550 likes | 1.28k Views
Microbial Diversity. Outline. 16S/18S rDNA and operational taxonomic units Clone libraries Intergenic Transcribed Spacer Analysis Denaturant Gradient Gel Electrophoresis Terminal Restriction Fragment Length Polymorphism.
E N D
Outline • 16S/18S rDNA and operational taxonomic units • Clone libraries • Intergenic Transcribed Spacer Analysis • Denaturant Gradient Gel Electrophoresis • Terminal Restriction Fragment Length Polymorphism
Bacterial species are described with chemical (including DNA sequences), physiological, and morphological characteristics
Bacteria maybe compared through DNA/DNA Hybridization or 16S rDNA sequencing
DNA sequence analysis The only approach that gives us a detailed description of microbial diversity
16S ribosomal RNA sequence is often used to determine taxonomic identity of microorganism
Why use 16S/18S ribosomal gene sequences? • All microorganisms, except viruses have them • Slow enough mutation rate that all organisms can be compared
Generating Fingerprints of Microbial Diversity • TRFLP • DGGE • RISA
Maui precipitation gradient • Collaboration with Ted Schuur (University of Florida) • Bacterial community profiles • Comparison of bacterial community profiles with ecosystem variables
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)
N mineralization and Nitrification - N mineralization - Nitrification g per g day
Redox Potential (15 cm) Redox Potential (mV) Mean Annual Precipitation (mm)
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
Terminal Restriction Fragment Length Polymorphism (TRFLP) PCR 27F 519R * Digest *
DGGE Principle Low denaturant High Denaturant
DGGE patterns Top Horizon Second Horizon 1 2 3 4 5 6 1 2 3 4 5 6 Dry Wet Dry Wet
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
What is Intergenic Transcribed Spacer Analysis (also known as RISA)? 16S primer 23S primer 16S rDNA Spacer 23SrDNA
Intergenic transcribed Spacer Analysis shows bacterial communities between the two soils are different tRNA’s 23SrDNA 16SrDNA Unique spacer sequences
ITS Patterns Top Horizon Second Horizon 1 2 3 4 5 6 1 2 3 4 5 6 M Dry Wet Dry Wet
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
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