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

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

2 About this Case: 1.These slides were created by the Evo-Ed Project : 2.Funding for the Evo-Ed Project is provided by the National Science Foundation and by Lyman Briggs College, Michigan State University. 3.These slides are provided as a teaching resource. You are encouraged to modify them to meet your specific teaching and learning needs. 4.Please adhere to the copyright conditions specified on the following slide. 5.There is a reference slide at the end of the presentation that lists the sources for the images we have used in this presentation. 6.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 3.0 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

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 × bacterial cells on Earth! The human body harbors upwards of 1000 different bacteria species!

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

8 Diversity in Shapes bacillus

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.

10 Phylogenetic Tree of Life

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? 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.

14 E. coli Under the Microscope (Scanning Electron Micrograph)

15 E. coli Under the Microscope (Transmission Electron Micrograph)

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!) 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

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.

20 Experimental Protocol

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…

23 Discussion Question: Why is population #9 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.

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?

26 Citrate vs Glucose

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 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

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

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.

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.

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.

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40 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

41 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

42 Advantage of Exchanging Succinate for Citrate? Citrate has more potential energy than succinate: – Citrate yields NADH, NADH, ATP – Succinate yields FADH 2, 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*.

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

44 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

45 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?

46 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/FADH 2 molecules are used to build up a proton gradient for 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.

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.

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

52 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

53 Negative control of the cit operon O2O2 repressor 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.

54 Negative control of the cit operon Gene not transcribed transport protein cannot be made. O2O2 repressor 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.

55 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

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

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

58 In oxic conditions, the cit operon promoter is still inhibited by a repressor protein. New Behavior in Oxic Conditions O2O2 repressor

59 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 O2O2 repressor

60 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 O2O2 repressor

61 O2O2 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.

62 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. O2O2

63 Review 1.How does the cit gene produce the Cit Transporter? 2.What normally happens in the presence of O 2 ? 3.What genetic mutation occurred? 4.What was the result of this mutation?

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.

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 GlucoseCitrate Cit-

69 The Flask #9 ecosystem, just after Cit+ evolves Cit + GlucoseCitrate Cit-

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

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.

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)(Abstract)

75 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

76 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

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? A.Glucose B.Galactose C.Citrate D.Ammonium E.Both A and C

78 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

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? A.Point mutation B.Chromosomal Inversion C.Deletion D.Gene Duplication

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? 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

81 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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