Answers to Review Questions (p. 37) 1.

Explain the apparent contradictions in defining microbiology as the study of microscopic organisms or the study of single-celled organisms. ANS: Most single-celled organisms require a microscope to be viewed, but not all. Some species are actually large enough to see with the naked eye. Many microbes form multicellular communities that render them visible. Two examples of these are mushrooms and biofilms. There are also some multicellular organisms that are microscopic, but do not fall into the category of a microorganism. 2. What is the genome of an organism? How do genomes of viruses differ from those of cellular microbes? ANS: The genome of an organism is the total genetic information contained in the organisms chromosomal DNA. For most cellular organisms it contains all the information necessary for the organisms self-replication. The genome of a virus is not always DNA. Furthermore, the viral genome does not contain all the information needed for self-replication. It relies on the cell machinery and its genome typically contains information to take over host cell processes to generate more virus particles. 3. Under what conditions might microbial life have originated? What evidence supports current views of microbial origin? ANS: The early Earth environment was composed mainly of highly reduced compounds. Living cells may have formed from spontaneous reactions sparked by UV absorption or electrical discharge. Miller found that when reduced compounds were subjected to an electrical discharge, several amino acids were observed. Or did a similar experiment and found the production of adenine. There is still debate as to where the first cells came from. Some scientists believe life has an extraterrestrial origin. 4. List the ways in which microbes have affected human life throughout history. ANS: Probably the first thing that will come to mind is a microbes disease-causing properties. Microbes have been used in food production, mining, for their insecticidal activity, and for antibiotic production, to name just a few uses. We also rely on organisms to cycle compounds such as carbon and nitrogen. 5. Summarize the key experiments and insights that shaped the controversy over spontaneous generation. What key questions were raised, and how were they answered? ANS: Spontaneous generation means that life arises spontaneously, without parental organisms. In the 1600s, Redi showed that maggots appearing on decaying meat were actually the offspring of flies. When flies could not gain access to the meat, no maggots were observed. In the 1700s, Spallanzani sterilized liquid broth and showed that no organisms could grow unless the medium was inoculated. Proponents of spontaneous generation argued that there was no growth due to lack of oxygen. In the 1800s, Pasteur created swan-necked flasks to illustrate that it was not the lack of oxygen that had prevented growth in Spallanzanis experiments. In the late 1800s, Tyndall recognized the presence of heat-resistant spores in some boiled media that resulted in the growth of microbes. 6. Explain how microbes are cultured on liquid and solid media. Compare and contrast the culture methods of Koch and Winogradsky. How did their different approaches to microbial culture address different questions in microbiology? ANS: Liquid and solid media can be identical, with the exception that agar has been added to solidify the medium in the latter case. A solid medium allows for an organism to be isolated in pure culture. Koch used a defined solid medium to isolate the causative agent of tuberculosis. This allowed the isolation of organisms that fed on organic materials.

Winogradsky studied microbes in their natural habitats. He was able to isolate organisms that fed solely on inorganic materials. The Winogradsky column is still used in labs today as a form of enrichment culture to isolate organisms of a desired nature. Koch was able to design synthetic media to culture and isolate specific organisms. Winogradsky used natures own enrichment culture in the form of a column to isolate organisms with very special traits. 7. Explain how a series of observations of disease transmission led to development of immunization to prevent disease. ANS: In Turkey, in the early 1700s, it was found that fluid from smallpox pustules could be used to immunize other people. In some cases, however, individuals contracted serious disease and became contagious. In the late 1700s, in England, Jenner used matter from cowpox lesions to immunize against smallpox. It had been recognized that milkmaids contracted cowpox and became mildly sick, but were then seemingly immune to smallpox. Pasteur then used attenuated, or weakened, viruses for immunization. Ultimately, it was discovered that one can use simply a molecular component of a pathogen to generate immunity. 8. Summarize key historical developments in our view of microbial taxonomy. What attributes of microbes have made them challenging to classify? ANS: Microscopy allowed for the visualization of microorganisms. Through the development of staining techniques and more sensitive forms of microscopy, we were able to begin categorizing organisms. With the advent of the analysis of various metabolic pathways, we were able to further categorize them. Ultimately, with the ability to sequence genomes, or partial genomes, taxonomic classification has gone to new levels. Some organisms have been renamed or moved to different locations on the phylogenetic tree. Many organisms are difficult to culture; in fact, only a very small percentage of organisms have been identified and sequenced. 9. Explain how various discoveries in natural bacterial genetics were used to develop recombinant DNA technology. ANS: Griffith first observed transformation when some material from dead bacteria caused previously harmless bacteria to turn into a deadly form. Approximately 15 years later, Avery identified the transforming material as DNA. It was found that organisms contained restriction endonucleases that cut DNA at specific sequences. These enzymes have been used to cut and paste DNA to make recombinant DNA for genetic transfer of information between organisms. Viruses, in their entirety or in part, may be used to transfer information into an organism. Answers to Review Questions (p. 112) 1. What are the major features of a bacterial cell, and how do they fit together for cell function as a whole? ANS: The bacterial cell has a relatively small genome that is condensed within a region called the nucleoid. It contains very little, if any, noncoding or extraneous DNA. The cytoplasm is packed with 70S ribosomes. Depending on the genus, there are various subcellular components that coordinate cell function. The outer envelope of the cell consists of the cell membrane, which is surrounded by a cell wall in most bacteria. Together they protect the cell, regulate exchange with its surroundings, and are involved in communication with other cells. 2. What fundamental traits do most prokaryotes have in common with eukaryotic microbes? What traits are different? ANS: Overall, they are very similar. The traits stated in question 1 could also be stated for eukaryotes, except the eukaryotes have more noncoding DNA and the genetic material is housed in a membrane-bounded nucleus. Whereas the cell membranes have similar structures and functions, some of their chemical makeup is very different. Cell walls, if present, are also chemically distinct. Eukaryotes also contain other organelles, which are membrane-bound, highly specialized entities.

3. Give examples of how our views of ribosome structure and function have emerged from microscopy, cell fractionation, X-ray diffraction crystallography, and genetic analysis. Explain the advantages and limitations of each technique. ANS: Microscopy gave us a general view of the ribosome, but no chemical or physical specifics. Cell-fractionation enabled us to learn that the ribosome consists of a small and a large subunit. By analyzing fractions, we detected polysomes. We can also use fractions in cell-free expression systems. This technique cannot address processes requiring cellular integrity. X-ray diffraction crystallography was used to determine the structure of the 30S ribosome with all of its ribosomal RNA (rRNA) and protein components. It also enabled discovery of the three transfer RNA (tRNA) binding sites, A, P, and E. Again, this only works on isolated entities whose full function cannot be observed. The actual function of cellular components can be dissected by genetic analysis. Genetic analysis, in conjunction with X-ray crystallography, enabled discovery of how the antibiotic streptomycin specifically inhibits protein synthesis. 4. Outline the structure of the peptidoglycan sacculus, and explain how it expands during growth. Cite two different kinds of experimental data that support our current views of the sacculus. ANS: The peptidoglycan sacculus is a single interlinked molecule that encloses the entire cell. It consists of parallel polymers of disaccharides called glycan chains cross-linked with peptide side chains. The layers are alternating units of N-acetylglucosamine and N-acetylmuramic acid, forming a large sheet. Layers of these sheets are held together by peptide side chains. Peptide extensions can form cross-bridges connecting parallel strands of glycans. In essence, the sacculus is a huge mesh bag, holding the cell together. Cell wall expansion and septation can be observed by EM and further dissected by using fluorescence microscopy to follow specific proteins during the growth and septation process. Genetic mutants have been used in conjunction with fluorescence microscopy to reveal an even greater understanding of the process. 5. Compare and contrast the structure of gram-positive and gram-negative cell envelopes. Explain the strengths and weaknesses of each kind of envelope. ANS: The gram-positive cell has only two layers, the cell membrane and the cell wall. The cell wall contains multiple layers of peptidoglycan. It also contains teichoic acids, which are not found in gram-negative cells. Gram-positive cells have much more structural integrity than gramnegative cells because of the thick, highly cross-linked peptidoglycan. A gram-negative cell can be considered to have four components. It has a cell membrane or inner membrane, peptidoglycan, periplasm, and outer membrane. The cell membranes of grampositive and gram-negative cells are very similar. The gram-negative cell wall has a very thin peptidoglycan layer that does not contain teichoic acids. The periplasm is the region bounded by the inner and outer membranes. It contains specialized proteins and enzymes. The outer membrane is a bilayer composed of phospholipids and LPS. It also contains transport proteins called porins. The outer membrane prevents uptake of certain toxic molecules and allows growth in harsher environments. The LPS acts as an endotoxin and the O-polysaccharide component helps the bacteria resist phagocytosis by white blood cells. Gram-positive and gram-negative cells are quite often covered by a slippery capsule composed of polysaccharides. Its presence may be observed by negative staining. Organisms freshly isolated from their natural environment may also possess a protein or glycoprotein surface layer, called an S-layer. 6. Outline the process of DNA replication, and explain how it is coordinated with cell wall septation. ANS: DNA replication begins at the origin of replication and proceeds bidirectionally, forming two copies, one for each progeny. A replication fork is propagated by helicase, which unwinds the DNA. Primase generates RNA primers necessary for synthesis to begin. DNA polymerase then begins the synthetic process. DNA polymerase, with its accessory proteins, forms a replisome, which includes two DNA polymerases. One polymerase replicates the leading strand and the other the lagging strand. Two replisomes are required since replication is bidirectional.

One cell becomes two daughter cells, each containing a complete copy of the chromosome. This entails coordination of septation and replication. As the replication termination site is replicated, septum growth is triggered. 7. Explain how DNA transcription to RNA is integrated with translation and protein processing and secretion. ANS: Since there is no nucleus in a prokaryotic cell, replication, transcription, and translation all occur within the same compartment, the cytoplasm. As soon as a ribosome binding site is transcribed, a ribosome binds and begins the translation process. Multiple ribosomes may translate one message. This is referred to as a polysome. Chaperones bind to proteins and ensure correct folding of the proteins as they are in the process of being synthesized. Proteins destined to be secreted bear a signal recognition sequence. Signal recognition particles (SRPs) bind to these proteins as they are being synthesized and ensure correct processing and secretion. 8. What kinds of subcellular structures are found in certain cells with different functions, such as magnetotaxis or photosynthesis? ANS: Magnetotactic organisms contain magnetosomes. These are magnetite-containing sacs found in anaerobic aquatic organisms. The magnetosomes orient the organism along the magnetic field. This directs them to the bottom of the water source, where less oxygen is present. Photosynthetic organisms contain phycobilisomes and thylakoids. Phycobilisomes are the light-harvesting complexes. The thylakoids are folded sheets packed with photosynthetic proteins and electron carriers. It is here that the light reaction of photosynthesis occurs. Carboxysomes are present for carbon dioxide fixation and gas vesicles keep the organisms higher in the water, closer to the needed light. 9. Compare and contrast bacterial structures for attachment and motility. Explain the molecular basis of chemotaxis. ANS: Flagella are used for motility. They tend to be longer than appendages for attachment, and they rotate like a propeller, at the expense of a proton gradient, to afford movement. They are also a quite complex structure composed of many different proteins. The most common structures for attachment are pili, composed of protein monomers of pilin. There are pili for attachment and pili for sex, or exchange of genetic information. Chemotaxis is a mechanism that affords an organism the ability to swim toward attractants and away from repellents. Simplistically, receptors sense a compound, and the receptor is either phosphorylated or dephosphorylated, which sends a signal to the flagella to rotate in either one direction or the other. This leads to swimming or tumbling, allowing the organisms to react to chemicals in its environment. Answers to Review Questions (p. 147) 1. What nutrients do microbes need to grow? ANS: Microbes require specific macronutrients including carbon, nitrogen, phosphorous, hydrogen, oxygen, sulfur, and the cations magnesium, iron, potassium, and calcium. They also require micronutrients such as cobalt, copper, manganese, nickel, and zinc. These usually contaminate glassware, so they rarely have to be added to the growth medium. The organisms may also require very specialized growth factors. 2. Explain the differences between autotrophy, heterotrophy, phototrophy, and chemotrophy. ANS: Heterotrophs obtain organic compounds from other organisms. They disassemble the carbon sources producing energy and then reassemble them to make cell constituents. When the entire molecule is oxidized, carbon dioxide is given off in the process. Autotrophs fix carbon dioxide to make complex cell constituents such as carbohydrates. Phototrophs obtain energy through the absorption of light. Chemotrophs obtain energy from oxidation/reduction reactions that transfer electrons from high-energy compounds to make products of lower energy.

Organisms may be phototrophs, chemoautotrophs, photoheterotrophs, or chemoheterotrophs. 3. Explain the basics of the carbon and nitrogen cycles. ANS: Carbon cycle: The carbon cycle requires both autotrophs and heterotrophs. The heterotrophs degrade complex organic compounds such as polysaccharides to monomers, which are then broken down to smaller compounds and, when completely oxidized, to carbon dioxide. The photosynthetic and lithotrophic autotrophs produce energy, which is used to fix carbon dioxide and produce glucose. Nitrogen cycle: Here bacteria and archaea collaborate to interconvert nitrogen gas, ammonium ions, and nitrate ions. Certain bacteria and archaea are able to convert atmospheric nitrogen to ammonia, which other organisms can then use for synthesis of proteins and other essential compounds. Nitrifying bacteria oxidize ammonia to produce nitrate. Denitrifying bacteria can use the nitrate to produce a series of compounds, ultimately resulting in the production of nitrogen gas. 4. Describe the various mechanisms of transporting nutrients in prokaryotes and eukaryotes. What are facilitated diffusion, coupled transport, ABC transporters, group translocation, and endocytosis? ANS: Facilitated diffusion is transport down a concentration gradient until equilibrium is reached. The aquaporin family of transporters is responsible for transport of water; small polar molecules such as glycerol also cross the membrane by facilitated diffusion. The transporter binds glycerol and changes shape to deliver it to the other side of the membrane. Coupled transport can move something against a gradient, but transport is coupled to ion movement. Symporters transport two things in the same direction. Antiporters transport ions in opposite directions. If a charge differential is created during transport it is termed electrogenic. ABC transporters (ATP Binding Cassette transporters) are powered by ATP and have been found in organisms from all three domains. These are used in both uptake and efflux systems. The uptake systems require a substrate-binding protein (SBP) that delivers the solute to the ABC transporter. The efflux system is generally used to transport a wide range of antibiotics, unfortunately resulting in antibiotic-resistant organisms. Group translocation, also referred to as the phosphotransferase system (PTS), is a well-studied system for the transport of some sugars. Phosphoenolpyruvate (PEP) begins the process by donating its phosphate to transport the sugar across the membrane. You cannot say that transport occurs against the concentration gradient because glucose, for example, is on the outside and glucose-6-phosphate is on the inside. Group translocation occurs only in prokaryotes. Endocytosis is a form of transport used only in eukaryotes. Regions of the cell membrane surround fluid or suspended particles and pinch off to form membrane-enclosed vesicles called endosomes. Nutrients trapped in these vesicles are brought into the cell. 5. Why is it important to grow bacteria in pure culture? ANS: It is essential to have a pure culture for many types of study. A pure culture represents numerous organisms, but all are the same. Any type of analysis with pure culture will yield a result specific to that particular organism. 6. Under what circumstances would you use a selective medium? A differential medium? ANS: A selecti e medium contains components that inhibit some groups of organisms, but favor others. For example, there are selective media that suppress the growth of gram-positive organisms. Using this strategy you can take a mixed culture and end up favoring only the growth of gram-negative organisms. A differential medium allows one to distinguish between types of organisms based on their metabolic activities resulting in different growth characteristics. Some media are a combination of both. MacConkey agar selects for gram-negative organisms and differentiates between lactose fermenters and lactose nonfermenters. Lactose fermenters will appear as red colonies on this medium.

7. What are the factors that define the growth phases of bacteria grown in batch culture? ANS: !ag: The characteristic of this phase is determined by the difference between the medium/environment the culture was grown in versus that to which it is transferred. A change in pH, temperature, or nutrient source requires additional gene synthesis to deal with the changes, and hence the lag period will be longer. Early log: Cells have retooled and can now grow exponentially. Cells grow and divide at the maximum rate, and the curve is linear. !ate log: As cell density increases, quorum sensing occurs and the cells undergo a lower doubling rate. This also requires synthesis of a new set of genes. "tationary: When nutrients become limiting or toxins build up, the growth curve levels off. Cell replication and cell death are equal. Many bacteria synthesize new components to strengthen themselves and become more resistant to environmental changes. #eath: The rate at which cells die is exponential. Some cells mutate and are able to survive longer than others. 8. Describe the important features of biofilms. ANS: Biofilms may be composed of a single species or collaborating species. Cells attach to a surface and begin to replicate. As their numbers increase they communicate by a chemical signaling process called quorum sensing. The bacteria secrete a thick extracellular matrix of polysaccharide. Channels are maintained in the biofilms, and nutrients flow through them. Genes expressed by sessile biofilm bacteria increase antibiotic resistance. Anaerobic processes occur in some biofilms, which indicates that anaerobic environments can be maintained in an otherwise aerobic environment. 9. Name three kinds of bacteria that undergo differentiation, and give highlights of the differentiation processes. ANS: The progeny of Caulobacter crescentus include a swimming form and a holdfast form. Only the holdfast form can replicate, so prior to replication the swimming form converts to the holdfast form. Some gram-positive organisms, such as Clostridium botulinum, the causative agent of botulism, are capable of spore formation. When conditions for vegetative growth become poor, the cell begins the sporulation process. An asymmetrical division process begins, which ultimately produces a forespore within the mother cell. The mother cell is destroyed over time and the endospore is ultimately released as a mature spore. It remains in this dormant, highly resistant form until conditions improve, at which time it germinates, converting to a vegetative cell that can then replicate and divide. $y%ococcus %anthus has a gliding form, which mobilizes using special forms of pili. The cells signal each other, coming together to form a motile, slug-like, community. As nutrients become depleted, the community becomes a large fruiting body. Within the body the cells differentiate into spherical spores, which will ultimately be released into the environment. Other examples are noted in the text, so answers may vary.

Answers to Review Questions (pp. 179180) 1. Explain the nature of extremophiles and discuss why these organisms are important. ANS: Extremophiles live in extreme environments. Some examples of extreme environments are high temperature, extremely high or low pH, and even high salt. These organisms have adapted to these conditions and even require them. They are important because we can use their enzymes, and they help us understand how cells work. 2. What are the parameters that define any growth environment? ANS: The parameters are temperature, pH, osmolarity, oxygen, and pressure, as well as nutrient availability.

3. List and define the classifications used to describe microbes that grow in different physical growth conditions. ANS: &emperature: Classification is based on optimum temperature for growth. Above 80C hyperthermophile; 5080Cthermophile; 2040Cmesophile; less than 20C psychrophile pH: Optimum pH for growth: pH > 9alkaliphile; pH 58neutralophile; pH < 5 acidophile 'smolarity: The concentration and type of solute molecules required for growth: halophile (> 2M NaCl) '%ygen: growth only in oxygenaerobe, growth with or without oxygenfacultative, growth only in reduced oxygen concentrationsmicroaerophile, growth only without oxygen anaerobe Pressure: growth at pressures above 380 atmbarophile 4. What do thermophiles have to do with the PCR reaction? ANS: It was from the thermophile &hermus a(uaticus that Taq polymerase was isolated. Taq polymerase is used in the PCR reaction since it can withstand the high temperatures required repeatedly during the cycling steps. 5. Why is water activity important to microbial growth? What changes water activity? ANS: Water activity relates to how much water is available to the organism. The more solutes there are in a solution, the less water is available for microbes to use for growth. 6. How do cells protect themselves from osmotic stress? ANS: Simple diffusion is not the primary means for movement across the cell membrane. Aquaporins are special membrane channels that transport water much faster than could simple diffusion. This alleviates the osmotic stress caused by differences in osmolarity across the membrane. In a hypertonic medium cells also have the capacity to synthesize or import compatible solutes to increase the internal osmolarity. When cells are immersed in a hypotonic medium, the internal pressure increases and they need to lower the internal osmolarity to compensate. Pressure-sensitive (mechanosensitive) channels are activated at these increased pressures, and solutes are allowed to escape, thereby lowering internal osmolarity. 7. Why do changes in H+ concentration affect cell growth? ANS: Enzymes, regardless of the pH at which the organism from which they were isolated thrives, tend to operate best at a pH between 5.0 and 8.5. The cells must have a mechanism to maintain a relatively neutral intracellular pH, even when the extracellular environment is well outside that range. 8. How do acidophiles and alkaliphiles manage to grow at the extremes of pH? ANS: Acidophiles have very different membrane lipids, containing high levels of tetraether lipids. This feature decreases proton permeability of the membrane. They also have mechanisms to pump protons out of the cell. Alkaliphiles possess a membrane composed of diether lipids, which add to its stability and prevent protons from leaking out of the cell. Most alkaliphiles have evolved to utilize a sodium motive force versus the traditional proton motive force, since there is a shortage of available protons. 9. Because an organism can live in an oxygenated environment, does that mean that the organism uses oxygen to grow? Because an organism can live in an anaerobic environment, does that mean it cannot use oxygen as an electron acceptor? Why or why not? ANS: A strict aerobe is an organism that not only is able to grow in the presence of oxygen, but uses it as a terminal electron acceptor. Some organisms can exist in oxygenated environments, but do not need it to grow.

A strict anaerobe does not have the capacity to utilize oxygen as a terminal electron acceptor and are killed by reactive oxygen species that form when they are exposed to oxygen. E) coli, however, is a facultative aerobe. It has the capacity to use oxygen as a terminal electron acceptor when it is in an aerobic environment, but also can undergo fermentative growth if it should find itself in an oxygen-poor, or anaerobic, environment. 10. What happens when a cell exhausts its available nutrients? ANS: One of the first things that happens is that signal molecules are produced. These molecules are involved in global control of gene expression. Genes are turned on to produce transport systems for alternative nutrients. This happens even if the matching substrate is not present. The organism may also begin to store glycogen as a potential backup reserve. As conditions get worse, the organism will proceed to ready itself for other stresses, like temperature or pH extremes. 11. List and briey explain the various means by which humans control microbial growth. What is a D-value? What is a phenol coefficient? ANS: There are physical, chemical, and biological methods to control microbial growth. Physical methods: The steam autoclave utilizes high temperature created by increased pressure to effectively sterilize liquids and solids. Pasteurization is performed by heating liquids to a temperature that kills pathogens but does not destroy the food product. Filtration can be used to remove cells from solutions or air. Filters with pore sizes of 0.2 micrometers can remove microbial cells, but not viruses. Irradiation is a strategy for sterilizing food after harvesting. Chemical methods: Various chemicals can be used to control growth. Phenol is the benchmark against which other disinfectants are measured in a phenol coefficient test. The results are compared to phenol and a phenol coefficient is determined. The higher the value, the more effective is the chemical. Biological methods: Microbes make chemical compounds that control growth of other microbes. These compounds are referred to as antibiotics. The D-value is obtained from a microbial death curve. The D-value corresponds to the time it takes to kill 90% of the microbial population. 12. How do microbes prevent the growth of other microbes? ANS: Some microbes prevent the growth of others by producing antibiotics. The simple presence of some organisms may be enough to slow the growth of others. Sometimes this is simply accomplished by competition for nutrients, other times it is a bit more complex. Sometimes organisms make changes to the environment that retard the growth of other organisms. Phage therapy was also introduced. A bacteriophage can slow growth or kill its target bacterium, depending upon the mode of infection it employs.

Answers to Review Questions (p. 216) 1. Compare and contrast the form of icosahedral and filamentous (helical) viruses, citing specific examples. ANS: *cosahedral iruses: Icosahedral viruses have twenty identical triangular faces. This affords the capsid three axes of rotational symmetry. The different axes have either twofold, threefold, or fivefold symmetry. The icosahedral capsid of herpes virus contains spooled DNA. Filamentous iruses: The filamentous viruses are also symmetrical, but they are helical in nature. The genome is coiled within the capsid. Whereas icosahedral viruses have a fixed-size capsid, the helical capsids can increase in length to incorporate a longer genome. Examples of filamentous viruses are bacteriophage M13 and the Ebola virus. 2. How do viral genomes gain entry into cells in bacteria, plants, and animals?

ANS: A virus must either insert its genome into a host or disassemble within the host so that the viral genome can be replicated. Whether a virus is infecting a bacterium or an animal cell, its interaction with its host is very specific. Some viruses have tail fibers that are responsible for attachment and injection of the genome into the host. Other viruses have glycoprotein spikes that interact with the viral host, initiating viral uptake. Both enveloped and naked animal viruses can be engulfed into the cell by endocytosis. Some enveloped viruses uncoat at the cell membrane by fusing their envelope with the host cell membrane. Plant viruses do not involve specific membrane receptors. They access the cell interior through physical damage to the tissue or are inoculated by a feeding insect. 3. Explain the structure and function of the seven Baltimore groups of viral genomes. ANS: The Baltimore classification of viruses is based upon not only the specific makeup of the viral genome, but also on the route used to generate messenger RNA (mRNA). This system of classification identifies seven fundamental groups of viral species. Double-stranded (ds) DNA viruses that utilize either their own or the hosts DNAdependent RNA pol to make mRNA. II. Single-stranded (ss) DNA viruses first utilize the hosts DNA pol to generate dsDNA. The hosts DNA-dependent RNA pol is then utilized to make mRNA. III. Double-stranded RNA viruses require a viral RNA-dependent RNA pol to make mRNA. IV. Positive (+) sense single-stranded RNA viruses do not require enzymes to make mRNA since the virus itself serves directly as mRNA. V. Negative (-) sense single-stranded RNA viruses require a viral RNA-dependent RNA pol to make mRNA. VI. RNA reverse-transcribing viruses or retroviruses. This group of viruses, like HIV, has a (+) strand RNA genome. A viral RNA-dependent DNA pol, reverse transcriptase, is used to generate dsDNA. The DNA is then inserted into the host genome where it can then be expressed using host DNA-dependent RNA pol. VII. DNA reverse-transcribing viruses, or pararetroviruses. The animal virus hepatitis B has a dsDNA genome that is first transcribed into RNA. A viral RNA-dependent DNA pol then generates more viral progeny. The plant viruses that fall into this category behave somewhat differently. They make an RNA intermediate that replicates using a plantencoded RNA-dependent DNA pol. Interestingly, many plants contain the genetic information to encode reverse transcriptase. 4. How do viral genomes interact with host genomes, and what are the consequences for host evolution? ANS: Some viruses integrate into the host chromosome. When they commence the lytic cycle they sometimes accidentally pick up some of the host genome. This is packaged into the capsid and exits the cell upon lysis. Upon infecting another cell, genetic information can be transferred. This genetic exchange or transfer, of course, could lead to evolutionary changes. 5. Compare and contrast the lytic, lysogenic, and slow-release life cycles of bacteriophages. What are the strengths and limitations of each? ANS: The lytic life cycle results in propagation of many viruses at the expense of the host as it lyses. A lysogenic life cycle ensures that a single copy of the viral genome will be replicated along with the host genome. This could result in a large population of host cells able to produce large amounts of the virus when it enters the lytic life cycle after some form of stress is detected by the host. The slow-release cycle also ensures plenty of host to produce more viral progeny, since the host does not lyse upon release of the virus. 6. Compare and contrast the life cycles of RNA viruses and DNA viruses in animal hosts. What are the strengths and limitations of each? I.

ANS: DNA viruses have the advantage of being able to use some or all of the host replication machinery. For most DNA viruses, the DNA genome enters the nucleus, where it is transcribed. Just as the hosts transcripts enter the cytoplasm for translation, so do the viral transcripts. The capsid proteins are, of course, synthesized in the cytoplasm, but then they are returned to the nucleus for assembly of the virion. Poxviruses are the only DNA viruses that replicate entirely in the cytoplasm. The life cycle for most RNA viruses occurs in the cytoplasm. The virus is either packaged with or encodes an RNA-dependent RNA polymerase. The capsid self-assembles in the cytoplasm, where packaging occurs. If the virus contains an envelope, the envelope proteins are produced in the cytoplasm and are then transported to the cell membrane. As the viruses bud off the host cell it surrounds itself with the viral envelope. The RNA retroviruses come packaged with their own reverse transcriptase, which makes a double-stranded DNA from the RNA genome. The DNA copy is transported into the nucleus, where it is integrated into the host chromosome. A host RNA pol will transcribe viral mRNA viral progeny and new RNA genomes. Translation occurs in the cytoplasm, where virions will self-assemble and bud out. 7. Explain the plate titer procedure for enumerating viable bacteriophages. How must this procedure be modified to titer animal viruses? Oncogenic viruses? ANS: A plaque assay for lytic bacteriophage is performed by plating a diluted suspension of bacteriophage with bacterial cells in soft agar, which is poured over a nutrient agar base layer. The bacterial cells will form a confluent lawn of cells over the surface of the plate. A plaque or clear zone is observed when a single virus infects a cell, replicates, and lyses the cell, releasing progeny to infect and kill adjacent cells. The number of plaques observed corresponds to the number of viruses in the given suspension of liquid culture. A phage particle is referred to as a plaque-forming unit (PFU). Animal viruses can be measured by plaque assay. The virus is added to tissue culture and allowed to attach to cells. The liquid is then removed and replaced with a gel medium, which will prevent the dispersal of viruses that are released from infected cells. Animal viruses that do not lyse cells can be detected using fluorescently labeled antibodies that will bind to the focus of virus-infected cells. When working with an oncogenic virus, the foci are referred to as transformed foci. They are very evident in that they have lost contact inhibition and grow in piles. 8. Explain the generation of the step curve of virus proliferation. Why is virus proliferation generally observed as a single step, or generation, in contrast to the life cycles of cellular microbes, outlined in Chapter 4? ANS: If the multiplicity of infection (MOI) is relatively high, all cells will be infected and you can observe the so-called one-step growth curve of bacteriophage. The viral titer in solution will drop to nearly zero after the initial infection due to the fact that they will all be absorbed to the host surface. There will then be a latent period when the virus is replicating in the cell. The first part of the latent period is the eclipse period. Eclipse ends when the first infectious virion is formed within the cell. The latent period continues as viruses accumulate and ends with the release of the first progeny viruses into the medium. There will be a rise period as viruses are being released from all infected cells, and then it will plateau. The burst size corresponds to the number of phages per ml (PFUs per ml) divided by the initial concentration of cells used for infection. In one-step, or one cycle, a cell infected by one virus releases many viral progeny. This differs from the exponential growth of bacteria. Each bacterial cell grows and divides by binary fission to generate two progeny. 9. Explain the key contributions of viruses to natural ecosystems. What may happen in an ecosystem where viruses are absent or fail to cause significant infection? ANS: Viruses play two very important roles in natural ecosystems. They limit host population density. As the host population increases, transmission of virus increases. This ultimately will lead to a situation where the virus will limit the host density without extinction of the host.

Every virus has a specific host. All viruses will maintain a specific host population density, so this ensures that one species will not dominate. If an ecosystem did not have any viruses, the organisms would reproduce unchecked. This would result in an avalanche of events, all of which would be problematic. Also, viruses play an important role in carbon cycling in the marine environment, so carbon would be depleted without viral participation.