Questions
1. Dr. Lenski’s study requires an understanding of how E. coli obtains metabolic energy
from food. Organisms
obtain this energy by aerobic respiration, anaerobic respiration or fermentation. Do a
little outside research and
compare and contrast each of these.
2. Is E. coli a “good” or “bad” bacterium? Explain your answer.
3. What attributes of E. coli make it a good organism for research purposes?
4. Describe the protocol for Dr. Lenski’s Long Term Evolution Experiment (LTEE)
studies.
5. What was observed in fl ask #9?
6. Application question: Provide a reasonable explanation, based on what you learned
from Question 1, for the
sudden increased growth of E. coli population in fl ask #9.
7. Challenge question: How does a change in growth rate lead to a change in fitness?
1) Compare and contrast aerobic respiration, anaerobic respiration, and
fermentation in terms of metabolic energy production.



Aerobic Respiration:
o
Requires oxygen.
o
Glucose is fully oxidized into CO₂ and water.
o
Yields the highest energy: ~36–38 ATP per glucose molecule.
o
Utilizes glycolysis, the citric acid cycle, and the electron transport chain.
Anaerobic Respiration:
o
Does not require oxygen.
o
Uses an alternative final electron acceptor (e.g., nitrate, sulfate).
o
Produces less energy than aerobic respiration: ~2–36 ATP per glucose.
o
Common in environments where oxygen is absent.
Fermentation:
o
Does not involve an electron transport chain.
o
Relies solely on glycolysis, with pyruvate being reduced to regenerate
NAD+.
o
Yields the least energy: ~2 ATP per glucose.
o
Produces by-products like ethanol, lactic acid, or gases depending on the
organism.
2) Is E. coli a “good” or “bad” bacterium? Explain.
E. coli can be both:


"Good":
o
Most strains are harmless and are essential for gut health, aiding in
digestion and vitamin production.
o
It is a model organism in research due to its simplicity, rapid reproduction,
and well-characterized genetics.
"Bad":
o
Some strains, like E. coli O157:H7, can cause severe foodborne illnesses,
including diarrhea and kidney damage.
3) What attributes make E. coli a good organism for research purposes?

Rapid reproduction: Short generation times (~20 minutes under optimal
conditions).

Ease of cultivation: Can grow in simple media and across a wide temperature
range (7–49°C, optimal at 37°C).

Genetic simplicity: Single circular chromosome, making genetic manipulation
straightforward.

Well-characterized genetics: Many tools and databases are available for E. coli
research.

Experimental flexibility: Grows under aerobic and anaerobic conditions.
4) Describe the protocol for Dr. Lenski’s Long-Term Evolution Experiment (LTEE).

Began in 1988 with 12 initially identical E. coli populations in nutrient broth.

Broth contained glucose (primary carbon source) and citrate (not usable under
aerobic conditions).

Populations were grown daily, diluted 1:100 into fresh broth, and incubated at
37°C.

Evolution was monitored by freezing samples every 500 generations for future
study.

Over 60,000 generations of growth have been achieved, equivalent to ~1 million
years of human evolution.
5) What was observed in Flask #9?

Around generation 33,000, the population in Flask #9 became significantly
cloudier, indicating increased bacterial growth.

Investigation revealed that the E. coli in this flask had evolved the ability to import
and metabolize citrate aerobically—a rare and novel trait.
6) Application: Provide a reasonable explanation for the increased growth of the
E. coli population in Flask #9.
The evolution of the ability to transport citrate into the cell in the presence of oxygen
provided a new energy source. Citrate metabolism via the CitT transporter allowed the
bacteria to:

Import citrate into the cell, where it could enter the citric acid cycle for ATP
production.

Exchange citrate for succinate, bypassing less efficient metabolic steps and
accessing additional energy. This mutation led to a significant competitive
advantage, resulting in higher growth rates and population densities.
7) Challenge: How does a change in growth rate lead to a change in fitness?
Fitness is defined as an organism's ability to survive and reproduce. Increased growth
rate affects fitness as follows:

Higher resource utilization: Faster growth allows organisms to outcompete
others for limited resources.

Greater reproduction: Rapid division leads to a higher number of offspring,
increasing the likelihood of passing on beneficial mutations.

Adaptation: In a selective environment like the LTEE, mutations that enhance
growth are strongly favored, driving evolutionary change.
1a. What does OD mean, and what does it represent?

OD (Optical Density): A measure of the turbidity or cloudiness of a liquid culture,
determined using a spectrophotometer.

It represents the density of bacterial cells in the culture: higher OD values
correspond to a larger population of E. coli.
1b. What do higher OD values at generation ~33,000 tell us about the population
of E. coli in Flask #9?
The increased OD values around generation 33,000 indicate a dramatic rise in
population density. This suggests that E. coli in Flask #9 gained the ability to utilize
citrate as a carbon source under aerobic conditions, leading to enhanced growth and
survival.
1c. What changes in cell function allowed this to happen?
The key change was the evolution of a mutation allowing the CitT transport protein to be
expressed under aerobic conditions:

CitT Transporter: This antiporter exchanges extracellular citrate for intracellular
succinate, enabling citrate uptake.

Once inside the cell, citrate enters the citric acid cycle, providing a new energy
source.
2. What is the original molecule that E. coli breaks down to produce Acetyl-CoA?
How does it get into the cell?

Original Molecule: Glucose.

Entry Mechanism:
o
Glucose enters the cell via facilitated diffusion or active transport using
specific glucose transporters (e.g., phosphotransferase system).
o
Inside the cell, glucose undergoes glycolysis to produce pyruvate, which is
then converted to Acetyl-CoA.
3. What are the inputs and outputs of the citric acid cycle?



Inputs:
o
Acetyl-CoA.
o
Oxaloacetate.
o
NAD+, FAD, ADP (or GDP) + Pi.
Outputs:
o
CO₂ .
o
NADH, FADH₂ (energy carriers).
o
ATP or GTP (direct energy).
Fate of Outputs:
o
NADH and FADH₂ transfer electrons to the electron transport chain,
driving ATP production.
o
CO₂ is released as a waste product.
4. What happens to citrate in E. coli when oxygen is not present?
Under anaerobic conditions, citrate is not typically metabolized because E. coli lacks the
necessary transport mechanism for citrate uptake and utilization in these conditions.
Instead, other anaerobic pathways, such as fermentation or anaerobic respiration, are
utilized.
5. How does citrate get into the cell under aerobic conditions in Dr. Lenski’s
studies?

The CitT Transporter Protein was mutated to function in aerobic conditions.

Mechanism:
o
CitT acts as an antiporter, exchanging extracellular citrate with intracellular
succinate.
o
This enables the active transport of citrate into the cell, where it
participates in the citric acid cycle.
6. Follow the matter and energy as citrate enters the cell and is incorporated into
the citric acid cycle under aerobic conditions.
1. Matter Flow:
o
Citrate is imported into the cell via the CitT transporter.
o
It is converted into isocitrate by aconitase in the citric acid cycle.
2. Energy Flow:
o
The citric acid cycle breaks citrate down into smaller molecules, producing
NADH and FADH₂ .
o
These carriers transfer high-energy electrons to the electron transport
chain, resulting in ATP generation.
7. What is the advantage to E. coli of importing citrate under conditions with and
without oxygen?

With Oxygen:
o

Citrate metabolism provides an additional carbon and energy source,
increasing growth and survival.
Without Oxygen:
o
While citrate is less commonly used anaerobically, mutations in CitT may
allow its utilization, providing a competitive edge in nutrient-limited
environments.
8. Why is succinate important when explaining the evolution of citrate uptake
under aerobic conditions?

Succinate-Citrate Exchange: The CitT transporter operates as an antiporter,
meaning citrate import is coupled with succinate export.

Significance:
o
Succinate cycling ensures continuous transport of citrate into the cell.
o
Succinate, as part of the citric acid cycle, is regenerated and can be
exchanged again, creating a feedback loop that optimizes citrate
utilization.
1) Roles of Operon Components in Prokaryotic Gene Expression

Structural Genes: Encode the proteins necessary for a specific function. In the
citrate operon, the structural genes include those for the CitT transporter protein
and other enzymes involved in citrate metabolism.

Promoter: The DNA sequence where RNA polymerase binds to initiate
transcription of the operon.

Repressor: A regulatory protein that binds to the operator to inhibit transcription
by blocking RNA polymerase.

Operator: A DNA sequence located near the promoter that acts as a binding site
for the repressor. Its binding prevents transcription.
2) Function of the E. coli Citrate Operon Under Anaerobic Conditions

Promoter: The RNA polymerase binds here to start transcription of the citrate
operon.

Repressor: Under anaerobic conditions, the repressor protein is inactive or
absent, allowing transcription of the citrate operon.

Operator: Without active repression, the operator does not block RNA
polymerase, enabling expression of the cit operon genes. These genes encode
proteins necessary for citrate uptake and metabolism under anaerobic
conditions.
3) Negative Control of the Citrate Operon

Definition: Negative control means transcription is turned off by a repressor
when certain conditions are met.

Mechanism in Citrate Operon:
o
In the presence of oxygen, the repressor binds to the operator, preventing
RNA polymerase from initiating transcription.
o
This ensures the operon is not expressed unnecessarily under conditions
where citrate metabolism is not advantageous.
4) Result of Negative Control Under Aerobic Conditions
When oxygen is present:

The repressor binds to the operator.

This prevents the transcription of the citrate operon genes, including the citT
gene.

Consequently, the CitT transporter is not synthesized, and citrate cannot be
imported into the cell.
5) Relationship Between the Citrate Operon and CitT Transporter Protein
Synthesis

The citT gene is part of the citrate operon and encodes the CitT transporter
protein.

When the operon is active (e.g., under anaerobic conditions), transcription of the
citT gene occurs, leading to the production of the CitT transporter.
6) Duplication of the Citrate Operon in Flask #9

Precisely Duplicated Region: A segment of the citrate operon, including the citT
gene and its regulatory sequences, was duplicated.

Structural Changes:
o
A new promoter was placed upstream of the duplicated citT gene.
o
This promoter was active under aerobic conditions, allowing the
transcription of the duplicated citT gene even in the presence of oxygen.
7) Pre- and Post-Mutation Citrate Operon Sketch
While I cannot sketch directly here, a description for your sketch:

Pre-Mutation:
o

Single citT gene downstream of the original promoter and operator, which
are controlled by the repressor.
Post-Mutation:
o
The original citT gene remains intact.
o
The duplicated citT gene has a new promoter that is not regulated by the
original repressor.
8) How the New Arrangement Facilitates CitT Transcription

Anaerobic Conditions: The original operon functions as before, allowing citT
transcription and CitT protein synthesis.

Aerobic Conditions: The new promoter upstream of the duplicated citT gene is
active, bypassing the negative control exerted by the repressor. This leads to
CitT protein production under aerobic conditions.
9) Was the Rearrangement a Single or Multiple Genetic Event?

The rearrangement was likely a multiple genetic event:
o
The duplication of the operon involves recombination or replication errors.
o
The placement of the new promoter upstream of the duplicated citT gene
would require an additional mutation or insertion event.
10) Advantages and Disadvantages of the Citrate Operon Mutation


Advantages:
o
Enables citrate utilization as an energy source under aerobic conditions,
providing a competitive edge in nutrient-limited environments.
o
Leads to increased population density and growth rates (as observed in
Flask #9).
Disadvantages:
o
Mutant E. coli cells must invest additional energy in expressing the
duplicated operon, which could be a disadvantage when citrate is absent
or in environments where citrate metabolism does not confer a benefit.
o
May introduce metabolic imbalances due to overproduction of CitT or
other operon-related proteins.
1) What molecule(s) did the original LTEE population use for food?

The original LTEE (E. coli) populations primarily used glucose as their main
energy and carbon source.

Glucose was supplied in limited amounts in the growth medium, creating
selective pressure for adaptations that improved resource utilization.
2) What molecule(s) did the newly evolved Cit+ population use for food?

The newly evolved Cit+ (citrate-positive) population developed the ability to
import and metabolize citrate under aerobic conditions.

Citrate, provided in the medium as a buffer, became an additional carbon source
for Cit+ cells, supplementing their glucose metabolism.
3) What molecule(s) did the established populations derived from Flask #9 use for
food?

Cit+ Populations: Utilized both glucose and citrate as food sources.

Cit− Populations: Continued to rely solely on glucose for energy, as they lacked
the ability to metabolize citrate under aerobic conditions.
4) Can the Cit- and Cit+ populations co-exist in the same flask? Explain your
answer.
Yes, the Cit− and Cit+ populations can co-exist in the same flask due to resource
partitioning:

Glucose Utilization: Cit− populations consume glucose, which is limited in the
medium. Cit+ populations also consume glucose but can survive on citrate after
glucose is depleted.

Citrate Utilization: Only Cit+ populations can metabolize citrate, providing them
with a competitive edge after glucose is exhausted.

Ecological Niches: These differences create separate niches for Cit− and Cit+
populations, allowing them to coexist despite competition.
5) Homogeneous vs. Heterogeneous Populations


Definitions:
o
Homogeneous Population: Consists of genetically identical or highly
similar individuals.
o
Heterogeneous Population: Consists of genetically diverse individuals
with significant differences in traits or adaptations.
Original LTEE Population (12 Flasks):
o

Likely homogeneous at the start of the experiment, as all flasks were
inoculated with the same ancestral strain of E. coli.
Flask #9 Population After 33,000 Generations:
o
Likely heterogeneous, as mutations led to the emergence of Cit+
populations alongside Cit− populations. The differing abilities to utilize
citrate created distinct subpopulations with separate ecological roles.
6) What if the Cit transporter was not an antiporter and succinate was not
exchanged for citrate?
If the CitT transporter was not an antiporter and did not exchange succinate for citrate:



Cit+ Population Growth:
o
Citrate import would still occur, but the absence of succinate export could
disrupt intracellular metabolic balance.
o
Cit+ populations might grow more slowly or fail to fully utilize citrate due to
metabolic bottlenecks.
Cit− and Cit+ Coexistence:
o
The lack of succinate export could reduce the availability of succinate in
the medium for Cit− populations.
o
This might lead to increased competition for resources between Cit+ and
Cit− populations, reducing their ability to coexist.
Overall Ecosystem Impact:
o
The antiport mechanism ensures a balanced exchange of metabolites,
allowing both populations to exploit their respective niches. Without this
mechanism, the relationship between Cit+ and Cit− populations would
likely become more competitive and less stable.