Download 1 Microbial Ecology Example of the Marine Carbon Cycle:

Microbial Ecology
• Learning Objectives:
– To learn how to study microbes in their natural environments
– To understand techniques used to investigate microbial ecology
• Outline:
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Overview of microbial ecology
Microbial ecology techniques
Example 1: Archaea in the ocean.
Example 2: Arsenic cycling in Mono Lake.
Example 3: Microbiome of the GI tract.
Microbiology and biogeochemical cycling
What is microbial ecology?
• Study of microbes and their interactions with the
environment.
• Some examples of microbial ecology:
– Quantifying sulfur oxidizers in a deep sea hydrothermal vent.
– Determining biodiversity of prokaryotes in the human GI tract.
– Monitoring the distribution of ctx gene in marine estuaries.
• The subject of investigation can be application based or
fundamental.
– Application based science (or applied science) is usually driven by
problems effecting society in some way.
– Fundamental science aims to advance the understanding of a
particular process in nature.
Example of the Marine Carbon Cycle:
• Microbes mediate transformation and recycling of
elements in nature.
– Carbon, sulfur, nitrogen, phosphorus are some examples.
– Toxic metals also undergo biogeochemical cycling (e.g. Hg, As)
• Biological, geological, and chemical processes work
together to alter fate and transport of elements.
• Element cycles usually involve oxidation-reduction
reactions during transport of an element in the
environment.
• Elements move through different trophic levels.
• Impact of biogeochemical cycling:
– Affect bioavailability of elements to higher organisms
– Control energy flow within the oceans.
– Nutrient cycling can also occur within an organism (GI tract)
Edward F. DeLong and David M. Karl, Genomic perspectives in microbial
oceanography, Nature 437, 336-342 (15 September 2005)
To culture or not to culture?
Common approaches used in microbial ecology:
Culture-dependent approach: grow organisms of a specific type
• Study it as a model organism for an environmental process; or
quantify abundance of specific organisms (disease causing or not).
• Pros:
1. Enumerating (counting) microorganisms
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– You now have a system that is useful for mechanistic studies.
– You can determine the abundance of a particular population of microbes
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2. Microcosm study
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Cons:
– You can’t grow every microorganism (culture bias).
– Is your model organism the one responsible for a particular process?
– You can never prove a sample is negative for a particular organism.
Culture-independent approach: use molecular techniques to observe
organisms or detect “signatures” of their activities without growth.
• Pros:
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Total counts by microscope
– DNA dye and epifluorescence microscopy
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– Uses 16S rRNA gene probes for bacteria or
archaea
– You can target specific genera
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Using FISH and microscopy it was
discovered that crenarchaea were highly
abundant in the ocean.
– The crenarchaea were thought to be either
extremophiles or methanogens.
Don’t culture. Instead, sequence the DNA straight from the
environment.
1. Enumeration: archaea and bacteria in the ocean
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– Usually underestimates the total count. (Why?)
– Called culture bias (bacterial enumeration
anomaly) because you can’t culture
everything.
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1. Enumeration of
microorganisms
FISH: fluorescence in situ hybridization (see
figure)
Viable counts: plate samples on media.
This can be a very rapid and useful approach to identifying
organisms and diversity within a particular environmental sample.
4. Metagenomic approaches
Cons:
– Environment is complex and hard to sort out
– Low abundance organisms are not well represented
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The mud in a bottle experiment
This is useful for determining rates of reactions
Unlike in the environment you can manipulate the environment
within the bottle.
3. 16S rRNA and functional gene analysis
– You can identify microbes without knowing their culturing conditions.
– The culture bias is no longer a problem
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The goal is to determine the abundance of microorganisms.
Some approaches use cultivation approaches others do not.
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Archaea were found to
abundant in the deep ocean.
This was unusual.
Archaea were also highly
abundant in coastal regions.
This raised questions about
the function of these archaeal
microorganisms.
FISH revealed the global
oceans contain:
Bacteria
– 1.3 x 1038 archaeal cells
– 3.1 x 1038 bacterial cells
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One group of archaea
comprises 1 x 1038 cells!
What is this organisms?
A representative microbe was
isolated in 2005
Archaea
Archaeal dominance in the m esopelagic zone of the Pacific
Ocean, Nature Karner 2001 vol:409 iss:6819 pg:507 -510
2. Microcosm studies
Isolation of marine Crenarchaeota SCM1
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Nitrosopumilus maritimus
Isolated from an aquarium in
Seattle.
It is a chemoautotroph
Isolated with:
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DAPI
FISH
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filtered aquarium water
ammonium chloride
Bicarbonate
streptomycin
It is the first ammonia
oxidizing crenarchaeota.
Very similar to the archaea in
the open ocean.
Collect a water or sediment
sample and incubate in a
medium that simulates the
environment.
Measure rate of substrate
utilization by:
http://www.mikelevin.com/MonoLake.html
– Direct chemical analysis. You
need a method for measuring
the chemical of interest
– Or using a chemical isotope.
You measure radioactivity
instead of the chemical.
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TEM
SEM
Isolation of an autotrophic ammonia-oxidizing marine
archaeon
Martin Könneke, Anne E. Bernhard, José R. de la Torre,
Christopher B. Walker, John B. Waterbury and David A. Stahl
Nature 437, 543-546 (22 September 2005)
3. PCR for 16S rRNA and functional genes
• 16S rRNA gene analysis:
– Used to asses the microbial diversity within a particular sample
without growing any organisms.
• Functional gene analysis:
Example: in Mono Lake
arsenic is really high. The
respiration of arsenate accounts
for ~14% of the total carbon
turnover in the lake.
3. Detection of the functional gene for arsenate
reduction, called arrA
Gel of PCR products carried out on DNA extracted from sediment samples at 8 different depths
within a sediment core. You can see the DNA bands become less intense for sediments that
are deeper in the core.
The next step is to figure out how many different kinds of arrA sequences are represented in the
DNA band. We do this by making a clone library and sequencing a lot of the clones.
– Use PCR to detect genes that encode for a protein that does
something of interest like ammonia oxidation, (amoA)
Increasing depth in core
• Diversity of PCR products can be assessed by:
– Making a clone library (brute force but low throughput)
– Using electrophoresis-based fingerprinting methods (higher
throughput): DGGE
• Sequence information is analyzed by making phylogenetic
trees.
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Blanks
plasmids
DNA inserts
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Add DNA from PCR to a
plasmid.
Ligate the two pieces together.
– One molecule of the
functional gene ligates to one
molecule of plasmid
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Transform into E. coli.
Each colony represents one
cloned DNA fragment.
Sequence the DNA insert.
– How many should we
sequence?
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Sequencing and Tree drawing
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Making a clone library
Bioinformatic analysis of the
DNA sequences.
– BLAST (online database
search program)
– Alignments and phylogenetic
trees.
ligate
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B
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transform
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plate
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DGGE analysis can give us a sense
of the diversity within a particular
sample without sequencing.
You can also analyze multiple
samples at the same time.
We put DNA from a PCR onto a
special gradient gel.
The DNA will migrate through the
gel and separate into individual
bands based on their GC content.
The bands represent individual
DNA sequences with different GC
content.
More bands = more diverse
The brighter bands also indicate a
more abundant organism.
You can cut out bands and
sequence the DNA.
Phylogenetic inferences to known
sequences and organisms from
online genetic databases:
– Genbank (functional genes)
– Ribosome Database Project (16S
rRNA genes)
Colonies
with cloned
PCR
products
It is now common to combine “classic” approaches
with modern genomic methods
DGGE: denaturing gradient gel electrophoresis
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Sequencing is usually done by dyeterminator sequencing by capillary
eletrophoresis with laser detector
You need purified plasmid DNA
or PCR products
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Determine geochemical profiles
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Do experiments with
environmental samples
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Mud, sediment, water
Get rate measurements
in situ activities.
Isolate pure cultures
Characterize physiology of the
strain, do genetic studies,
biochemistry, etc.
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Cyanobacteria
Chromatiaceae
Beta-proteobacteria,
Emphasis on geochemistry
need to measure chemical
parameters
Genome sequence
Microarrays: gene expression
Microbial ecology tools
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Develop gene detection tools
to investigate diversity of
functional genes for a process
Identification of the pure
culture in natural populations
Oremland et al. (2005) Whither or wither geomicrobiology in the
era of 'community metagenomics'.Nat Rev Microbiol. 3(7):572-8
High throughput sequencing has spawned the
“modern” approach to microbial ecology.
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Metagenomic projects
Extract environmental DNA
Make large insert DNA clone
libraries
– Bacterial artificial chromosome
(300 kb)
– fosmid (plasmid ~50 kb)
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Sequence lots of DNA
Bioinformatic analyses of sequence
data.
What to do with this data?
Goal is to understand something
about the environment:
– Must develop follow-up studies
– Are the genes expressed?
– Are the encoded products
functional in situ?
– Are there significant cycling of
elements, nutrients or energy flux
within an ecosystem.
Human Microbiome Project
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This is called the next genomic frontier for humans.
Human microbiota: the microorganisms that live in and on us.
Microbiome: the genes of the individual microbial symbionts
Gut microbiota are important to us:
– Help harvest energy from our diet and synthesize vitamins.
– Drug and toxin metabolism might predispose us to certain diseases or
cancer.
– Aid in the renewal of gut epithelial cells.
– Affects our innate immune and adaptive immune system. Could
influence immune disorders.
– Cardiac size and human physiology (germ free mice have smaller hearts)
– Behavior (germ free mice are more active).
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Disruption or alteration in one or more of these gut microbial
processes might affect our health in positive or negative ways.
The microbiome needs to be defined.
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Metagenomics: using massive
high throughput DNA
sequencing technology to
sequence genomes of
organisms in an environmental
DNA sample.
Here are few projects:
• Yellowstone hot springs
(various places)
• Dechlorinating bioreactor
• Biogas reactor
• Compost
• Bovine Rumen
• Acid mine drainage
• Marsupial (wallaby) gut
• Waste water
• Termite gut
• Viral communities
• Lots of different human guts
• Neanderthal
Human microbiota
The human microbiome
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Two parts: the core and variable
microbiomes
Core human microbiome (red):
– Set of microbial genes present in a given
habitat in all of humans.
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diet
Habitat can be defined over a range of
spatial scales:
– The entire body.
– The gut or part of the gut
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How stable and resilient is an
individual’s microbiota?
We don’t know the core and variable
human microbiome yet.
Large intestine has about 1010-1011 microorganisms in the human
colon.
From 16S rRNA surveys 90% of the prokaryotes belong to just 2
divisions (70 total)
– Firmicutes and Bacteroidetes
Variable human microbiome (blue):
– Set of microbial genes in a given habitat
in a smaller subset of humans.
– These genes differ among individuals
and for different diseases.
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What we do know about the human microbiome
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Among individuals it appears that there is a high degree of differences
in microbial community structure (the abundance and types of taxa
present).
– The differences appear to be stable.
– How is high inter-individual diversity sustained?
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The first application of functional attributes of the human microbiome
showed the gut genes were enriched for metabolic pathways:
xenobiotics (foreign substances), glycans and amino acids; the
production of methane; and the biosynthesis of vitamins.
Is there a link between obesity and the microbiome?
(Box 26.3)
How similar are gut microbiome to other
microbiomes?
• Study done in 2006 showed that germ-free mice
inoculated with microbiota from normal human got bigger
without eating more food.
• In comparison to a decaying whale carcass, ocean water,
and agricultural soil, gut microbiomes have similar
genetic composition.
• However, gut microbiomes appear to have more genes for
carbohydrate and glycan metabolism (see fig below).
– The human microbiota was more efficient in extracting energy.
• Gut microbiota from genetically obese mice were more
efficient than normal mice in releasing calories from food.
– Obese mice gain more fat than wild-type mice on the same diet.
• The obese mice had more Firmicutes.
• An experiment with humans that restricted fat and carbs
that also lost weight (6% of body weight) had less
Firmicutes in their gut microbiota.
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