Workshop: The Evolution of Cells by Dana Krempels

WORKSHOP LEADERS! If you don’t remember how to read a phylogenetic tree and need a refresher on classification, please review: http://www.bio.miami.edu/dana/160/160S12_5.html http://www.bio.miami.edu/dana/160/160S12_10.html The first living things on earth were simple, single-celled organisms enclosed by a plasma membrane. These first cells contained proteinaceous, fluid matrix that housed simple organelles (ribosomes) and a DNA double helix that served as a blueprint for inheritance of the cell’s traits. Such cells are commonly said to be prokaryotic, and prokaryotic organisms are called prokaryotes. Cells evolved much later to contain a membrane bounded nucleus and membrane-bounded organelles. These such cells are eukaryotic, and organisms composed of eukaryotic cells are known as eukaryotes. In this workshop, we will explore the similarities and differences of these types of cells (and organisms), and discuss their evolutionary origins. Origins and the Fossil Record 1. What is the literal translation of this word from its Greek roots? pro = “before” karyon = “nut” (referring to the appearance of the stained nucleus) 2. To what does this descriptive term refer? The absence of a membrane-bounded nucleus. 3. Do prokaryotes have a structure analogous to the nucleus? If so, what is it? Describe its morphology. The nucleoid. This is a region near the center of the cell that contains most of the organism’s genetic material. The DNA is found in the form of a circular chromosome (there may be several identical copies of the genome), and is “naked” (not associated with any proteins). The DNA is loosely coiled in the nucleoid. 4. Modern classification separates all living things into three domains. them: Bacteria, Archaea, Eukarya

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5. The oldest known fossil remnants of living organisms resemble which of the above most closely? What does this say about which type of organisms evolved first? Stromatolites are often cited as the oldest fossil signs of life, and have been dated back as far as 2.7 billion years. These rock formations show striations very similar to those made in sediments by extant cyanobacteria, which are classified as true Bacteria. However, this does not necessarily mean that Bacteria evolved before Archaens. In fact, they may have evolved at close to the same time. Controversial fossil remnants (what appear to be remants of ancient bacteria and archaens—depending on who’s looking) have been found together in rocks as old as 3.4 billion years. See: http://arstechnica.com/science/news/2011/08/new-find-provideswindow-on-earths-oldest-life.ars and then: http://www.physorg.com/news/2011-03-overturns-oldest-evidence-lifeearth.html

6. Of the three domains, which contain prokaryotic organisms? Bacteria and Archaea 7. Do any of the Domains contain both prokaryotes and eukaryotes? If so, which? No Domains contain both. Phylogenetic Relationships Consider the phylogenetic tree below. 8. Examine the tree. Which taxon shares the most recent common ancestor with Bacteria? The ancestor of Archaea/Eukarya branched from the same ancestor as Bacteria. Neither Archaea nor Eukarya can be said to be more closely related to Bacteria. 9. Which Domain shares the most recent common ancestor with Archaea? Eukarya 10. What evidence was used to make this phylogenetic connection,and who was the first person to propose it? Carl Woese determined that Archaean DNA was very different from that of Bacterial DNA, and was in fact more similar to Eukaryota DNA. 12. In the context of the origin of life, who—or what—is LUCA? Last Universal Common Ancestor The hypothetical ancestor of all living things. 11. Carl Woese cautions us that “finding” LUCA may be a far more difficult task than anyone suspects. List possible difficulties scientists may face in trying to identify LUCA. Discuss - Even the oldest fossil microorganisms may not be the first living things. They may be the remnants of a massive, global bottleneck that occurred when asteroids bombarded our planet about 4 billion years ago, leaving little of the earth’s crust intact. Any remnant of the first life on earth may be destroyed and beyond recovery. - Horizontal gene transfer clouds the picture. The endosymbiosis of various lineages of prokaryotes gave rise to ancestral forms of life, and such endosymbiosis may have occurred several different times. - Many scientists believe that thermophilic archaeans (deep sea vent communities) may be the descendants of LUCA. But because thermophilic archaeans in the deep sea vent communities may have been in almost the

only location that would be largely immune to the effects of surface environmental changes and even asteroid storms, they might have arrived later than LUCA, and survived longer. - According to Carl Woese: "The universal ancestor may not be a single lineage at all. At the universal ancestor stage, horizontal gene transfer may have been so dominant that the ancestor may in effect have been a community of cell lineages that evolved as a whole. We will be able to trace all life back to an ancestor, but that state will not be some particular cell lineage." (http://www.spacedaily.com/news/life-01zm.html)

12. List some of the reasons that prokaryotes are so diverse in form and function. Discuss. -

a vast number of different habitats and niches are available on earth for such small, rapidly reproducing organisms -

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prokaryotes reproduce very quickly. This means that any mutations that are adaptive can rapidly spread and produce a large, well-adapted population of microbes. -

prokaryotes readily undergo genetic recombination via conjugation, transformation, and transduction (viral intermediate). This coupled with their rapid rate of reproduction can result in the fast spread of adaptive alleles (antibiotic resistance, toxin production, protective gel capsule, etc.) -

prokaryotes exhibit vast metabolic diversity and can live just about anywhere. Their genomes have been labile in the metabolism department! They don’t need much to survive: minimal medium -

Endospores allow them to survive extreme conditions and bounce back when things are more hospitable. -

Application and Analysis: Symbiosis 1. What is meant by the term symbiosis? (What is the literal translation from Greek?) sym = “together” bios = “life” It means “living together” Define each of the following types of symbiosis in terms of two hypothetical populations, Population A and Population B. For each one, give an example using a prokaryotic species as one of your two interacting populations. a. mutualism – Population A and Population B benefit each other example: You can come up with some, I’m sure. b. commensalism – Population A benefits, Population B is not affected. example: Make them work for it! c. parasitism – Population A (parasite) benefits at the expense of B (host) example: List all the bacterial diseases you can think of… 2. When people think of bacteria, they usually think “germs” and “disease.” Yet we now know that bacteria are not only ubiquitous, but usually harmless or even

beneficial. Go around the group, and—one at a time—each person must identify one thing that bacteria have done today to make their lives better. (This will get more difficult with each turn, so the later people will have to be creative. HINT: Try researching with key words “beneficial bacteria”.) Intestinal bacteria (e.g., Bacteroides thetaiotaomicron) that provide micronutrients; Nitrogen-fixing bacteria that make life possible Pathogenic bacteria that suppress populations of competing species (rats, etc.); the list goes on… (They can get more and more specific and individual as the round-robin proceeds) 3. These days, it’s becoming more popular to switch to a vegetarian diet. When a person changes to an all-plant-matter diet, s/he may experience some intestinal perturbations. The same is true for a vegetarian who “backslides” and eats meat. Discuss why this might be so. (HINT: Think about your cellular passengers, not your own tissues.) Different intestinal residents (bacteria and archaeans) are better adapted to exploit different food sources. As a person’s diet changes, the bacterial population will change accordingly. Individual bacterial strains might also change, and in the interim, some food may not be broken down as quickly as it is when the bacterial populations are “well tuned” to the food source. Gas and intestinal discomfort can result, but it’s usually only temporary. 4. A human being infected with HIV may develop AIDS (Acquired Immune Deficiency Syndrome). In an AIDS patient, the virus invades immune system cells known as T-cells, which are central in guiding cell-mediated immunity to pathogens. A person suffering from AIDS will thus not die from the direct effects of the virus, but from invading pathogens (bacterial, viral, and fungal). Would it be possible to stave off the effects of AIDS by administering antibiotics and anti-fungal drugs to AIDS patients? Why or why not? Even anti-microbial drugs cannot kill every single individual pathogen. It’s like having a police force with limited numbers of officers: they can’t be everywhere all the time. The host’s immune system must be the primary attacking force, with the antimicrobial drugs serving temporary “fire power” while the immune system “catches up” and gets the pathogen overgrowth under control. In an AIDS patient, there is no immune response, so anti-microbial therapy will be of limited efficacy, especially as the pathogens are subjected to natural selection by the drugs themselves. Resistant strains will often evolve, and then even antibiotics will not help. If anti-microbial drugs were to be administered to such a patient, and you were the prescribing physician, what protocol would you design for greatest efficacy? (HINT: Consider that different anti-microbial drugs have different modes of action against the pathogens, and are not effective against every species or strain. Also consider the possible effects of natural selection.) Discuss. As hinted above, a rotating schedule of different anti-microbial drugs might be most effective at preventing the evolution of resistant strains of pathogens. 5. You are a veterinarian treating two different rabbits (Peanut and Petunia) for dental abscesses. Petunia is a purebred Holland Lop, and Peanut is a “mutt” who was adopted from the Broward Humane Society. You took a sample of pus from the tooth socket of each rabbit and sent them to a pathology lab for identification.

Both cultures revealed overgrowth of Pasteurella multocida, sensitive to the antibiotics ciprofloxacin, penicillin, and amikacin. You put both rabbits on a combination of ciprofloxacin and amikacin. After a one month treatment, the abscesses appeared to be gone in both rabbits, and the owners went home happy. A month later, both rabbits were back with recurred abscesses. You put them both back on ciprofloxacin and amikacin, this time for two months. Peanut’s abscess resolved completely and did not come back. Petunia’s, however, did not respond to the same antibiotics, and you had to switch to penicillin injections (oral penicillins are deadly to rabbits) before it finally was resolved for good. List all the possible reasons--at the cellular, individual organism, and evolutionary levels--that might have been responsible the different results in the two patients. Side notes on antibiotic mode of action: Ciprofloxacin is a fluoroquinolone. It kills bacteria by deactivating DNA gyrase, the enzyme that relaxes the DNA strand just in front of the replication fork during DNA replication. Without DNA gyrase, the bacterial DNA tangles up and cannot be replicated. Amikacin is a aminoglycoside. It suppresses bacterial growth by inhibiting ribosome activity. At very high doses, it can kill bacteria, but it is generally considered bacteriostatic (i.e., growth-inhibiting), not bacteriocidal (i.e., lethal to bacteria). Pencillin is a Beta-lactam. It acts by inhibiting cell wall biosynthesis. Cellular level: As I mentioned in the AIDS patient scenario: antibiotics are not the cure for an infection. They are essentially a tool we use to “buy time” for the host’s immune system to ramp up, recognize, and fight the invading pathogens. You can’t flood the body with so much antibiotic that it saturates the system. The number of antibiotic molecules in the body is finite, and usually smaller than the number of bacteria. So there will always be bacteria that are “missed” by the drug, and are left to procreate after the antibiotics are taken away. In Peanut’s case, he was lucky enough that the bacterial pathogens did not undergo a mutation that rendered them resistant to ciprofloxacin and/or amikacin. This is pretty much luck of the draw: bacteria DO NOT evolve resistance because they need it! Mutations are random, and adaptive mutations (such as antibiotic resistance) have to be there first in order to be selected. In Petunia’s case, she wasn’t so lucky. She had a population with some members bearing one or more mutations that made them resistant to the drug combination. A different antibiotic was needed to thwart the newly evolved bacteria that were resistant to cipro and amikacin. Individual level: It is possible that Peanut had a stronger immune system than Petunia at the start. Petunia may have had other things going on that compromised her immune system. Because she is a purebred, she is likely more inbred than Peanut a “mutt.” Greater heterozygosity is strongly associated with a more versatile immune system. Was Petunia older (immune system is not as effective as we age)? What other contributing factors can you hypothesize? Evolutionary level: Both natural selection and genetic drift can play a role here. Antibiotics act as a selective agent, removing the susceptible

bacteria, but unable to remove those with resistance to that particular antibiotic. Once all the susceptible bacteria are gone, the resistant members of the population can proliferate in the absence of competition, and a new antibiotic is necessary to fight them. Also, genetic drift (random chance!) can play a role: In a body where not all bacteria come into contact with antibiotic molecules, it’s possible that some sensitive individuals will survive by sheer luck. It is pretty much impossible to kill off every single bacterium in the body with antibiotics (nor would you want to!), so there will always be a “seed” population left behind to re-grow, should the host’s immune system be compromised again. Whether that population is resistant or not depends on the interaction of natural selection with genetic drift.