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The Case of Citrate Metabolism Evolution in E

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1 The Case of Citrate Metabolism Evolution in E
The Case of Citrate Metabolism Evolution in E. coli Bacteria slide version 1.0

2 About this Case: These slides were created by the Evo-Ed Project: Funding for the Evo-Ed Project is provided by the National Science Foundation and by Lyman Briggs College, Michigan State University. These slides are provided as a teaching resource. You are encouraged to modify them to meet your specific teaching and learning needs. Please adhere to the copyright conditions specified on the following slide. There is a reference slide at the end of the presentation that lists the sources for the images we have used in this presentation. If you would be willing to be in involved in our research study examining how the use of these case studies impacts learning, please contact us at

3 Copyright: Creative Commons Attribution-NonCommercial-ShareAlike 3
Copyright: Creative Commons Attribution-NonCommercial-ShareAlike 3.0 Unported (CC BY-NC-SA 3.0) You are free: to Share — to copy, distribute and transmit the work to Remix — to adapt the work Under the following conditions: Attribution — You must attribute the work to the Evo-Ed Project at Michigan State University using the following url: Noncommercial — You may not use this work for commercial purposes. Share Alike — If you alter, transform, or build upon this work, you may distribute the resulting work only under the same or similar license to this one. With the understanding that: Waiver — Any of the above conditions can be waived if you get permission from Jim Smith, Merle Heidemann or Peter White at Michigan State University, Public Domain — Where the work or any of its elements is in the public domain under applicable law, that status is in no way affected by the license. Other Rights — In no way are any of the following rights affected by the license: Your fair dealing or fair use rights, or other applicable copyright exceptions and limitations; The author's moral rights; Rights other persons may have either in the work itself or in how the work is used, such as publicity or privacy rights. Notice — For any reuse or distribution, you must make clear to others the license terms of this work. The best way to do this is with a link to the web page *We do not own the copyright to any images that have been attributed to other authors/sources.

4 Introduction These slides are provided as a teaching resource for the E. coli citrate metabolism case as described on A fuller description of the case can be found on the website. Teaching notes can be found in the notes section beneath each slide when viewing the slides in “Normal View” in PowerPoint. To select this option in PowerPoint, go to the main menu, choose “View” and then “Normal.”

5 Background: Citrate Metabolism in E. coli Bacteria
Image is public domain.

6 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! Image is public domain.

7 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 E. coli is a chemoorganoheterotroph, meaning it gets food and energy from organic carbon sources (rather than from the sun, using photosynthesis, or from non-organic sources).

8 Diversity in Shapes coccus bacillus spirochete vibrio
An example of bacterial diversity. The 4 major cell types observed in bacteria: coccus, bacillus, spirochaete, and vibrio Top left image: Stained MRSA, methicillin-resistant Staphylococcus aureus. Taken from (Public Domain) Top right image: Bacillus subtilis; a bacteria typically found in the human intestinal microbiome Taken from Bottom left image: mixed bacterial population, large cell is a type of spirochaete (spyro-keet). Taken from Bottom right image: Vibrio cholerae; some strains of this bacterium cause Cholera taken from (Public Domain)

9 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. A μm is 1/1000 of a mm The following few slides are included as an overview and background on Escherichia coli. For more information visit:

10 Phylogenetic Tree of Life
Taken from https://commons.wikimedia.org/wiki/File:Tree_of_life_SVG.svg English: A phylogenetic tree of life, showing the relationship between species whose genomes had been sequenced as of The very center represents the last universal ancestor of all life on earth. The different colors represent the three domains of life: pink represents eukaryota (animals, plants and fungi); blue represents bacteria; and green represents archaea. Note the diversity of bacteria compared to that of the eukaryotes and archaea. This tree is representative of the diversity of life on earth and does not include all known species (of course).

11 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.

12 How is E. coli transmitted among species?
This shows how E. coli is spread among from cattle to humans. This slide is included to provide students with more background about E. coli and how widespread it is in the environment. The above was downloaded from “How does E. coli get from organism to organism if it is in the gut of animals most of the time? Model for transmission of E. coli O157:H7 from cattle to humans. The figure represents data from numerous studies and depicts examples of the major classes of foods and other sources of E. coli O157:H7 infection that have been reported. The contamination of crops and water sources is associated with the use of manure in fertilizer or with potential fecal contamination from nearby cattle. The sources of human infection with E. coli O157:H7 were identified first by epidemiological methods. In some cases, E. coli O157:H7 was isolated from the suspected food or other source; in many of these cases, including outbreaks associated with ground meat, PFGE or phage typing provided additional confirmation that the bacteria isolated from the patient and the suspected food or other source were the same strain. PFGE (pulse field gel electrophoresis) typing has also been useful in linking geographically separated outbreaks to a common source of contaminated meat. The finding of E. coli O157:H7 in birds, deer, and other animals has led to speculation that these organisms may also be vehicles for O157:H7 transmission.” (the above is a quote from the link provided) Gansheroff L J , and O'Brien A D PNAS 2000;97: ©2000 by National Academy of Sciences

13 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 prefers body temperature for growth (natural habitat is the gut of warm-blooded animals), but can grow at a wide range of temperatures. The actual cells are quite small but can grow to very large population sizes (millions to billions of cells) in a relatively small space like a few milliliters of broth. Under the right conditions, cells can reproduce quite rapidly, on the scale of minutes (compared to days/months/years/decades for other organisms). WHO: A population can be up to 10^9 or 10^10 cells per ml of rich medium. Medium is yeast extract and amino acids/protein. I.e. it is LB broth. Photo taken from Colonies are generally how most people observe bacteria without the use of microscope. These colonies are a few millimeters in size and readily viewable by the naked eye. This plate shows what would be observed after 24 hours of growth at optimal temperature, 37°C. There are approximately 1 million bacterial cells in a single colony, all derived from a single cell multiplying over and over. All of the cells in a single colony are theoretically the same, genotypically and phenotypically.

14 E. coli Under the Microscope (Scanning Electron Micrograph)
Image is Public Domain Cells are ~2 μm X ~0.5 μm (micrometers, 1x10-6 m); invisible to the naked eye

15 E. coli Under the Microscope (Transmission Electron Micrograph)
Image taken from Wadsworth Center, New York State Department of Health

16 The Long-Term Evolution Experiment on E. coli

17 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!) Photo from E. coli generate around 6.67 generations per day in the conditions present in the broth cultures used by Lenski. Dr. Richard Lenski, Distinguished Professor of Microbial Ecology, Michigan State University

18 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 Citrate is included as a binding agent to help E. coli obtain iron from the broth for various cellular functions. Citrate is only transported into E. coli cells in anoxic conditions. The LTEE is conducted in oxic conditions. Since Citrate cannot be transported into the cell, it cannot be used for ATP production.

19 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. Notes: The strain of E. coli used is asexual, so no recombination of DNA between cells. Evolution occurs by mutation and natural selection. *Lenski actually had two sets of six colonies rather than one set of twelve colonies (as we show above). In Lenski’s colonies, the first group of six and the second group of six were differentiated by their ability to metabolize arabinose – one set of colonies could metabolize arabinose and the other set could not. This is a fitness-neutral condition and, as such, it has no bearing on the evolution of citrate metabolism. In order to avoid any confusion about this detail, we represent the colonies as one set of twelve rather than two groups of six.

20 Experimental Protocol
Every 24 hours 1% of each population transferred to fresh flasks of nutrient broth Population sizes of ~300 – 500 million cells Every 500 generations, a portion of each population is frozen Frozen samples remain viable, creating a frozen fossil record for study at any time This is done for each of the 12 populations (note: only 2 populations are shown). Growth is indicated by the cloudiness of the broth. Each day a bacteria population goes through ~6.67 generations. In a given year, each population goes through around 2400 generations (i.e * 365).

21 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!)

22 16 years into the experiment, something very unexpected happened…
At around 33,000 generations, one of the populations started to get markedly cloudier than the others.

23 Discussion Question: Why is population #9 cloudier than the others?
At around 33,000 generations, one of the populations started to get markedly cloudier than the others.

24 The increased cloudiness indicated that the bacteria population in flask #9 was reaching significantly higher abundance than the populations in the other flasks. At around 33,000 generations, one of the populations started to get markedly cloudier than the others.

25 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 is included as a binding agent to help E. coli obtain iron from the broth for various cellular functions. Citrate is only transported into E. coli cells in anoxic conditions. The LTEE is conducted in oxic conditions. Since Citrate cannot be transported into the cell, it cannot be used for ATP production.

26 Citrate vs Glucose The nutrient broth has 139 μM glucose and 1700 μM citrate. 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 to the individuals in the E coli population. Glucose enters the bacteria through a glucose transporter – see the following paper for more details on how glucose enters the bacterial cell: Even though glucose is a far more energy rich molecule, the ability to transport citrate into the cell would nonetheless increase energy stores. The transport of citrate into the E coli cell is not possible when oxygen is present in the environment.

27 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

28 Cell Biology of Citrate Metabolism in E. coli Bacteria

29 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.

30 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.

31 The Long-Term Evolution Experiment
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. Glucose Citrate is included as a binding agent to help E. coli obtain iron from the broth for various cellular functions. Citrate is only transported into E. coli cells in anoxic conditions. The LTEE is conducted in oxic conditions. Since Citrate cannot be transported into the cell, it cannot be used for ATP production. Image Sources: Wikipedia Citrate

32 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 Left image taken from (Public Domain) Right image taken from (Public Domain) White spheres: Hydrogen atoms Red spheres: Oxygen atoms Black spheres: Carbon atoms

33 Citrate vs Glucose The nutrient broth contains more
citrate (1700 μM) than glucose (139 μM).

34 Citrate vs Glucose The transport of citrate into the E coli cell is not possible when oxygen is present in the environment. Glucose enters the bacteria through a glucose transporter – see the following paper for more details on how glucose enters the bacterial cell: Even though glucose is a far more energy rich molecule, the ability to transport citrate into the cell would nonetheless increase energy stores. The transport of citrate into the E coli cell is not possible when oxygen is present in the environment.

35 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. Even though glucose is a far more energy rich molecule, the ability to transport citrate into the cell would nonetheless increase energy stores. However, the transport of citrate into the E coli cell is not possible when oxygen is present in the environment. Glucose enters the bacteria through a glucose transporter – see the following paper for more details on how glucose enters the bacterial cell:

36 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.

37 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.

38 Overview of Citric Acid Cycle
Red and blue molecules are are molecules used for storing energy. Cit+ cells are able to utilize citrate in the citric acid cycle immediately rather than having to produce it from acetyl-CoA and oxaloacetate. Succinate is eventually produced and is moved out of the cell by the CitT antiporter protein in exchange for more citrate. Citrate has more potential energy than succinate, as it is able to produce more of reduced NAD (NADH, H+) for use in oxidative phosphorylation versus succinate.

39 Overview of Citric Acid Cycle
Red and blue molecules are are molecules used for storing energy. Cit+ cells are able to utilize citrate in the citric acid cycle immediately rather than having to produce it from acetyl-CoA and oxaloacetate. Succinate is eventually produced and is moved out of the cell by the CitT antiporter protein in exchange for more citrate. Citrate has more potential energy than succinate, as it is able to produce more of reduced NAD (NADH, H+) for use in oxidative phosphorylation versus succinate.

40 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. By exchanging excess succinate for citrate, the cell can extract more energy and store it in ATP.

41 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. By exchanging excess succinate for citrate, the cell can extract more energy and store it in ATP.

42 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*. *return to previous slide for clarification, if needed

43 More available energy results in more growth, and ultimately a denser culture with a higher population of E. coli.

44 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. This graph shows the optical density of population #9 over a span of 5000 generations. There is a sudden increase in density around generation 33000, identifying this as the region in time when the population evolved the ability to transport citrate in oxic conditions. OD = optical density, it is a measurement of the number of cells present in the population. The more cells there are, the greater the OD. OD is determined by passing light through the broth containing cells and measuring the amount of light that is able to pass through the broth. The more cells there are in the broth, the cloudier the broth will be, and therefore less light will pass through. The amount of light measured that passes through the broth is inversely proportional to OD (less light = greater OD).

45 Review How is citrate used as a source of energy?
How is citrate imported into the cell? How is succinate involved? What is unique about the cells from #9? Where does the citrate come from? Where does the succinate go? See slide 32, 39 See slide 36, 40, 42 See slide 30, 37 See slide 41

46 Optional Cellular Respiration Calculations:
How many moles of ATP can be made per mole of glucose? How many moles of ATP can be made per mole of citrate? How many moles of ATP can be made per mole of succinate? 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. IMPORTANT: OPTIONAL *If you are studying cellular respiration, then students can calculate how many moles of ATP can be made from 1 mole of glucose. This requires knowledge of glycolysis, the citric acid cycle and oxidative phosphorylation.

47 The Molecular Genetics of Citrate Metabolism in E. coli Bacteria

48 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. Important definitions for this section

49 The cit operon The citrate-succinate transporter gene, citT is a gene within the cit operon.

50 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 diagram shows RNA polymerase having just transcribed the cit gene into cit-mRNA, and a ribosome attaching to cit-mRNA, readying to translate it into a protein.

51 The cit operon For simplicity’s sake, we will represent the cit operon as pictured below.

52 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 Transcription is stopped by negative control GENE –transcription mRNA –translation PROTEIN GENE –transcription mRNA –translation PROTEIN

53 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

54 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. Gene not transcribed transport protein cannot be made. O2 repressor

55 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.

56 Genetic mutation The duplication changed how the genes and promoters in this region of DNA were arranged.

57 Genes and promoters rearranged
As a result of the duplication event, the genes and promoters in this region of DNA were rearranged. new arrangement

58 New Behavior in Oxic Conditions
In oxic conditions, the cit operon promoter is still inhibited by a repressor protein. O2 repressor

59 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

60 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

61 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.

62 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. Gene is transcribed and translated; transmembrane protein is made. O2

63 Review 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? See slide 50-52 See slide 53-54 See slide 56 See slide 57-62

64 The Ecology and Phylogenetics of Citrate Metabolism in E. coli Bacteria

65 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.

66 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. Niche: how an organism or population responds to an environment’s distribution of resources and competitors

67 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

68 The Flask #9 ecosystem, before Cit+ evolves
Glucose Citrate The entire population (Cit-) feeds exclusively on glucose, leaving the abundance of citrate available as a potential carbon source.

69 The Flask #9 ecosystem, just after Cit+ evolves
Glucose Citrate After Cit+ evolves, Cit- still feeds on glucose; however Cit+ can feed on citrate as well as glucose

70 The Flask #9 ecosystem, some time after Cit+ evolves
Succinate Glucose Citrate As a consequence of Cit+ evolving, a pool of succinate accumulates in the environment as the Cit+ portion of the population exports succinate in exchange for importing citrate. Both Cit+ and Cit- have the ability to obtain energy from succinate, and those abilities improved over time. Ecological coexistence can occur because only Cit+ cells can use the citrate, while the Cit- type is better at exploiting the glucose.

71 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

72 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

73 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. Phylogeny adapted from Zachary Blount PhD Defense Presentation. Note the 3 clades coexisting ~10,000 generations before Cit+ evolves.

74 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 Genomic analysis of a key innovation in an experimental Escherichia coli population. Nature 489: (Abstract)

75 Clicker Question 1 Normally, can E. coli metabolize citrate?
Yes, in conditions with oxygen Yes, in conditions without oxygen Yes, in conditions with or without oxygen No, not in any conditions Answer: B Normally, as a species, E. coli cannot utilize citrate in conditions with oxygen. This aspect is a major diagnostic criterion for E. coli in science and medicine.

76 Clicker Question 2 In conditions without oxygen, how does E. coli bring citrate into the cell? Citrate enters the cell via active transport Citrate diffuses freely across the cell membrane Citrate is brought in through an antiporter protein in exchange for succinate Trick question, E. coli can’t utilize citrate without oxygen Answer: C The citrate-succinate antiporter, CitT, brings citrate into the cell while exporting succinate. A is not correct because no energy (ATP) is required to move citrate across the cell membrane. B is not correct because citrate is far too large to freely diffuse across the cell membrane. D is incorrect because normally E. coli can only use citrate without oxygen present.

77 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? Glucose Galactose Citrate Ammonium Both A and C Answer: E Both glucose and citrate are the only forms of carbon available in the nutrient broth used in the LTEE. There is no galactose in the recipe, and Ammonium, while present in the nutrient broth, does not have any carbon atoms in it (NH4+).

78 Clicker Question 4 During the Long-Term Evolution Experiment, what interesting phenotype evolved after ~33,000 generations? The E. coli evolved virulence and are now able to infect people The E. coli evolved the ability to utilize citrate in the presence of oxygen The E. coli evolved multicellularity None of the above Answer: B In the LTEE, there was no selection for either A or C dictated by the experimental environment, so it is highly unlikely that either of these phenotypes would evolve, not to mention the complexity of attaining these traits would probably take a very a long time to evolve. The massive pool of unused citrate provided an ecological opportunity that was eventually exploited by the Cit+ clade that evolved.

79 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? Point mutation Chromosomal Inversion Deletion Gene Duplication Answer: D

80 Clicker Question 6 After the gene duplication event, what was the genetic basis for citrate getting into the cell in the presence of oxygen? citT was inserted into the E. coli genome allowing CitT to be translated, allowing citrate into the cell A hybrid gene was generated, which brings citrate into the cell A plasmid was taken up from the environment with the genes required for citrate metabolism 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 Answer: D After the gene duplication, citT, which codes for the citrate-succinate antiporter CitT, was placed under the expression control of the promoter for rnk. Normally, citT is only expressed in the absence of oxygen; its promoter is repressed. However, after the gene duplication, citT is expressed in the presence of oxygen, allowing CitT to be produced and citrate is brought into the cell.

81 Clicker Question 7 Why is it practical for the Cit+ cells to export succinate in exchange for importing citrate? Succinate cannot be metabolized further by E. coli, so the cells get rid of it More energy can be acquired from citrate than succinate, so metabolizing citrate over succinate is more energetically-favorable for the cell Citrate can be fermented by the cells for energy while succinate cannot Importing citrate allows the cells to attain more glucose for energy Answer: B


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