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The Case of
Citrate Metabolism
Evolution in E. coli Bacteria
slide version 1.0
http://www.evo-ed.com
About this Case:
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Introduction
These slides are provided as a teaching resource for
the E. coli citrate metabolism case as described on
www.evo-ed.com. A fuller description of the case
can be found on the website.
Teaching notes can be found in the notes section
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Background:
Citrate Metabolism in E. coli
Bacteria
Bacteria Basics
Bacteria are considered one of the
earliest forms of life.
Found anywhere and
everywhere: animal guts,
oceanic hydrothermal vents,
even radioactive waste!
Estimated that there are 4–6 × 1030 bacterial cells
on Earth!
The human body harbors upwards of 1000 different
bacteria species!
Bacteria Basics
Structure: Prokaryotes
• Single copy of their circular chromosome per cell.
• No membrane-bound organelles (nucleus, mitochondria,
etc.)
Metabolism: Aerobic AND Anaerobic
• Can grow aerobically, using the same glycolysis and the
citric acid cycle pathways found in eukaryotes to generate
energy from carbon-containing molecules like glucose,
sucrose, or lactose.
• Many can also grow anaerobically through the processes of
fermentation
Diversity in Shapes
coccus
spirochete
bacillus
vibrio
E. coli basics:
Full name: Escherichia coli
Shape: Bacillus
Size: 2 μm X 0.5 μm
Found in the digestive tracts
of most warm-blooded
animals
Generally harmless except
for a few strains that can
cause foodborne illness
(e.g. E. coli O157:H7)
One of the most studied and well-characterized organisms in
existence.
Phylogenetic Tree of Life
Escherichia coli is a proteobacterium
• Proteobacteria is a major
phylum within the bacterial
domain.
• All proteobacteria are gramnegative, that is, they have a
thin peptidoglycan layer
between the cell membrane
and an outer membrane.
• E. coli is a member of the
class gammaproteobacteria.
How is E. coli transmitted among species?
Gansheroff L J , and O'Brien A D PNAS 2000;97:2959-2961
©2000 by National Academy of Sciences
E. coli Research is Common
• Easy to grow and maintain - can grow
between ~7 and 49°C, optimal growth at 37°C
• Very small organism,
but grows to form very
large populations.
• Reproduce rapidly,
with generation times
as low as of 20 minutes
under optimal conditions.
E. coli Under the Microscope
(Scanning Electron Micrograph)
E. coli Under the Microscope
(Transmission Electron Micrograph)
The Long-Term Evolution
Experiment on E. coli
The Long-Term Evolution Experiment
• Began in February of 1988 by Dr.
Richard Lenski using E. coli to study
evolution in action.
• Very simple idea: grow E. coli in
serial broth cultures for a long time
and see what happens.
• Cultures have been growing nearly
every day since, resulting in over
60,000 generations of growth and
Dr. Richard Lenski, Distinguished
counting (equivalent to over 1
Professor of Microbial Ecology,
million years of human evolution!) Michigan State University
The Long-Term Evolution Experiment
• The broth contains a small amount of glucose
for the bacteria to use as a food source.
• Another possible food source, citrate, is also
present in the broth, but the bacteria cannot
grow on it under the conditions of the
experiment.
Glucose
Citrate
How does evolution happen in the
experiment?
• Dr. Lenski began twelve, initially identical populations, each
in its own flask*.
• The populations are kept completely isolated from one
another, preventing any gene flow between them.
Experimental Protocol
• The populations have been transferred almost
every day since, evolving for over 60,000
generations and counting (equivalent to over
1.2 million years of human evolution!)
16 years into the experiment, something
very unexpected happened…
Discussion Question:
Why is population #9 cloudier than the
others?
The increased cloudiness indicated that the bacteria
population in flask #9 was reaching significantly higher
abundance than the populations in the other flasks.
The growing medium…
• Remember, the bacteria grow in a medium that
contains both glucose and citrate molecules.
• Normally, E. coli cannot use citrate in the
experimental environment.
• Could the bacteria
in flask #9 have
evolved the ability
to grow on citrate?
Citrate vs Glucose
How does evolution happen in the
experiment?
• Evolution occurs by mutation and natural selection.
• Mutation is when a change in the DNA sequence occurs in an
individual. This change may or may not affect a trait, and may
have a neutral, beneficial, or detrimental effect.
• Natural selection is a process in which organisms with
favorable traits are better able to survive and reproduce, and
are therefore more likely to pass on their traits to the next
generation. Similarly, organisms with detrimental traits are less
able to survive and reproduce, and are therefore less likely to
pass on their traits to the next generation. Consequently, over
time the population becomes better able to survive and
reproduce in the environment in which it lives
Cell Biology of Citrate
Metabolism in E. coli Bacteria
Aerobic Citrate Metabolism Evolves
• After ~33,000 generations (16 years into the
experiment), population #9 was observed to
be cloudier than any other population.
– This means there was a significant increase in
bacterial growth in population #9.
Aerobic Citrate Metabolism Evolves
• Investigations indicated that the cells in
population #9 were able to import citrate
from the medium.
• This is unusual given that E. coli generally
cannot import citrate in the oxic (aerobic)
conditions present in the experiment.
• Once citrate enters the E. coli cell, it can be
metabolized in Citric Acid cycle reactions.
The Long-Term Evolution Experiment
In the broth, the energy
molecule used by the
bacteria is glucose.
Glucose
A second energy
molecule called citrate
is also present in the broth
but it can only be metabolized
in the absence of oxygen.
Citrate
How is Citrate used for energy?
• Citrate is important in biology, as it is an
intermediate in the citric acid cycle
– Citric Acid cycle generates cellular energy in all
aerobic organisms (yes, even humans!)
– When imported, citrate is incorporated into the
Citric Acid cycle
Citrate vs Glucose
The nutrient broth contains more
citrate (1700 μM) than glucose (139 μM).
Citrate vs Glucose
The transport of citrate into the E coli cell is not
possible when oxygen is present in the environment.
Citrate vs Glucose
If citrate could get into the cell, the bacterium could
metabolize it in Citric Acid cycle reactions, resulting in a
significant increase in the energy available.
The CitT Transport Protein
• The CitT protein (in green) is
an antiporter, meaning
that it can transport
citrate molecules into the
cell in exchange for
succinate molecules.
• It operates via passive
transport.
The CitT Transport Protein
• In Lenski’s E. coli
evolution experiments,
the cells in population #9
evolved a way to produce
the CitT transport protein
in oxic (aerobic)
conditions.
CitT Transport Protein Evolution
In Lenski’s E. coli evolution
experiments, the cells in population
#9 evolved a way to produce the
CitT transport protein in oxic
conditions.
CitT Transport Protein Evolution
This allows them to import citrate
into the citric acid cycle, gain 1ATP
and 2NADH from it, and export it as
succinate in return for more citrate.
Advantage of Exchanging Succinate for
Citrate?
• Citrate has more potential energy than
succinate:
– Citrate yields NADH, NADH, ATP
– Succinate yields FADH2, NADH
• By exporting succinate to import more citrate,
the second half of the Citric Acid cycle is
bypassed and the first half can be repeated*.
More available energy results in more
growth, and ultimately a denser culture with
a higher population of E. coli.
Consequences of Citrate Metabolism
• The abundance of citrate was a large potential
food source waiting to be exploited until
generations when the ability to transport
citrate into the cell evolved.
Review
1. How is citrate used as a source of energy?
2. How is citrate imported into the cell? How is
succinate involved?
3. What is unique about the cells from #9?
4. Where does the citrate come from? Where
does the succinate go?
Optional Cellular Respiration
Calculations:
1. How many moles of ATP can be made per mole
of glucose?
2. How many moles of ATP can be made per mole
of citrate?
3. How many moles of ATP can be made per mole
of succinate?
4. Calculate the percent increase in energy stores if
E coli trades out succinate for citrate.
Assume that the above reactants are fully oxidized to
oxaloacetate and that NADH/FADH2 molecules are used to
build up a proton gradient for oxidative phosphorylation.
The Molecular Genetics of Citrate
Metabolism in E. coli Bacteria
Bacterial Genetics Terms and
Definitions
• Operon: Cluster of genes under the regulatory
control of a promoter.
• Promoters: DNA sequences that bind RNA
polymerase and transcription factors. Promoters
initiate transcription (turn on genes) for
production of mRNA; usually located upstream of
the gene it controls.
• Operators: regions of DNA associated with
promoters that bind regulatory proteins to either
promote or hinder RNA polymerase binding to
promoter.
The cit operon
• The citrate-succinate transporter gene, citT is
a gene within the cit operon.
The cit operon
• The genes of the cit operon are transcribed
from a single promoter located at the
beginning of the operon.
• The transcribed mRNA (bottom) is then
translated by a ribosome into proteins.
The cit operon
• For simplicity’s sake, we will represent the cit
operon as pictured below.
Negative control of the cit operon
• If citT is not transcribed, the citT transport
protein cannot be made, and E. coli cannot
transport environmental citrate into the cell.
Central Dogma of protein synthesis
GENE –transcription mRNA –translation PROTEIN
Transcription is stopped by negative control
GENE –transcription mRNA –translation PROTEIN
Negative control of the cit operon
• In the presence of oxygen a repressor protein
binds to the cit promoter and blocks transcription.
• Therefore, when E. coli is in an aerobic
environment, the genes in the cit operon,
including citT, are not transcribed.
O2
repressor
Negative control of the cit operon
• In the presence of oxygen a repressor protein
binds to the cit promoter and blocks transcription.
• Therefore, when E. coli is in an aerobic
environment, the genes in the cit operon,
including citT, are not transcribed.
O2
repressor
Gene not transcribed
transport protein
cannot be made.
Genetic mutation
• A stretch of DNA in the region of the citT gene
was duplicated.
• This mutation occurred randomly within E. coli
population #9. It has not, to our knowledge,
occurred in any of the other 11 E. coli
populations.
Genetic mutation
• The duplication changed how the genes and
promoters in this region of DNA were arranged.
Genes and promoters rearranged
• As a result of the duplication event, the genes
and promoters in this region of DNA were
rearranged.
new arrangement
New Behavior in Oxic Conditions
• In oxic conditions, the cit operon promoter is still
inhibited by a repressor protein.
O2
repressor
New Behavior in Oxic Conditions
• In oxic conditions, the cit operon promoter is still
inhibited by a repressor protein.
• The genes in the cit operon, including the original
copy of citT are not transcribed.
O2
repressor
New Behavior in Oxic Conditions
• In oxic conditions, the cit operon promoter is still
inhibited by a repressor protein.
• The genes in the cit operon, including the original
copy of citT are not transcribed.
• However, the promoters downstream of the cit
operon are not (and never were) repressed by
oxygen.
O2
repressor
New Behavior in Oxic Conditions
• In oxic conditions, the cit operon promoter is still
inhibited by a repressor protein.
• The genes in the cit operon, including the original
copy of citT are not transcribed.
• However, the promoters downstream of the cit
operon are not (and never were) affected by oxygen.
O2
• This promoter facilitates the transcription of the
downstream DNA, including the duplicated copy
of the citT gene.
Citrate-Succinate Transporter
• citT is now transcribed and translated into the
citrate-succinate transporter.
• Bacteria that can make this protein have an
advantage over those that cannot because they
can transport energy rich molecules into the cell.
O2
Gene is transcribed and
translated; transmembrane
protein is made.
Review
1.
2.
3.
4.
How does the cit gene produce the Cit Transporter?
What normally happens in the presence of O2?
What genetic mutation occurred?
What was the result of this mutation?
The Ecology and Phylogenetics of
Citrate Metabolism in E. coli Bacteria
Ecology of Flask #9
The bacterial population in Flask #9 that evolved
the ability to express the citrate transporter in
aerobic conditions is called Cit+
Cit+ was not the only population in Flask #9: the
predecessor population, called Cit-, was still there.
These two populations, in the environment of the
flask, created a little ecosystem that can be studied.
Ecology of Flask #9
One might expect Cit+ to take over quickly due
to the abundance of citrate available, but the
two strains developed two different niches, so
they were able to coexist.
Basic ecological principle: for two organisms to
coexist in the same environment, they need to
exploit two different niches.
Which Niches Developed?
Recall that the CitT protein exchanges
citrate for succinate (citrate comes in
and succinate goes out)
After Cit+ evolved, a pool of succinate
accumulated in the environment as
succinate moved out of the Cit+ cells
in exchange for citrate.
Within the experimental populations, E. coli cells (both Cit- and
Cit+) have the ability to grow on succinate, so there were now
three different carbon compounds available in the environment:
glucose, citrate, and succinate
The Flask #9 ecosystem,
before Cit+ evolves
Cit-
Glucose
Citrate
The Flask #9 ecosystem,
just after Cit+ evolves
Cit-
Glucose
Cit+
Citrate
The Flask #9 ecosystem,
some time after Cit+ evolves
Cit-
Succinate
Cit+
Glucose
Citrate
Phylogenetics Terms and Definitions
• Phylogenetics: the study of the evolutionary
relationships between groups of organisms
• These relationships are determined by comparing
DNA sequence data for the organisms under
study
• Phylogenetic trees are used to show these
relationships in a visual way
• Clades are groups of closely-related organisms
that share a common ancestor, which is
represented as a node on the tree
Population #9 Phylogenetics
• Entire population was heterogeneous for much of
its history
– Possibly indicative of more complex ecological
interaction within the population than previously
thought
• 3 different clades coexisted with one another for
at least 10,000 generations prior to the evolution
of Cit+
• Cit+ forms a fourth clade around 33,222
generations
•This figure is a phylogeny
of Flask #9 up to 40,000
generations of evolution. It
was generated by using
whole-genome DNA
sequencing and comparing
sequence of individuals
within the population.
Clades 1-3 are Cit-, with
Clade 4 being the Cit+
lineage.
•Branches that have
stopped are indicative of
that lineage becoming rare
or extinct.
•Note that this figure only
shows data up to 40,000
generations, the
experiment is now past
60,000 generations.
Advanced Study
Read the following article and make a list of
questions to bring to class:
Blount, Z. D., J. E. Barrick, C. J. Davidson, and R. E.
Lenski. 2012. Genomic analysis of a key innovation in
an experimental Escherichia coli population. Nature
489:513-518. (Abstract)
Clicker Question 1
• Normally, can E. coli metabolize citrate?
A.
B.
C.
D.
Yes, in conditions with oxygen
Yes, in conditions without oxygen
Yes, in conditions with or without oxygen
No, not in any conditions
Clicker Question 2
• In conditions without oxygen, how does E. coli
bring citrate into the cell?
A. Citrate enters the cell via active transport
B. Citrate diffuses freely across the cell membrane
C. Citrate is brought in through an antiporter
protein in exchange for succinate
D. Trick question, E. coli can’t utilize citrate without
oxygen
Clicker Question 3
• In the Long-Term Evolution Experiment, prior
to the evolution of Cit+, what are the available
carbon sources in the nutrient broth?
A.
B.
C.
D.
E.
Glucose
Galactose
Citrate
Ammonium
Both A and C
Clicker Question 4
• During the Long-Term Evolution Experiment,
what interesting phenotype evolved after
~33,000 generations?
A. The E. coli evolved virulence and are now able to
infect people
B. The E. coli evolved the ability to utilize citrate in
the presence of oxygen
C. The E. coli evolved multicellularity
D. None of the above
Clicker Question 5
• To get citrate into the cell, the E. coli in the
LTEE experienced what type of mutation that
allowed them to express CitT in the presence
of oxygen?
A.
B.
C.
D.
Point mutation
Chromosomal Inversion
Deletion
Gene Duplication
Clicker Question 6
• After the gene duplication event, what was the genetic
basis for citrate getting into the cell in the presence of
oxygen?
A. citT was inserted into the E. coli genome allowing CitT to
be translated, allowing citrate into the cell
B. A hybrid gene was generated, which brings citrate into
the cell
C. A plasmid was taken up from the environment with the
genes required for citrate metabolism
D. citT was placed under the control of another promoter
which is active in the presence of oxygen, allowing CitT
to be produced when normally it would not be
Clicker Question 7
• Why is it practical for the Cit+ cells to export
succinate in exchange for importing citrate?
A. Succinate cannot be metabolized further by E. coli,
so the cells get rid of it
B. More energy can be acquired from citrate than
succinate, so metabolizing citrate over succinate is
more energetically-favorable for the cell
C. Citrate can be fermented by the cells for energy
while succinate cannot
D. Importing citrate allows the cells to attain more
glucose for energy

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