1.89, Environmental Microbiology
Prof. Martin Polz
Lecture 16
Recap
Hybridization
Environmental sample
extraction
Q PCR
Quantification
DNA
PCR
Specific genes
(rRNA)
cloning
Phylogenetic relationships
Diversity with in samples
Compare community
Sequences
Structure between samples
Overall
Major phylogenetic lineages have remained uncultured ⇒ existence only known from
clone libraries
Neutral
mutation – in
3rd codon so
don’t matter
Number of unique sequences found
Estimation of Diversity
Example: coastal ocean bacterioplankton
Statistical tools: chao 1 test
S = Sobs +
Total
number of
sequences
Number clones sequenced
a2
2b
a=number sequences found once
b=number sequences found twice
1000
1600 rRNA genes
1.89, Environmental Microbiology
Prof. Martin Polz
Lecture 16
Page 1 of 3
"unstructured tree"
average branch length is approximately the same
Clusters of closely related sequences
(similar clusters observed for different genes sequenced)
Functional cluster of organisms; carry out same
ecological functions because arose from common organism.
Functional cluster of
organisms; carry out same ecological functions because arose from
common organism
Question: How can such structures arise? What does it mean in an ecological context?
see here
Organisms takes
Adaptive mutation
can be point or
lateral gene
transfer or …
ove
r
Adaptive mutation
Adaptive mutation
Same niche
Time
Clonal diversification
from mutant; have
similar function
(via neutral mutations)
Time
selective sweep
Same niche
We can detect/quantify microbial diversity:
Community Fingerprinting: Only works on abundant organisms.
Quick way of seeing
if two communities
contain the same
types of organisms
(temporal or spacial
heterogeneity)
Techniques:
1. ARDRA (Restriction digestion of PCR amplified rDNA)
2. T-RF (introduce RE, cut at various places, specific patterns revealed on
electropherograms)
Fluorescent
molecule
RE cut
Primer
rDNA
1.89, Environmental Microbiology
Prof. Martin Polz
Lecture 16
Page 2 of 3
3. DGGE (Denaturant Gradient Gel Electrophoresis) will not denature. Run on
gel to get patterns that reveal ecologically significant patterns.
-
Increasing
denaturant
gradient
GC Region
G
C
H-bond
[
+
Melting
Get specific patterns
Detection/Quantification in Environment
Techniques:
1. In situ hybridization
Ribosomes (rRNAs)
Filters
Mix with cells carrying
fluorescent labels
(oligonucleotides)
Fix cells with format dehyde.
Must kill cells before live cells
impermeable to probes and would
digest probes.
Wash away
unbound probe
Binding of probe to
rRNA in ribosome
Count labeled cells
2. QPCR (Quantitative PCR) see handout
1.89, Environmental Microbiology
Prof. Martin Polz
Lecture 16
Page 3 of 3