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

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/DNA Hybridization

DNA/DNA Hybridization

DNA/DNA Hybridization

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

Comparing DNA sequences

Comparing DNA sequences

Phylogenetic trees

Universal Phylogenetic Tree

Variation in E.Coli and Archaeal 16S rRNA

Making clone libraries

Diversity analysis of deep sea sample

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

Hawaiian Precipitation Gradient

Precipitation Gradient

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)

Net Primary Productivity

N mineralization and 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 *

TRFLP pattern

TRFLP close-up

Cluster analysis of TRFLP patterns

DGGE Principle Low denaturant High Denaturant

DGGE patterns Top Horizon Second Horizon 1 2 3 4 5 6 1 2 3 4 5 6 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