The Case of

Citrate Metabolism
Evolution in E. coli
Bacteria
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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.
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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 46 10 30
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 m X 0.5 m
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 49C, optimal growth at
37C
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 counting (equivalent Dr. Richard Lenski,
to over 1 million years of human Distinguished Professor of
Microbial Ecology, Michigan
evolution!)
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 M) than glucose (139
M).

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 Lenskis 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 Lenskis 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 simplicitys 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
GENE
transcription mRNA translation
control
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.
Represso
r protein
is
activate
d by O

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.
Repress
or
protein
Gene not
transcribed
transport protein

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.

represso
r

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.
represso
r

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.

represso
r

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.

repress
or

The repositioned rnk promoter

facilitates the transcription of the
downstream DNA, including the

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.
repress
or

Gene is transcribed and the

mRNA is translated;
transmembrane protein is

Review
1. How does the cit gene produce the Cit
Transporter?
2. What normally happens in the presence
of O2?
3. What genetic mutation occurred?
4. 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-

Succinat
e

Glucose

Cit+

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

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 489:513518. (Abstract)

Clicker Question 1
Normally, can E. coli metabolize
citrate?
A. Yes, in conditions with oxygen
B. Yes, in conditions without oxygen
C. Yes, in conditions with or without
oxygen
D. 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 cant 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