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 p

er g

day

- N mineralization - Nitrification

Redox Potential (15 cm)

Mean Annual Precipitation (mm)

Red

ox P

oten

tial

(mV

)

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)

27F 519RPCR

Digest*

*

TRFLP pattern

TRFLP close-up

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

DGGE Principle

Low denaturant

High Denaturant

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

Dry Wet Dry Wet

DGGE patterns

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

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What is Intergenic Transcribed Spacer Analysis (also known as

RISA)?

16S rDNA Spacer 23SrDNA

16S primer 23S primer

Intergenic transcribed Spacer Analysis shows bacterial communities between the two soils are

different

16SrDNAtRNA’s

23SrDNA

Unique spacer sequences

Top Horizon Second Horizon1 2 3 4 5 6 1 2 3 4 5 6 M

Dry Wet Dry Wet

ITS Patterns

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

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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

R2 = 0.6501

02468

10

0.1 10 1000 100000

KCl extractable Al (ng/g dry soil)

pH

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