Needing help with quetions 2 & 3 on page 11 of attached document. The question is pertaining to polyurethane as a huge polymer and why is it important that the polyurethanse is a secreted enzyme and the net loss of ATP...etc... Case Study/Application Assignment Number One Spring 2014 General Instructions: The Case Study beginning on page 2 will involve concepts you learn in Chapters 3, 4, 7, 2 and 8. Use those chapters and related references outside your text to thoroughly and thoughtfully complete each question you are asked in the case. Read each section carefully. A grading rubric for this case will be posted by Feb 10th in Shared Files. Online Students: Print a copy of the case study first to use as a reference and to give yourself a “full-picture” of where you’re headed as you work your way through it. As you work your way through this Case, please type all of your answers directly into the document in the spaces provided. Follow the required word count. For the poster graphic, mark your suggested changes right on the page. My expectation is that each student complete their own work. You are welcome to bounce ideas off one another and work on solving ideas together but the writing must be yours and yours alone. For online students in particular, all submitted writing goes automatically through “turnitin.com” and is scored for originality (including if the work is original to other students in the class). 1 ELVIS Meltdown! Microbiology Concepts of Culture, Growth and Metabolism Adapted from the Department of Cell Biology and Molecular Genetics University of Maryland - College Park, MD Part One: Return to Sender Fresh out of college, with your degree in microbiology, you have landed your first “real job” as a scientist with DuPunt, a company that specializes in the development and production of polyurethane derivatives (specialized plastics). You (and your boss) are not quite sure why DuPunt has a microbiologist on staff, but you are both about to find out why. Your boss has called you into his office. “Read this article!” he says, pushing the front page of a national newspaper across his desk to you. Stifling your initial reaction to the article, you manage to mumble, “What a tragedy.” 2 “Yes. Yes. And this could take an ugly turn for DuPunt!” your boss says. “I’m not sure what caused this mess, but I do know a couple of things that didn’t make it into that news article: (1) the only plastics showing damage in the ELV were polyurethanes; and (2) our company provided those polyurethane products to NASA at a cost of $15,000,000. We’re in big trouble if we can’t prove that something from that planet is responsible for destroying ELVIS!” He continues, “The polyurethane we provided was first-rate. We didn’t cut any corners. Products from the same batches of polyurethane have been sent into outer space before, and returned with no damage. There must be some explanation other than our incompetence. This is where you come in. I need you to find that explanation!” “Why me?” you ask. “Because of the stink!” your boss exclaims. “What do mean by “the stink”? you ask. Your boss replies. “Some of the scientists present at the ELVIS disaster said the smell reminded them of an old fermenter or an autoclave. Those are microbiology terms, aren’t they? Those comments tell me that this whole stinking mess might be caused by microorganisms – you know, bacteria, fungi, viruses, germs…something like that. Get right to work on this! You and I will have to work closely on this, you know. I’ll handle all the communications with the press, and you handle the science. Just make sure that you explain everything clearly to me so that I can speak about it to the press without making a fool of myself and DuPunt!” Lets Get Started: As a microbiologist for DuPunt you must determine what has happened to the polyurethane. Here is your hypothesis: 3 The degradation of polyurethane products was caused by a microorganism or microorganisms present in the soil samples collected by ELVIS. Questions: 1. Using the light microscope, you examine all the soil samples and the “goo” from the degraded polyurethane. Will this approach allow you to observe all the potential microorganisms in the sample? Why or why not? If not, what are the limitations of this approach? (Minimum 300-word response) There are limitations with using a light microscope. In order for a microscope to work to the best of its ability it needs to have adequate magnification, resolution and clarity of the image. The light microscope provides a magnification from 40x to 2000x. Magnification is how much bigger a sample appears under microscope than in real life. The resolution power of the microscope defines the object. It helps distinguish between two points on an image. The resolution of an image is limited by wavelength because when objects in the specimen are smaller they don’t interrupt the waves so they go undetected. You can magnify the image all you want, but if you don’t have the resolution you can’t see detail and the image will just appear blurry. Next comes the type of light microscope used. They are described by the nature of their field. There is the bright-field in which it forms its image as light is transmitted through the specimen. The specimen will appear darker than the surrounding illuminated field. A bright-field microscope can be changed to a dark-field by adding a disc to stop all the light from entering the objective lens. This will illuminated specimens surrounded by a dark field. The next field microscope is phase-contrast microscope. Phasecontrast microscope contains devices that change the light to make subtle changes as its passes through the specimen. This is used to detected internal cellular detail. The final light microscope to mention is the differential interference. The DIC provides a detailed view of unstained, live specimens. There are bacteria that are too small to be detected and those require electron microscopy. There are bacteria that are distorted and not be stained by normal methods. All these factors need to play into finding the right approach to find the microorganism that caused the degradation of polyurethane. 2. You next use phase contrast microscopy to observe a wet mount of a soil sample (see picture below) and a “goo” sample (see picture below) from the ELVIS. Based only on the pictures below, in what ways are the samples you see in both the soil and “goo” similar to microbes previously characterized on Earth? In what ways are they different? (Minimum 350-word response) Let’s start by describing the make-up of soil. Soil is made up of minerals, organic matter, water, air and soil organisms. Soil is a big ecosystem that supports complex relationships between geologic, chemical and biological factors. Organic matter, in soil, is plant and animal residue at various stages of decomposition, cells and tissues of soil organisms, and other substances that are synthesized by microorganisms. There are many organisms that find their home in the soil such as fungi and bacteria. When comparing the slides there are many things to examine. Microbial sizes, shapes and arrangements are all things that help classify the sample out into categories as microbes-bacteria, archae, fungi, algae, protozoa, and viruses come in all different sizes and shapes. When we are comparing the slides we have to remember that the images are under a light microscope. Viruses are the smallest of microbes and not usually 4 detectable with a light microscope . Archae are single-celled simple organisms. They contain many bacterial characteristics but their ribosomal RNA contains unique signature sequences. Fungi are very popular in soil. They are single celled or complex multicellular. Their role is beneficial in cycling carbon and other elements. Algae can also be single celled or multicellular. They are the eukaryotic protists that photosynthesize with chlorophyllia. Protozoa are unicellular eukaryotic protists. This now leads us to bacteria. Bacteria have no nucleus. Their genetic information is contained in a single loop of DNA. Bacteria are classified into five groups based on shape. Sperical (cocci), Rod (bacilli), spiral (spiralla), comma (vibrios) or corkscrews (spirochaetes) are all shapes of bacteria. Bacteria can be then in single, pairs, chains or clusters. Let’s go back to the samples, the images of the Earth samples have flagella where the goo does not. The goo sample is actually much simpler as it has much less organelles. Distant microbes were less complex and did not have flagella to move to new locations and find nutritional sources. One thing to consider is the complaint of smell when they returned. This would imply that the microorganisms underwent fermentation versus respiration. Figure 1 – Soil Sample Figure 2 – Goo Sample Your boss has done a little reading about microorganisms but he finds it all pretty complicated. “It’s like a foreign language!” he complains. “I have to face the press to explain our idea that microbes might be responsible for all the damage to ELVIS. Clearly, I’m going to need some visual aids or the press won’t have a clue as to what I am saying. I think I’ll need to explain what a bacterium looks like and how it might be possible that they can degrade polyurethane. I really can’t waste time showing them all the potential types of bacteria (archea, mycoplasma, etc), so I think I’ll just show them what a gram negative bacterium looks like as an example. I’ve already put together a poster with a diagram of a typical gram-negative bacterium. Will you take a look at it, to make sure I haven’t made any mistakes? I labeled all the features and also indicated the major biochemical composition of each feature. I’m sure this figure will wind up in lots of newspapers and magazines, so it really needs to be scientifically correct. We wouldn’t want to make DuPunt look stupid, would we? Just proofread it and make any necessary corrections, okay? Go ahead and mark up the poster as needed.”(see Figure 3 next page) 5 Make any changes you wish to make to the actual graphic. Highlight those changes. 6 Part Two – Suspicious Minds Your direct microscopic observation of microorganisms in the soil samples has sparked your boss’s interest. He is eager to determine what type of microorganism(s) is/are present. Though he only presented a model of a bacterium for his public press conference, there is still not enough evidence to determine if the microorganism is a prokaryote, eukaryote, or even some type of organism never seen before. He asks you to take a sample of the soil to the electron microscope for further analysis. To be the most objective, he asks you to run a sample through the electron microscope and decides to do the same himself for comparative analysis. You are just as interested in the nature of the microorganism(s) as your boss, but instead of directly analyzing the soil, you use both the soil sample and the “goo” sample and, using pure culture techniques, isolate a similar organism from each for testing. Question: 1. How would you go about isolating a pure and matched culture from each of your samples? How would you know that you have isolated the same organism from both the soil and the “goo” sample? (Be specific) (minimum 450-word response) First thing first…definition of culture is to cultivate microorganisms. It is a process to take seeds (microbes) and plant them into an environment (medium) to let them thrive and grow. To do so one would introduce a sample into a nutrient also known as medium. This provides an environment in which the sample can grow or multiply. A medium is the foundation of culturing. As all microbes are different some require a few inorganic compounds for growth versus others who require a complex list of specific inorganic and organic compounds. Media (plural for medium) come in over 500 different types. They can be contained in test tubes, flasks, or Petri dishes. There are three main categories of media. Those categories are based on their properties: physical state, chemical composition and functional type. Physical state of media is the consistency of the medium. It can be liquid, semisolid, solid that converts to a liquid or solid that can’t be liquefied. The chemical composition is either synthetic or chemically defined or nonsynthetic with is complex. The functional purpose is the purpose of the medium such as general purpose, enriched, selective, differential, anaerobic growth, specimen transport, assay or enumeration. Some media can serve more than one function. Isolation techniques are used to help separate microbes and spread them apart to create isolated colonies that contain a single microbe. This method helps define making a pure culture. Proper isolation is required that a small number of cells be inoculated into a large area of the medium. The tools needed to perform isolation would be a medium as described earlier, Petri dish or place media is controlled and inoculating tools. Inoculating tools can be loops, needles, pipettes and swabs. For a controlled sample, sterile technique needs to be used. Sterile technique is when you start with a sterile medium, sterile inoculating tools and nothing non-sterile contaminates the specimen. Things that would be considered non-sterile would be room air, fingers non-sterile materials. So now that we have a background on what we are using let’s carry on to that actual process. We have collected our sample of the goo and of the soil. We placed those samples into a container of medium that will support the growth. After placing the sample in the media we place it into an incubator. An incubator is a controlled environment that helps promote growth. After optimal growth in the medium we use isolation techniques to spread the colonies apart to grow colonies 7 that contain a single microbe. Those isolation techniques could be techniques such as the streak plate method or pour plate (loop dilution) method. There is also the spread plate technique. All these methods use various ways to spread the surface promoting individual colonies. Once our subculture is separated into a pure culture of the bacteria we observe for the macroscopic appearance and then under the microscope for basic details such as cell type and shape. Those basic details should be seen in the soil and goo samples. This is reassurance that you don’t have a contaminated culture. Later, you and your boss compare samples (see table below). Test 80 S ribosomes 70S ribosomes Circular DNA Linear DNA RNA Phospholipid membrane Peptidoglycan Lipotechoic acid Flagellar basal body proteins Cytoskeleton proteins Mitochondria Histone proteins Nuclear pore proteins Boss’s sample + + + + + + + + + + + + + Your sample + + + + + + + - “I’m not sure what’s wrong with your sample, but my results prove that we are dealing with a new kind of life form here…I’m calling it the “preukaryote” because it has components characteristic of both prokaryotes and eukaryotes. It’s time for a press conference!” boasts your boss. Question: 2. If your goal is to isolate this “new microorganism”, which results are more informative: yours or your boss’s? Why? What do your results indicate about the nature of this microbe? Does its structure closely resemble that of a prokaryote or a eukaryote? Do you agree with your boss’s conclusion that this new microorganism is a prokaryoticeukaryotic hybrid? Why or why not? (Minimum 450-word response). As you notice the results vary greatly. This goes back to the pure culture or axenic method that I used. My boss took the sample and viewed it under an electronic microscope and found that all the elements were viewed. This is due to the fact that soil contains many organisms and since they weren’t separated out, he was able to view all sorts of things. He was viewing a mixture and nothing can be excluded or included as it isn’t about a single microbe. I had gone through the work of isolating the soil and the goo to find the microorganism that was found and able to grow in both samples. My boss states that the new organism is a “preukaryote” due to the fact that the components characterize both prokaryotes and eukaryotes. Let’s work this through. Due to the fact that my boss’s sample 8 was in my eyes not pure, I am going to look at my positive viewings to see if his speculation is true. The first thing is the 70S ribosome. Ribosome is made up of RNA and protein and is the site of protein synthesis. 70S ribosome means that it is heavier more compact structure that sediment faster. Prokaryotic ribosome is 70S as it actually composed of two smaller subunits. The next positive finding is circular DNA. Most bacteria’s hereditary material is in the form of circular DNA which is designated as the bacterial chromosome. Moving on to RNA, this is found in both prokaryotes and eukaryotes. It is a single strand containing ribose sugar instead of deoxyribose and uracil instead of thymine. Phospholipids are fatty acids plus glycerol plus phosphate that are found in membranes; both prokaryotic and eukaryotic cells have a cell membrane. Although those cell membranes are made up differently and have different thicknesses but are present in both. Peptidoglycan is a special class of compounds in which glycans are lined to peptide fragments, also known as a short chain of amino acids. Eukaryotic cells do not contain peptidoglycan as of this point we could say that my boss’s theory has proven false but let’s carry on and see what the last findings show. I couldn’t help but notice that within my findings I didn’t have a positive lipotechoic acid. Lipotechoic acid is found on the cell wall of gram-positive cells. Gramnegative cells don’t have this acid. Flagellar basal body proteins are that provide motility or self-propulsion. They are found in some not all prokaryotic cells but also some eukaryotic cells as well. They help move the bacteria and this mobility helps growth to spread. The last positive finding is that of cytoskeleton proteins. Cytoskeleton was known to the eukaryotic cells as an intracellular framework of fibers and tubules that bind and support the cell. Prokaryotic cells have more recently been found to house a cytoskeleton into the fine structure of certain rod and spiral shaped bacteria. Upon reviewing my findings compared to my boss’s I would still have to say that I disagree with his theory as the findings are more closely related to a prokaryotic cell. Later on, as you are getting ready to head home, you hear your boss bellow, “What the H-E double hockey sticks is going on here!” You ask him what happened. “This morning I put a few thousand cells from your pure culture onto two slides in water. Because I had to leave for the press conference and didn’t want them to dry out, I sealed the coverslips. When I left, they ere clearly distributed evenly on the slides. Now look! On this slide I used a rubber gasket to make the seal. On this slide, I used a Lycra gasket. Now look at the cell distribution! On the rubber-sealed slide, the cells are evenly distributed, but on the Lycra-sealed slide all the cells are congregated around the edge of the coverslip. Look…they are all over the edges; none are left in the middle part of the slide”. Question: 3. Come up with at least two possible explanations for the “amazing” redistribution of the new microorganism on the Lycra-sealed slide. (Minimum 400-word response) There are a couple possibilities on the explanation of why the redistribution of the unknown bacteria on the Lycra-sealed slide. Two of those explanations would be motility and taxis. 9 Motility means the movement of the bacteria by the flagella that are driven by a proton gradient. Taxis mean a motile response to an environmental stimulus. Bacteria under the microscope often have appendages noted from their surface. Appendages are divided into two groups, motility and attachments/channels. If the pure culture was put onto two slides of water and evenly distributed and sealed and movement happened, motility has occurred. There can be movement of some cells without it actually making progress. That is why electronic microscopic examination and sometimes staining of the specimen is necessary to see if the species is motile. Flagella provide the power of motility or propulsion within bacteria cells. Flagellum is an appendage that allows a cell to swim freely though an aqueous habitat. There are three parts to the flagellum; the filament, hook, and basal body. In prokaryotic cells the hook and filament are free to rotate 360 degrees. This is a differential when comparing eukarotic cells and prokaryotic cells. The flagella are arranged by two patterns; the polar arrangement and the peritrichous arrangement. In a polar arrangement the flagella are attached at one or both ends of the cell. In a pertrichous arrangement the flagella are randomly placed over the cell surface. There is growth spread though out the entire medium that is an indication of motility as it is widespread and didn’t just stay in the same spot. The flagella can guide the bacteria in the wanted direction because of the system for detecting chemicals is linked to the drive of the flagellum. How this works is on the cell surface molecules that bind specifically with other molecules, receptors, bind specific molecules from the environment. The attachment of sufficient numbers of these molecules transmits signals to the flagellum which in return set the flagellum into a rotary motion. As the flagellum rotate counterclockwise the cell itself moves in a linear motion called a run. Runs are interrupted by tumbles. Tumbles are when the flagellum reverses direction and rotates clockwise causing a tumble. When placed in a medium a cell moves randomly in short runs and tumbles until it gets closer to an attractant and then it spends more time in runs. So what causes a cell to run and tumble toward or away from something? That would be called chemotaxis. Chemotaxis is the tendency of organisms to move in response to a chemical gradient, either towards or away from a chemical stimulus. The chemical stimulus is usually known as a nutrient or away from a potentially harmful compound. There are other taxis such as phototaxis which the movement is in response to light rather than chemicals. Part Three – All Shook Up You have found media that support growth of the pure cultures you isolated (now named by your boss as the Extraterrestrial Polyurethane-Degrading Microbe (EPTUM)). The recipes for these media are shown below: Medium 1 10 Medium 2 5 g yeast extract 20 g tryptone extract 0.5 g NaCl 3.6 g glucose 1 liter H2O Growth EPTUM Growth - aerobic EPTUM Growth - anaerobic E. coli - aerobic E. coli - anaerobic 10.5 g K2HPO4 4.5 g KH2PO4 1 g MgSO4 10 g polyurethane 1 liter H2O Medium 1 + + + + Medium 2 + - You are excited because, in Medium 2, EPTUM utilizes polyurethane as its energy source and its sole source of carbon and nitrogen, a finding that raises the possibility that EPTUM could be a useful tool for bioremediation of polyurethane-containing wastes (in landfills etc.). You have also made some progress in characterizing the central metabolic pathways and related biochemical properties of EPTUM. In particular, you have discovered that: ? EPTUM secretes an enzyme (polyurethanase) that catalyzes the degradation of polyurethane and generates citric acid (citrate) as a product. ? The cytoplasmic membrane of EPTUM contains a transport system capable of transporting citrate across the membrane and into the cell at the expense of 4 ATP molecules (hydrolyzed to form ADP and phosphate) per molecule of citrate transported. ? The cytoplasm of EPTUM contains all the enzymes necessary for glycolysis and the citric acid (Kreb’s) cycle. ? The cytoplasmic membrane of EPTUM contains proteins that form a functional electrontransport system that utilizes oxygen as the final electron acceptor. Questions: 1. Which medium would you consider to be “non-selective” and which “selective”? Is it possible that one of the media is “differential?” (Minimum 350-word response) Non-selective and selective are terms used when describing medium. Microbiologists can mix and match agents to fine-tune a medium for any purpose. Selective medium is described as a media that contains one or more agents that hinder the growth of certain microbe or microbes. Selective media allows a certain microbe to grow by itself. This method subdues the unwanted organisms and allows for growth of the desired ones. A selective medium is 11 one that all the chemicals used are known and no yeast, animal or plant tissue is present such as in Medium 2. Some selective media contain strong inhibitory agents to favor the growth of a pathogen that would otherwise be overlooked because of its low numbers in a specimen. Examples of selective media are mannitol salt agar with is used for the isolation of staphylococcus aureus from infected material. Phenylethanol agar which is used for isolation of staphylococci and streptococci. MacConkey agar (MAC) is used to isolate gram-negative enteric. Sabourad’s agar (SAB) is media that has an acid that inhibits bacteria so it is used to isolate fungi. The list goes on and on of the medium, selective agent and what they are used for. Non-selective media is a medium in which all species will grow. It is a medium that will contain water, various salts, carbon source and a source of amino acids and nitrogen such as yeast extract as noted in Medium 1. Differential media grow several types of microorganisms but are designed to bring out visible differences amongst them. The differences that may appear in the differential media are such things as colony size or color, formation of gas bubbles and precipitates. The changes can also appear as changes in media colors too. The changes come from the types of chemicals contained in the media and the way the microbes react to them. Dyes are effective differential agents because of the pH indicators that change color in response to the production of an acid or base. So since not all bacteria will grow on the same media, the question is what bacteria grow where? Medium 2 was an environment that grew EPTUM in an aerobic situation. This would make Medium 2 a selective media. 2. Given that polyurethane is a huge polymer, why is it important that the polyurethanase is a secreted enzyme? If we assume that polyurethane is the source of energy for the organism, how can carbon atoms from the polyurethane find their way into the central metabolic pathways of the microbe? What is the “entry point”? (Minimum 400-word response) 3. Why does the growth of EPTUM in Medium 2 require oxygen? Address each of the following questions in your answer: (at least 3 full sentences per question) ? Would there be a net gain or loss of ATP in the transport of the citrate across the EPTUM membrane? Explain. ? According to the growthr observations, will glycolysis be useful for generating any ATP during growth of EPTUM on medium 2? Why or why not? ? Could glycolysis be useful for generating any ATP during growth of EPTUM on medium 1? Why or why not? ? How many ATPs can be generated during the citric acid cycle? Where would the citrate generated in the breakdown of polyurethane on Medium 2 enter the cycle? 12 ? What is the relationship between the citric acid cycle and substrate-level phosphorylation (or the electron transport system)? Can EPTUM generate any ATP via this cycle when grown on Medium 2? If it can, how many overall ATP is EPTUM capable of generating when grown on Medium 2? If it can’t, explain why. ? How many overall ATP is EPTUM capable of generating when grown on Medium 1? ? What is the importance of oxygen as it relates to the ATP tally for EPTUM on either medium 1 or medium 2? Part Four – A Little Less Conversation At a press conference announcing your company’s isolation and characterization of EPTUM, a reporter raises an important question: “How do you know that this microbe actually came from the Nearby Previously Invisible Planet (NPIP) and not from Earth? Could this be a microbe be an Earth microbe that was present in/on ELVIS before it was launched into space?” Question: 1. What kinds of experiments would need to be done to determine an answer to the reporter’s question prior to a new attempt to launch ELVIS into outer space? Briefly suggest a plan of action. (Minimum 450-word response) Why that is a very good question, how do we know that the microbe actually came from the Nearby Previously Invisible Planet and not from Earth? There are many things that would need to happen before we can attempt a new launch of ELVIS into outer space. We would first have to find a process of decontamination of the structure prior to launch. That is, if there is such a thing. The method of microbial control is called decontamination. There are many methods to decontamination such as heat or radiation, chemical agents like disinfectants and antiseptics. There are some microbial forms that constantly present in the external environment. So what is the difference in disinfection versus sterilization versus antisepsis? Disinfection is the destruction or removal of vegetative pathogens but no endospores. Sterilization is the complete removal and destruction of all viable microorganisms and antisepsis is chemicals that are applied to body 13 surfaces to destroy or inhibit vegetative pathogens. To achieve microbial death the cell is exposed to an agent that promotes cell structures to break down and the entire cell sustains irreversible damage. The permanent loss of reproductive capability even under optimum growth conditions is the definition of cell death. The plan of action to determine this would be to go back to the beginning and sample the areas of interest. In the specific case those two areas would be Earth and the NPIP. We would have to inoculate the sample into a container of medium. The inoculated media is then placed in a controlled environment to grow. After the growth, the known bacteria will be isolated out to obtain a pure culture. Then information will be gathered to see about finding a suitable way to promote cell death that is feasible and physically able. 14
