The Case of Citrate Metabolism

Evolution in E. coli Bacteriaslide version 1.0

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Introduction

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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 Shapescoccus bacillus

vibriospirochete

E. coli basics:Full name: Escherichia coliShape: Bacillus

Size: 2 μm X 0.5 μm

Found in the digestive tractsof 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 gram-negative, 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 verylarge 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 to over 1 million years of human evolution!)

Dr. Richard Lenski, Distinguished Professor of Microbial Ecology,

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 bacteriain 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.

In the broth, the energy molecule used by the bacteria is glucose.

A second energy molecule called citrate is also present in the broth but it can only be metabolized in the absence of oxygen.

The Long-Term Evolution Experiment

Glucose

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 GlucoseIf 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.

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.

CitT Transport Protein Evolution

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.

• 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.

Consequences of Citrate Metabolism

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.

• 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

Transcription is stopped by negative control

Negative control of the cit operon

GENE –transcription mRNA –translation PROTEIN

GENE –transcription mRNA –translation PROTEIN

Negative control of the cit operon

O2repressor

• 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.

Negative control of the cit operon

Gene not transcribed transport protein cannot be made.

O2repressor

• 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.

• 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.

Genetic mutation

• As a result of the duplication event, the genes and promoters in this region of DNA were rearranged.

Genes and promoters rearranged

new arrangement

• In oxic conditions, the cit operon promoter is still inhibited by a repressor protein.

New Behavior in Oxic Conditions

O2

repressor

• 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.

New Behavior in Oxic Conditions

O2

repressor

• 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.

New Behavior in Oxic Conditions

O2

repressor

O2

• 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.

New Behavior in Oxic Conditions

• This promoter facilitates the transcription of the downstream DNA, including the duplicated copy of the citT gene.

• 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.

Citrate-Succinate Transporter

Gene is transcribed and translated; transmembrane

protein is made.

O2

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+ cellsin 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

Glucose Citrate

Cit-

The Flask #9 ecosystem, just after Cit+ evolves

Cit+

Glucose Citrate

Cit-

The Flask #9 ecosystem, some time after Cit+ evolves

Cit+

Succinate Glucose Citrate

Cit-

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. Yes, in conditions with oxygenB. Yes, in conditions without oxygenC. Yes, in conditions with or without oxygenD. 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 transportB. Citrate diffuses freely across the cell membraneC. Citrate is brought in through an antiporter

protein in exchange for succinateD. 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. GlucoseB. GalactoseC. CitrateD. AmmoniumE. 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 peopleB. The E. coli evolved the ability to utilize citrate in

the presence of oxygenC. The E. coli evolved multicellularityD. 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. Point mutationB. Chromosomal InversionC. DeletionD. 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 cellB. A hybrid gene was generated, which brings citrate into the

cellC. A plasmid was taken up from the environment with the

genes required for citrate metabolismD. 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 itB. 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