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

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

18. Making clone libraries

22. Diversity analysis of deep sea sample

23. Generating Fingerprints of Microbial Diversity
TRFLP
DGGE
RISA

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

25. Hawaiian Precipitation Gradient

26. Precipitation Gradient

27. 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)

28. Net Primary Productivity

29. N mineralization and Nitrification
g per g day

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

31. 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

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

33. TRFLP pattern

34. TRFLP close-up

36. Cluster analysis of TRFLP patterns

37. DGGE Principle
Low denaturant
High Denaturant

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

39. 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

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

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

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

43. 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

45. effective cation exchange capacity
exchangeable H+
Percent of Maximum Cation Holding Capacity
exchangeable Ca2+
exchangeable
Al3+
Soil pH