Transcript 4/27/12 Tools for microbial ecology

22.1 Enrichment
• Isolation
– The separation of individual organisms from
the mixed community
• Enrichment Cultures
– Select for desired organisms through
manipulation of medium and incubation
conditions
• Inocula
– The sample from which microorganisms will be
isolated
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Figure 22.1
Mineral salts medium
containing mannitol but
lacking NH4, NO3, or
organic nitrogen.
Soil
+NH4 plate
Incubate
aerobically
NH4 plate
NH4
+NH4 plate
NH4 plate
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22.1 Enrichment
• Enrichment Cultures
– Can prove the presence of an organism in a
habitat
– Cannot prove an organism does not inhabit an
environment
• The ability to isolate an organism from an
environment says nothing about its ecological
significance
Animation: Enrichment Cultures
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22.1 Enrichment
• The Winogradsky Column
– An artificial microbial ecosystem (Figure 22.2)
– Serves as a long-term source of bacteria for
enrichment cultures
– Named for Sergei Winogradsky
– First used in late 19th century to study soil
microorganisms
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Figure 22.2
Gradients Column
O2
Lake or
pond water
Mud
supplemented
with organic
nutrients
and CaSO4
Foil cap
Algae and
cyanobacteria
Purple nonsulfur
bacteria
Sulfur
chemolithotrophs
Patches of purple
sulfur or green
sulfur bacteria
H2S
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Anoxic
decomposition
and sulfate
reduction
22.1 Enrichment
• Enrichment bias
– Microorganisms cultured in the lab are
frequently only minor components of the
microbial ecosystem
• Reason: the nutrients available in the lab culture
are typically much higher than in nature
• Dilution of inoculum is performed to eliminate
rapidly growing, but quantitatively insignificant,
weed species
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22.2 Isolation
• Pure cultures contain a single kind of
microorganism
– Can be obtained by streak plate, agar shake, or
liquid dilution (Figure 22.3)
• Agar dilution tubes are mixed cultures diluted in
molten agar
– Useful for purifying anaerobic organisms
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Figure 22.3
Colonies
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Paraffin–mineral
oil seal
22.2 Isolation
• Most-probable-number technique
– Serial 10 dilutions of inocula in a liquid media
– Used to estimate number of microorganisms in
food, wastewater, and other samples (Figure 22.4)
Animation: Serial Dilutions and a Most Probable Number Analysis
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Figure 22.4
1 ml
(liquid)
or 1 g
(solid)
Enrichment culture
or natural sample
Dilution
1 ml
1 ml
1 ml
1 ml
No
growth
Growth
Growth
1 ml
9 ml of
broth
1/10
(101)
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102
103
104
105
106
22.2 Isolation
• Flow cytometry
– Uses lasers
– Suspended cultures passed through
specialized detector
– Cells separated based on fluorescence
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22.5 PCR Methods of Microbial Community
Analysis
• Specific genes can be used as a measure of
diversity
– Techniques used in molecular biodiversity
studies (Figure 22.12)
•
•
•
•
•
DNA isolation and sequencing
PCR
Restriction enzyme digest
Electrophoresis
Molecular cloning
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Figure 22.12
Microbial
community
Extract total
community DNA
Amplify by PCR
using fluorescently
tagged primers
DNA
PCR
Restriction
enzyme
digest and
run on gel
Sample
1 2 3 4
Sample
1 2 3 4
Gel
Amplify 16S RNA
genes using general
primers (for example,
Bacteria-specific) or
more restrictive
primers (to target
endospore-forming
Bacteria)
All 16S
rRNA
genes
T-RFLP
gel
Sample
1 2 3 4
Excise bands
and clone 16S
rRNA genes
Different
16S rRNA
genes
DGGE gel
Excise
bands
Sequence
Sequence
Bacillus subtilis
Generate
tree from
results
using
endosporespecific
primers
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Generate
tree from
Bacillus cereus
results
Bacillus megaterium
using
Env 2
endosporeClostridium histolyticum specific
primers
Env 1
Env 3
22.5 PCR Methods of Microbial Community
Analysis
• DGGE: denaturing gradient gel
electrophoresis separates genes of the same
size based on differences in base sequence
(Figure 22.13)
– Denaturant is a mixture of urea and
formamide
– Strands melt at different denaturant
concentrations
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Figure 22.13
2
1
3
4
5
6
7
8
6
7
8
PCR amplification
1
2
DGGE
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3
4
5
22.5 PCR Methods of Microbial Community
Analysis
• T-RFLP: terminal restriction fragment length
polymorphism
– Target gene is amplified by PCR
– Restriction enzymes are used to cut the PCR
products
• ARISA: automated ribosomal intergenic spacer
analysis (Figure 22.14)
– Related to T-RFLP
– Uses DNA sequencing
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Figure 22.14
Position 1
1
1540
23S rRNA gene
ITS region
16S rRNA gene
Forward PCR primer
containing fluorescent
tag ( )
2900
Reverse PCR primer
50–1500bp
Position 1513
Position 23
PCR
Community DNA
Fluorescence
Gel analysis
1000
750
500
250
0
400
450
500
550
600
650
700
750
800
850
900
950
Fragment size (base pairs)
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1000
1050
1100
1150
1200
1250
22.5 PCR Methods of Microbial Community
Analysis
• Results of PCR phylogenetic analyses
– Several phylogenetically distinct prokaryotes
are present
• rRNA sequences differ from those of all
known laboratory cultures
– Molecular methods conclude that less than
0.1% of bacteria have been cultured
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22.6 Microarrays and Microbial Diversity:
Phylochips
• Phylochip: microarray that focuses on
phylogenetic members of microbial community
(Figure 22.15)
– Circumvents time-consuming steps of DGGE and
T-RFLP
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Figure 22.15
Positive
Negative
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Weak positive
22.7 Environmental Genomics and
Related Methods
• Environmental genomics (metagenomics)
– DNA is cloned from microbial community and
sequenced
– Detects as many genes as possible
– Yields picture of gene pool in environment
– Can detect genes that are not amplified by current
PCR primers
– Powerful tool for assessing the phylogenetic and
metabolic diversity of an environment
(Figure 22.16)
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Figure 22.16
Microbial
community
Extract total
community DNA
DNA
Community
sampling approach
Environmental
genomics approach
Restriction digest total DNA and
then shotgun sequence, OR
sequence directly (without
cloning) using a high throughput
DNA sequencer
Amplify single gene,
for example, gene
encoding 16S rRNA
Sequence and
generate tree
Assembly and
annotation
Outcomes
Single-gene phylogenetic tree
1. Phylogenetic snapshot
of most members of
the community
2. Identification of novel
phylotypes
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Partial
genomes
Total gene pool of the community
1. Identification of all gene categories
2. Discovery of new genes
3. Linking of genes to phylotypes
III. Measuring Microbial Activities in Nature
• 22.8 Chemical Assays, Radioisotopic Methods,
and Microelectrodes
• 22.9 Stable Isotopes
• 22.10 Linking Specific Genes and Functions to
Specific Organisms
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22.8 Chemical Assays, Radioisotopes, &
Microelectrodes
• In many studies, direct chemical measurements
are sufficient (Figure 22.18)
– Higher sensitivity can be achieved with
radioisotopes
• Proper killed cell controls must be used
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Figure 22.18
Sulfate reduction
Lactate
incorporation
H2S
14CO
2
Lactate or H2S
Formalin-killed control
Photosynthesis
14C-Glucose
Dark
H2 absent
14CO
2
H2 present
respiration
evolution
Sulfate reduction
H2 35S
Killed
Time
Time
Killed
Light
Killed
Time
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Time
22.8 Chemical Assays, Radioisotopes, &
Microelectrodes
• Microelectrodes
– Can measure a wide range of activity
– pH, oxygen, CO2, and others can be
measured
– Small glass electrodes, quite fragile
(Figure 22.19)
– Electrodes are carefully inserted into the
habitat (e.g., microbial mats)
• Measurements taken every 50–100mm
(Figure 22.20)
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Figure 22.19
Gold
Glass
Platinum
5 mm
Membranes
50–100 mm
NO3
2e 1 N2O  H2O
N2O
Bacteria
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Glass
Cathode
N2  2 OH
Nutrient solution
Figure 22.20
Oxygen (O2) concentration (mM)
100
200
0
300
Depth in sediment (mm)
Seawater
0
NO3
O2
Oxic sediment
5
Anoxic sediment
10
0
8
4
Nitrate (NO3) concentration (mM)
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