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Microbial evolution is in the cards: horizontal gene transfer in the classroom.

As humans produce new compounds and release them into the environment, bacteria are presented with a vast array of unique carbon sources. Some of these compounds are easily degraded by metabolic pathways that already exist. Others linger in the environment, with the necessary metabolic tools for their degradation apparently unavailable (Alexander, 1999; Seffernick & Wackett, 2001). Over time, however, some of these lingering compounds begin to disappear (Alexander, 1999; Seffernick & Wackett, 2001).

What event triggers this apparently sudden onset of degradation? One hypothesis is that the exchange of genetic material between bacteria allows for new metabolic pathways to be assembled, resulting in the ability to exploit unique carbon sources (van der Meer et al., 1998; Wackett & Hershberger, 2001). This exchange of genetic material, called horizontal gene transfer, passes genes that encode the enzymes necessary for the metabolism of compounds from a donor bacterium to a recipient bacterium with a different genetic background. It is from this new combination of genes that the ability to degrade novel compounds can emerge (van der Meer et al., 1998).

Horizontal gene transfer is not only implicated in the evolution of metabolic pathways. The resistance plasmids (R plasmids) are genetic elements that replicate independently of the chromosome, can be transferred from one bacterium to another, and carry resistance to multiple antibiotics (Madigan et al., 2000). The R plasmid R100, for example, carries genes conferring resistance to sulfonamides, streptomycin, spectinomycin, fusidic acid, chloramphenicol, tetracycline, and mercury and can be transferred between many of the enteric bacteria (Madigan et al., 2000). Horizontal gene transfer can also lead to the acquisition of pathogenicity islands, large clusters of genes required for pathogen virulence. One such example is Vibrio cholerae, the causative agent of cholera. Two pathogenicity islands are required for full virulence of V cholera. Both are encoded in the genetic material of bacterial viruses that can be passed from virulent V. cholera to avirulent V. cholera (Karaolis et al., 1999), thus spreading the virulent phenotype.

This game-based activity models horizontal gene transfer in action and illustrates some of its main concepts, acting as a starting point for discussion. This activity was originally developed as an introductory microbiology classroom activity to demonstrate the assembly of novel metabolic pathways via horizontal gene transfer in the environment. The activity consists of a simple card game with the objective of assembling a complete degradation pathway in your hand of cards. A discussion of concepts demonstrated and questions raised follows the completion of the game. Students will gain from this activity:

* a better understanding of evolution by horizontal gene transfer

* an exposure to metabolic pathways and their variety

* insight into the more dynamic aspects of bacterial life.

Materials & Methods

Overview

The objective of this game is to assemble a functional degradation pathway of a target compound to tricarboxylic acid (TCA) cycle intermediates. There is one deck of cards that represents the compounds to be degraded and a second deck of cards that represents portions of degradative pathways. The trading of the pathway cards by students represents the exchange of genetic material that occurs during horizontal gene transfer. A winning hand occurs when a student holds a complete pathway in his/ her hand for the degradation of the chosen compound. The assembled pathway must outline the degradation of the target compound all the way to the point at which its metabolites can enter the tricarboxylic acid (TCA) cycle, the central catabolic pathway of life, thereby becoming a useful carbon and/or energy source.

Preparation

This activity requires the preparation of two decks of self-made cards: a pathway deck (about 40 cards) and a compounds deck (about a dozen). The original cards were developed using various pathways for the degradation of aromatic compounds. Because the chosen compounds were closely related, the different pathways intersect at various points, such as catechol or salicylate. This allows several different pathways to be assembled that allow for degradation of the same compound. To emphasize the role of horizontal gene transfer, it is important that no compounds be chosen which could have their ultimate degradation diagramed on a single pathway card. One set of decks will be needed for every four to six students in the class, or students may work in teams of three to four students if class size is larger.

Guidelines for Design of the Decks

1. Focus on a group of compounds whose metabolisms converge on common intermediates. We chose simple aromatic compounds, but the metabolism of other chemically-related compounds such as amino acids or sugars could be addressed.

2. Pick a variety of pathways that intersect. Some helpful resources for researching metabolic pathways are the University of Minnesota Biocatalysis/Biodegradation Database (http://umbbd.ahc.umn.edu/) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) (http:// www.genome.ad.jp/kegg/).

3. If a pathway splits, make one card for each branch of the pathway (Figure 1A).

4. When making the cards for the pathway deck, ensure the product on one card is the substrate on the next (Figure 1B).

[FIGURE 1 OMITTED]

5. Pick compounds for the compound deck that need at least two pathway cards to be degraded, but preferably no more than four.

6. Include as much information on the cards as you feel is appropriate (compound names, enzyme names, gene names, etc.), but try not to make them too busy or crowded.

7. Only one to three enzymatic reactions should appear on each pathway card. If less detail is required, several successive arrows can represent multi-step transformations when intermediate metabolites are inconsequential to the game.

After designing and printing your cards, test them out on a small group of people before using them in class. You may find that a few modifications are in order.

To further engage students in this activity, you may want to involve them in the preparation of the cards* For example, each student could choose or be assigned a pathway to illustrate on standardized cards*

During the Activity

1. Have the students play an open hand first, being able to see each other's cards. When they think they have pieced together a pathway, check to make sure the pathway is appropriate before they continue.

2. Give the students 15-20 minutes (or more) to play the game. Sometimes someone is dealt a complete pathway immediately and other times it seems to take forever for anyone to assemble a pathway. When it is taking a long time, you might want to check to make sure no one is holding a pathway without realizing it. If no one actually has a pathway, you may want to let the game continue or you could intervene constructively (i.e., help them construct the pathway and ask why it was taking so long to piece together).

3. Depending on the level of the students, it might be helpful to check in on their games periodically to make sure they have the concept of the game and discuss potential misunderstandings.

Rules of the Game

1. Divide into groups of four to six people. Pick one person to record the game results*

2. Deal out the entire deck of pathway cards* Look at your cards, but prevent others from seeing your hand. Each card represents enzymatic reactions in a degradative pathway that are encoded by a gene or a group of genes. These reactions are assumed to be irreversible. You will be using the cards in your hand to construct a degradation pathway. A complete pathway starts with the target compound and ends with metabolites that can enter the TCA cycle (Figure 2).

[FIGURE 2 OMITTED]

3. The dealer picks a single target compound from the compound deck. This is the compound you are attempting to degrade.

4. Take a minute to examine the cards in your hand. Someone may already have the complete pathway in his/her hand. If so, go to Step 8.

5. Play starts with the person to the left of the dealer blindly taking one pathway card from anyone (s)he chooses*

6. The person from whom the card was taken then takes one card from anyone (s)he chooses. Take time to examine your hand for possible pathways before you continue playing.

7. Play continues in this way until someone believes (s)he has a complete pathway.

8. At this point, the person who believes (s)he has a complete pathway tells the group and lays his/her cards for the pathway on the table. The other members of the group examine the pathway for accuracy. If the group agrees the pathway is feasible, the recorder copies down the pathway.

9. If time remains, collect the cards, reshuffle both decks, and start another hand.

10. When the allotted amount of time is up, discussion will follow. Think of (and remember!) questions and issues the game raises while you are playing.

11. One copy of the pathways generated during the game will be collected from each group at the end of class.

Discussion

* There are many topics that can be addressed with this activity. It is important for the students to generate questions and ideas for discussion while playing the game. You might ask them to write down thoughts and questions as they think of them during the game. Some thoughts and questions you might want to address are:

* How is this game similar to horizontal gene transfer in the environment? How is it different?

* Why do you have to pick cards randomly rather than asking for a specific portion of a pathway?

* What complexities arise when bacteria are exchanging DNA and genes as compared to students exchanging cards?

* What other features of horizontal gene transfer might be incorporated into the game? How might they influence the rules?

* What might be some of the potential limits to genetic exchange in the environment?

* It sometimes takes a long time to assemble an entire degradative pathway in one organism. How do some communities that degrade novel compounds circumvent this problem? How could people trying to use bacteria to degrade a specific compound circumvent this problem?

* In the environment, sometimes a certain compound persists for a long time, but then begins to disappear quickly. What might some explanations for this phenomenon be, especially considering today's activity?

* If bacteria were unable to transfer genetic material to each other and utilize new genetic information, what would the world be like? How would it affect the environment, microbial ecology, evolution, human beings, etc.?

* Molecular biologists and industrial engineers often try to isolate or develop strains of bacteria to degrade specific compounds. Does this activity give you any ideas about how they might make their efforts more efficient?

Results

Students are instructed to record the pathways generated during the game and hand them in to the instructor. The instructor could use them to simply make sure the students were performing the activity or could go over the pathways to make sure they were feasible and return them to the students for further reference. Because of the variability of the game, these should not be graded except for simple class participation purposes. Groups also might like to share their results with the rest of the class as part of the discussion.

Conclusions

There are many ways this activity can be modified to fit the specific goals of the instructor. The subject matter of the cards and the level of discussion can both be modified. For example, students could be assigned a research paper describing the actual pathway(s) used to degrade one of the target compounds selected during the game. The paper could include a discussion of the results from the game and why the pathway developed through the card game might or might not work.

In another variation of the game, students could aim to degrade the most compounds possible. Students could trade cards for a specified amount of time. At the end of trading, students would then examine their hands and try to piece together as many degradative pathways as possible (individual cards could be used in more than one pathway). This works especially well in larger classes where groups can work together and then compare results with the other groups at the end of the exercise.

Discussion following the game is critical to the success of this activity. Because there are inherent differences between this simulation of horizontal gene transfer and the biology, misconceptions can arise. By addressing the differences between the game and environmental horizontal gene transfer, potential misconceptions can be clarified, even transformed into insight. Also, discussion acts as a forum for the consideration of the game-inspired topics of the instructor's (or students') choosing, such as evolution or metabolic engineering, that are not explicitly dealt with in the game itself.

This activity has been successfully implemented at the undergraduate introductory level, and was by far the highlight of the course for both the students and instructor. It was also used in an introductory graduate level class for non-microbiologists. In both classes, student discussion was lively and insightful. When the class discussed differences and similarities between the card game and real horizontal gene transfer, comments were thoughtful and constructive. The game illustrated the intended concepts, sparked discussion, and was enjoyed by the students.

References

Alexander, M. (1999). Biodegradation and Bioremediation. San Diego, CA: Academic Press, Inc.

Karaolis, D.K., Somara, S., Maneval, DR. Jr., Johnson, J.A. & Kaper, J.B. (1999). A bacteriophage encoding a pathogenicity island, a type IV pilus and a phage receptor in cholera bacteria. Nature, 399(6734), 375-379.

Madigan, M.T., Martinko, J.M. & Parker, J. (2000). Brock Biology of Microorganisms. Upper Saddle River, NJ: Prentice Hall.

Seffernick, J. L. & Wackett, L. F. (2001). Rapid evolution of bacterial catabolic enzymes: A case study with atrazine chlorohydrolase. Biochemistry, 40(43), 12747-12753.

van der Meer, J. R., Werlen, C., Nishino, S.F. & Spain, J.C. (1998). Evolution of a pathway for chlorobenzene metabolism leads to natural attenuation in contaminated groundwater. Applied and Environmental Microbiology, 64(11), 4185-4193.

Wackett, L. P. & Hershberger, D.C. (2001). Biocatalysis and Biodegradation. Washington, DC: ASM Press.

JEANNE KAGLE is Professor of Biology, Mansfield University, Mansfield, PA 16933; e-mail: jkagle@mansfield.edu. ANTHONY G. HAY is Assistant Professor, Department of Microbiology, Cornell University, Ithaca, NY 14850; e-mail: agh5@cornell_ edu.
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Title Annotation:INQUIRY & INVESTIGATION
Author:Kagle, Jeanne; Hay, Anthony G.
Publication:The American Biology Teacher
Geographic Code:1USA
Date:Jan 1, 2007
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