Microbial Ecology What is microbial ecology?

• Learning Objectives: • Study of microbes and their interactions with the

– To learn how to study microbes in their natural environments
– To understand techniques used to investigate microbial ecology
• Outline:
– 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.
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.

Microbiology and biogeochemical cycling 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?
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:
– You now have a system that is useful for mechanistic studies.
– You can determine the abundance of a particular population of microbes
• 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:
– You can identify microbes without knowing their culturing conditions.
– The culture bias is no longer a problem
• Cons:
– Environment is complex and hard to sort out
– Low abundance organisms are not well represented
Common approaches used in microbial ecology:
1. Enumerating (counting) microorganisms
– The goal is to determine the abundance of microorganisms.
– Some approaches use cultivation approaches others do not.
2. Microcosm study
– 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
– This can be a very rapid and useful approach to identifying
organisms and diversity within a particular environmental sample.
4. Metagenomic approaches
– Don’t culture. Instead, sequence the DNA straight from the
environment.

1. Enumeration of
• Total counts by microscope
microorganisms 1. Enumeration: archaea and bacteria in the ocean
– DNA dye and epifluorescence microscopy
• FISH: fluorescence in situ hybridization (see • Archaea were found to
figure) abundant in the deep ocean.
– Uses 16S rRNA gene probes for bacteria or • This was unusual.
archaea • Archaea were also highly
– You can target specific genera abundant in coastal regions.
• Viable counts: plate samples on media. • This raised questions about
– Usually underestimates the total count. (Why?) the function of these archaeal
microorganisms. Bacteria
– Called culture bias (bacterial enumeration
anomaly) because you can’t culture • FISH revealed the global
everything. oceans contain:
• Using FISH and microscopy it was – 1.3 x 1038 archaeal cells
– 3.1 x 1038 bacterial cells
discovered that crenarchaea were highly
abundant in the ocean. • One group of archaea
comprises 1 x 1038 cells!
– The crenarchaea were thought to be either Archaea
extremophiles or methanogens. • What is this organisms?
• A representative microbe was
isolated in 2005 Archaeal dominance in the m esopelagic zone of the Pacific
Ocean, Nature Karner 2001 vol:409 iss:6819 pg:507 -510
 Isolation of marine Crenarchaeota SCM1 2. Microcosm studies
• Nitrosopumilus maritimus • Collect a water or sediment
sample and incubate in a
• Isolated from an aquarium in
Seattle. DAPI FISH medium that simulates the http://www.mikelevin.com/MonoLake.html

environment.
• It is a chemoautotroph
• Measure rate of substrate
• Isolated with: utilization by:
– filtered aquarium water – Direct chemical analysis. You
– ammonium chloride need a method for measuring
– Bicarbonate the chemical of interest
– streptomycin – Or using a chemical isotope.
• It is the first ammonia You measure radioactivity
instead of the chemical.
oxidizing crenarchaeota.
• Very similar to the archaea in • Example: in Mono Lake
arsenic is really high. The
the open ocean. TEM SEM respiration of arsenate accounts
Isolation of an autotrophic ammonia-oxidizing marine
archaeon for ~14% of the total carbon
Martin Könneke, Anne E. Bernhard, José R. de la Torre, turnover in the lake.
Christopher B. Walker, John B. Waterbury and David A. Stahl
Nature 437, 543-546 (22 September 2005)

3. Detection of the functional gene for arsenate

3. PCR for 16S rRNA and functional genes
reduction, called arrA
• 16S rRNA gene analysis:
Gel of PCR products carried out on DNA extracted from sediment samples at 8 different depths
– Used to asses the microbial diversity within a particular sample within a sediment core. You can see the DNA bands become less intense for sediments that
without growing any organisms. are deeper in the core.

• Functional gene analysis: 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)
• Diversity of PCR products can be assessed by: Increasing depth in core

– Making a clone library (brute force but low throughput)

– Using electrophoresis-based fingerprinting methods (higher
throughput): DGGE 1 2 3 4 5 6 7 8 Blanks
• Sequence information is analyzed by making phylogenetic
trees.
 DNA inserts
Making a clone library
• Add DNA from PCR to a
plasmid.
• Ligate the two pieces together.
– One molecule of the
functional gene ligates to one
molecule of plasmid
• Transform into E. coli.
• Each colony represents one
cloned DNA fragment.
• Sequence the DNA insert.
– How many should we
sequence?
• Bioinformatic analysis of the
DNA sequences.
– BLAST (online database
search program)
– Alignments and phylogenetic
trees.
DGGE: denaturing gradient gel electrophoresis
• 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.
plasmids
+ Sequencing and Tree drawing
ligate • Sequencing is usually done by dye-
terminator sequencing by capillary
eletrophoresis with laser detector
A B C
• You need purified plasmid DNA
or PCR products
transform
• Phylogenetic inferences to known
sequences and organisms from
online genetic databases:
plate – 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
• Determine geochemical profiles Emphasis on geochemistry
– need to measure chemical
parameters
• Do experiments with
environmental samples
– Mud, sediment, water
– Get rate measurements
– in situ activities.
• Isolate pure cultures
• Characterize physiology of the
strain, do genetic studies,
biochemistry, etc.
– Genome sequence
– Microarrays: gene expression
Cyanobacteria • Microbial ecology tools
– Develop gene detection tools
Chromatiaceae to investigate diversity of
Oremland et al. (2005) Whither or wither geomicrobiology in the
era of 'community metagenomics'.Nat Rev Microbiol. 3(7):572-8
functional genes for a process
Beta-proteobacteria, – Identification of the pure
culture in natural populations
 High throughput sequencing has spawned the
Metagenomic projects
“modern” approach to microbial ecology.
Here are few projects:
• Extract environmental DNA
• Make large insert DNA clone • Yellowstone hot springs
libraries (various places)
– Bacterial artificial chromosome • Dechlorinating bioreactor
(300 kb) • Biogas reactor
– fosmid (plasmid ~50 kb)
• Compost
• Sequence lots of DNA
• Bioinformatic analyses of sequence • Bovine Rumen
data. • Acid mine drainage
• What to do with this data? • Marsupial (wallaby) gut
• Goal is to understand something • Waste water
about the environment: • Metagenomics: using massive • Termite gut
– Must develop follow-up studies
high throughput DNA • Viral communities
– Are the genes expressed?
sequencing technology to • Lots of different human guts
– Are the encoded products
functional in situ? sequence genomes of
• Neanderthal
– Are there significant cycling of organisms in an environmental
elements, nutrients or energy flux DNA sample.
within an ecosystem.

Human Microbiome Project Human microbiota

• 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).
• 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.
 The human microbiome
• Two parts: the core and variable
microbiomes
• Core human microbiome (red):
– Set of microbial genes present in a given
habitat in all of humans.
• 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.
• Habitat can be defined over a range of
spatial scales:
– The entire body.
– The gut or part of the gut
• How stable and resilient is an
individual’s microbiota?
• We don’t know the core and variable
human microbiome yet.
What we do know about the human microbiome
• Large intestine has about 1010-1011 microorganisms in the human
colon.
• From 16S rRNA surveys 90% of the prokaryotes belong to just 2
diet divisions (70 total)
– Firmicutes and Bacteroidetes
• 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?
• 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? How similar are gut microbiome to other
(Box 26.3) microbiomes?
• Study done in 2006 showed that germ-free mice • In comparison to a decaying whale carcass, ocean water,
inoculated with microbiota from normal human got bigger and agricultural soil, gut microbiomes have similar
without eating more food. genetic composition.
– The human microbiota was more efficient in extracting energy. • However, gut microbiomes appear to have more genes for
• Gut microbiota from genetically obese mice were more carbohydrate and glycan metabolism (see fig below).
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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