT
- 32. OBJECTIVES CRP TAXONOMIC ORIGIN AND FUNCTION CHARACTERIZATION ? Hypothesis : Cystein Rich Proteins (CRPs) = metallothioneins (MTs) ARE CRPs METALLOTHIONEINS ?1 CRPs FUNCTIONAL ROLE ?2 CRPs TAXONOMIC ORIGIN ?3
- 33. ARE CRPs METALLOTHIONEINS ? 1 2 3
- 34. ARE CRPs METALLOTHIONEINS ? MTs amino acids sequences is characteristic at family level (Capdevila 2012) MVGCNCGSSCKCGDQCKC MVGCNCGSSCQCGDQCKC MKGCNCGSSCQCGDQCKC MKGCNCGSSCKCGDQCKC
- 35. MVGCNCGSSCKCGDQCKC MVGCNCGSSCQCGDQCKC MKGCNCGSSCQCGDQCKC MKGCNCGSSCKCGDQCKC Length, cysteine content ARE CRPs METALLOTHIONEINS ? MTs amino acids sequences is characteristic at family level (Capdevila 2012)
- 36. Cys% 1520253035 Length (a.a.) 20 40 60 80 100 120 140 160 Metazoan (F01-06) CRPs vs MTs : LENGTH AND CYSTEINE CONTENT Plants (F15) Fungi (F08-13) Ciliates (F07) Bacteria (F14) 24-162 a.a. 15-35 % Cys
- 37. Cys% 1520253035 Length (a.a.) 20 40 60 80 100 120 140 160 CRPs vs MTs : LENGTH AND CYSTEINE CONTENT 110-133 a.a. 20-23 % Cys CRPs Metazoan (F01-06) Plants (F15) Fungi (F08-13) Ciliates (F07) Bacteria (F14) 24-162 a.a. 15-35 % Cys
- 38. Cys% 1520253035 Length (a.a.) 20 40 60 80 100 120 140 160 CRPs vs MTs : LENGTH AND CYSTEINE CONTENT 110-133 a.a. 20-23 % Cys CRPs in frame of large and low cysteine content MTs CRPs CRPs are in a specific frame compared with MTs Metazoan (F01-06) Plants (F15) Fungi (F08-13) Ciliates (F07) Bacteria (F14) 24-162 a.a. 15-35 % Cys
- 39. MVGCNCGSSCKCGDQCKC MVGCNCGSSCQCGDQCKC MKGCNCGSSCQCGDQCKC MKGCNCGSSCKCGDQCKC CxC Length, cysteine content Cysteine position are conserved in cysteine motifs ARE CRPs METALLOTHIONEINS ? MTs amino acids sequences is characteristic at family level (Capdevila 2012)
- 40. CRPs vs MTs : CYSTEIN MOTIF OCCURENCE
- 41. CRPs vs MTs : CYSTEIN MOTIF OCCURENCE CRPs have a specific cysteine motif occurrence compared with MTs CRPs and MTs have the same kind of Cys motifs
- 42. MVGCNCGSSCKCGDQCKC MVGCNCGSSCQCGDQCKC MKGCNCGSSCQCGDQCKC MKGCNCGSSCKCGDQCKC CxC Length, cysteine content Cysteine position are conserved in cysteine motifs ARE CRPs METALLOTHIONEINS ? MTs amino acids sequences is characteristic at family level (Capdevila 2012)
- 43. MSGCNCGSSCNCGDQCKCNKRSGLSYVEAGETTETVVLGVGPTKIHFEGAEMSVAAEDGGCKCGSSCTCDPCNCK MVGCNCGSSCKCGDQCKC MVGCNCGSSCQCGDQCKC MKGCNCGSSCQCGDQCKC MKGCNCGSSCKCGDQCKC CxC Length, cysteine content Cysteine position are conserved in cysteine motifs Cysteine motifs organization in sequence ARE CRPs METALLOTHIONEINS ? MTs amino acids sequences is characteristic at family level (Capdevila 2012)
- 44. MSGCNCGSSCNCGDQCKCNKRSGLSYVEAGETTETVVLGVGPTKIHFEGAEMSVAAEDGGCKCGSSCTCDPCNCK 2. BIMODULAR MGCDDKCGCAVPCPGGTGCRCTSARSGAAAGEHTTCGCGEHCGCNPCACGREGTPSGRANRRANCSCGAACNCASCGSATA 3. TRIMODULAR MDTQTQTKVTVGCSCNPCKCQPLCKCGTTAACNCQPCENCDPCSCNPCKCGVTESCGCNPCKCAECKCGSHTEKTSACKCNPCACNPCNCGSTSNCKCNPCKCAECKC 4. REPETITIVE MODULES MDPNCSCSTGGSCTCTSSCACKNCKCTSCKKSCCSCCPVGCSKCAQGCVCKGAADKCTCCA 1. NO MODULAR ORGANIZATION CRPs vs MTs : CYSTEINE MOTIF ORGANIZATION
- 45. CRPs vs MTs : CYSTEINE MOTIF ORGANIZATION CRPs Bimodular (?) organization and a short linker CRPs N domain CRPs C domainLinker
- 46. CRPs vs MTs : CYSTEINE MOTIF ORGANIZATION CRPs CCC CC CxCC xxCxx CxC xxCxx Cys motifs are different in N and C domains CRPs N domain CRPs C domainLinker Bimodular (?) organization and a short linker
- 47. CRPs vs MTs : CYSTEINE MOTIF ORGANIZATION CRPs CCC CC CxCC xxCxx CxC xxCxx CRPs have specific cysteine motif organization compared with MTs CRPs N domain CRPs C domainLinker Bimodular (?) organization and a short linker Cys motifs are different in N and C domains
- 48. Expression in E. coli IN VITRO CHELATION ASSAY 5 cpr genes (4 sub-family) pGEX-4T-1-CRP (Tag GST)
- 49. Cd Zn Cu Culture and overexpression Metal statured medium Proteins purification IN VITRO CHELATION ASSAY 5 cpr genes (4 sub-family) pGEX-4T-1-CRP (Tag GST) Expression in E. coli
- 50. Proteins ICP-OES ESI-TOF-MS (Acid/neutral) Biochemical analysis Cd Zn Cu 5 cpr genes (4 sub-family) pGEX-4T-1-CRP (Tag GST) Culture and overexpression Metal statured medium Proteins purification IN VITRO CHELATION ASSAY Expression in E. coli
- 51. Proteins ICP-OES ESI-TOF-MS (Acid/neutral) Biochemical analysis Cd Zn Cu Culture and overexpression Metal statured medium Proteins purification IN VITRO CHELATION ASSAY 5 cpr genes (4 sub-family) pGEX-4T-1-CRP (Tag GST) Expression in E. coli
- 52. CRPs METAL BINDING ABILITIES pH 2.4 Intensity Molecular mass CRP5-Zn quantification by mass spectrometry
- 53. CRPs METAL BINDING ABILITIES pH 2.4 Intensity Molecular mass Bind to mixture of Zn species CRP5-Zn quantification by mass spectrometry
- 54. CRPs METAL BINDING ABILITIES pH 2.4 CRP5-Zn quantification by mass spectrometry Intensity Molecular mass Bind to mixture of Zn species CRP 5 is mainly bind to 9 Zn
- 55. CRPs METAL BINDING ABILITIES Zn Cd Cu 0 2 4 6 8 10 12 14 METALS/PROTEINS CRP1.1 CRP1.2 CRP3 CRP4 CRP5 9 9 9 9 8 8 8 8 8 11 12 13 10 9 N.A.
- 56. CRPs METAL BINDING ABILITIES 0 2 4 6 8 10 12 14 METALS/PROTEINS CRP1.1 CRP1.2 CRP3 CRP4 CRP5 9 9 9 9 8 8 8 8 8 11 12 13 10 9 N.A. CRPs can bind Zn, Cd, Cu Zn Cd Cu
- 57. CRPs METAL BINDING ABILITIES 0 2 4 6 8 10 12 14 METALS/PROTEINS CRP1.1 CRP1.2 CRP3 CRP4 CRP5 9 9 9 9 8 8 8 8 8 11 12 13 10 9 N.A. CRPs can bind Zn, Cd, Cu Different binding properties by CRP Zn Cd Cu
- 58. ARE CRPs METALLOTHIONEINS ? 2 31
- 59. ARE CRPs METALLOTHIONEINS ? 2 31 CRPs = Hypothetical metallothioneins
- 60. ARE CRPs METALLOTHIONEINS ? 2 3 CRPs = metallothioneins 1 CRPs = Hypothetical metallothioneins In vitro chelation of Zn, Cd and Cu
- 61. ARE CRPs METALLOTHIONEINS ? 2 3 With properties never observed in MTs large length and low cysteine residues Cysteine motif occurrence Bimodular organization with a short linker CRPs = metallothioneins 1 CRPs = Hypothetical metallothioneins In vitro chelation of Zn, Cd and Cu CRPs = new metallothionein familly
- 62. ARE CRPs METALLOTHIONEINS ? 2 3 With properties never observed in MTs large length and low cysteine residues Cysteine motif occurrence Bimodular organization with a short linker CRPs = Environmental metallothioneins (EMT) Ziller et al. 2017 CRPs = metallothioneins 1 CRPs = Hypothetical metallothioneins CRPs = new metallothionein familly In vitro chelation of Zn, Cd and Cu
- 63. CRPs FUNCTIONAL ROLE ? 2 31
- 64. FUNCTIONNAL ORIENTATION Cd Zn Cu S. cerevisiae transformation *ABC GSH-Cd vacuolar transporter *Zn vacuolar transporter *Cup1 & Crs5 : Metallothioneins
- 65. Mutant + CRP Decrease in cell concentration Strong Cd tolerance – Metal + Metal CRP 1.1 CRP 1.2 CRP 3 CRP 4 CRP 5 FUNCTIONNAL ORIENTATION Cd Zn Cu S. cerevisiae transformation Tolerant control *Sensitive control (mutant) *ABC GSH-Cd vacuolar transporter *Zn vacuolar transporter *Cup1 & Crs5 : Metallothioneins
- 66. Decrease in cell concentration Strong Cd tolerance – Cd + Cd CRP 1.1 CRP 1.2 CRP 3 CRP 4 CRP 5 FUNCTIONNAL ORIENTATION Cd Zn Cu S. cerevisiae transformation Tolerant control *Sensitive control (mutant) *ABC GSH-Cd vacuolar transporter *Zn vacuolar transporter *Cup1 & Crs5 : Metallothioneins Strong Cd tolerance Mutant + CRP
- 67. Mutant + CRP Decrease in cell concentration Strong Cd tolerance – Cd + Cd CRP 1.1 CRP 1.2 CRP 3 CRP 4 CRP 5 FUNCTIONNAL ORIENTATION Cd Zn Cu S. cerevisiae transformation Tolerant control *Sensitive control (mutant) *ABC GSH-Cd vacuolar transporter *Zn vacuolar transporter *Cup1 & Crs5 : Metallothioneins Strong Cd tolerance Weak Zn tolerance No Cu tolerance
- 68. FUNCTIONNAL ORIENTATION Cd Zn Cu S. cerevisiae transformation *ABC GSH-Cd vacuolar transporter *Zn vacuolar transporter *Cup1 & Crs5 : Metallothioneins Strong Cd tolerance Weak Zn tolerance No Cu tolerance In vitro Cu binding Ziller et al. 2017
- 69. FUNCTIONNAL ORIENTATION Cd Zn Cu S. cerevisiae transformation *ABC GSH-Cd vacuolar transporter *Zn vacuolar transporter *Cup1 & Crs5 : Metallothioneins Strong Cd tolerance Weak Zn tolerance No Cu tolerance In vitro Cu binding Ziller et al. 2017 CRPs restore Cd and Zn tolerance
- 70. Soil with crp sequences TRANSCRIPTIONAL REGULATION OF CRP GENES
- 71. Soil with crp sequences crppromoters MRE, ARE ? ? Organism unknown Promoter region not available TRANSCRIPTIONAL REGULATION OF CRP GENES
- 72. +Cd In situ gene induction quantification of soil microcosms Soil with crp sequences crppromoters MRE, ARE ? ? Organism unknown Promoter region not available TRANSCRIPTIONAL REGULATION OF CRP GENES
- 73. 3 ppm 6 ppm 12 ppm 24 ppm CADMIUM TREATMENTS TOTAL AND SOLUBLE Cd QUANTIFICATION BY ICP-MS AVAILABLE METALS ESTIMATION TOTAL METALS
- 74. 3 ppm 6 ppm 12 ppm 24 ppm CADMIUM TREATMENTS TOTAL AND SOLUBLE Cd QUANTIFICATION BY ICP-MSCdmeasured (ppm) Cd added (ppm) AVAILABLE METALS ESTIMATION TOTAL METALS
- 75. 3 ppm 6 ppm 12 ppm 24 ppm CADMIUM TREATMENTS AVAILABLE METALS ESTIMATION TOTAL METALS Cdmeasured (ppm) Cd added (ppm) Leaching : 17% Cd initially amended is lost TOTAL AND SOLUBLE Cd QUANTIFICATION BY ICP-MS
- 76. 3 ppm 6 ppm 12 ppm 24 ppm CADMIUM TREATMENTS Cdmeasured (ppm) Cd added (ppm) Total Cd is largely available (soil properties) Leaching : 17% Cd initially amended is lost TOTAL AND SOLUBLE Cd QUANTIFICATION BY ICP-MS AVAILABLE METALS ESTIMATION TOTAL METALS
- 77. 3 ppm 6 ppm 12 ppm 24 ppm CADMIUM TREATMENTS Cdmeasured (ppm) Cd added (ppm) Natural Cd quantities 0,1-1 ppm (Alloway et al. 2013) Biological effects 3-10 ppm ( Smolders 2002) Total Cd is largely available (soil properties) Leaching : 17% Cd initially amended is lost TOTAL AND SOLUBLE Cd QUANTIFICATION BY ICP-MS AVAILABLE METALS ESTIMATION TOTAL METALS
- 78. TRANSCRIPTIONAL REGULATION OF CRP GENES +Cd In situ gene induction quantification of soil microcosms Soil with crp sequences crppromoters MRE, ARE ? ? Organism unknown Promoter region not available crp sequences are in low abundance Soil DNA extract crp
- 79. +Cd Droplet digital PCR ? In situ gene induction quantification of soil microcosms Soil with crp sequences crppromoters MRE, ARE ? ? Organism unknown Promoter region not available crp sequences are in low abundance Soil DNA extract crp TRANSCRIPTIONAL REGULATION OF CRP GENES
- 80. Droplet digital PCR is a quantitative PCR based on Poisson statistic, and microfluidic chip DROPLET DIGITAL PCR PCR mix partition in nanoliter droplet Standard PCR and flow cytometer Poisson law
- 81. Droplet digital PCR is a quantitative PCR based on Poisson statistic, and microfluidic chip ddPCR is more sensitive than qPCR in detecting low quantities of target bacterial DNA in soils (Kim et al. 2014) DROPLET DIGITAL PCR PCR mix partition in nanoliter droplet Standard PCR and flow cytometer Poisson law
- 82. Droplet digital PCR is a quantitative PCR based on Poisson statistic, and microfluidic chip ddPCR is more sensitive than qPCR in detecting low quantities of target bacterial DNA in soils (Kim et al. 2014) Never use for soil eukaryotic model DROPLET DIGITAL PCR PCR mix partition in nanoliter droplet Standard PCR and flow cytometer Poisson law ddPCR vs qPCR ?
- 83. +Cd Droplet digital PCR ? In situ gene induction quantification of soil microcosms Soil with crp sequences TRANSCRIPTIONAL REGULATION OF CRP GENES Plasmid crp1 range 101, 102, 103, 104 copy number
- 84. +Cd Droplet digital PCR ? In situ gene induction quantification of soil microcosms Soil with crp sequences TRANSCRIPTIONAL REGULATION OF CRP GENES Plasmid crp1 range 101, 102, 103, 104 copy number ddPCR > qPCR Better efficiency Lowest measure variability
- 85. Plasmid crp1 range 101, 102, 103, 104 copy number +Cd Droplet digital PCR ? In situ gene induction quantification of soil microcosms Soil with crp sequences TRANSCRIPTIONAL REGULATION OF CRP GENES ddPCR > qPCR Better efficiency Lowest measure variability Plasmid crp1 range 101, 102, 103, 104 copy number + Microcosm DNA extracts
- 86. Plasmid crp1 range 101, 102, 103, 104 copy number +Cd Droplet digital PCR ? In situ gene induction quantification of soil microcosms Soil with crp sequences TRANSCRIPTIONAL REGULATION OF CRP GENES ddPCR > qPCR Better efficiency Lowest measure variability ddPCR and qPCR amplification perturbations + Microcosm DNA extracts Plasmid crp1 range 101, 102, 103, 104 copy number
- 87. CRPs FUNCTIONAL ROLE ? 2 31 crp overexpression in Cd, Zn and Cu sensitive yeast CRPs restore Cd and Zn tolerance
- 88. CRPs FUNCTIONAL ROLE ? 2 31 crp overexpression in Cd, Zn and Cu sensitive yeast We have tested crp quantification from microcosms DNA extracts Need more optimizations to be used in situ ddPCR is promising method CRPs restore Cd and Zn tolerance
- 89. CRPs ORIGINE ? 2 31
- 90. Soil with crp sequences crp ?Organism unknown CRP TAXONOMIC ORIGIN ?
- 91. Genome walking Soil with crp sequences crp ?Organism unknown CRP TAXONOMIC ORIGIN ?
- 92. Genome walking Soil with crp sequences crp ?Organism unknown Phylogenetic conserved gene CRP TAXONOMIC ORIGIN ?
- 93. Genome walking Soil with crp sequences crppromoters MRE, ARE ? ? Organism unknown Promoter region not available Phylogenetic conserved gene CRP TAXONOMIC ORIGIN ?
- 94. Genome walking Soil with crp sequences crppromoters MRE, ARE ? ? Organism unknown Promoter region not available crp sequences are in low abundance Soil DNA extract crp Phylogenetic conserved gene CRP TAXONOMIC ORIGIN ?
- 95. Targeted gene captureGenome walking Soil with crp sequences crppromoters MRE, ARE ? ? Organism unknown Promoter region not available crp sequences are in low abundance Soil DNA extract crp Phylogenetic conserved gene CRP TAXONOMIC ORIGIN ?
- 96. Sonde ARN Biotine labeled probe set Targeted sequence selective enrichment in environmental DNA extracts (Denonfoux et al. 2013) TARGETED GENE CAPTURE crp Fragmented environmental DNA extract
- 97. Sonde ARN Biotine labeled probe set Targeted sequence selective enrichment in environmental DNA extracts (Denonfoux et al. 2013) TARGETED GENE CAPTURE crp DNA-probes hybridization Fragmented environmental DNA extract
- 98. Sonde ARN Biotine labeled probe set Targeted sequence selective enrichment in environmental DNA extracts (Denonfoux et al. 2013) TARGETED GENE CAPTURE crp DNA-probes hybridization Washing and elutionFragmented environmental DNA extract Fragmented environmental DNA extract enriched in targeted sequences
- 99. Sonde ARN Biotine labeled probe set Fragmented environmental DNA extract Targeted sequence selective enrichment in environmental DNA extracts (Denonfoux et al. 2013) Critical point : probes set design TARGETED GENE CAPTURE Fragmented environmental DNA extract enriched in targeted sequences crp DNA-probes hybridization Washing and elution
- 100. CRP SEQUENCES CAPTURE : SET OF PROBES DESIGNED crp consensus from a 45 sequence alignment ≈ 490 pb 40 probes 50 pb Specificity Sensitivity
- 101. crp consensus from a 45 sequence alignment ≈ 490 pb 40 probes 50 pb No enrichmentSpecificity Sensitivity CRP SEQUENCES CAPTURE : SET OF PROBES DESIGNED
- 102. crp consensus from a 45 sequence alignment ≈ 490 pb 40 probes 50 pb No enrichmentSpecificity Sensitivity Probes too shorts for long seize DNA captured ? CRP SEQUENCES CAPTURE : SET OF PROBES DESIGNED
- 103. crp consensus from a 45 sequence alignment ≈ 490 pb 40 probes 50 pb 16 probes 327-343 pb No enrichmentSpecificity Sensitivity Specificity Sensitivity Probes too shorts for long seize DNA captured ? Enrichment x 1 000 CRP SEQUENCES CAPTURE : SET OF PROBES DESIGNED
- 104. PCR SCREENING AND SEQUENCING RESULTS Captured DNA Cloning and PCR screening inserts>1kb 50395 Sequencing
- 105. PCR SCREENING AND SEQUENCING RESULTS Captured DNA Cloning and PCR screening inserts>1kb 50395 Sequencing 33 Crp 4 342 43 Crp 1 333 9 17 crp DNA sequences captured with bording region = success 2 crp sequences
- 106. PCR SCREENING AND SEQUENCING RESULTS Captured DNA Cloning and PCR screening inserts>1kb 50395 Sequencing crp DNA sequences captured with bording region = success But they are too short Gene capture process favor short fragments ? 2 crp sequences 33 Crp 4 342 43 Crp 1 333 9 17
- 107. CRPs ORIGINE ? 2 31 Crp sequences enrichment of soil DNA extracts
- 108. CRPs ORIGINE ? 2 31 Cloning/sequencing approach : 2 crp sequences with flanking regions Crp sequences enrichment of soil DNA extracts
- 109. CRPs ORIGINE ? 2 31 Genome walking High throughput sequencing Cloning/sequencing approach : 2 crp sequences with flanking regions Crp sequences enrichment of soil DNA extracts
- 110. Origin(s) and function(s) of CRPs ? GENERAL CONCLUSION
- 111. Origin(s) and function(s) of CRPs ? GENERAL CONCLUSION 1 Ziller et al. 2017 ► Environmental metallothionein (EMT) CRPs vs MTs CRPs = new metallothionein family
- 112. Origin(s) and function(s) of CRPs ? GENERAL CONCLUSION 2 1 Ziller et al. 2017 Functional characterization CRPs = role in metal tolerance Quantitative PCR development (ddPCR) from soil microcosms ► Environmental metallothionein (EMT) CRPs vs MTs CRPs = new metallothionein family
- 113. Origin(s) and function(s) of CRPs ? GENERAL CONCLUSION 3 2 Taxonomic origin crp sequence enrichment by gene capture 1 ► Environmental metallothionein (EMT) Ziller et al. 2017 CRPs vs MTs CRPs = new metallothionein family Functional characterization CRPs = role in metal tolerance Quantitative PCR development (ddPCR) from soil microcosms
- 114. PERSPECTIVES IN SILICO Isolation of other new CRP in databases ?
- 115. CRP SCREENING IN DATABASES
- 116. CRP SCREENING IN DATABASES 2 CRPs Dictyostelium discoideum
- 117. PERSPECTIVES IN SILICO Isolation of other new CRP in databases ? IN VITRO
- 118. PERSPECTIVES CELLULAR SYSTEM crp organism isolation and identification Other crp families functional role ? IN SILICO Isolation of other new CRP in databases ? IN VITRO
- 119. PERSPECTIVES CELLULAR SYSTEM NATURAL SYSTEM crp organism isolation and identification Other crp families functional role ? IN SILICO Isolation of other new CRP in databases ? Importance of organisms that contain CRP ? Molecular biosensor tool for metal pollution detection ?IN VITRO
- 120. MTs cellular functions are multiple (Capdevila 2012) MetallothioneinsMetallothioneins DISCUSSION ON METALLOTHIONEINS CELLULAR FUNCTION
- 121. MTs cellular functions are multiple (Capdevila 2012) Reactive metals ROS NOS Metalloprotéines Autres Molécules Metallothioneins Reactive metals ROS NOS Metalloprotéines Autres Molécules Metallothioneins DISCUSSION ON METALLOTHIONEINS CELLULAR FUNCTION
- 122. MTs cellular functions are multiple (Capdevila 2012) Reactive metals ROS NOS Metalloprotéines Autres Molécules Cell signaling Cell detoxification Redox equilibrium Metals equilibrium Transcription regulation Enzymatic activity Metallothioneins Reactive metals ROS NOS Metalloprotéines Autres Molécules Metallothioneins DISCUSSION ON METALLOTHIONEINS CELLULAR FUNCTION
- 123. MTs cellular functions are multiple (Capdevila 2012) Reactive metals ROS NOS Metalloprotéines Autres Molécules Cell signaling Cell detoxification Redox equilibrium Metals equilibrium Transcription regulation Enzymatic activity Metallothioneins Reactive metals ROS NOS Metalloprotéines Autres Molécules Metallothioneins Are MTs a metabolic hub ? DISCUSSION ON METALLOTHIONEINS CELLULAR FUNCTION
- 124. MTs cellular functions are multiple (Capdevila 2012) Metabolic hub properties (Cumberworth et al. 2013) Small linear motifs Low complexity regions Reactive metals ROS NOS Metalloprotéines Autres Molécules Cell signaling Cell detoxification Redox equilibrium Metals equilibrium Transcription regulation Enzymatic activity Metallothioneins Reactive metals ROS NOS Metalloprotéines Autres Molécules Metallothioneins Are MTs a metabolic hub ? Cys rich Cys motifs DISCUSSION ON METALLOTHIONEINS CELLULAR FUNCTION
- 125. ACKNOWLEDGMENTS FINANCIAL SUPPORT SUPERVISORS Laurence Roland TEAM AMEE Delphine Florian Goeffroy Jeanne Laurent Marie-André Martino Mylène Patricia TEAM DMTV Catherine Claire Colin Guillaume Margot Morgane Patrick² Stéphanie Van Yoann COLLEAGUES All the persons who I've forgotten to mention
- 126. ? ? THANK YOU Supervisor : Laurence Fraissinet-Tachet Co-supervisor : Roland Marmeisse ANTOINE ZILLER
Editor's Notes
- We have a double problematic. if go outside and take a handful of soil, you will have huge biodiversity on your hand because it’s mainly invisible, microscopic and unkwown. Poeple talk about biodiversity dark matter.
- We have a double problematic. if go outside and take a handful of soil, you will have huge biodiversity on your hand because it’s mainly invisible, microscopic and unkwown. Poeple talk about biodiversity dark matter.
- We have a double problematic. if go outside and take a handful of soil, you will have huge biodiversity on your hand because it’s mainly invisible, microscopic and unkwown. Poeple talk about biodiversity dark matter.
- We have a double problematic. if go outside and take a handful of soil, you will have huge biodiversity on your hand because it’s mainly invisible, microscopic and unkwown. Poeple talk about biodiversity dark matter.
- We have a double problematic. if go outside and take a handful of soil, you will have huge biodiversity on your hand because it’s mainly invisible, microscopic and unkwown. Poeple talk about biodiversity dark matter.
- We have a double problematic. if go outside and take a handful of soil, you will have huge biodiversity on your hand because it’s mainly invisible, microscopic and unkwown. Poeple talk about biodiversity dark matter.
- We have a double problematic. if go outside and take a handful of soil, you will have huge biodiversity on your hand because it’s mainly invisible, microscopic and unkwown. Poeple talk about biodiversity dark matter.
- We have a double problematic. if go outside and take a handful of soil, you will have huge biodiversity on your hand because it’s mainly invisible, microscopic and unkwown. Poeple talk about biodiversity dark matter.
- We have a double problematic. if go outside and take a handful of soil, you will have huge biodiversity on your hand because it’s mainly invisible, microscopic and unkwown. Poeple talk about biodiversity dark matter.
- We have a double problematic. if go outside and take a handful of soil, you will have huge biodiversity on your hand because it’s mainly invisible, microscopic and unkwown. Poeple talk about biodiversity dark matter.
- We have a double problematic. if go outside and take a handful of soil, you will have huge biodiversity on your hand because it’s mainly invisible, microscopic and unkwown. Poeple talk about biodiversity dark matter.
- We have a double problematic. if go outside and take a handful of soil, you will have huge biodiversity on your hand because it’s mainly invisible, microscopic and unkwown. Poeple talk about biodiversity dark matter.
- We have a double problematic. if go outside and take a handful of soil, you will have huge biodiversity on your hand because it’s mainly invisible, microscopic and unkwown. Poeple talk about biodiversity dark matter.
- We have a double problematic. if go outside and take a handful of soil, you will have huge biodiversity on your hand because it’s mainly invisible, microscopic and unkwown. Poeple talk about biodiversity dark matter.
- We have a double problematic. if go outside and take a handful of soil, you will have huge biodiversity on your hand because it’s mainly invisible, microscopic and unkwown. Poeple talk about biodiversity dark matter.
- We have a double problematic. if go outside and take a handful of soil, you will have huge biodiversity on your hand because it’s mainly invisible, microscopic and unkwown. Poeple talk about biodiversity dark matter.
- We have a double problematic. if go outside and take a handful of soil, you will have huge biodiversity on your hand because it’s mainly invisible, microscopic and unkwown. Poeple talk about biodiversity dark matter.
- We have a double problematic. if go outside and take a handful of soil, you will have huge biodiversity on your hand because it’s mainly invisible, microscopic and unkwown. Poeple talk about biodiversity dark matter.
- We have a double problematic. if go outside and take a handful of soil, you will have huge biodiversity on your hand because it’s mainly invisible, microscopic and unkwown. Poeple talk about biodiversity dark matter.
- We have a double problematic. if go outside and take a handful of soil, you will have huge biodiversity on your hand because it’s mainly invisible, microscopic and unkwown. Poeple talk about biodiversity dark matter.
- We have a double problematic. if go outside and take a handful of soil, you will have huge biodiversity on your hand because it’s mainly invisible, microscopic and unkwown. Poeple talk about biodiversity dark matter.
- We have a double problematic. if go outside and take a handful of soil, you will have huge biodiversity on your hand because it’s mainly invisible, microscopic and unkwown. Poeple talk about biodiversity dark matter.
- We have a double problematic. if go outside and take a handful of soil, you will have huge biodiversity on your hand because it’s mainly invisible, microscopic and unkwown. Poeple talk about biodiversity dark matter.
- We have a double problematic. if go outside and take a handful of soil, you will have huge biodiversity on your hand because it’s mainly invisible, microscopic and unkwown. Poeple talk about biodiversity dark matter.
- We have a double problematic. if go outside and take a handful of soil, you will have huge biodiversity on your hand because it’s mainly invisible, microscopic and unkwown. Poeple talk about biodiversity dark matter.
- We have a double problematic. if go outside and take a handful of soil, you will have huge biodiversity on your hand because it’s mainly invisible, microscopic and unkwown. Poeple talk about biodiversity dark matter.
- We have a double problematic. if go outside and take a handful of soil, you will have huge biodiversity on your hand because it’s mainly invisible, microscopic and unkwown. Poeple talk about biodiversity dark matter.
- We have a double problematic. if go outside and take a handful of soil, you will have huge biodiversity on your hand because it’s mainly invisible, microscopic and unkwown. Poeple talk about biodiversity dark matter.
- We have a double problematic. if go outside and take a handful of soil, you will have huge biodiversity on your hand because it’s mainly invisible, microscopic and unkwown. Poeple talk about biodiversity dark matter.
- We have a double problematic. if go outside and take a handful of soil, you will have huge biodiversity on your hand because it’s mainly invisible, microscopic and unkwown. Poeple talk about biodiversity dark matter.
- We have a double problematic. if go outside and take a handful of soil, you will have huge biodiversity on your hand because it’s mainly invisible, microscopic and unkwown. Poeple talk about biodiversity dark matter.
- CRP alignment reveals that cysteines are conserved as defined as MT family criteria.
- CRP alignment reveals that cysteines are conserved as defined as MT family criteria.
- CRP alignment reveals that cysteines are conserved as defined as MT family criteria.
- CRP alignment reveals that cysteines are conserved as defined as MT family criteria.
- CRP alignment reveals that cysteines are conserved as defined as MT family criteria.
- CRP alignment reveals that cysteines are conserved as defined as MT family criteria.
- The same set of clusters are found in crustacean and ciliate MT families. We can wonder if the CRPs are just MT sequences from family 3 or 7 which have diverged?
- The same set of clusters are found in crustacean and ciliate MT families. We can wonder if the CRPs are just MT sequences from family 3 or 7 which have diverged?
- CRP alignment reveals that cysteines are conserved as defined as MT family criteria.
- CRP alignment reveals that cysteines are conserved as defined as MT family criteria.
- CRP alignment reveals that cysteines are conserved as defined as MT family criteria.
- CRP alignment reveals that cysteines are conserved as defined as MT family criteria.
- CRP alignment reveals that cysteines are conserved as defined as MT family criteria.
- CRP alignment reveals that cysteines are conserved as defined as MT family criteria.
- We analyzed CRP metal binding abilities. and try to found what are the difference between CRPs and MTs concerning this characteristic ?
- We analyzed CRP metal binding abilities. and try to found what are the difference between CRPs and MTs concerning this characteristic ?
- We analyzed CRP metal binding abilities. and try to found what are the difference between CRPs and MTs concerning this characteristic ?
- We analyzed CRP metal binding abilities. and try to found what are the difference between CRPs and MTs concerning this characteristic ?
- We analyzed CRP metal binding abilities. and try to found what are the difference between CRPs and MTs concerning this characteristic ?
- We analyzed CRP metal binding abilities. and try to found what are the difference between CRPs and MTs concerning this characteristic ?
- We analyzed CRP metal binding abilities. and try to found what are the difference between CRPs and MTs concerning this characteristic ?
- We analyzed CRP metal binding abilities. and try to found what are the difference between CRPs and MTs concerning this characteristic ?
- We analyzed CRP metal binding abilities. and try to found what are the difference between CRPs and MTs concerning this characteristic ?
- We analyzed CRP metal binding abilities. and try to found what are the difference between CRPs and MTs concerning this characteristic ?
- Functional complementation was analyzed by drop test. Drop test consists in spreading serial dilutions of liquid yeast cultures on solid media supplemented with metals. You always have a control medium without metals. At the top, positive and negative controls : -Wild type able to grow -Sensitive mutant unable to grow And you have your complemented mutants to test. Here, they show a better development than the negative control mutant.
- Functional complementation was analyzed by drop test. Drop test consists in spreading serial dilutions of liquid yeast cultures on solid media supplemented with metals. You always have a control medium without metals. At the top, positive and negative controls : -Wild type able to grow -Sensitive mutant unable to grow And you have your complemented mutants to test. Here, they show a better development than the negative control mutant.
- Functional complementation was analyzed by drop test. Drop test consists in spreading serial dilutions of liquid yeast cultures on solid media supplemented with metals. You always have a control medium without metals. At the top, positive and negative controls : -Wild type able to grow -Sensitive mutant unable to grow And you have your complemented mutants to test. Here, they show a better development than the negative control mutant.
- Functional complementation was analyzed by drop test. Drop test consists in spreading serial dilutions of liquid yeast cultures on solid media supplemented with metals. You always have a control medium without metals. At the top, positive and negative controls : -Wild type able to grow -Sensitive mutant unable to grow And you have your complemented mutants to test. Here, they show a better development than the negative control mutant.
- Functional complementation was analyzed by drop test. Drop test consists in spreading serial dilutions of liquid yeast cultures on solid media supplemented with metals. You always have a control medium without metals. At the top, positive and negative controls : -Wild type able to grow -Sensitive mutant unable to grow And you have your complemented mutants to test. Here, they show a better development than the negative control mutant.
- Functional complementation was analyzed by drop test. Drop test consists in spreading serial dilutions of liquid yeast cultures on solid media supplemented with metals. You always have a control medium without metals. At the top, positive and negative controls : -Wild type able to grow -Sensitive mutant unable to grow And you have your complemented mutants to test. Here, they show a better development than the negative control mutant.
- We analyzed CRP metal binding abilities. and try to found what are the difference between CRPs and MTs concerning this characteristic ?
- We analyzed CRP metal binding abilities. and try to found what are the difference between CRPs and MTs concerning this characteristic ?
- We analyzed CRP metal binding abilities. and try to found what are the difference between CRPs and MTs concerning this characteristic ?
- We analyzed CRP metal binding abilities. and try to found what are the difference between CRPs and MTs concerning this characteristic ?
- We analyzed CRP metal binding abilities. and try to found what are the difference between CRPs and MTs concerning this characteristic ?
- We analyzed CRP metal binding abilities. and try to found what are the difference between CRPs and MTs concerning this characteristic ?
- We analyzed CRP metal binding abilities. and try to found what are the difference between CRPs and MTs concerning this characteristic ?
- We analyzed CRP metal binding abilities. and try to found what are the difference between CRPs and MTs concerning this characteristic ?
- We analyzed CRP metal binding abilities. and try to found what are the difference between CRPs and MTs concerning this characteristic ?
- We analyzed CRP metal binding abilities. and try to found what are the difference between CRPs and MTs concerning this characteristic ?
- We analyzed CRP metal binding abilities. and try to found what are the difference between CRPs and MTs concerning this characteristic ?
- We analyzed CRP metal binding abilities. and try to found what are the difference between CRPs and MTs concerning this characteristic ?
- We analyzed CRP metal binding abilities. and try to found what are the difference between CRPs and MTs concerning this characteristic ?
- We analyzed CRP metal binding abilities. and try to found what are the difference between CRPs and MTs concerning this characteristic ?
- We analyzed CRP metal binding abilities. and try to found what are the difference between CRPs and MTs concerning this characteristic ?
- We analyzed CRP metal binding abilities. and try to found what are the difference between CRPs and MTs concerning this characteristic ?
- We analyzed CRP metal binding abilities. and try to found what are the difference between CRPs and MTs concerning this characteristic ?
- We analyzed CRP metal binding abilities. and try to found what are the difference between CRPs and MTs concerning this characteristic ?
- We analyzed CRP metal binding abilities. and try to found what are the difference between CRPs and MTs concerning this characteristic ?
- We analyzed CRP metal binding abilities. and try to found what are the difference between CRPs and MTs concerning this characteristic ?
- We analyzed CRP metal binding abilities. and try to found what are the difference between CRPs and MTs concerning this characteristic ?
- We analyzed CRP metal binding abilities. and try to found what are the difference between CRPs and MTs concerning this characteristic ?
- We analyzed CRP metal binding abilities. and try to found what are the difference between CRPs and MTs concerning this characteristic ?
- We analyzed CRP metal binding abilities. and try to found what are the difference between CRPs and MTs concerning this characteristic ?
- We analyzed CRP metal binding abilities. and try to found what are the difference between CRPs and MTs concerning this characteristic ?
- We analyzed CRP metal binding abilities. and try to found what are the difference between CRPs and MTs concerning this characteristic ?
- We analyzed CRP metal binding abilities. and try to found what are the difference between CRPs and MTs concerning this characteristic ?
- We analyzed CRP metal binding abilities. and try to found what are the difference between CRPs and MTs concerning this characteristic ?
- We analyzed CRP metal binding abilities. and try to found what are the difference between CRPs and MTs concerning this characteristic ?
- We analyzed CRP metal binding abilities. and try to found what are the difference between CRPs and MTs concerning this characteristic ?
- We analyzed CRP metal binding abilities. and try to found what are the difference between CRPs and MTs concerning this characteristic ?
- We analyzed CRP metal binding abilities. and try to found what are the difference between CRPs and MTs concerning this characteristic ?
- We analyzed CRP metal binding abilities. and try to found what are the difference between CRPs and MTs concerning this characteristic ?
- We analyzed CRP metal binding abilities. and try to found what are the difference between CRPs and MTs concerning this characteristic ?
- We have a double problematic. if go outside and take a handful of soil, you will have huge biodiversity on your hand because it’s mainly invisible, microscopic and unkwown. Poeple talk about biodiversity dark matter.
- We have a double problematic. if go outside and take a handful of soil, you will have huge biodiversity on your hand because it’s mainly invisible, microscopic and unkwown. Poeple talk about biodiversity dark matter.
- We have a double problematic. if go outside and take a handful of soil, you will have huge biodiversity on your hand because it’s mainly invisible, microscopic and unkwown. Poeple talk about biodiversity dark matter.
- We have a double problematic. if go outside and take a handful of soil, you will have huge biodiversity on your hand because it’s mainly invisible, microscopic and unkwown. Poeple talk about biodiversity dark matter.
- We have a double problematic. if go outside and take a handful of soil, you will have huge biodiversity on your hand because it’s mainly invisible, microscopic and unkwown. Poeple talk about biodiversity dark matter.
- We have a double problematic. if go outside and take a handful of soil, you will have huge biodiversity on your hand because it’s mainly invisible, microscopic and unkwown. Poeple talk about biodiversity dark matter.
- We have a double problematic. if go outside and take a handful of soil, you will have huge biodiversity on your hand because it’s mainly invisible, microscopic and unkwown. Poeple talk about biodiversity dark matter.
- We have a double problematic. if go outside and take a handful of soil, you will have huge biodiversity on your hand because it’s mainly invisible, microscopic and unkwown. Poeple talk about biodiversity dark matter.
- We have a double problematic. if go outside and take a handful of soil, you will have huge biodiversity on your hand because it’s mainly invisible, microscopic and unkwown. Poeple talk about biodiversity dark matter.
- We have a double problematic. if go outside and take a handful of soil, you will have huge biodiversity on your hand because it’s mainly invisible, microscopic and unkwown. Poeple talk about biodiversity dark matter.
- We have a double problematic. if go outside and take a handful of soil, you will have huge biodiversity on your hand because it’s mainly invisible, microscopic and unkwown. Poeple talk about biodiversity dark matter.
- We have a double problematic. if go outside and take a handful of soil, you will have huge biodiversity on your hand because it’s mainly invisible, microscopic and unkwown. Poeple talk about biodiversity dark matter.
- We have a double problematic. if go outside and take a handful of soil, you will have huge biodiversity on your hand because it’s mainly invisible, microscopic and unkwown. Poeple talk about biodiversity dark matter.
- We have a double problematic. if go outside and take a handful of soil, you will have huge biodiversity on your hand because it’s mainly invisible, microscopic and unkwown. Poeple talk about biodiversity dark matter.
- We have a double problematic. if go outside and take a handful of soil, you will have huge biodiversity on your hand because it’s mainly invisible, microscopic and unkwown. Poeple talk about biodiversity dark matter.