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Microbial Diversity

Microbial Diversity

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Microbial Diversity

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Presentation Transcript

  1. Microbial Diversity

  2. Outline • 16S/18S rDNA and operational taxonomic units • Clone libraries • Intergenic Transcribed Spacer Analysis • Denaturant Gradient Gel Electrophoresis • Terminal Restriction Fragment Length Polymorphism

  3. Bacterial species are described with chemical (including DNA sequences), physiological, and morphological characteristics

  4. Bacteria maybe compared through DNA/DNA Hybridization or 16S rDNA sequencing

  5. DNA/DNA Hybridization

  6. DNA/DNA Hybridization

  7. DNA/DNA Hybridization

  8. DNA sequence analysis The only approach that gives us a detailed description of microbial diversity

  9. 16S ribosomal RNA sequence is often used to determine taxonomic identity of microorganism

  10. Why use 16S/18S ribosomal gene sequences? • All microorganisms, except viruses have them • Slow enough mutation rate that all organisms can be compared

  11. Comparing DNA sequences

  12. Comparing DNA sequences

  13. Phylogenetic trees

  14. Universal Phylogenetic Tree

  15. Variation in E.Coli and Archaeal 16S rRNA

  16. Making clone libraries

  17. Diversity analysis of deep sea sample

  18. Generating Fingerprints of Microbial Diversity • TRFLP • DGGE • RISA

  19. Maui precipitation gradient • Collaboration with Ted Schuur (University of Florida) • Bacterial community profiles • Comparison of bacterial community profiles with ecosystem variables

  20. Hawaiian Precipitation Gradient

  21. Precipitation Gradient

  22. Similarities among all 6 sites along the precipitation gradient • Mean Annual Temperature (160C) • Dominant Vegetation (Metrosideros forest) • Parent Material (Lava and Ash) • Substrate Age (410,000 yrs)

  23. Net Primary Productivity

  24. N mineralization and Nitrification  - N mineralization  - Nitrification g per g day

  25. Redox Potential (15 cm) Redox Potential (mV) Mean Annual Precipitation (mm)

  26. How does the bacterial community change across the precipitation gradient? • Intergenic Transcribed Spacer analysis • Denaturant Gradient Gel Electrophoresis • Phospho Lipid Fatty Acid Analysis • Terminal Restriction Fragment Length Polymorphism analysis

  27. Terminal Restriction Fragment Length Polymorphism (TRFLP) PCR 27F 519R * Digest *

  28. TRFLP pattern

  29. TRFLP close-up

  30. Cluster analysis of TRFLP patterns

  31. DGGE Principle Low denaturant High Denaturant

  32. DGGE patterns Top Horizon Second Horizon 1 2 3 4 5 6 1 2 3 4 5 6 Dry Wet Dry Wet

  33. Cluster Analysis of Denaturant Gradient Gel Electrophoresis patterns 3-2 2-2 5-1 2-1 3-1 1-1 1-2 4-2 4-1 6-1 6-2 5-2

  34. What is Intergenic Transcribed Spacer Analysis (also known as RISA)? 16S primer 23S primer 16S rDNA Spacer 23SrDNA

  35. Intergenic transcribed Spacer Analysis shows bacterial communities between the two soils are different tRNA’s 23SrDNA 16SrDNA Unique spacer sequences

  36. ITS Patterns Top Horizon Second Horizon 1 2 3 4 5 6 1 2 3 4 5 6 M Dry Wet Dry Wet

  37. Cluster Analysis of Ribosomal Intergenic Spacer patterns 2-2 1-2 5-1 3-1 4-2 3-2 2-1 4-1 1-1 6-1 6-2 5-2

  38. effective cation exchange capacity exchangeable H+ 0 25 50 75 100 Percent of Maximum Cation Holding Capacity exchangeable Ca2+ exchangeable Al3+ 3 4 5 6 7 8 Soil pH