1.

89, Environmental Microbiology

Prof. Martin Polz
Lecture 16

Recap

Hybridization
Environmental sample

extraction
Q PCR
DNA
Quantification

PCR

Specific genes
(rRNA)
Phylogenetic relationships

cloning Diversity with in samples

Compare community
Sequences Structure between samples

Overall
Major phylogenetic lineages have remained uncultured ⇒ existence only known from
clone libraries

Estimation of Diversity
Example: coastal ocean bacterioplankton
Number of unique sequences found

Statistical tools: chao 1 test

a2
S = Sobs +
2b

Total a=number sequences found once

Neutral number of b=number sequences found twice
mutation – in sequences
3rd codon so
don’t matter

Number clones sequenced 1000

1600 rRNA genes

1.89, Environmental Microbiology Lecture 16

Prof. Martin Polz Page 1 of 3
 "unstructured tree" average branch length is approximately the same

Clusters of closely related sequences

Functional cluster of
organisms; carry out
(similar clusters observed for different genes sequenced)
same ecological functions
Functional cluster of organisms; carry out same
because arose from
ecological functions because arose from common organism.
common organism

see here Question: How can such structures arise? What does it mean in an ecological context?

Organisms takes
ove
r
Adaptive mutation Adaptive mutation Clonal diversification
can be point or from mutant; have
lateral gene Adaptive mutation similar function
transfer or … (via neutral mutations)
Same niche
Time Time
selective sweep

Same niche

We can detect/quantify microbial diversity:

Community Fingerprinting: Only works on abundant organisms.

Techniques:
1. ARDRA (Restriction digestion of PCR amplified rDNA)
Quick way of seeing
if two communities
contain the same 2. T-RF (introduce RE, cut at various places, specific patterns revealed on
types of organisms electropherograms)
(temporal or spacial
heterogeneity)
Fluorescent
molecule

RE cut

Primer

rDNA

1.89, Environmental Microbiology Lecture 16

Prof. Martin Polz 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
- GC Region
denaturant

gradient

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 Wash away
(oligonucleotides) unbound probe

Binding of probe to
rRNA in ribosome
Fix cells with format dehyde.
Must kill cells before live cells
impermeable to probes and would
digest probes.

Count labeled cells

2. QPCR (Quantitative PCR) see handout

1.89, Environmental Microbiology Lecture 16

Prof. Martin Polz Page 3 of 3