Download - Microbial Diversity
![Page 1: Microbial Diversity](https://reader035.vdocuments.us/reader035/viewer/2022062222/56815c1f550346895dc9f3ec/html5/thumbnails/1.jpg)
Microbial Diversity
![Page 2: Microbial Diversity](https://reader035.vdocuments.us/reader035/viewer/2022062222/56815c1f550346895dc9f3ec/html5/thumbnails/2.jpg)
Outline
• 16S/18S rDNA and operational taxonomic units
• Clone libraries• Intergenic Transcribed Spacer Analysis• Denaturant Gradient Gel Electrophoresis• Terminal Restriction Fragment Length
Polymorphism
![Page 3: Microbial Diversity](https://reader035.vdocuments.us/reader035/viewer/2022062222/56815c1f550346895dc9f3ec/html5/thumbnails/3.jpg)
Bacterial species are described with chemical (including DNA sequences), physiological,
and morphological characteristics
![Page 4: Microbial Diversity](https://reader035.vdocuments.us/reader035/viewer/2022062222/56815c1f550346895dc9f3ec/html5/thumbnails/4.jpg)
Bacteria maybe compared through DNA/DNA Hybridization or 16S rDNA
sequencing
![Page 5: Microbial Diversity](https://reader035.vdocuments.us/reader035/viewer/2022062222/56815c1f550346895dc9f3ec/html5/thumbnails/5.jpg)
DNA/DNA Hybridization
![Page 6: Microbial Diversity](https://reader035.vdocuments.us/reader035/viewer/2022062222/56815c1f550346895dc9f3ec/html5/thumbnails/6.jpg)
DNA/DNA Hybridization
![Page 7: Microbial Diversity](https://reader035.vdocuments.us/reader035/viewer/2022062222/56815c1f550346895dc9f3ec/html5/thumbnails/7.jpg)
DNA/DNA Hybridization
![Page 8: Microbial Diversity](https://reader035.vdocuments.us/reader035/viewer/2022062222/56815c1f550346895dc9f3ec/html5/thumbnails/8.jpg)
DNA sequence analysis
The only approach that gives us a detailed description of
microbial diversity
![Page 9: Microbial Diversity](https://reader035.vdocuments.us/reader035/viewer/2022062222/56815c1f550346895dc9f3ec/html5/thumbnails/9.jpg)
16S ribosomal RNA sequence is often used to determine taxonomic identity of microorganism
![Page 10: Microbial Diversity](https://reader035.vdocuments.us/reader035/viewer/2022062222/56815c1f550346895dc9f3ec/html5/thumbnails/10.jpg)
Why use 16S/18S ribosomal gene sequences?
• All microorganisms, except viruses have them
• Slow enough mutation rate that all organisms can be compared
![Page 11: Microbial Diversity](https://reader035.vdocuments.us/reader035/viewer/2022062222/56815c1f550346895dc9f3ec/html5/thumbnails/11.jpg)
Comparing DNA sequences
![Page 12: Microbial Diversity](https://reader035.vdocuments.us/reader035/viewer/2022062222/56815c1f550346895dc9f3ec/html5/thumbnails/12.jpg)
Comparing DNA sequences
![Page 13: Microbial Diversity](https://reader035.vdocuments.us/reader035/viewer/2022062222/56815c1f550346895dc9f3ec/html5/thumbnails/13.jpg)
Phylogenetic trees
![Page 14: Microbial Diversity](https://reader035.vdocuments.us/reader035/viewer/2022062222/56815c1f550346895dc9f3ec/html5/thumbnails/14.jpg)
Universal Phylogenetic Tree
![Page 15: Microbial Diversity](https://reader035.vdocuments.us/reader035/viewer/2022062222/56815c1f550346895dc9f3ec/html5/thumbnails/15.jpg)
![Page 16: Microbial Diversity](https://reader035.vdocuments.us/reader035/viewer/2022062222/56815c1f550346895dc9f3ec/html5/thumbnails/16.jpg)
Variation in E.Coli and Archaeal 16S rRNA
![Page 17: Microbial Diversity](https://reader035.vdocuments.us/reader035/viewer/2022062222/56815c1f550346895dc9f3ec/html5/thumbnails/17.jpg)
![Page 18: Microbial Diversity](https://reader035.vdocuments.us/reader035/viewer/2022062222/56815c1f550346895dc9f3ec/html5/thumbnails/18.jpg)
Making clone libraries
![Page 19: Microbial Diversity](https://reader035.vdocuments.us/reader035/viewer/2022062222/56815c1f550346895dc9f3ec/html5/thumbnails/19.jpg)
![Page 20: Microbial Diversity](https://reader035.vdocuments.us/reader035/viewer/2022062222/56815c1f550346895dc9f3ec/html5/thumbnails/20.jpg)
![Page 21: Microbial Diversity](https://reader035.vdocuments.us/reader035/viewer/2022062222/56815c1f550346895dc9f3ec/html5/thumbnails/21.jpg)
![Page 22: Microbial Diversity](https://reader035.vdocuments.us/reader035/viewer/2022062222/56815c1f550346895dc9f3ec/html5/thumbnails/22.jpg)
Diversity analysis of deep sea sample
![Page 23: Microbial Diversity](https://reader035.vdocuments.us/reader035/viewer/2022062222/56815c1f550346895dc9f3ec/html5/thumbnails/23.jpg)
Generating Fingerprints of Microbial Diversity
• TRFLP• DGGE• RISA
![Page 24: Microbial Diversity](https://reader035.vdocuments.us/reader035/viewer/2022062222/56815c1f550346895dc9f3ec/html5/thumbnails/24.jpg)
Maui precipitation gradient
• Collaboration with Ted Schuur (University of Florida)
• Bacterial community profiles• Comparison of bacterial community
profiles with ecosystem variables
![Page 25: Microbial Diversity](https://reader035.vdocuments.us/reader035/viewer/2022062222/56815c1f550346895dc9f3ec/html5/thumbnails/25.jpg)
Hawaiian Precipitation Gradient
![Page 26: Microbial Diversity](https://reader035.vdocuments.us/reader035/viewer/2022062222/56815c1f550346895dc9f3ec/html5/thumbnails/26.jpg)
Precipitation Gradient
![Page 27: Microbial Diversity](https://reader035.vdocuments.us/reader035/viewer/2022062222/56815c1f550346895dc9f3ec/html5/thumbnails/27.jpg)
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)
![Page 28: Microbial Diversity](https://reader035.vdocuments.us/reader035/viewer/2022062222/56815c1f550346895dc9f3ec/html5/thumbnails/28.jpg)
Net Primary Productivity
![Page 29: Microbial Diversity](https://reader035.vdocuments.us/reader035/viewer/2022062222/56815c1f550346895dc9f3ec/html5/thumbnails/29.jpg)
N mineralization and Nitrification
g p
er g
day
- N mineralization - Nitrification
![Page 30: Microbial Diversity](https://reader035.vdocuments.us/reader035/viewer/2022062222/56815c1f550346895dc9f3ec/html5/thumbnails/30.jpg)
Redox Potential (15 cm)
Mean Annual Precipitation (mm)
Red
ox P
oten
tial
(mV
)
![Page 31: Microbial Diversity](https://reader035.vdocuments.us/reader035/viewer/2022062222/56815c1f550346895dc9f3ec/html5/thumbnails/31.jpg)
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
![Page 32: Microbial Diversity](https://reader035.vdocuments.us/reader035/viewer/2022062222/56815c1f550346895dc9f3ec/html5/thumbnails/32.jpg)
Terminal Restriction Fragment Length Polymorphism (TRFLP)
27F 519RPCR
Digest*
*
![Page 33: Microbial Diversity](https://reader035.vdocuments.us/reader035/viewer/2022062222/56815c1f550346895dc9f3ec/html5/thumbnails/33.jpg)
TRFLP pattern
![Page 34: Microbial Diversity](https://reader035.vdocuments.us/reader035/viewer/2022062222/56815c1f550346895dc9f3ec/html5/thumbnails/34.jpg)
TRFLP close-up
![Page 35: Microbial Diversity](https://reader035.vdocuments.us/reader035/viewer/2022062222/56815c1f550346895dc9f3ec/html5/thumbnails/35.jpg)
![Page 36: Microbial Diversity](https://reader035.vdocuments.us/reader035/viewer/2022062222/56815c1f550346895dc9f3ec/html5/thumbnails/36.jpg)
Cluster analysis of TRFLP patterns
Distance (Objective Function)
Information Remaining (%)
4.6E-02
100
1.2E+00
75
2.5E+00
50
3.7E+00
25
4.9E+00
0
SITE 1-1SITE 2-3SITE 3-2SITE 1-2SITE 3-1SITE 2-1SITE 2-2SITE 1-3SITE 3-3SITE 4-1SITE 4-2SITE 4-3SITE 5-1SITE 5-2SITE 5-3SITE 6-1SITE 6-2SITE 6-3
![Page 37: Microbial Diversity](https://reader035.vdocuments.us/reader035/viewer/2022062222/56815c1f550346895dc9f3ec/html5/thumbnails/37.jpg)
DGGE Principle
Low denaturant
High Denaturant
![Page 38: Microbial Diversity](https://reader035.vdocuments.us/reader035/viewer/2022062222/56815c1f550346895dc9f3ec/html5/thumbnails/38.jpg)
Top Horizon Second Horizon 1 2 3 4 5 6 1 2 3 4 5 6
Dry Wet Dry Wet
DGGE patterns
![Page 39: Microbial Diversity](https://reader035.vdocuments.us/reader035/viewer/2022062222/56815c1f550346895dc9f3ec/html5/thumbnails/39.jpg)
Jaccard (Opt:1.50%) (Tol 1.0%-1.0%) (H>0.0% S>0.0%) [0.0%-100.0%]
100
50
h3-2
h2-2
h5-1
h2-1
h3-1
h 1-1
h1-2
h4-2
h4-1
h6-1
h6-2
h5-2
Cluster Analysis of Denaturant Gradient Gel Electrophoresis patterns
3-22-25-12-13-11-11-24-24-16-16-25-2
![Page 40: Microbial Diversity](https://reader035.vdocuments.us/reader035/viewer/2022062222/56815c1f550346895dc9f3ec/html5/thumbnails/40.jpg)
What is Intergenic Transcribed Spacer Analysis (also known as
RISA)?
16S rDNA Spacer 23SrDNA
16S primer 23S primer
![Page 41: Microbial Diversity](https://reader035.vdocuments.us/reader035/viewer/2022062222/56815c1f550346895dc9f3ec/html5/thumbnails/41.jpg)
Intergenic transcribed Spacer Analysis shows bacterial communities between the two soils are
different
16SrDNAtRNA’s
23SrDNA
Unique spacer sequences
![Page 42: Microbial Diversity](https://reader035.vdocuments.us/reader035/viewer/2022062222/56815c1f550346895dc9f3ec/html5/thumbnails/42.jpg)
Top Horizon Second Horizon1 2 3 4 5 6 1 2 3 4 5 6 M
Dry Wet Dry Wet
ITS Patterns
![Page 43: Microbial Diversity](https://reader035.vdocuments.us/reader035/viewer/2022062222/56815c1f550346895dc9f3ec/html5/thumbnails/43.jpg)
Jaccard (Tol 1.0%-1.0%) (H>0.0% S>0.0%) [0.0%-100.0%]
100
80604020
h2-2
h1-2
h5-1
h3-1
h4-2
h3-2
h2-1
h4-1
h1-1
h6-1
h6-2
h5-2
Cluster Analysis of Ribosomal Intergenic Spacer patterns
2-21-25-13-14-23-22-14-11-16-16-25-2
![Page 44: Microbial Diversity](https://reader035.vdocuments.us/reader035/viewer/2022062222/56815c1f550346895dc9f3ec/html5/thumbnails/44.jpg)
![Page 45: Microbial Diversity](https://reader035.vdocuments.us/reader035/viewer/2022062222/56815c1f550346895dc9f3ec/html5/thumbnails/45.jpg)
exchangeable Al3+
exchangeable Ca2+
effective cation exchange capacity
exchangeable H+
Soil pHPerc
ent o
f Max
imum
Cat
ion
Hol
ding
Cap
acity
3 4 5 6 7 8
0
25
5
0
75
10
0
![Page 46: Microbial Diversity](https://reader035.vdocuments.us/reader035/viewer/2022062222/56815c1f550346895dc9f3ec/html5/thumbnails/46.jpg)
R2 = 0.6501
02468
10
0.1 10 1000 100000
KCl extractable Al (ng/g dry soil)
pH
![Page 47: Microbial Diversity](https://reader035.vdocuments.us/reader035/viewer/2022062222/56815c1f550346895dc9f3ec/html5/thumbnails/47.jpg)
R2 = 0.5344
2
2.5
3
3.5
4
0.1 10 1000 100000
KCl extractable Al (ng/g dry soil)
Div
ersit
y